Causative agents of anaerobic infections. LAboratory diagnosis of anaerobic infections

Causative agents of zoonose diseases.  laboratory  diagnosis of plague, other yersiniosis.  laboratory diagnosis of brucellosis,  tularaemia, and anthrax.

 

 

Causative agents of anaerobic infections. LAboratory diagnosis of anaerobic infections

 

Clostridia Responsible for Anaerobic Infections. Anaerobic infections (gas gangrene) are polybacterial. They are caused by several species of clostridia in association with various aerobic microorganisms (pathogenic staphylococci and streptococci).

The organisms responsible for anaerobic infections are: (1) Cl. perfringens, (2) Cl. novyi, (3) Cl. septicum, (4) C. histolyticum, and (5) Cl. sordellii. Cl. chauvoei, Cl. fallax, and Cl. sporogenes are pathogenic for animals. Cl. aerofoetidum and Cl. tertium are non-pathogenic organisms which have significance in the pathogenesis of anaerobic infections only in association with pathogenic bacteria.

 

Cl. perfringens                                                 Cl. novyi

 

 

Cl. septicum                                             C. histolyticum

 

Anaerobic infections may be caused by any one of the first four species mentioned above but usually several members of a parasitocoenosis acting in a particular combination are responsible for them. The less pathogenic and non-pathogenic species cannot be responsible for anaerobic infections by themselves, but they cause tissue destruction, lower the oxidation-reduction potential, and thus create favourable conditions for the growth of pathogenic species.

 

Clostridium perfringens. The causative agent was discovered in 1892 by W. Welch and G. Nut-tall. This organism occurs as a commensal in the intestine of man and animals. Outside of the host’s body it survives for years in the form of spores. It is almost always found in the soil. The organism was isolated from 70-80 per cent of anaerobic infection cases during World War I, and from 91-100 per cent of cases during World War II.

Morphology. Cl. perfringens is a thick pleomorphous non-motile rod with rounded ends 3-9 mcm in length and 0.9-1.3 mcm in breadth (Fig.). In the body of man and animals it is capsulated, and iature it produces an oval, central or subterminal spore which is wider than the vegetative cell. Cl. perfringens stains readily with all aniline dyes and is Gram-positive but in old cultures it is usually Gram-negative.

 

Figure. Pure culture and colonies of Clostridium perfringens

 

Electron microscopy demonstrates a homogeneous cell wall with no clearly demarcated layers. The cytoplasmatic membrane consists of one layer, the cytoplasm is granular and contains ribosomes and polyribosomes. The nucleoid is in the centre of the cell. Spore formation begin safter 3 to 3.5 hours of growth, the spores are enclosed by sporangia. The G+C content in DNA ranges from 24 to 27 per cent.

Cultivation. Cl. perfringens is less anaerobic than the other causative agents of anaerobic infections. It grows on all nutrient media which are used for cultivation of anaerobes. The optimum temperature for growth is 35-37 °0 (it does not grow below 16 and above 50 °C), and optimal pH of medium is 6.0-8.0. A uniform turbidity and large volumes of gas are produced in cultures grown on Kitt-Tarozzi medium.

 

 

Brain medium is not blackened (Tabl. 1). The colonies resemble discs or lentils deep in agar stabcultures (see Fig. 1). On blood agar containing glucose smooth disc-like grey colonies are formed, with smooth edges and a raised centre.

Many strains of Cl. perfringens lose their anaerobic properties on exposure to antibiotics, bacteriophage, and X-rays and may be cultivated under aerobic conditions. Catalase and peroxidase, enzymes typically present in aerobic organisms, were revealed in the variants thus obtained. The aerobic variants are non-toxic and non-pathogenic for laboratory animals.

Fermentative properties. Cl. perfringens slowly liquefies gelatin, coagulated blood serum and egg albumen (Tabl.). The organism reduces nitrates to nitrites and normally no indole or only traces are produced. Volatile amines, aldehydes, ketones, and acetyl methyl carbinol, are produced. Milk is vigorously coagulated and a sponge-like clot is formed. In meat medium the organism yields butyric and acetic acids and large quantities of gases (CO₂ H₂, H₂S, NH₃). It ferments glucose, levulose, galactose, maltose, saccharose, lactose, starch, and glycogen with acid and gas formation. Mannitol is not fermented.

 

Toxin production. The organism produces a toxin which has a complex chemical structure (lethal toxin, haemotoxin, neurotoxin, and necrotic toxin). The toxins and enzymes produced by the various species of the gas gangrene group are similar from one species to another. Actually, many of them have not been purified or characterized, and are grouped together under the general name lethal toxins. The products produced by C perfringens have received the most study: at least 12 different toxins and enzymes have been described and labeled with Greek letters (Table), but not all serologic strains of C perfringens produce all 12 products or even similar quantities of certain toxins and enzymes.

Table

Toxins and Toxigenic Types of Clostridium perfringens

Note: “+++” – most strains, “++” – some strains, “+” – a few strains,  “–“ – not produced

 

The most extensively studied toxin is the alpha-toxin, a phospholipase-C (lecithinase) that hydrolyzes the phospholipid, lecithin, to a diglyceride and a phosphorylcholine. Because lecithin is a component of cell membranes, its  hydrolysis can result in cell destruction throughout the body. Lecithinase C acts as digestant enzyme in human intestine.Another toxin produced by this group is the m, toxin, a lethal hemolytic product characterized by its effect on the heart—more precisely, its cardiotoxic properties. C. perfringens type E is the only one of this group to produce the iota (i) toxin, which is believed to be responsible for an acute enterotoxemia in both domestic animals and humans. iToxin is a binary product in which two nonlinked proteins are required for activity. One molecule binds to a cell (iota–b), functioning as a receptor to transport the active toxin molecule (iota–a) across the membrane. Like botulism C2 toxin, i toxin will ADP–ribosylate poly L–arginine and skeletal muscle and nonmuscle actin, but its true substrate within the cell is unknown. Other toxic enzymes produced by the gas gangrene group include a collagenase that hydrolyzes the body’s collagen; a hyaluronidase; a fibrinolysin, which breaks down blood clots; a DNase; and a neuraminidase, which can remove the neuraminic acid from a large number of glycoproteins. With such an array of toxic sub–stances, it is no wonder that gas gangrene was one of the major causes of death in the American Civil War, and, undoubtedly, in many other wars.

 

Lecithinase production

 

Due to such a complex of toxic substances and enzymes Cl. perfringens is capable of causing rapid and complete necrosis of muscular tissue. This process is the result of a combined effect of lecithinase, collagenase, and hyaluronidase on the muscles. Collagenase and hyaluronidase destroy the connective tissue of the muscles, and lecithinase C splits lecithin, a component in the muscle fibre membranes. Haemolysis in anaerobic infection is due to the effect of lecithinase on lecithin of the erythrocyte stroma. The animal dies from rapidly developing asphyxia which is the result of intensive erythrocyte destruction and disturbance of the nerve centres.

In addition to battlefield casualties, automobile and farm equipment accidents also may cause traumatic wounds resulting in gas gangrene. Also, because C. perfringens can be part of the normal flora of the female genital tract, induced abortions may result in uterine gas gangrene.

Clostridia may also cause a diffuse spreading cellulitis accompanied by an overwhelming toxemia. Such infections probably originate from the large intestine, either from a bowel perforation or from a contaminated injection site. Gas may be produced, but the cellulitis differs from the classic gas gangrene in that muscle necrosis is not involved.

Antigenic structure and classification. Six variants of Cl. perfringens are distinguished: A, B, C, D, E, and F. These variants are differentiated by their serological properties and specific toxins.

Variant A is commonly found as a commensal in the human intestine, but it produces anaerobic infections when it penetrates into the body by the parenteral route. Variant B is responsible for dysentery in lambs and other animals. Variant C causes hemorrhagic enterotoxaemia in sheep, goats, sucking pigs, and calves. Variant D is the cause of infectious enterotoxaemia in man and animals, and variant E causes enterotoxaemia in lambs and calves. Variant F is responsible for human necrotic enteritis.

Resistance. The spores withstand boiling for period of 8 to 90minutes. The vegetative forms are most susceptible to hydrogen peroxide, silver ammonia, and phenol in concentrations commonly employed for disinfection.

Pathogenicity for animals. Among laboratory animals, guinea pigs, rabbits, pigeons, and mice are most susceptible to infection. Postmortem examination of infected animals reveals oedema and tissue necrosis with gas accumulation at the site of penetration of the organism. Most frequently clostridia are found in the blood.

 

Clostridium novyi. The organism was discovered by F. Novy in 1894. Its role in the aetiology of anaerobic infections was shown in 1915 by M. Weinbergand P. Seguin. It ranks second among the causative agents of anaerobic infections. Soil examination reveals the presence of the organism in 64per cent of the cases.

Morphology. Cl. novyi is a large pleomorphous rod with rounded ends, 4.7-22.5 mcm in length and 1.4-2.5 mcm in width, and occurs often in short chains (Fig.). The organism is motile, peritrichous, and may possess as many as 20 flagella. It forms oval, normally subterminal spores in the external environment. In the body of man and animals it is non-capsulated. The organism is Gram-positive. The G+C content in DNA amounts to 23 per cent.

 

Figure. Pure culture and deep colonies of Clostridium novyi

 

Cultivation. C/. novyi is the strictest of the anaerobes. Its optimal growth temperature is 37-45 C (growth temperature ranges from 16 to 50 C), and optimal pH of medium is 7.8. Growth on Kitt-Tarozzi medium is accompanied by gas accumulation, precipitation, and clearance of the medium. On sugar-blood agar the colonies are rough, raised in the centre, and have fringed edges surrounded by zones of haemolysis. In agar stab cultures the organisms produce flocculent colonies with a dense centre from which thin filaments grow outwards.

Fermentative properties. The organisms slowly liquefy and blacken gelatin. They coagulate milk, producing small flakes. Glucose, maltose, and glycerin are fermented with acid and gas formation. Acetic, butyric, and lactic acids as well as aldehydes and alcohols are evolved as a result of the breakdown of carbohydrates.

Toxin production. Cl. novyi A produces alpha, gamma, delta, and epsilon toxins; Cl. novyi B produces alpha, beta, zeta, and eta toxins. Cl.novyi C is marked by low toxigenicity. In cultures Cl. novyi liberates active haemolysin which possesses the properties of lecithinase.

Antigenic structure and classification. Cl. novyi is differentiated into four variants A, B, C and D. Variant A is responsible for anaerobic infections in man, and type B causes infectious hepatitis, known as the black disease of sheep. Variant C produces bacillary osteomyelitis in buffaloes, and variant D is responsible for haemoglobinuria in calves.

Resistance. Spores survive iature for a period of 20-25 years with-out losing their virulence. Direct sunlight kills them in 24 hours, boiling destroys them in 10-15 minutes. Spores withstand exposure to a 3 percent formalin solution for 10 minutes. Coal-tar is an extremely active disinfectant.                  

Pathogenicity for animals. Cl. novyi causes necrotic hepatitis (black disease) in sheep. In association with non-pathogenic clostridia it produces bradsot (acute hemorrhagic inflammation of the mucous membranes of the true stomach and duodenum, attended with formation of gases in the alimentary canal and necrotic lesions in the liver) and haemoglobinuria in calves.

A subcutaneous injection of the culture into rabbits, white mice, guinea pigs, and pigeons results in a jelly-like oedema usually without the formation of gas bubbles. Postmortem examination displays slight changes in the muscles; the oedematous tissues are pallid or slightly hyperaemic.

 

Clostridium septicum. The organism was isolated from the blood of a cow in 1877 by L. Pasteur and J. Joubert. In 1881 R. Koch proved the organism to be responsible for malignant oedema. It is found in 8 per cent of examined soil specimens.

Morphology. The clostridia are pleomorphous and may be from3.1-14.1 mcm long and from 1.1-1.6 mcm thick; filamentous forms, measuring up to 50 mcm in length, also occur. The organisms are motile, peritrichous, and produce no capsules in the animal body. The spores are central or subterminal. The clostridia are Gram-positive but Gram-negative organisms occur in old cultures.

Cultivation. Cl. septicum are strict anaerobes. Their optimal growth temperature is 37-45° C, and they do not grow below 16° C. The pH of medium is 7.6. The organisms grow readily in meat-peptone broth and meat-peptone agar to which 5 per cent glucose has been added. On glucose-blood agar they produce a continuous thin film of intricately interwoven filaments lying against a background of haemolysed medium. In agar stab cultures the colonies resemble balls of wool. In broth a uniform turbidity is produced, and an abundant loose, whitish, and mucilaginous precipitate later develops.

Fermentative properties. Cl. septicum liquefies gelatin slowly, produces no indole, reduces nitrates to nitrites, and decomposes proteins, with hydrogen sulphide and ammonia formation. Force-meat is reddened but not digested; the culture evolving a rancid odour. Levulose, glucose, galactose, maltose, lactose, and salicin are fermented with acid and gas formation. Milk is coagulated- slowly.

Toxin production. Cl. septicum produces a lethal exotoxin, necrotic toxin, haemotoxin, hyaluronidase, deoxyribonuclease, and collagenase. The organism haemolyses human, horse, sheep, rabbit, and guinea pig erythrocytes.

Antigenic structure and classification. On the basis of the agglutination reaction, serovars of Cl. septicum can be distinguished, which produce identical toxins, the differential properties being associated with the structure of the H-antigen Cl. septicum possesses antigens common to Cl. chauvoei which is responsible for anaerobic infections in animals.

Resistance is similar to that of Cl novyi.

Pathogenicity for animals. Among domestic animals horses, sheep, pigs, and cattle may contract the disease. Infected guinea pigs die in18-48 hours. Postmortem examination reveals crepitant haemorrhagic oedema and congested internal organs. The affected muscles have a moist appearance and are light brown in colour. Long curved filaments which consist of clostridia are found in impression smears of microscopical sections of the liver.

 

Clostridium histolyticum. The organism was isolated in 1916 by M. Wemberg and P. Segum. It produces fibrinolysin, a proteolytic enzyme, which causes lysis of the tissues in the infected body. An intravenous injection of the exotoxin into an animal is followed shortly by death. The fact that the organisms are pathogenic for man has not met with general acceptance in the recent years The organism’s responsibility for anaerobic infections during World War II was insignificant.

Pathogenesis and diseases in man. Anaerobic infections are characterized by a varied clinical picture, depending on a number of factors. These include the number of pathogenic anaerobic species and their concomitant microflora, i. e. non-pathogenic or conditionally pathogenic anaerobes and aerobes which occur in particular association reflecting the complex process of parasitocoenosis. The type of wound and the immunobiological condition of the body are also among the factors.

The causative agents of anaerobic infections require certain conditions for their development after they have gained entrance into the body, i. e. favourable medium (the presence of dead or injured tissues)and a low oxidation-reduction potential (state of anaerobiosis) which arises due to the presence of necrotized cells of the affected tissues and aerobic microflora. Later the pathogenic anaerobes cause the necrosis of the healthy tissues themselves.

This process develops particularly intensively in the muscles owing to the fact that they contain large amounts of glycogen which serves as a favourable medium for pathogenic anaerobes responsible for anaerobic infections. Oedema is produced during the first phase of the infection, and gangrene of the muscles and connective tissue, during the second phase.

The exotoxins which are produced by clostridia anaerobic infections exert not only a local effect, causing destruction of muscular and connective tissues, but affect the entire body. This results in severe toxaemia. The body is attacked also by toxic substances produced by the decaying tissues. Investigations have shown that exotoxins produced by the causative agents of anaerobic infections possess potentiation activity. Simultaneous injections of one-fourth of a lethal dose of both Cl. perfringens and Cl. novyi toxins produce a reaction which is more marked than that produced by separate injections of the toxins into different parts of the body.

As a result of the vasoconstrictive effect of the toxins, development of oedema, and gas formation, the skin becomes pale and glistening at first and bronze-coloured later. The temperature of the affected tissues is always lower than that of the healthy areas. Deep changes occur in the subcutaneous cellular, muscle, and connective tissues, and degenerative changes take place in the internal organs.

The organisms themselves play an essential part in the pathogenesis of anaerobic infections owing to their high invasive activity. An extremely important role in the development of the disease is attributed to the reactivity state of the macro-organism (trauma, concomitant diseases, etc.).

Ingestion of food (sheep’s milk cheese, milk, curds, sausages, cod, etc.) contaminated abundantly with C/. perfringens results in toxinfections and intoxications. These conditions are characterized by a short incubation period (from 2 to 6 hours), vomiting, diarrhoea, headache, chills, heart failure, and cramps in the gastrocnemius muscle; the body temperature may either be normal, or elevated to   38 °C.

Immunity. The immunity produced by anaerobic infections is associated mainly with the presence of antitoxins which act against the most commonly occurring causative agents of the wound infection. For example, Cl. perfringens loses its lecithinase activity completely in the presence of a sufficient amount of antitoxin against its alpha-toxin.

The toxin-antitoxin reaction depends to a great extent on the presence of lecithin which acts as substratum for toxin activity. The antitoxin cannot neutralize lecithinase if the former is added at certain periods of time after the toxin had been in the presence of lecithin, the reaction being simply somewhat delayed in such cases. A definite role is played by the antibacterial factor, since the existence of bacteraemia in the pathogenesis of anaerobic infections has been shown.

Laboratory diagnosis. Material selected for examination include spieces of affected and necrotic tissues, oedematous fluid, dressings, surgical silk, catgut, clothes, soil, etc. The specimens are examined in stages:

(1) microscopic examination of the wound discharge for the presence of C/. perfringens;

(2) isolation of the pure culture and its identification according to the morphological characteristics of clostridia, capsule production, motility, milk coagulation, growth on iron-sulphite agar, gelatin liquefaction, and fermentation of carbohydrates (see Table 1);

(3) inoculation of white mice with broth culture filtrates or patient’s blood for toxin detection;

(4) performance of the antitoxin-toxieutralization reaction on white mice (a rapid diagnostic method).

C. perfringens is found in 70% to 80% of all cases of gas gangrene, and of the five serologic types of this organism, type A is the most prevalent. Any exudate is cultivated on thioglycolate broth and on blood–agar plates that are incubated both aerobically and anaerobically. The presence of large gram–positive rods that grow only anaerobically is strong evidence for clostridia C. perfringens is characterized by a stormy fermentation in milk, in which the coagulated milk is blown apart by gas formed during the fermentation of the lactose in milk. Organisms producing an a toxin hydrolyze the lecithin in an egg yolk medium, breaking down the lipid emulsion and, in turn, causing an opaque area to appear around the colony. Individual clostridial species are identified by a series of biochemical tests.

 

Treatment and prophylaxis comprise the following procedures:

–       surgical treatment of wounds (surgical cleansing of wounds to eliminate extraneous material or necrotic tissue is, undoubtedly, the most important control mechanism for gas gangrene);

–       early prophylactic injection of a polyvalent purified and concentrated antitoxin “Diaferm 3” in a dose of 10000 units against Cl. perfringens, Cl. novyi, Cl. septicum. For treatment the doses of antitoxin are increased five-fold;

–       use of antibiotics (streptomycin, penicillin, chlortetracycline, and gramicidin), sulphonamides, anaerobic bacteriophages, diphage, antistaphylococcal plasma and antistaphylococcal gamma globulin. In a number of cases treatment with antitoxin alone does not give the desired effect, while the combined use of antitoxin and antibiotics significantly lowers the mortality rate.

Transfusion of blood, oxygen therapy, administration of inhibitors of proteolytic enzymes and biologically active preparations which normalize metabolism are auxiliary therapeutic measures. Hyperbaric oxygen chambers, in which an infected area is placed in a chamber containing pure oxygen under pressure, have been used with some success to stop the growth of these obligate anaerobes.

 

CLOSTRIDIUM PERFRINGENS AND FOOD POISONING

In addition to being the major etiologic agent in wound infections, C perfringens also is an important cause of food poisoning. Most outbreaks follow the ingestion of meat or gravy dishes that are heavily contaminated with vegetative cells of C perfringens. Interestingly, C perfringens type A strains produce a heat–labile enterotoxin only when the vegetative cells form spores in the small intestine, releasing the newly synthesized enterotoxin. Symptoms of acute abdominal pain and diarrhea begin 8 to 24 hours after ingestion of the contaminated food and usually subside within 24 hours. The toxin appears to bind to specific receptors on the surface of intestinal epithelial cells in the ileum and jejunum. The entire molecule then is inserted into the cell, membrane, but does not enter the cell. This induces a change in ion fluxes, affecting cellular metabolism and macromolecular synthesis. As the intracellular Ca²⁺ levels increase, cellular damage and altered membrane permeability occurs, resulting in the loss of cellular fluid and ions.

Rare, but severe, cases of food poisoning, characterized by hemorrhagic enteritis and a high mortality rate, usually are caused by C perfringens type C. Such cases have been reported primarily from Germany and New Guinea. Those in New Guinea (known as pig–bel) have been associated with the eating of pork or other high–protein foods. Type C organisms produce a sporulation enterotoxin indistinguishable from that produced by C. perfringens type A, but they also produce large amounts of a toxin and the lethal necrotizing b toxin. It seems that the severe hemorrhagic enteritis is primarily a result of the action of the b toxin.

C perfringens type C has been reported to occur in the feces of over 70 % of the villagers in Papua, New Guinea. Because of a diet low in protein, the organisms in the gut do not ordinarily grow and produce sufficient toxin to cause any pathologic manifestations. Meat and other high–protein foods (which are seldom eaten), however, stimulate growth and toxin production by the clostridia. The disease occurs primarily in young children because of their poor immunity to b toxin. Also, because of a diet low in proteins, such children have an abnormally low level of intestinal proteases that could destroy the toxin before intestinal damage occurs. Furthermore, even this low level of protease activity is inhibited by protease inhibitors present in sweet potatoes that are consumed in large quantities at New Guinea feasts.

Because of the severity and high incidence of this disease, a program of active immunization with C perfringens b toxoid was initiated in 1980. Data indicate that the use of this vaccine has resulted in a dramatic decrease in the incidence of pig–bel in the New Guinea highlands.

C perfringens also has been reported to cause an infectious diarrhea, in which the organisms seem to be spread from person to person. Such infections are characterized by large numbers of C perfringens and high titers of enterotoxin in stool specimens, as well as a considerably longer duration of illness.

 

CLOSTRIDIUM DIFFICILE. Pseudomembranous colitis, a severe, necrotizing process that may occur in the large intestine after antibiotic therapy and produces severe diarrhea, has been associated with a number of antimicrobial agents, but the antibiotics clindamycin, ampicillin, amoxicillin, and the cephalosporins have been incriminated most often. One mechanism of this diarrhea was elucidated in 1978, when it was observed that the use of these antibiotics resulted in an over growth of an organism in the intestine identified as Clostridium difficile. C difficile can cause a spectrum of symptoms, ranging from asymptomatic carriage, mild to severe cholera–like diarrhea with 20 or more watery stools per day, and, in its most serious form, pseudomembranous colitis. Evidence indicates that C difficile is responsible for virtually all cases of pseudomembranous colitis and for up to 20% of cases of antibiotic–associated diarrhea without colitis. C difficile seems to be part of the normal intestinal flora of about 7% to 10% of adults; but only when antibiotic–sensitive organisms are eliminated from the intestine is it able to grow to sufficient numbers to produce disease. Interestingly, as many as 50% to 75% of neonates may become colonized with C difficile acquired as a nosocomial infection. Fortunately, most infants re–main asymptomatic, but they do serve as a reservoir for the spread of toxigenic C difficile to others both in the hospital and at home.

 

 

 

To demonstrate the nosocomial acquisition of this organism in adult patients, the University of Washington (Seattle) carried out a study in which 428 consecutive patients were cultured for C difficile over an 11–monthperiod. They reported that 7% had positive results on admission, but of the patients with negative culture re–sults, 21% acquired the organism during their hospital stay. Of these, 37% had diarrhea. Moreover, of the hospi–tal personnel carrying for the patients, 59% were positive for C difficile.

C difficile produces disease by the elaboration of two distinct exotoxins, which have been designated as A and B. Toxin A is an enterotoxin that is primarily responsible for the diarrhea associated with this disease. Its mechanism of action seems to result from tissue damage after an inflammatory process induced by the toxin. Toxin A acts as a strong chemoattractant for neutrophils, and it is thought that the release of inflammatory cytokines from these cells results in altered membrane permeability, fluid secretion, and hemorrhagic necrosis. Toxin B is a cytotoxin that demonstrates a lethal effect on cultured tissue cells. Its cytotoxic action is thought to involve depolymerization of filamentous actin, resulting in a change in the cell cytoskeleton and a rounding of the cell.

In addition, an enzyme with ADP–ribosylating activity has been described in one strain of C difficile. This toxin has been shown to modify cell actin in a manner similar to that of Clostridium botulinum C^ and C per–fringens t toxin.

The diagnosis of C difficile diarrhea usually is based on the demonstration of the presence of toxin A, toxin B, or both. Toxin B can be detected by its effect on cell cultures, but this requires 18 to 24 hours. Latex beads coated with antibody to toxin A also are commercially available, as is an enzyme–linked immunosorbent assay kit, for detecting both toxins A and B.

The primary treatment is to discontinue the implicated antibiotic. Most patients then recover spontaneously. An agent can be substituted that is unlikely to cause an antibiotic–associated diarrhea such as a quinoline, sulfonamide, parenteral aminoglycoside, metronidazole, or trimethoprim–sulfomethoxazole.

Clostridium sordellii occasionally is one of the etiologic agents of clostridial myonecrosis. It is mentioned here because pathogenic strains of C sordellii produce two toxins that share biologic and immunologic properties with toxins A and B of C difficile, and it may be responsible for some cases of antibiotic–associated diarrhea.

 

Diagnosis in detail

GAS ANAEROBIC INFECTION. Gas anaerobic infection is a disease developing in the wake of extensive deeply penetrating wounds of muscles and other tissues provided they are contaminated with anaerobes from the environ­ment, particularly from soil. The pathogens responsible for this disease are Clostridium perfringens, Cl. septicum, Cl. sordellii, Cl. novyi, etc.

The material to be studied is damaged and necrotic tissues taken at the borderline between pathologically-altered and healthy tissues, exudate, pus, secretions from wounds, and blood. Post-mortem ma­terial examined includes secretions from wounds, pieces of altered muscles, blood from the heart, and pieces of the spleen and liver. In food poisoning vomits, waters of stomach lavage, faeces, blood, and food remains are examined.

Bacteriological and biological examination. The material is stained by the Gram technique, examined microscopically, paying at­tention to the presence of gross Gram-positive spore rods or individ­ual spores, and then introduced into casein or meat liquid and solid media (blood agar, Wilson-Blair medium).

The inoculated cultures are cultivated in an anaerobic jar, while columns with medium are placed into a 37 °C incubator.

Make preparations from the inoculated cultures, stain them by Gram’s method, note the nature of the growth on liquid nutrient media, and subculture the material onto solid media.

Filtrates of the cultures or centrifugates are examined for the presence of toxin in experiments on mice or guinea pigs and utilized for conducting the neutralization reaction with diagnostic sera of Cl. perfringens, Cl. septicum, Cl. sordellii, Cl. oedematiens of A and B types.

The nature of growth on solid nutrient media is determined on the third day. Using a needle, pick up colonies and inoculate, with the help of column technique, into a semi-solid agar containing 0.5 per cent of glucose. Assay the morphology of the bacteria isolated, their motility, capacity to ferment carbohydrates, change the colour of litmus milk, liquefy gelatin, and coagulated serum or yolk. For this purpose emulsify the colony on a glass slide in a drop of acridine orange, cover it with a cover slip, and examine under the immersion objective of a luminescent microscope. Detection of only green rods is indicative of toxigenic species.

The presence of red rods or those of a green colour with red frag­ments points to weak or no toxigenicity of bacteria.

For rapid diagnosis the material tested is centrifuged and the pellet is used to make the in vitro neutralization test with specific antitoxic sera. Other rapid methods of the diagnosis include demonstration of lecithinase in filtrates and its neutralization with type-specific sera.

The material is centrifuged, diluted with isotonic sodium chloride solution 1:2, 1:4 . . ., an activator (0.005 M CaCl₂) is added, and 1 ml of each dilution is mixed with 0.1 ml of type-specific serum. The mixture is incubated at 20 °C for 40 min, and then 0.2 ml of lecithovitellin is added, and the mixture is reincubated at 37 °C for 2 hrs. The same mixture, only without serum, serves as the control. In the control a filtrate of the lecithinase-positive microorganism Cl. perfringens forms turbidity and opacification, with no such changes being observed in test tubes with the serum.

 

FOOD POISONING CAUSED BY CLOSTRIDIUM PERFRINGENS

Food poisoning in man is most often caused by Clostridium per­fringens of types A and C.

The material used for examination is food remains, such clinical specimens as vomited matter, faeces, and blood in anaerobic sepsis, and such autopsy samples as blood and pieces of the internal or­gans.

Bacteriological examination is conducted for the isolation and identification of the causative agent, determination of the degree of colonization of the material examined by this microorganism and the type of the toxin produced by the latter.

Day 1. The material to be examined is diluted ten-fold with pep­tone water to 10^–10 and 1-ml portions from the respective dilutions are transferred into the melted Wilson-Blair medium which has been cooled to 45 °G. In some cases the material is introduced into blood or yolk agar which is then decanted into plates. After the agar has solidified, the inoculated culture is immersed with a 2 per cent meat-peptone agar and incubated for 6-8 hrs at 45-46 “C or for 20 hrs at 37 °C.

In addition, homogenates of the materials examined are streaked onto liquid nutrient media (Kitt-Tarozzi’s medium). The inoculated cultures are incubated at     37 °C. Growth of Clostridia may he noted in 6-8 hrs. If this is the case, further examinations are carried out within 24 hours.

Day 2. Count black colonies in the Wilson-Blair medium, select the specimen where some 10-30 colonies have formed (20-100 per plate) and recalculate the number per ml (taking into account the dilution and the dose of the inoculum).

To obtain a pure culture after microscopy, subculture 3-5 colonies into the Kitt-Tarozzi medium and 2-3 colonies onto litmus milk. The inoculated cultures are incubated at 37 °C for 24 hrs.

Day 3. Study the nature of growth in the Kitt-Tarozzi medium. Cl. perfringens grow with intense gas formation. On litmus milk one can observe characteristic fermentation with lightening of the serum and formation of a sponge clot of brick colour.

To detect exotoxin and determine its type, the neutralization reac­tion is performed with a filtrate of the broth culture. The test is performed and the results are read as it is done in botulism.

The diagnosis is considered confirmed if the food products re­sponsible for the disease contain large numbers of Clostridium (10⁶ and more per g), if the cultures of the material examined show Cl. perfringens of types A and C. if the Clostridia isolated produce exotoxins and strains of Cl. perfringens of any type (A, B, C, D, E) are found in the patient’s blood.

To speed up the diagnosis, examination is carried out according to the following scheme.

1. The material is heated for 15 min at 80 °C, introduced into defatted milk containing 0.5 per cent of glucose, and cultivated at 37 °C.

If the material harbours Cl. perfringens, milk peptonization is seen in several hours.

2. After a clot has formed, the serum is centrifuged and 0.5-1.0 ml administered intraperitoneally to white mice.

If a toxin is demonstrated, the neutralization test with serum against Cl. perfringens of type A only is performed. The toxin formed in the serum treated with trypsin (proteolytic activation of toxin) is also determined.

 

Tetanus Clostridia

A. Nicolaier discovered the causative agent of tetanus in 1884, and S.Kitasato isolated the pure culture in 1889.

Morphology. The causative agent of tetanus (Clostridium tetani) is a thin motile rod, 2.4-5 mcm in length and 0.5-1.1 mcm in breadth. It has pentrichous flagellation and contains granular inclusions which occur centrally and at the ends of the cell. The organism produces round terminal spores which give it the appearance of a drumstick (Fig.). Cl. tetani is Gram-positive.

 

Figure. Clostridium tetani with terminal spores ..

 

Electron microscopy shows that the cell wall is composed of five layers and the cytoplasmatic membrane of three layers; the cytoplasm is dense, granular and contains ribosomes and polysomes. During maxi-mum liberation of the exotoxin, the cytoplasmatic membrane draws away from the cell wall and the main bulk of the cell is lysed. The nucleoid is compact and occupies a small part of the cell. The spores are enclosed by a sporangium. The G+C content in DNA is 25 per cent.

Cultivation. The organisms are obligate anaerobes. They grow on sugar and blood agar at pH 7.0-7.9 and at a temperature of 38 °C (no growth occurs below 14 and above 45 °C) and produce a pellicle with a compact center and thread-like outgrowths at the periphery. Some-times a zone of haemolysis is produced around the colonies. Brain medium and bismuth-sulphite agar are blackened by Cl. tetani. Agar stab cultures resemble a fir-tree or a small brush and produce fragile colonies which have the appearance of tufts of cotton wool or clouds (Fig.). A uniform turbidity is produced on Kitt-Tarozzi medium with liberation of gas and a peculiar odour as a result of proteolysis.

Figure. Clostridium tetani. Colonies in stab agar culture.

 

Fermentative properties. Cl. tetani causes slow gelatin liquefaction and produces no indole. Nitrates are rapidly reduced to nitrites. The organisms coagulate milk slowly, forming small flakes. No carbohydrates are usually fermented (see Table 1, Mettodological  instructions).

 

Toxin production. Cl tetani produces an extremely potent exotoxin which consists of two fractions, tetanospasmin, which causes muscle contraction, and tetanolysin, which haemolyses erythrocytes.

A  0.0000005 ml dose of toxin obtained from a broth culture filtrate kills a white mouse which weighs 20 g; and  0.000000005 g of dry toxin obtained by ammonium sulphate precipitation is fatal to the mouse. Several million lethal mouse doses are contained in 1 mg of crystalline toxin.

The mode of action of the tetanus toxin is similar to that of enzymes which catalyse chemical reactions in the bodies of affected animals.

Tetanus toxin (also termed tetanospasmin) is synthesized in the bacterium as a single polypeptide chain, but after its release by lysis of the organism, a bacterial protease cleaves one peptide bond to yield two chains that remain linked together through a disulfide bond. The larger chain (H chain) has a molecular weight of 100,000 daltons, and it possesses the specific receptors that bind the toxin to the neuronal gangliosides. The smaller peptide (L chain) has a molecular weight of 50,000 daltons and is thought to exert the biologic effect of the toxin.

The mechanism of action of the toxin is not fully understood, but it is known that the toxin is first bound to neuronal cells at the neuromuscular junction. The complete toxin then crosses the nerve cell membrane and is transported retrogradely to the inhibitory interneurons. There, by an as yet unknown mechanism, the toxin enters the interneurons and blocks the exocytosis of inhibitory transmitters, namely, glycine and gamma-aminobutyric acid. In an analogous situation, tetanus toxin has been reported to inhibit the secretion of lysosomal contents from stimulated human macrophages. The final effect is a spastic paralysis characterized by the convulsive contractions of voluntary muscles. Because the spasms frequently involve the neck and jaws, the disease had been referred to as lockjaw. Death ordinarily results from muscular spasms affecting the mechanics of respiration.

Interestingly, all toxin-producing strains of C tetani possess a large plasmid, which encodes for the synthesis of the toxin. Loss of the plasmid converts the cell to an avirulent, non-toxin-producing organism.

A second toxin produced by C tetani is called tetanolysin. This toxin is related functionally and serologically to streptolysin O and belongs to a large group of oxygensensitive hemolysins from a variety of bacteria. In addition to erythrocytes, tetanolysin lyses a variety of cells such as polymorphonuclear neutrophils, macrophages, fibroblasts, ascites tumor cells, and platelets. It is unknown, however, whether it plays any significant role in infections by C tetani.

Antigenic structure and classification. Cl. tetani is not serologically homogeneous and 10 serological variants have been recognized. All 10variants produce the same exotoxin. The I, III, VI, and VII types exhibit a manifest specificity. The motile strains contain the H-antigen, and the non-motile strains contain only the O-antigen. Variant specificity is associated with the H-antigen and group specificity with the O-antigen.

Resistance. Vegetative cells of the tetanus organism withstand a temperature of 60-70° C for 30 minutes and are destroyed quite rapidly by all commonly used disinfectants. The spores are very resistant, and survive in soil and on various objects over a long period of time and with-stand boiling for 10-90 minutes or even, as with spores of certain strains, for 1-3 hours. The spores are killed by exposure to a 5 per cent phenol solution for 8-10 hours, and by a 1 per cent formalin solution, for 6 hours. Direct sunlight destroys them in 3-5 days.

Pathogenicity for animals. Horses and small cattle acquire the disease naturally, and many animals may act as carriers of Cl. tetani.

Among experimental animals, white mice, guinea pigs, rats, rabbits, and hamsters are susceptible to tetanus.

The disease in animals is manifested by tonic contractions of the striated muscles and lesions in the pyramid cells of the anterior cornua of the spinal cord. The extremities are the first to be involved in the process, the trunk being affected later (ascending tetanus).

Pathogenesis and disease in man. Healthy people and animals, who discharge the organisms in their faeces into the soil, are sources of the infection. Spores of Cl. tetani can be demonstrated in 50-80 per cent of examined soil specimens, and some soils contain the spores in all test samples (manured soil is particularly rich in spores). The spores may be spread in dust, carried into houses, and fall on clothes, underwear, foot-wear, and other objects.

 

The majority of tetanus cases in adults occur among farm workers, and more than 33 per cent of the total incidence of the disease is associated with children from 1 to 15 years old. In more than 50 per cent of cases tetanus is acquired as the result of wounds of the lower extremities inflicted by spades, nails, and stubbles during work in the orchard or in the field.

Cl. tetani may gain entrance into the body of a newborn infant through the umbilical cord and into a woman during childbirth, through the injured uterine mucosa.

The organisms produce exotoxins (tetanospasmin and tetanolysin) at the site of entry. In some cases tetanus is accompanied by bacteraemia.

Microbes and spores, washed-off from the toxin, normally produce no disease and are rapidly destroyed by phagocytes.

The tetanus toxin reaches the motor centres of the spinal cord via the peripheral nerves (it moves along the axial nerve cylinders or along the ecto- and endoneural lymphatics).

According to the school of thought of A. Speransky, the specificity of the tetanus toxin is manifest only at the onset of the disease. In its further stages the infection is governed by other phenomena, primarily by the neurodystrophic factors. Sites of high and increasing excitation develop under the influence of irritation stimuli.

The toxin enters the blood and is thus distributed throughout the whole body, causing subsequent excitation of the peripheral nerve branches and the cells of the anterior cornua of the spinal cord.

Receptors situated in the neuromuscular apparatus play a significant role in the development of tetanus. Impulses sent out from these receptors give rise to a dominant excitation focus in the central nervous sys-tem The effect of the toxin produces an increased reflex excitation of the motor centres, and this, in its turn, leads to the development of attacks of reflex tonic muscular spasms which may occur often in response to any stimuli coming from the external environment (light, sound, etc.).

The onset of the disease is characterized by persistent tonic muscular spasms at the site of penetration of the causative agent. This is followed by tonic spasms of the jaw muscles (trismus), face muscles (risus sardonicus), and occipital muscles. After this the muscles of the back (opisthotonus) and extremities are affected. Such is the development of the clinical picture of descending tetanus. The patient lies in bed, resting on his head and hips with his body bent forward like an arc. The death rate varies from 35 to 70 per cent, being 40 per cent on the average and 65 per cent in the USA. More than 50000 people die every year from tetanus in the world. According to incomplete WHO data, more than one million people contracted the disease within a period of 10 years (1951-1960) and about 500000 of them died.

Immunity following tetanus is mainly antitoxic in character, and of low grade. Reinfections may occur.

Laboratory diagnosis is usually not carried out because clinical symptoms of the disease are self-evident. Objects of epidemiological significance (soil, dust, dressings, preparations used for parenteral injections)are examined systematically.

Wounds, dressings, and medicaments used for parenteral injections are examined for the presence of Cl. tetani and their spores by the following procedures. Specimens are inoculated into flasks or test tubes. The sowings are kept at a temperature of 80° C for 20 minutes to sup-press the growth of any non-sporeforming microflora which may be present. After 2-10 days’ incubation at 35° C, the culture is studied microscopically and tested for the presence of toxin by injection into mice. If Cl. tetani is present, tetanus of the tail develops during 24-48 hours, followed by tetanus of the body and death. The disease does not occur in mice which have been inoculated with antitetanus serum.

If no tetanus toxin is detected in the first inoculation but microscopic examination reveals the presence of organisms morphologically identical with Cl. tetani, the initial culture is inoculated into a condensated water of coagulated serum. A thin film will appear over the entire surface of the medium after 24 hours’ growth in anaerobic conditions. Experimental animals are infected with a culture grown on liquid nutrient medium and kept for 4-5 days at 35° C.

A biological test is employed for detecting the exotoxin in the test material extract. Two white mice are given intramuscular injections of0.5-1.0 ml of a centrifuged precipitate or filtrate of the extract. An equal amount of the filtrate is mixed with antitoxic serum, left to stand for 40 minutes at room temperature, and then injected into another two mice in a dose of 0.75 or 1.5 ml per mouse. If the toxin is present in the filtrate, the First two mice will die in 2-4 days while the other two (control mice) will survive.

Treatment. Intramuscular injections of large doses of antitoxic antitetanus serum are employed. The best result is produced by gamma-globulin obtained from the blood of humans immunized against tetanus. Anticonvulsant therapy includes intramuscular injections of 25 per cent solutions of magnesium sulphate, administration of diplacine, condelphine, aminazine, pipolphen or andaxine and chloral hydrate introduced in enemas. To reproduce active immunity, 2 ml of toxoid is administered two hours before injecting the serum; the same dose of toxoid is repeated within 5-6 days. Uninoculated persons are subjected to active and passive immunization. This is achieved by injecting 0.5 ml of toxoid and 3.000 units of antitoxic serum and then 5 days later, another 0.5 ml of toxoid. The tetanus antitoxin is also introduced into previously inoculated individuals suffering from a severe wound. Injection of the total dose of antitoxin is preceded by an intracutaneous test for body sensitivity to horse protein. This is carried out by introducing 0.1 ml of antitoxin, previously diluted 1 :100, into the front part of the forearm. If the intracutaneous test proves negative, 0.1 ml of whole antitoxin is injected subcutaneously and if no reaction is produced in 30 minutes, the total immunization dose is introduced.

The complex of prophylactic measures includes adequate surgical treatment of wounds. The organisms are sensitive to penicillin, but the antibiotic has no effect on the neutralization of the toxin. However, after surgical cleansing of the wound, antibiotic therapy can be helpful in preventing any additional growth of the organisms.

Prophylaxis is ensured by prevention of occupational injuries and traumas in everyday life. Active immunization is achieved with tetanus toxoid. It is injected together with a tetravalent or polyvalent vaccine or maybe a component of an associated adsorbed vaccine. The pertussis-diphtheria-tetanus vaccine and associated diphtheria-tetanus toxoid are employed for specific tetanus prophylaxis in children. Immunization is carried out among all children from 5-6 months to 12 years of age, individuals living in certain rural regions (in the presence of epidemiological indications), construction workers, persons working at timber, water-supply, cleansing and sanitation, and peat enterprises, and railway transport workers.

Immunization with tetanus toxoid stimulates the production of sufficient amounts of antitoxin. Immunity lasts for a period of 2 or 3 years.

The effectiveness of the immunization has made tetanus a relatively rare disease in the developed countries(36 cases in the United States during 1994). In Third World countries, however, most persons are not immunized, and infections of the umbilical cord give rise to large numbers of neonatal tetanus. For example, special surveillance programs indicate death rates from neonatal tetanus to be 2% of all live births in Bangladesh, 6% in Papua, New Guinea, and 14.5% in Haiti. One study relating the proportion of pregnant females with tetanus antitoxin titres adequate to provide protection for the newborn found 96% protected in New Haven, Connecticut, and only 19% in Santiago, Chile.

Oral immunization may become possible using a live attenuated strain of Salmonella typhimurium that was transfected with a plasmid engineered to express a 50–kdfragment of the tetanus toxin. Given orally, this strain provided protective immunity in mice.

After an injury, human tetanus immune globulins should be administered to those who have never been immunized with tetanus toxoid or to those who did not receive the full three doses of toxoid. Booster injections of toxoid also are given if the immune status of the patient is unknown, or if it has been over 5 years since the last dose of toxoid.

 

Clostridia Responsible for Botulism

The causative agent of botulism (L. botulus sausage, botulism poisoning by sausage toxin), Closlridium botulinum, was discovered in Hollandin 1896 by E. van Ermengem. The organism was isolated from ham which had been the source of infection of 34 people and from the intestine and spleen on post-mortem examination. In Western Europe botulism was due to ingestion of sausages, while in America it was caused by canned vegetables, and in Russia, by ingestion of red fish. In the recent 50 years 5635 persons contracted botulism, 1714 of them died.

Morphology. Cl. botulinum is a large pleomorphous rod with rounded ends, 4.4-8.6 mcm in length and 0.3-1.3 mcm in breadth. The organism sometimes occurs in short forms or long threads. Cl. botulinum is slightly motile and produces from 4 to 30 flagella per cell. In the external environment Cl. botulinum produces oval terminal or subterminal spores which give them the appearance of tennis rackets (Fig.). The organisms are Gram-positive.

 

Figure. Pure culture and deep colonies of Closlridium botulinum

 

 

 

On ultrathin sections the cell wall in A, B, and E types consists of five layers, the cytoplasmatic membrane of three layers. By the time of maxi-mum exotoxin liberation (on the 5th-7th day) cell lysis with the discharge of crystalline structure occurs. The cytoplasm is granular and contains inclusions of various size. The nucleoid is compact and occupies a small part of the cytoplasm. Spore formation takes place on the3rd or 4th day of cultivation 1 he G +C content in DNA ranges between 26 and 28 per cent.

Cultivation. Cl. botulinum are strict anaerobes. The optimal growth temperature for serovars A, B, C, and D is 30-40 C, for serovar E 25-37 ºC, for serovar G 30-37 ºC They grow on all ordinary media at pH 7.3-7.6 Cultivation is best on minced meat or brain which the organisms turn darker. The cultures have an odour of rancid butter.

On Zeissler’s sugar-blood agar irregular colonies are produced which possess filaments or thin thread-like outgrowths. The colonies are surrounded by a zone of haemolysis.

 

In agar stab cultures the colonies resemble balls of cotton wool or compact clusters with thread-like filaments (Fig. ).

On gelatin the organisms form round translucent colonies surrounded by small areas of liquefaction. Later the colonies turn turbid, brownish, and produce thorn-like filaments.

In liver broth (Kitt-Tarozzi medium) turbidity is produced at first, but a compact precipitate forms later, and the fluid clears.

Fermentative properties. Cl. botulinum (serovars A and B) are proteolytic organisms, and decompose pieces of tissues and egg albumin in fluid medium. The organisms liquefy gelatin, produce hydrogen sul-phide, ammonia, volatile amines, ketones, alcohols, and acetic, butyric, and lactic acids. Milk is peptonized with gas formation. Glucose, levulose, maltose, and glycerin are fermented, with acid and gas formation (see Table 1, Mettodological  instructions no 43).

Toxin production. Cl. botulinum produces an extremely potent exotoxin. The toxin is produced in cultures and foodstuffs (meat, fish, and vegetables) under favourable conditions in the body of man and animals. Multiplication of the organism and toxin accumulation are inhibited in the presence of a 6-8 per cent concentration of common salt or in media with an acid reaction. Heating at 90 C for 40 minutes or boiling for 10 minutes destroys the toxin.

The toxin produced by Cl. botulinum, as distinct from the tetanus and diphtheria toxins, withstands exposure to gastric juice and is absorbed intact. The toxin produced by serovar A Cl. botulinum can kill 60000million mice having a total weight of 1 200 000 tons. The toxin has been obtained in crystalline form and is the most potent of all toxins known to date. Curiously, the toxins seem to be secreted as progenitor toxins which, even though some have been crystallized, are composed of two polypcptide subunits linkedby disulfide bonds. Also, the toxicity of those toxins thathave been extensively studied can be increased from four–fold to 250–fold by treatment with trypsin. This phenomenon is not understood at the molecular level.

The botulinum toxin is a globulin and does not change on recrystallization. Its activity is similar to that of enzymes which catalyse chemical processes in the body of man and animals with formation of large amounts of toxic substances. These substances produce the clinical manifestations of poisoning.

The toxin acts primarily as a neurotoxin, inducing paralysis in three basic steps (1) binding of the toxin to a receptor on the nerve synapse, (2) entrance of the toxin(or possibly one polypeptide subunit) into the nerve cell, and (3) blocking of the release of acetylcholine from the cell, resulting m a flaccid muscle paralysis.

C botulinum type C produces two distinct toxins that have been designated Cl and C2 The Cl toxin functions like other botulism toxins to block the release of acetylcholine at the myoneural junction C2 toxin, however, is a binary complex consisting of two unlinked components designated as I and II Component II recognizes the cell receptor and thus facilitates the entrance of component I into the cytoplasm The C2 toxin causes a necrotic enteritis, which seems to result in an increase in vascular leakage of the intestinal mucosa. Its mechanism of action is unclear, but it has been shown to ADP-ribosylate G-actin as well as the synthetic substrate, homo-poly l arginine

C botulinum organisms, types C and D, also produce an additional toxin which has been termed exoenzymeC3 The DNA encoding C3 is located on both phage C and phage D, the phages that also encode for botulism toxins C and D, respectively Its function is to ADP-ribosylates Rho protein, a eucaryotic member of the ras superfamily of proteins Because the ras superfamily of proteins are GTP-binding proteins involved in enzyme regulation, this exoenzyme could function as a virulence factor, but the exact consequence of the C3 ADP-ribosylation is unknown.

Antigenic structure and classification. Six serovars of Cl. botulinum are known: A, B, C, D, E, and F, serovars A, B, and F being the most toxic. Each of the serovars is characterized by specific immunogenicity associated with the H-antigen and is neutralized by the corresponding antitoxin. Variants C and D are responsible for neuroparalytic lesions in animals. As has been proved recently, serovar C may produce diseases also in man. The 0-antigen is common to all variants.

Resistance. The vegetative forms of the organisms are killed in 30minutes at 80 C, while the spores withstand boiling for periods from 90minutes to 6 hours-and survive 115″ C for 5-40 minutes and 120° C, for3-22 minutes. Spores remain viable in large pieces of meat and in large cans even after autoclaving for 15 minutes at 120° C. In 5 per cent phenol solutions they survive for up to 24 hours and in cultures they may live for a year.

Pathogenicity for animals. Horses, cattle, minks, birds, and among the laboratory animals, guinea pigs, white mice, cats, rabbits, and dogs are susceptible to the botulinum toxin.

Paralysis of the deglutitive, mastication, and motor muscles is usually produced in horses 3 days after infection. The mortality rate reaches 100per cent. Botulism in bovine cattle is accompanied with bulbar paralysis, and in birds it causes limbemeck and paresis of the legs.

Infection of guinea pigs results in muscular weakness which appears in 24 hours, followed by death in 3-4 days. Autopsy displays hyperaemia of the intestine, gastric flatulence, and a urinary bladder filled beyond capacity. White mice die on the second day after infection manifesting relaxed abdomen muscles and paresis of the hind limbs. Paralysis of the eye muscles, disturbances of accommodation, aphonia, pendulous and protruding tongue, and diarrhoea are caused in cats.

Pathogenesis and disease in man. Botulism is contracted by ingesting meat products, canned vegetables, sausages, ham, salted and smoked fish (red fish more frequently), canned fish, chicken and duck flesh, and other products contaminated with Cl. botulinum. The organisms enter the soil in the faeces of animals (horses, cattle, minks, and domes-tic and wild birds) and fish and survive there as spores.

Natural nidality of botulism among ducks and other wild birds has been ascertained. Extremely widespread epizootics occur in the western regions of Canada, in Uruguay, and in the USA. Natural foci develop in territories where there are stagnant reservoirs rich in vegetable debris which provides favourable conditions for anaerobiosis in warm weather. Intensive processes of decay are accompanied with oxygen uptake which contributes to the growth of serovar C botulism clostridia and production of high concentrations of the exotoxin in the water and alkaline mud in swamps. Besides birds, muskrats and frogs may also contract botulism. Migrating birds spread the causative agent of botulism from the natural foci during their flights.

Cl. botulinum spores occur both in cultivated and virgin soil. They were isolated from 70 per cent of examined soil samples in California, and from 40 percent of samples in the Northern Caucasus. Spores have been isolated from littoral soil at the Sea of Azov, sea water, and silt. They have also been found on the surface of vegetables and fruit, in the intestine of healthy animals, in 5.4 per cent of cases in the guts of fresh red fish (sturgeon, beluga, etc.), and in 15-20 per cent of cases in the guts and in 20 per cent of cases in the tissues of dead fish.

The infectious condition is caused by the exotoxin which is absorbed in the intestine, from where it invades the blood, and affects the medulla oblongata nuclei, cardiovascular system, and muscles. It has been ascertained that Cl. bolulinum may enter the body through wounds. Usually, the wounds themselves were not serious, but wound botulism should be suspected in any persons with even minor wounds who present the typical symptoms of botulism: blurred vision, weakness, and difficulty in swallowing. In the past botulism was considered to be only of a toxic nature. Recent investigations have proved the Cl. botulinum to be present in various organs of individuals who have died from botulism. Therefore, this disease is a toxinfection. The incubation period in botulism varies from 2 hours to10 days, its usual duration is 18 to 24 hours.

Botulism symptoms include dizziness, headache, and, sometimes, vomiting. Paralysis of the eye muscles, accommodation disturbances, dilatation of the pupils, and double vision occur. Difficulty in swallowing, aphonia, and deafness also arise. The death rate is very high (40-60 per cent).

 

Botulism in child

 

Immunity. The disease does not leave a stable anti-infectious immunity (antitoxic and antibacterial).

Laboratory diagnosis. Remains of food which caused poisoning, blood, urine, vomit, faeces, and lavage waters are examined. Post-mortem examination of stomach contents, portions of the small and large intestine, lymph nodes, and the brain and spinal cord is carried out.

The test specimens are inoculated into Kitt-Tarozzi medium which has previously been held at 100 C for 10-20 minutes. To free the cultures from foreigon-sporeforming microflora, 50 per cent of the test tubes containing the inoculated medium is heated at 80 C for 20minutes and then incubated in anaerobic conditions. The isolated pure culture is identified by its cultural, biochemical, and toxigenic properties.

For toxin detection a broth culture filtrate, patient’s blood or urine, or extracts of food remains, are injected subcutaneously or intraperitoneally into guinea pigs, white mice, or cats. One of the control animals is infected with unheated material, while the other animal is injected with the heated specimen. In addition, 3 laboratory animals are given injections .of the filtrate together with serovar A antitoxin, with serovar B antitoxin, and with serovar E antitoxin.

The indirect haemagglutination reaction and determination of the phagocytic index are also performed. This index is significantly lowered in the presence of the toxin.

A rapid method of detection of serovar A, B, C, D, and E toxins in water has been developed in which the toxin is absorbed by talc and a suspension of the talc and toxin is injected into the animals.

Treatment. The stomach is lavaged with potassium permanganate or soda solutions Polyvalent botulinum antitoxin is injected intramuscularly (intravenously or into the spinal canal) m doses of 10000 IU (serovars A, C, and E) and 5000 IU (serovar B). If there is no improvement, the injection is repeated at the same dosage within 5-10 hours. All individuals who had used food which caused even a single case of food poisoning are given 1000-2000 IU of antitoxin as a preventive measure. Simultaneously with the antitoxin, 0 5 ml of each serovar of botulinum toxoid is injected three times at intervals of 3-5 days, for production of active immunity. Penicillin and tetracycline are recommended

General measures include subcutaneous injections of saline and glucose solutions Camphor, caffeine, vitamin C, and thiamine are prescribed if necessary. Strychnine is given 2-3 times a day as a stimulant.

Prophylaxis. Proper organization of food processing technology at food factories, meat, fish, and vegetable canning in particular, and preparation of smoked and salted fish and sausages is essential for the prevention of botulism. Home-preserved fish products (smoked and salted)as well as canned mushrooms and canned vegetables of a low acid con-tent (cucumbers, peppers, eggplant), stewed apricots, etc. are very dangerous since they are usually prepared without observance of sanitary rules.

Fish should be gutted after being caught, and placed in the refrigerator. The established temperature regimen must be observed during transportation, and the fish must be protected from pollution with soil and bowel contents. Vegetables must be washed thoroughly. The cooking of meat and fish in small pieces is recommended. Foodstuffs (ham, fish) should not be stored in large hunks and in many layers. The weight of a canned product should not exceed 0.5 kg. Cl botulinum which have with stood sterilization cause swelling of the can lids. The contents of such cans have an odour of rancid butter Such canned goods must not be put on the market and must be withdrawn and thoroughly examined. Fish must be salted in strong salt solutions (brine) with a minimal concentration of 10 per cent. Canned goods must be stored in a cool place.

Active immunization of man, horses, and cows with the toxoid is recommended by many authors in view of Cl botulinum being wide-spread in nature

Botulism occurrence in the USSR is extremely rare as a result of continuous improvement of the standard of living of the population, and of technological methods in food processing and canning industries, observance of the rules of hygiene and strict state and sanitary control of the production, storage, and sale of foodstuffs.

Infant Botulism

A new variety of botulism was recognized during 1976with the report of five cases of infant botulism. These cases occurred in babies as young as 5 weeks, some of whom were breast fed, although all had had some exposure to other foods. Since then, hundreds of additional cases of infant botulism have been diagnosed, and it has become a significant paediatric clinical entity.

Epidemiology and pathogenesis of infant botulism. Infant botulism has been diagnosed in infants ranging from 3to 35 weeks of age. It is well-established that the disease is acquired by the ingestion of C botulinum spores that subsequently germinate in the intestine and produce botulism toxin. Such spores are ubiquitous and, in fact, soil and dust samples from many homes have been shown to contain such spores. Thus, even breast-fed infants are susceptible through contaminated dust. Honey also has been shown to contain spores of C botulinum, and a number of cases of infant botulism have followed the ingestion of honey.

The major initial symptom of infant botulism is 2to 3 days of constipation followed by flaccid paralysis, resulting in difficulty iursing and a generalized weakness that has been described as “overtly floppy.”

The mortality rate of infants admitted to the hospital has been about 3%, and some patients have required mechanical respirators because of respiratory distress. Death, however, may occur more frequently in undiagnosed cases, and considerable data link infant botulism to at least some cases of the sudden infant death syndrome.

Because, by definition, infant botulism is the result of toxin production by organisms that have colonized the gut, it is not surprising that there have been a few cases of adult-infant botulism that occurred after antibiotic therapy or gastric surgery. Even in cases of food-borne botulism, it is usual for the gut to be colonized with C. botulinum, providing a continuing source of toxin.

Diagnosis and treatment of infant botulism. A tentative clinical diagnosis of botulism can be made for an infant with several days of constipation, an unexplained weakness, difficulty in swallowing, or respiratory distress. A laboratory diagnosis, however, requires the demonstration of botulism toxin in the feces, which is determined by the injection of fecal extracts intraperitoneally into a mouse. Death of the mouse within 96 hours (which did not occur in controls in which the fecal extracts were first neutralized with botulism antitoxin) is taken as positive evidence for the presence of the toxin.

Infants are not usually treated with antitoxin, primarily because it is a horse product and may induce lifelong hypersensitivity. Attempts to eradicate the bacteria are not recommended because of the fear that the organisms might lyse in the intestine, releasing large amounts of toxin. Treatment thus far has been mostly symptomatic, requiring an average of 1 month of hospitalization.

 

Diagnosis in details

TETANUS

Tetanus is an acute infectious disease caused by Clostridium tetani and attended by tonic and clonic muscular contractions. The clinical picture is so typical that, as a rule, the bacteriological examination for diagnosis is unnecessary. To detect the causative agent, surgical dressings and various preparations intended for parenteral adminis­tration are usually checked.

In cases of an obscure course of the disease examine pus, blood, pieces of tissue cut from the wound, as well as post-mortem specimens of organs, tissues, and blood. From tissues and thick pus prepare suspensions in isotonic sodium chloride solution. Cotton wool and gauze are cut with scissors and placed into nutrient media.

Bacterioscopic examination. Detection of thin long Gram-positive rods with round terminal spores in smears from the material obtained from the patient or corpse suggests the presence of Cl. tetani.-. Yet, one cannot derive the conclusion as to the presence of Clostridia of tetani on the basis of bacterioscopic findings alone since the material tested may contain other morphologically similar microorganisms, e.g., Cl. tetanomorphum, Cl. paratetanomorphum, etc.

Bacteriological examination. The material to be examined is streaked on the Kitt-Tarozzi enrichment medium and placed in the incubator for 3-4 days after which it is subcultured to solid media to obtain separate colonies. Following incubation in a microanaerostatic jar, Cl. tetani colonies on a blood sugar agar appear as small spiders or dew-drops, whereas in the column of sugar agar they re­semble balls of wool or cotton.

The isolated pure culture is identified and examined for toxigenicity. Cl. tetani form a toxin on the 4th-5th day of cultivation. The culture formed on the Kitt-Tarozzi medium is centrifuged and 0.3-0.4 ml of the supernatant is injected intramuscularly (at the root of the tail) to two white mice. Two control mice receive the same amount of the tested liquid which is mixed with an antitoxic anti-tetanus serum, following the incubation for 1 h at 37 °C. The mice are observed for 4-5 days. In 2-4 days infected animals present signs of tetanus (rigidity of the tail and muscles at the site of the toxin administration) and soon die, while the control mice survive un­affected.

If white mice are injected the material tested (together with the inoculated culture), they present the same clinical picture of tetanus that is seen after administration of the toxin.

Isolation of a pure culture of Cl. tetani by the Fildes technique. For this purpose a 3-4-day culture in the enrichment medium is heated for 1.5 hrs at 60 °C, after which several drops are inoculated into condensation water of the coagulated serum. The inoculated culture is kept under strictly anaerobic conditions. In 1-2 days, a “creeping” growth of Cl. tetani is observed. From the upper part of the inocu­lated culture it is again subcultured with a loop into condensation water of the coagulated serum. Such passages are reiterated until a pure culture is isolated.

BOTULISM

Botulism is acute food poisoning characterized by the predomi­nant damage to the central and vegetative nervous system. The causal organism of this disease is Clostridium botulinum.

To carry out the examination, one usually collects at least 10-12 ml of blood, which is supplemented with sodium citrate in a 3:1 ratio, 100-200 ml of vomited matter, lavage waters of the stom­ach, faeces, and urine, as well as 200-300 g of remains of the food-stuffs that are suspected to be the cause of the disease. All materials should be taken prior to the administration of a therapeutic serum. At necropsy blood, pieces of the internal organs, lymph nodes, con­tents of the stomach and intestines, brain, and spinal cord are examined.

Examination has a double purpose: detection in the material tested of the botulin toxin (two-thirds of the specimen) and isolation from the material of the causative agent (one-third of the specimen).

Demonstration of the botulin toxin and identification of its type with the help of the neutralization reaction are very important with regard to the prescription of a specific therapy.

Preparation of material. Lavage waters of the stomach (25-30 ml) containing food lumps are ground in a sterile mortar; two-thirds of the sample are kept at room temperature for 1 h for extracting and then filtered through a cotton-gauze filter or centrifuged at 3000 X g for 15-20 min.

Citrate blood or serum obtained from the patient should not be diluted be­fore the examination; it is administered to mice only in the form of intraperitoneal injection.

Patients’ faeces (20-25 g) are ground in a sterile mortar with a double vol­ume of isotonic sodium chloride solution and kept at room temperature for 1-1.5 hrs, and then filtered through a water-gauze filter. Extracts from post-mortem material are prepared in a similar manner.

Procedure of the test. To perform the neutralization test, use dry diagnostic antitoxic sera of A, B, C, and E types, which are diluted with isotonic sodium chloride solution to 100-200 lU/ml, which ensures neutralization of the homolo­gous toxin in the specimen tested.

The neutralization reaction is carried out with either a mixture of sera (a preliminary reaction) or with monovalent sera (for detection of a specific type of toxin).

The prepared material to hp studied (in the form of a filtrate or pullet) or blood is dispensed in 1-mt volumes into live test tubes; into each of the first four tubes 1 ml of anti-botulinal serum of types A, B, C, and E is added respec­tively, into the last one, 1 ml of the normal serum is introduced. The tubes are incubated for 30 min after which 1-ml amounts of the mixture from each test tube are introduced to five pairs of white mice weighing 1(3-18 g (blood is inject­ed intraperitoneally; other biomaterials, subcutaneously. The animals are observed for four days.

If the material studied contains the botulinal toxin, only one pair of mice survives due to the neutralization of the toxin by the antitoxic serum of the corresponding type (a positive reaction). If all mice die, the neutralization test should be repeated after diluting the biomaterial by 5-, 10-, 20- and even 100­fold. If the material tested contains the botulinal toxin, mice develop paresis of the limbs. Autopsy findings include hyperaemia of the internal organs, pneu­monic foci in the lungs, overfilling of the stomach, bladder, and gallbladder.

The laboratory conclusion about the presence in the material examined of the botulinal toxin should refer to its particular type.

Bacteriological examination. Prior to inoculation, the material to be tested is ground in a porcelain mortar. Some 10-12 ml of the material are introduced into the Kitt-Tarozzi medium, casein-acid, or casein-mycotic medium. One specimen is inoculated into four vials, two of which are heated: one at 60 °C for 15 min (for the selection of E type Cl. botulinum), the other at 80 °C for 20 min. Enrichment for cultures of Clostridia of types E and F occurs in a 28 °C incubator. To grow Clostridia of types A, B, and C, the cultures are cultivated at 35 °C for 48 hrs.

To activate toxin E from the protoxin, add trypsin to the nutrient medium to achieve the final concentration (0.1 per cent). The remains of samples of the material studied are stored in a refrigerator till the end of the analysis.

After 24-48 hrs of incubation, the enrichment medium becomes turbid and gas formation is observed. From the medium presenting growth prepare smears and stain them by the Gram technique. Upon detection of typical Clostridia with spores subculture them to solid nutrient media for obtaining separate colonies. Isolation of a pure culture presents some difficulty as Cl. botulinum often form associa­tions with some aerobic bacteria. Sometimes, only multiple passages make it possible to obtain a pure culture. On a sugar blood agar Cl. botulinum form irregularly-shaped colonies with a smooth or rough surface surrounded by a zone of haemolysis. Deep in the sugar agar column these colonies appear as fluffs or lentils.

To identify the obtained pure culture, it is inoculated into Hiss’s media. Cl. botulinum displays proteolytic properties: it liquefies gelatin and serum and splits yolk and pieces of meat. Most strains ferment glucose, mannitol, maltose, and other carbohydrates with acid and gas formation. Antigenic attributes are studied with the help of the agglutination test, using type specific sera.

Simultaneously with the investigation of the fermentative prop­erties the botulinal toxin is demonstrated in the filtrate of a broth culture, and its type is identified.

Detection of the botulinal toxin with the help of the phagocytic param­eter. The botulinal toxin inhibits the phagocytic activity of leuco­cytes, whereas specific sera eliminate this action by neutralizing the toxin. The employment of this method is particularly advisable for detecting the toxin in blood.

The rapid method of detecting the botulinal toxin in drinking water is based on the adsorption of toxin from water with the help of talcum powder and the subsequent administration to mice of the talc suspension obtained.

Epidemiological data and characteristic clinical manifestations (the paralytic syndrome) play an important role in the diagnosis of botulism. Negative results of laboratory studies do not exclude the presence of botulism.

 

Peptostreptococcus. Clinically significant anaerobic cocci include peptostreptococci, Veillonella species, and microaerophilic streptococci. The genus Peptostreptococcus contains very small bacteria that grow in chains. Peptostreptococcus is a genus of anaerobic, Gram-positive, non-spore forming bacteria. The cells are small, spherical, and can occur in short chains, pairs or individually. Peptostreptococcus are slow-growing bacteria with increasing resistance to antimicrobial drugs.These anaerobic counterparts of Streptococcus are usually not harmfull. They are known to be normal flora of the skin, urethra, and the urogenital tract. If given an opportunity, however, they can cause infections of bones, joints and soft tissue. Their increasing resistance to such antibiotics as penicillin G and clindamycin makes them especially important to clinical work. P. magnus is the species that is most often isolated from infected sites.

Peptostreptococcus infections can occur in all body sites, including the CNS, head, neck, chest, abdomen, pelvis, skin, bone, joint, and soft tissues. Inadequate therapy against these anaerobic bacteria may lead to clinical failures. Because of their fastidiousness, peptostreptococci are difficult to isolate and are often overlooked. Isolating them requires appropriate methods of specimen collection, transportation, and cultivation. Their slow growth and increasing resistance to antimicrobials, in addition to the polymicrobial nature of the infection, complicate treatment.

Peptostreptococcus is the only genus among anaerobic gram-positive cocci encountered in clinical infections. This group also includes species within the genus formerly known as Peptococcus, with the exception of Peptococcus niger. This change in taxonomy was based on the results of a guanine-plus-cytosine content analysis. Additionally, Gaffkya anaerobia was renamed Peptostreptococcus tetradius. The species of anaerobic gram-positive cocci isolated most commonly are Peptostreptococcus magnus, Peptostreptococcus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus prevotii, and Peptostreptococcus micros.

Anaerobic gram-positive cocci that produce large amounts of lactic acid during the process of carbohydrate fermentation were reclassified as Streptococcus parvulus and Streptococcus morbillorum from Peptococcus or Peptostreptococcus. Most of these organisms are anaerobic, but some are microaerophilic. Based on DNA homology and whole-cell polypeptide-pattern study findings supported by phenotypic characteristics, the DNA homology group of microaerobic streptococci that was formerly known as Streptococcus anginosus or Streptococcus milleri is now composed of 3 distinct species: S anginosus, Streptococcus constellatus, and Streptococcus intermedius. The microaerobic species S morbillorum was transferred into the genus Gemella. A new species within the genus Peptostreptococcus is Peptostreptococcus hydrogenalis; it contains the indole-positive, saccharolytic strains of the genus.

Pathophysiology: Peptostreptococcus organisms are part of the normal florae of human mucocutaneous surfaces, including the mouth, intestinal tract, vagina, urethra, and skin. They are isolated with high frequency from all specimen sources. Anaerobic gram-positive cocci are the second most frequently recovered anaerobes and account for approximately one quarter of anaerobic isolates. Anaerobic gram-positive cocci are usually recovered mixed with other anaerobic or aerobic bacteria from infections at different sites of the body.

Many of these infections are synergistic. Bacterial synergy, the presence of which is determined by mutual induction of sepsis enhancement, increased mortality, increased ability to induce abscesses, and enhancement of the growth of the bacterial components in mixed infections, is found between anaerobic gram-positive cocci and their aerobic and anaerobic counterparts. The ability of anaerobic gram-positive cocci and microaerophilic streptococci to produce capsular material is an important virulence mechanism, but other factors also may influence the interaction of these organisms in mixed infections.

Frequency:  In the US: The exact frequency of Peptostreptococcus infections is difficult to calculate because of inappropriate methods of collection, transportation, and cultivation of specimens. These infections are found more commonly in patients with chronic infections. Recovery rates in blood cultures are 2-5% and are higher in patients who have predisposing conditions. In 1974, Martin reported that anaerobic cocci were isolated in 8.5-31% of clinical specimens that yielded any anaerobic bacteria at the Mayo Clinic.

In 2 studies published in 1988 and 1989, Brook reported that anaerobic gram-positive cocci accounted for 26% of all anaerobic bacteria recovered at Bethesda Navy Hospital and Walter Reed Army Hospital from 1973-1985. The infected sites where the organisms predominated were ears (53% of all anaerobic isolates), cysts (40%), bones (39%), and obstetrical and gynecological sites (35%). They were occasionally found in the CNS, abdomen, lymph nodes, bile, and eyes. Most isolates were found in abscesses, wounds, and obstetrical and gynecological infections.

The recovery rates differed for the different anaerobic gram-positive cocci. In descending order of frequency, the most common anaerobic gram-positive cocci were P magnus (18% of all anaerobic gram-positive cocci and microaerophilic streptococci), P asaccharolyticus (17%), P anaerobius (16%), P prevotii (13%), P micros (4%), Peptostreptococcus saccharolyticus (3%), and Peptostreptococcus intermedius (2%).

The highest recovery rates of P magnus were in bone and chest infections. The highest recovery rate of P asaccharolyticus and P anaerobius were with obstetrical/gynecological and respiratory tract infections and wounds. Isolates of each of the most frequently recovered anaerobic gram-positive cocci were recovered from abscesses, wounds, and obstetrical and gynecological infections.

Although most of the infections were polymicrobial when anaerobic and facultative cocci were recovered, these organisms were isolated in pure culture in 45 (8%) of 559 children who had infections involving anaerobic gram-positive cocci, in 12 (10%) of 121 children who had infections due to microaerophilic streptococci, and in 15 (9%) of 176 patients who had P magnus infection. The most frequent types of infections from which anaerobic gram-positive cocci were isolated in pure culture were soft tissue infections, osteomyelitis, arthritis (especially in the presence of a prosthetic implant), and bacteremia. Most patients from whom microaerophilic streptococci were recovered in pure culture had abscesses (eg, dental, intracranial, pulmonary), bacteremia, meningitis, or conjunctivitis.

P magnus is the most commonly isolated anaerobic cocci. It is most often recovered in pure culture. The most common peptostreptococci in the different infectious sites are P anaerobius in oral infections; P magnus and P micros in respiratory tract infections; P magnus, P micros, P asaccharolyticus, Peptostreptococcus vaginalis, and P anaerobius in skin and soft tissue infections; P magnus and P micros in deep organ abscesses; P magnus, P micros, and P anaerobius in gastrointestinal tract–associated infections; P magnus, P micros, P asaccharolyticus, P vaginalis, P tetradius, and P anaerobius in female genitourinary infections; and P magnus, P asaccharolyticus, P vaginalis, and P anaerobius in bone and joint infections and leg and foot ulcers.

Internationally: The frequency of these infections appears to be higher in developing countries, where therapy is often inadequate or delayed.

Mortality/Morbidity: Mortality has decreased over the past 3 decades.

Age: Peptostreptococcus infections can occur in patients of all ages; however, head and neck infections occur more frequently in children than in adults.

Physical: Although anaerobic cocci can be isolated from infections at all body sites, a predisposition for certain sites has been observed. In general, Peptostreptococcus species, particularly P magnus, have been recovered more often from subcutaneous and soft tissue abscesses and diabetes-related foot ulcers than from intra-abdominal infections. Peptostreptococcus infections occur more often in chronic infections and in association with the predisposing conditions below.

CNS infections

Anaerobic gram-positive cocci and microaerophilic streptococci can be isolated from subdural empyema and from brain abscesses that develop as sequelae of chronic infections of the ears, mastoid, sinuses, and teeth.

Anaerobic gram-positive cocci and microaerophilic streptococci have been isolated from 18 (46%) of 39 brain abscesses.

Upper respiratory tract and dental infections

The high rate of anaerobic cocci colonization of the oropharynx accounts for the organisms’ significance in these infections. Anaerobic gram-positive cocci and microaerophilic streptococci are often recovered from acute and chronic upper respiratory tract infections. These organisms have been recovered in 15% of patients with chronic mastoiditis, 30% of patients with chronic sinusitis, 33% of patients with peritonsillar and retropharyngeal abscesses, and 50% of patients with purulent parotitis. They have also accounted for two thirds of isolates from periodontal abscesses.

In more than 90% of cases, other organisms also present in the oral florae have been found mixed with anaerobic gram-positive cocci and microaerophilic streptococci. These include Staphylococcus aureus, Streptococcus species, Fusobacterium species, and pigmented Prevotella and Porphyromonas species.

Anaerobic pleuropulmonary infections

Anaerobic gram-positive cocci and microaerophilic streptococci account for 10-20% of anaerobic isolates recovered from properly obtained specimens of pulmonary infections. The pulmonary infections in which these organisms have been found most frequently include aspiration pneumonia, empyema associated with aspiration pneumonia, lung abscesses, and mediastinitis.

Obtaining appropriate culture specimens of these organisms requires the use of transtracheal aspiration, aspiration through double-lumen catheterization, or direct lung puncture.

Intra-abdominal infections

Because anaerobic gram-positive cocci are part of the normal gastrointestinal florae, they can be isolated in approximately 20% of specimens from intra-abdominal infections, such as peritonitis and abscesses of the liver, spleen, and abdomen.

Anaerobic gram-positive cocci are generally recovered mixed with other organisms of intestinal origin that include Escherichia coli, Bacteroides fragilis group, and Clostridium species.

Female pelvic infections

Anaerobic gram-positive cocci and microaerophilic streptococci can be isolated in 25-50% of patients with endometritis, pyoderma, pelvic abscess, Bartholin gland abscess, postsurgical pelvic infections, or pelvic inflammatory disease. The origin of these organisms is probably the vaginal and cervical florae.

The predominant anaerobic gram-positive cocci are P asaccharolyticus, P anaerobius, and P prevotii.

Bacteremias with anaerobic gram-positive cocci and microaerophilic streptococci are often associated with septic abortion.

Anaerobic gram-positive cocci are generally found mixed with Prevotella bivia and Prevotella disiens.

Osteomyelitis and arthritis

Anaerobic gram-positive cocci are frequently isolated from anaerobically infected bones and joints. In studies, they accounted for 40% of anaerobic isolates of osteomyelitis caused by anaerobic bacteria and 20% of anaerobic isolates of arthritis caused by anaerobic bacteria.

P magnus and P prevotii are the predominant bone and joint isolates. In a 1980 study by Bourgault and colleagues, most patients with infections involving these organisms underwent orthopedic surgery and had foreign prosthetic material in place at the time of infection. Management of these infections requires prolonged courses of antimicrobials and is enhanced by removal of the foreign material.

Skin and soft tissue infections

Anaerobic gram-positive cocci and microaerophilic streptococci are often recovered in polymicrobial skin and soft tissue infections (eg, necrotizing synergistic gangrene; necrotizing fasciitis; decubitus ulcers; diabetes-related foot infections; paronychia; burns; human or animal bites; infected cysts; abscesses of the breast, rectum, and anus). Anaerobic gram-positive cocci and microaerophilic streptococci are generally found mixed with other aerobic and anaerobic florae that originate from the mucosal surface adjacent to the infected site or that have been inoculated into the infected site.

Gastrointestinal florae can cause infections such as gluteal decubitus ulcers, diabetes-related foot infections, and rectal abscesses.

Vaginal and cervical florae can cause scalp wound infections iewborns after fetal monitoring.

Because anaerobic gram-positive cocci and microaerophilic streptococci are part of the normal skin florae, care must be used when obtaining specimens to avoid contamination by these florae.

Bacteremia and endocarditis

Anaerobic gram-positive cocci and microaerophilic streptococci may be responsible for 4-15% of anaerobic bacteria isolated from blood cultures of patients with clinically significant anaerobic bacteremia. They are often recovered in persons with puerperal sepsis.

Peptostreptococci can cause fatal endocarditis, paravalvular abscess, and pericarditis.

The most frequent source of bacteremia due to Peptostreptococcus is infections of the oropharynx, lower respiratory tract, female genital tract, abdomen, skin, and soft tissues.

Predisposing factors for bacteremia due to Peptostreptococcus include malignancy; recent gastrointestinal, obstetrical, or gynecological surgery; immunosuppression; dental procedures; and oropharyngeal, female genital tract, abdominal, and soft tissue infections.

Microaerophilic streptococci typically account for 5-10% of cases of endocarditis; however, peptostreptococci have only rarely been isolated.

Causes:

The following are the major predisposing conditions to infection with anaerobic gram-positive cocci and microaerophilic streptococci:

Previous surgery

Immunodeficiency

Malignancy

Trauma

Diabetes

Steroid therapy

Presence of a foreign body

Sickle cell anemia

Reduced blood supply

Vascular disease

Infection with aerobic bacteria can make the local tissue conditions more favorable for the growth of anaerobes, including anaerobic cocci. Anaerobic conditions and anaerobic bacteria can impair host defenses. Anaerobic infection often manifests as suppuration, thrombophlebitis, abscess formation, and gangrenous destruction of tissue associated with gas. Anaerobes, including peptostreptococci, are common in chronic infections. Therapy with antimicrobials (eg, aminoglycosides, trimethoprim-sulfamethazine, older quinolones) often does not eradicate anaerobes.

Lab Studies:

Microbiology

Anaerobic, microaerophilic, and facultative gram-positive cocci have minor morphological differences. P magnus has a larger diameter than other anaerobic gram-positive cocci. P micros has a smaller diameter than other anaerobic gram-positive cocci and usually forms short chains. P anaerobius and Peptostreptococcus productus are elongated and often appear in pairs or chains.

 

LABORATORY INDICATIONS:

Esculin hydrolysis –

Hydrogen sulfide –

Catalase –

Lactose –

 

Gas-liquid chromatography and biochemical tests are required for genus-level identification and separation of most anaerobic gram-positive cocci. These organisms are fastidious, and their complete identification is often difficult. Because of ill-defined differences in the pathogenic potential for the different species, the need for exact specification is controversial.

Anaerobic cocci show slow but adequate growth on all nonselective anaerobic growth media. Vancomycin-containing selective media inhibit their growth.

Recovery in clinical specimens

Anaerobic and facultative gram-positive cocci are often isolated from clinical specimens mixed with other anaerobic or aerobic bacteria and, on rare occasions, are isolated as the sole pathogen. As a group, these organisms are the most frequently recovered anaerobes in cutaneous, oral, respiratory tract, and female genital tract infections.

Collecting anaerobic bacteria specimens is important because documentation of an anaerobic infection is through culture of organisms from the infected site. Documentation requires proper collection of appropriate specimens, expeditious transportation, and careful laboratory processing.

Obtain uncontaminated specimens. Inadequate culture techniques or media can lead to faulty results and the incorrect conclusion that only aerobic organisms are present in a mixed infection. Specimens must be obtained free of contamination. Inadequate techniques or media can lead to missing the presence of anaerobic bacteria or the assumption that only aerobic organisms are present in a mixed infection.

Because anaerobes are present on mucous membranes and skin, even minimal contamination with normal florae can be misleading.

Unacceptable or inappropriate specimens can also yield normal florae and therefore have no diagnostic value. Obtain appropriate specimens using techniques that bypass the normal florae.

Direct-needle aspiration is the best method of obtaining a culture. Direct-needle aspiration is probably the best method of obtaining a culture, and the use of swabs is much less desirable.

Specimens obtained from normally sterile sites, such as blood, spinal, joint, or peritoneal fluids, are collected after thorough skin decontamination.

Two approaches are used to culture the maxillary sinus following sterilization of the canine fossa or the nasal vestibule, either via the canine fossa or via the inferior meatus.

Urine collected is collected by percutaneous suprapubic bladder aspiration.

Other specimens can be collected from abscess contents, from deep aspirates of wounds, and by special techniques, such as transtracheal aspirates or direct lung puncture.

Specimens of the lower respiratory tract are difficult to obtain without contamination with indigenous florae. Double-lumen catheter bronchial brushing and bronchoalveolar lavage, cultured quantitatively, can be useful.

Culdocentesis fluid obtained after decontamination of the vagina is acceptable.

Transportation of specimens should be expeditious. Place specimens into an anaerobic transporter as soon as possible. These devices generally contain oxygen-free environments provided by a mixture of carbon dioxide, hydrogen, and nitrogen plus an aerobic condition indicator.

Liquid or tissue specimens are always preferred to swabs.

Inoculate liquid specimens into an anaerobic transport vial or a syringe. All air bubbles are expelled from the syringe. Insertion of the needle tip into a sterile rubber stopper is no longer recommended. Because air gradually diffuses through the plastic syringe wall, specimens should be processed in less than 30 minutes.

Transport tissue specimens in an anaerobic jar or a sealed plastic bag rendered anaerobic.

If swabs are used, place them in sterilized tubes containing carbon dioxide or prereduced, anaerobically sterile Carey and Blair semisolid media.

Gram stain of a smear of the specimen provides important preliminary information regarding types of organisms present, suggests appropriate initial therapy, and serves as a quality control. Immediately place cultures under anaerobic conditions and incubate for 48 hours or longer. An additional 36-48 hours is usually required for species- or genus-level identification using biochemical tests; kits containing these tests are commercially available.

A rapid enzymatic test enables identification after only 4 hours of aerobic incubation. Gas-liquid chromatography of metabolites is often used. Nucleic acid probers and polymerase chain reaction methods are also being developed for rapid identification. Detailed procedures of laboratory methods can be found in microbiology manuals.

Antimicrobial susceptibility test results of peptostreptococci have become less predictable because of the increasing resistance of peptostreptococci to several antimicrobials. Routine susceptibility testing is time consuming and often unnecessary; however, it is important to test the susceptibility of isolates recovered from sterile body sites, those that are clinically important and have variable susceptibilities, and especially those isolated in pure cultures from properly collected specimens. These include isolates associated with bacteremia; endocarditis; and bone, joint, or skull infections.

Perform testing with antibiotics. Recommended methods include agar microbroth and macrobroth dilution. Newer methods include the E-test and the spiral gradient end point system. Agents that should be tested include penicillin, broad-spectrum penicillin, penicillin plus a beta-lactamase inhibitor, clindamycin, chloramphenicol, second-generation cephalosporins (eg, cefoxitin), newer quinolones, metronidazole, and carbapenems.

Imaging Studies:

Radiological or imaging studies are helpful. The presence of gas in the infected site is a strong indication of anaerobic infection.

Medical Care: A patient’s recovery from anaerobic infection depends on prompt and proper treatment according to the following principles: (1) neutralizing toxins produced by anaerobes, (2) preventing local bacterial proliferation by changing the environment, and (3) limiting the spread of bacteria.

Control the environment by debriding necrotic tissue, draining pus, improving circulation, alleviating obstruction, and increasing tissue oxygenation. Certain types of adjunctive therapy, such as hyperbaric oxygen therapy, may be useful but remain unproven.

In many cases, antimicrobial therapy is the only form of therapy required, but it can also be an adjunct to a surgical approach. Because anaerobic bacteria, including peptostreptococci, are generally recovered mixed with aerobic organisms, choose antimicrobial agents that treat both types of pathogens, taking into consideration their aerobic and anaerobic antibacterial spectrum and their availability in oral or parenteral form.

Penicillin G is most effective for treating anaerobic gram-positive cocci and microaerophilic streptococci.

Other effective agents include other penicillins, cephalosporins, chloramphenicol, clindamycin, vancomycin, telithromycin, linezolid, quinupristin/dalfopristin, and carbapenems.

The efficacy of macrolides (eg, erythromycin) and imidazoles (eg, metronidazole) is variable and unpredictable. Imidazoles are ineffective against some anaerobic gram-positive cocci and all aerotolerant strains.

The newer quinolones are effective against more than 90% of anaerobic cocci; ciprofloxacin is less effective.

Occasionally, certain strains are resistant to antimicrobials, especially after administration of these agents.

When mixed with other beta-lactamase–producing bacteria, anaerobic gram-positive cocci and microaerophilic streptococci may survive penicillin or cephalosporin therapy because of the protection provided by the free enzyme. In such instances, antimicrobials with wider spectrums of activity may be more effective.

Surgical Care: In most cases, surgical therapy is critically important. Surgical therapy includes (1) draining abscesses, (2) debriding necrotic tissues, (3) decompressing closed-space infections, and (4) relieving obstructions. If surgical drainage is not used, the infection may persist and serious complications may develop.

BACTEROIDES

Bacteroides are not E. coli! They are not even that closely related to eachother. However they can both be found in the same place: the intestine. Each and every one of us contain many billions of these bugs inside their gut. Bacteroides are specialists in this environment as they are adapted to grow where there is no oxygen. E. coli can grow both with and without oxygen and is consequently a generalist and not as good at growing in either condition as a true anaerobe (B. fragilis) or a true aerobe (Bacillus subtillus). In fact Bacteroides are one of the most numerous of the intestinal bugs and we get to see a great many everyday as about 30 % of what comes out of the intestine is bacteria! Most of the time we get on perfectly well with Bacteroides, in fact they assist in breaking down food products and supply some vitamins and other nutrients that we cannot make ourselves. The problem with Bacteroides is when they get out of the intestine and into our bodies. One of the most common results of this is an abscess, which is a big ball of puss comprised mostly of bacteria (especially B. fragilis). If the ball breaks then billions of bacteria wreak havok in the body often resulting in death. Luckily this dosn’t happen too often as bacteria are susceptable to antibiotics. Unfortunately the Bacteroides are very good at finding ways to become resistant to all of the antibiotics that we use so developing new ways to fight the bugs is a great importance.

Phylogeny

Anaerobes comprise the majority of bacteria in the human colon; the most numerically predominant of these are members of the genus Bacteroides. Originally described in 1898, for many years the Bacteroides were a vague conglomeration of host-associated, obligately anaerobic, gram-negative, pleomorphic rods that could not be convincingly assigned to any other genera. Physiological analysis of this genus revealed considerable heterogeneity with regard to their biochemical properties, indicating these bacteria did not represent a true phylogenetic grouping. With the advent of phylogenetic analysis techniques, several investigators have tried to redefine this group of bacteria using physiological characteristics, serotyping, bacteriophage typing, lipid analysis, oligonucleotide cataloging, and 5S – 16S rRNA sequence comparisons. Based on this information, the original Bacteroides members have been partitioned into three genera: Bacteroides, Prevotella, and Porphyromonas. The Bacteroides are found predominantly in the colon of mammals, while the Prevotella and Porphyromonads generally are associated with the oral cavity and the rumen. The current definition of Bacteroides species is as follows: a) obligately anaerobic, Gram-negative, b) saccharolytic, producing acetate and succinate as the major metabolic end products, c) contain enzymes of the hexose monophosphate shunt-pentose phosphate pathway, d) have a DNA-base composition in the range 40-48 mol% GC, e) membranes contain sphingolipids, and contain a mixture of long-chain fatty acids, mainly straight chain saturated, anteiso-methyl, and iso-methyl branched acids, f) possess menaquiones with MK-10 and MK-11 as the major components, and g) contain meso-diaminopimelic acid in their peptidoglycan. This definition restricts the Bacteroides to ten species: B. fragilis, B. thetaiotaomicron, B. vulgatus, B. ovatus, B. distasonis, B. uniformis, B. stercoris, B. eggerthii, B. merdae, and B. caccae, with B. fragilis as the type strain. The Bacteroides, along with Prevotella and Porphyromonas, form one major subgroup in the bacterial phylum Cytophaga-Flavobacter-Bacteroides. This phylum diverged quite early in the evolutionary lineage of bacteria, and thus the Bacteroides, although gram-negative organisms, are not closely related to the enteric gram-negatives such as Escherichia coli.

  Bacteroides as Commensal Organisms

  The Bacteroides inhabit the human colon, which contains the largest, most complex bacterial population of any colonized area of the human body. The colonic contents contain in excess of 1011 organisms per gram of wet weight, representing over 400 species.

The Bacteroides are the most numerous members of the normal flora, representing nearly 1011 organisms per gram of feces (dry weight). Gut organisms are involved in numerous metabolic activities in the colon, including fermentation of carbohydrates, utilization of nitrogenous substances, and biotransformation of bile acids and other steroids. In order to maintain their high numbers, the Bacteroides are evidently able to compete with other members of the flora, as well as transient organisms, for utilization of these resources. While the role of the microflora in the physiology of the human intestine is not well studied, it is clear that the anaerobic members of this ecosystem play a fundamental role in the processing of complex molecules into simpler compounds, and through their metabolic activities the human microflora participate in the complex physiology of the host.

Most intestinal bacteria are saccharolytic, obtaining carbon and energy by hydrolysis of host and dietary carbohydrate molecules. Simple sugars are rarely encountered in the colon as most are absorbed in the small intestine, however it is estimated that approximately 2% of simple sugars can pass through the upper gastrointestinal tract when large amounts of starch and complex carbohydrates are also present during digestion. Bacteroides species are able to utilize simple sugars when present, but due to their limited availability, simple sugars are probably not the main source of energy for the Bacteroides. Much more prevalent in the colon are polysaccharides, from dietary sources and host cells. Polysaccharides from plant fibers, such as cellulose, xylan, arabinogalactan, and pectin, and vegetable starches such as amylose and amylopectin contain Bacteroides have been shown to have a variety of glucosidase activities, including a beta-1,3-glucosidase activity responsible for laminarin degradation, and a variety of a and b-1, 4 and -1, 6 xylosidase and glucosidase activities induced by the presence of hemicellulose. Originally it was believed that these enzymatic activities were extracellular, and the short oligosaccharides and monosaccharides produced by hydrolysis were taken up into the cell for fermentation. Analysis of the B. thetaiotaomicron starch utilization system (sus), has revealed the polysaccharides to be bound to an outer membrane receptor system, and pulled into the periplasm for degradation into monosaccharides. The Bacteroides use a similar approach for uptake and degradation of chondroitin sulfate, indicating this technique may provide a competitive advantage in the human gut, as polysaccharides sequestered in the periplasm are less likely to be “stolen” by other intestinal organisms or lost by diffusion.

Interestingly, utilization of chondroitin sulfate by Bacteroides thetaiotaomicron is repressed in the presence of glucose, while utilization of other sugars in B. thetaiotaomicron is tightly regulated in the presence of mannose. This implies the Bacteroides may have a catabolite repression mechanism to allow for the utilization of select carbon sources in preference to others. If so, this system is probably not similar to the catabolite repression systems of enteric bacteria, as the Bacteroides do not possess cyclic AMP. It is likely that most Bacteroides polysaccharide utilization systems are controlled by repressor/inducer mechanisms, as B. ovatus and B. thetaiotaomicron are able to utilize several sugars simultaneously, and several polysaccharide utilization genes have been shown to be activated in the presence of their substrate.  Carbohydrate fermentation by the Bacteroides and other intestinal bacteria results in the production of a pool of volatile fatty acids, predominately acetate, propionate (from succinate), and butyrate. These short chain fatty acids are reabsorbed through the large intestine, and utilized by the host as an energy source. It has been estimated that absorption of the short chain fatty acids could provide up to 540 kcal/d, a significant proportion of the host’s daily energy requirement.

The utilization of nitrogen sources by the intestinal Bacteroides is not well understood, as most work in the area of nitrogen uptake has been done with rumen organisms. However, several parallels may be drawn between intestinal and rumen bacteria, providing a paradigm of nitrogen utilization in the human gut. There are three major sources of nitrogen in the mammalian intestine: dietary protein, epithelial cell and mucin glycoproteins, and ammonia. Most dietary protein is degraded and absorbed before reaching the large intestine, but once in the colon, these peptides and amino acids are not able to be absorbed by the host. Instead, a two step degradation process occurs, during which peptides are proteolysed to amino acids, which are subsequently deaminated to form ammonia, CO2, volatile fatty acids, and branched chain fatty acids. The ammonia is utilized by the intestinal bacteria as a nitrogen source. Bacteroides fragilis has been shown to produce three major proteases, with activity against a variety of proteins, including casein, trypsin, and chymotrypsin, but not collagen, elastin, or gelatin. The Bacteroides also encode glutamine synthetase and glutamate dehydrogenase, which are important for ammonia assimilation but the regulation of these activities is not yet understood. 

The Bacteroides play a key role in the enterohepatic circulation of bile acids. Cholic acid and chenodeoxycholic acid are the two main bile acids synthesized in the human liver, where they are conjugated to taurine or glycine polar side groups before secretion in bile. Once bile enters the gut, the conjugated bile acids assist in the absorption of dietary fats. If the bile acids are not reabsorbed in association with fat in the upper intestine, they are deconjugated by bacteria to secondary bile acids, primarily deoxycholic and lithocholic acid, although the microflora can generate 15-20 other secondary bile acids from these same precursors. Deconjugation allows the bile acids to reenter the enterohepatic circulation via the portal system, where they are returned to the liver and reconjugated for further use. The secondary bile acids deoxycholic and lithocholic acid are produced by 7 alpha-dehydrogenation of the primary bile acids; once these secondary bile acids are produced, a variety of other bacterial reactions can occur, including oxidation-reduction, desulphation, and dehydrogenation, producing a variety of isomers of secondary bile acids. The Bacteroides have been found to play a major role in the biotransformation of bile acids, and contain many enzymes required for these reactions, including a hydrolase, dehydrogenase, and dehydroxylase. The direct benefit to the host is obvious, as deconjugation of the primary bile acids allow them to be reabsorbed in the large intestine instead of lost in the feces. The benefit to the Bacteroides and other intestinal bacteria is not clear, but may contribute to energy metabolism.

Aside from their metabolic activities, the Bacteroides and other anaerobes provide an additional benefit to their host in excluding pathogenic organisms from colonizing the intestine. Colonization resistance mediated by anaerobes is thought to occur by four mechanisms: competition for nutrients, competition for intestinal wall attachment sites, production of volatile fatty acids, and release of free bile acids. The intestinal microflora adhere to the surface of epithelial cells and mucin associated with the intestinal wall, with Bacteroides being the most common anaerobic colonizer. By coating the walls of the intestine, it is believed that the microflora prevent transient bacteria from obtaining a binding site on the intestinal surface, and the transients are subsequently lost with the luminal contents during peristalsis. The volatile fatty acids produced as metabolic end products by the Bacteroides are also believed to play a role in colonization resistance, by lowering the pH and oxidation-reduction potential of the intestinal milieu, resulting in unfavorable growth conditions for transient bacteria. The most notable pathogens inhibited under these conditions are Salmonella enteritidis, and Shigella flexineri. Production of free bile acids also plays a role in inhibition of pathogens, as bile salts are toxic to many organisms, including Clostridium botulinum.

  Pathogenicity and Virulence

  While the Bacteroides occupy a significant position in the normal flora, they also are opportunistic pathogens, primarily in infections of the peritoneal cavity. B. fragilis is the most notable pathogen; although it makes up only 1-2% of the normal flora, it is the Bacteroides species isolated from 81% of anaerobic clinical infections. B. fragilis is not overtly invasive, but is capable of participating in intraabdominal infections in the event the mucosal wall of the intestine is disrupted. Incidences during which Bacteroides infections may be initiated include gastrointestinal surgery, perforated or gangrenous appendicitis, perforated ulcer, diverticulitis, trauma, and inflammatory bowel disease.

The current model for development of abdominal infections is based on the concept of synergism, during which cooperation between different species of bacteria aids in the establishment of persistent infection. Synergism has been most clearly established in infections involving both E. coli and B. fragilis, although other combinations of aerobes and anaerobes also are synergistic. After disruption of the intestinal wall, members of the normal flora infiltrate the normally sterile peritoneal cavity, and during the early, acute stage of infection (approximately 20 hours), the aerobes, such as E. coli, are the most active members of infection, establishing preliminary tissue destruction and reducing the oxidation-reduction potential of the oxygenated tissue. Once sufficient oxygen has been removed to allow the anaerobic Bacteroides to replicate, these bacteria begin to predominate during the second, chronic stage of infection.

The Bacteroides contribute to development of a synergistic infection in three ways: stimulation of abscess formation, reduced phagocytosis by polymorphonuclear leukocytes (PMN’s), and inactivation of antibiotics by b lactamase production. Abscess formation is a major complication of intestinal infections, and results in the formation of a fibrous membrane surrounding a mass of cellular debris, dead PMN’s, and a mixed population of bacteria. If not removed, the abscess will expand, possibly causing intestinal obstruction, erosion of resident blood vessels, and ultimately fistula formation. Abscesses may also metastasize, resulting in bacteremia and disseminated infection. Formation of the abscess is a pathological response of the immune system to the presence of the Bacteroides capsular polysaccharide. B. fragilis is the only bacterium that has been shown to induce abscess formation as the sole infecting organism. Purified capsule can stimulate formation of a histologically identical abscess, indicating that it is this component of the bacterium which stimulates the host immune system to deposit fibrin, forming the outer membrane of the abscess. The Bacteroides capsule has been shown to have an unusual structure, composed of repeating units of two distinct polysaccharides, each of which contains exposed positively and negatively charged side-chains. Most bacterial polysaccharides stimulate an antibody-mediated immune response, but the B. fragilis capsule stimulates a T cell-mediated response. Presumably, the intention of the cell-mediated immune response is to wall off the infection and protect the host from dissemination, but in fact, formation of an abscess protects the Bacteroides and neighboring bacteria from exposure to high concentrations of antibiotics and further attack from the immune system.

Another important synergistic virulence factor of B. fragilis is the ability to inhibit phagocytosis. Once the Bacteroides actively begin to replicate, they are able to interfere with attack by the immune system in two ways. First, production of the capsule itself is able to reduce the ability of the PMN’s to phagocytose the bacterial cells. Secondly, the Bacteroides are able to secrete an as yet uncharacterized factor which degrades complement proteins, and thus inhibits both chemotaxis of PMN’s and opsonization of itself and neighboring bacteria.

A final contribution of the Bacteroides to a successful synergistic infection is the production of b-lactamase. Most Bacteroides strains express constitutive b-lactamase activity; the enzyme is extra-cellular, and thus is capable of diffusing within an abscess or other site of infection. Production of extra-cellular b-lactamases has been shown to protect other organisms in the vicinity during a mixed infection. These bacteria have several other features that contribute to their pathogenicity. The Bacteroides are among the most aerotolerant of anaerobes, able to tolerate atmospheric concentrations of oxygen for up to three days. During initiation of an intraabdominal infection, oxygen tolerance is believed to allow the bacteria to survive in the oxygenated tissue of the abdominal cavity until E. coli and other synergistic organisms are able to reduce the redox potential at the site of infection. Additionally, this oxygen tolerance may help in surviving free radical production by the immune system PMNs. Bacteroides have been found to encode two major oxidative stress response genes, catalase and superoxide dismutase, as well as approximately 28 other oxygen-induced proteins.

Although a commensal organism, Bacteroides can occasionally cause diarrhea. Strains of Bacteroides isolated from some patients with undiagnosed diarrhea were found to be enterotoxigenic, and in patients less than three years age they were associated with intestinal cramping, vomiting, and bloody stools. The purified toxin, fragilysin, was found to be a metalloprotease capable of hydrolysing gelatin, actin, tropomyosin, and fibrinogen. In a study comparing the frequency of B. fragilis enterotoxigenic and non-enterotoxigenic bacteria involved in various infection sites, the enterotoxic strains were found in higher frequencies in bacteremias. It is possible that fragilysin is involved in releasing the organism from an abscess or other site of infection and allowing it to enter the blood stream, thus disseminating infection throughout the body.

The Bacteroides genus of anaerobic bacteria comprise the majority of microorganisms that inhabit the digestive tract. 50% of most fecal matter is actually Bacteroides fragilis cells! Bacteroides organisms are the anaerobic counterpart of E. coli except they are somewhat smaller. They grow well on blood agar, and under the microscope, they may contain large vacuoles that are similar in appearance to spores. Members of Bacteroides species are not spore-forming, but they do produce a very large capsule. Their pathogenicity is limited, however, because they possess no endotoxin in their cell membrane. Infection only occurs after severe trauma to the abdominal region. Infection could lead to abscess formation and possibly fever. Antibiotic treatment usually consists of metronidazole or clindamycin.

  Antibiotic Resistance

  B. fragilis is the most common anaerobic organism isolated from clinical infections, and untreated has a mortality rate of 60%. This mortality rate can be greatly improved, however, with use of appropriate antimicrobial therapy. The Bacteroides are potentially resistant to a broad range of antibiotics, and resistance to a given antimicrobial can vary greatly between institutions. Resistance to any antimicrobial agent may occur by three mechanisms: altered target binding affinity, decreased permeability for the antibiotic, or the presence of an inactivating enzyme. The Bacteroides are adept at antimicrobial evasion, and may use any or all of the above mechanisms to thwart effective clinical therapy. Antimicrobial agents may target several areas of bacterial physiology: protein translation, nucleic acid synthesis, folic acid metabolism, or cell wall synthesis. Protein synthesis inhibitors bind either the 30s subunit of the ribosome (aminoglycosides, tetracycline), or the 50s subunit (macrolides, lincosamides, chloramphenicol). Bacteroides are inherently resistant to aminoglycosides, as uptake of this drug is energy dependent, and requires an oxygen or nitrate dependent electron transport chain which is lacking in these anaerobes. The Bacteroides have acquired resistances to the other protein synthesis inhibitors; resistance to clindamycin/erythromycin (macrolide-lincosamide antibiotics), and tetracycline will be discussed as pertinent examples.

Laboratory Identification:

Collection of specimens of anaerobic bacteria is important because documentation of an anaerobic infection is through culture of organisms from the infected site. Appropriate documentation of anaerobic infection requires proper collection of appropriate specimens, expeditious transportation, and careful laboratory processing.

Specimens must be obtained free of contamination. Inadequate techniques or media can lead to missing the presence of anaerobic bacteria or the assumption that only aerobic organisms are present in a mixed infection.

Because anaerobes are present on skin and mucous membranes, even minimal contamination with normal florae can be misleading.

Unacceptable or inappropriate specimens can yield normal florae and, therefore, have no or little diagnostic value.

Appropriate materials should be obtained by using techniques that bypass the normal florae.

Direct-needle aspiration is the best method of obtaining a culture; the use of swabs is much less desirable.

Specimens obtained from normally sterile sites, such as blood or spinal, joint, or peritoneal fluids, are collected after thorough skin decontamination.

Two approaches are used to culture the maxillary sinus following sterilization of the canine fossa or the nasal vestibule, via either the canine fossa or the inferior meatus.

Urine is collected by percutaneous suprapubic bladder aspiration.

Other specimens can be collected from abscess contents, from deep aspirates of wounds, and by special techniques, such as transtracheal aspirates or direct lung puncture.

Specimens of the lower respiratory tract are difficult to obtain without contamination with indigenous florae. Double-lumen catheter bronchial brushing and bronchoalveolar lavage, cultured quantitatively, can be useful.

Culdocentesis fluid obtained after decontamination of the vagina is acceptable.

Transportation of specimens should be prompt unless transport devices are available. Transport devices generally contain oxygen-free environments provided by a mixture of carbon dioxide, hydrogen, and nitrogen, plus an aerobic condition indicator. Specimens should be placed into an anaerobic transporter as soon as possible.

Liquid or tissue specimens are always preferred to swabs.

Liquid specimens are inoculated into an anaerobic transport vial or a syringe and a needle.

All air bubbles are expelled from the syringe. Insertion of the needle tip into a sterile rubber stopper is no longer recommended. Because air gradually diffuses through the plastic syringe wall, specimens should be processed in less than 30 minutes.

Swabs are placed in sterilized tubes containing carbon dioxide or prereduced anaerobically sterile Carey and Blair semisolid media.

Tissue specimens can be transported in an anaerobic jar or in a sealed plastic bag rendered anaerobic.

Gram stain of a smear of the specimen provides important preliminary information regarding the types of organisms present, suggests appropriate initial therapy, and serves as a quality control.

Cultures should be immediately placed under anaerobic conditions and should be incubated for 48 hours or longer. An additional 36-48 hours is generally required for species- or genus-level identification by using biochemical tests. Kits containing these tests are commercially available.

A rapid enzymatic test enables identification after only 4 hours of aerobic incubation.

Gas-liquid chromatography of metabolites is often used.

Nucleic acid probers and polymerase chain reaction methods are also being developed for rapid identification.

Detailed procedures of laboratory methods can be found in microbiology manuals.

Antimicrobial susceptibility testing of AGNB has become less predictable because their resistance to several antimicrobials has increased. Screening of AGNB isolates for beta-lactamase activity may be helpful. However, occasional strains may resist beta-lactam antibiotics through other mechanisms.

Routine susceptibility testing is time-consuming and often unnecessary. However, testing the susceptibility of isolates recovered from sterile body sites and/or those that are clinically important (ie, blood cultures, bone, CNS, serious infections) and have variable susceptibilities, especially those isolated in pure culture from properly collected specimens, is important.

Antibiotics that should be tested include penicillin, a broad-spectrum penicillin, a penicillin plus a beta-lactamase inhibitor, clindamycin, chloramphenicol, a second-generation cephalosporin (eg, cefoxitin), newer quinolones, metronidazole, and a carbapenem.

The recommended methods include agar microbroth and macrobroth dilution.

Newer methods include the E-test and the spiral gradient end point system.

 

  Specimen collection to avoid contamination with normal flora

  Oxygen-free transport medium system

  Avoid drying

  Bacteroides spp. grow rapidly (within two days) but most other anaerobes are slow growers on selective media

  B. fragilis are resistant to kanamycin, vancomycin and colistin

  B. fragilis growth is stimulated in the presence of 20% bile

 

LABORATORY INDICATIONS (B. fragilis):

Indole –

Catalase +

Esculin hydrolysis +

Glucose fermentation

Lactose +

Treatment, Prevention & Control:

  Surgical drainage of abscess(es) and removal of necrotic tissue(s)

  Long-term course of antibiotics

  Prophylatic use of antibiotics

  Prior to invasive surgical procedures that disrupt mucosal barriers

  Immediately following trauma that disrupts mucosal barriers

 

PREVOTELLA

P. albensis; P. baroniae; P. bergensis; P. bivia; P. brevis; P. bryantii; P. buccae; P. buccalis; P. corporis; P. dentalis; P. denticola; P. disiens; P. enoeca; P. genomosp. C1; P. intermedia; P. loescheii; P. marshii; P. melaninogenica; P. multiformis; P. multisaccharivorax; P. nigrescens; P. oralis; P. oris; P.oulorum; P. pallens; P. ruminicola; P.ruminicola 23; P. aff. ruminicola Tc2-24; P. salivae; P. shahii; P. tannerae; P. veroralis; P. sp

Prevotella sp. are among the most numerous microbes culturable from the rumen and hind gut of cattle and sheep, where they help the breakdown of protein and carbohydrate foods. They are also present in humans, where they can be opportunistic pathogens. Prevotella, credited interchangably with Bacteroides melaninogenicus, has been a problem for dentists for years. As a human pathogen known for creating periodontal and tooth problems, Prevotella has long been studied in order to counteract its pathogenesis (AAP).

Prevotella strains are Gram-negative, non-motile, rod-shaped, singular cells that thrive in anaerobic growth conditions. They are known for being host-associated, colonizing the human mouth. Prevotella bacteria colonize by binding or attaching to other bacteria in addition to epithelial cells, creating a larger infection in previously infected areas. Another survival mechanism is Prevotella cells’ natural antibiotic resistant genes, which prevent extermination.

Pathology

About twenty identified species of Prevotella are known to cause infection, including Prevotella dentalis, which was previously known as Mitsuokella dentalis. Prevotella species cause infections such as abscesses, bacteraemia, wound infection, bite infections, genital tract infections, and periodontitis (Pavillion). Specific infections caused by Prevotella include the disease of tissues surrounding an individuals teeth (see photo at right) and of the supporting tooth and gingivitis (TIGR). Symptoms of Prevotella infections can include pain, swelling, and in some cases a “wet” canal (Gomes).

 

Disease shown in Xray cause by Prevotella oralis.

Antibiotics for treating Prevotella include metronidazole, amoxycillin/clavulanate, ureidopenicilins, carbapenems, cephalosporins, clindamycin, and chloramphenicol (Pavillion).

Prevotella is also well-known as a preventative agent for the bovine disease of rumen acidosis. Rumen acidosis greatly affects milk production of cattle by disrupting the typical digestive processes of the stomach. This leads to an increased susceptibility to other pathogenic forces which also affect the health of food provided from the cattle. With an estimated twenty percent of all American cattle suffering from some form of acidosis, it has been calculated that the bovine market loses one billion dollars annually (ARS).

CLINICAL MANIFESTATIONS: Bacteroides and Prevotella species from the oral cavity can cause chronic sinusitis, chronic otitis media, dental infection, peritonsillar abscess, cervical adenitis, retropharyngeal space infection, aspiration pneumonia, lung abscess, empyema, or necrotizing pneumonia. Species from the gastrointestinal tract are recovered in patients with peritonitis, intra-abdominal abscess, pelvic inflammatory disease, postoperative wound infection, or vulvovaginal and perianal infections. Soft tissue infections include synergistic bacterial gangrene and necrotizing fasciitis. Invasion of the bloodstream from the oral cavity or intestinal tract can lead to brain abscess, meningitis, endocarditis, arthritis, or osteomyelitis. Skin involvement includes omphalitis iewborn infants, cellulitis at the site of fetal monitors, human bite wounds, infection of burns adjacent to the mouth or rectum, and decubitus ulcers. Neonatal infections, such as conjunctivitis, pneumonia, bacteremia, or meningitis, occur rarely. Most Bacteroides infections are polymicrobial.

ETIOLOGY: Most Bacteroides and Prevotella organisms associated with human disease are pleomorphic, nonspore-forming, facultatively anaerobic, gram-negative bacilli. Bacteroides and Prevotella species produce enzymes that play a role in the pathogenesis of disease.

EPIDEMIOLOGY: Bacteroides and Prevotella species are part of the normal flora of the mouth, gastrointestinal tract, or female genital tract. Members of the Bacteroides fragilis group predominate in the gastrointestinal tract flora; members of the Prevotella melaninogenica (formerly Bacteroides melaninogenicus) and Prevotella oralis (formerly Bacteroides oralis) groups are more common in the oral cavity. These species cause infection as opportunists, usually after an alteration of the body’s physical barrier, and in conjunction with other endogenous species. Endogenous transmission results from aspiration, spillage from the bowel, or damage to mucosal surfaces from trauma, surgery, or chemotherapy. Mucosal injury or granulocytopenia predispose to infection. Except in infections resulting from human bites, no evidence for person-to-person transmission exists.

The incubation period is variable and depends on the inoculum and the site of involvement but usually is 1 to 5 days.

DIAGNOSTIC TESTS: Anaerobic culture media are necessary for recovery of Bacteroides or Prevotella species. Because infections usually are polymicrobial, aerobic cultures also should be obtained. A putrid odor suggests anaerobic infection. Use of an anaerobic transport tube or a sealed syringe is recommended for collection of clinical specimens.

TREATMENT: Abscesses should be drained when feasible; abscesses involving the brain or liver may resolve with effective antimicrobial therapy. Necrotizing lesions should be débrided surgically.

The choice of antimicrobial agent(s) is based on anticipated or known in vitro susceptibility testing. Bacteroides infections of the mouth and respiratory tract generally are susceptible to penicillin G, ampicillin sodium, and broad-spectrum penicillins, such as ticarcillin disodium or piperacillin sodium. Clindamycin is active against virtually all mouth and respiratory tract Bacteroides and Prevotella isolates and is recommended by some experts as the drug of choice for anaerobic infections of the oral cavity and lungs. Some species of Bacteroides and Prevotella produce ß-lactamase. A ß-lactam penicillin active against Bacteroides combined with a ß-lactamase inhibitor can be useful to treat these infections (ampicillin-sulbactam sodium, amoxicillin-clavulanate potassium, ticarcillin-clavulanate, or piperacillin-tazobactam sodium). Bacteroides species of the gastrointestinal tract usually are resistant to penicillin G but are predictably susceptible to metronidazole, chloramphenicol, and usually, clindamycin. More than 80% of isolates are susceptible to cefoxitin sodium, ceftizoxime sodium, and imipenem. Cefuroxime, cefotaxime sodium, and ceftriaxone sodium are not reliably effective.

ISOLATION OF THE HOSPITALIZED PATIENT: Standard precautions are recommended.

CONTROL MEASURES: None.

 

Porphyromonas

Porphyromonas, which are commonly found in the human body and especially in the oral cavity, were originally classified in the Bacteroides genus. Porphyromonas gingivalis are an oral anaerobe associated with periodontal lesions, infections, and adult periodontal disease. Approximately 70-90% of people pubescent and older have gingivitis, an oral inflammatory process and a possible precursor to adult periodontal disease, which is associated with Porphyromonas gingivalis. Gingivitis allows Porphyromonas gingivalis to further infect the areas near the root of the teeth causing tooth decay and infection.

 

 

Porphyromonas gingivalis possesses an armamentarium of cell-surface associated and extracellular activities, which are studied intensively for their virulence potential. Several are putative adhesins which interact with other bacteria, epithelial cells, and extracellular matrix proteins. Secreted or cell-bound enzymes, toxins, and hemolysins may play a significant role in the spread of the organism through tissue, in tissue destruction, and in evasion of host defenses.

Three groups reported oral epithelial cell invasion by laboratory and clinical isolates of P. gingivalis. Recently it was reported that P. gingivalis invasion was accompanied by intracellular calcium-fluxes and inhibited by cytochalasin D and nocodazole, indicating that rearrangements in the cytoskeleton of the epithelial cell are necessary for internalization. The authors also report that protein kinase signaling pathways within epithelial cells are associated with P. gingivalis invasion as in other systems. The genes associated with invasion are being investigated.

Biochemical, immunological and genetic evidence indicates that P. gingivalis fimbriae are involved in adhesion to both saliva-coated hydroxy-apatite and to human oral epithelial cells. Animal studies suggest fimbriae play a role in host colonization.

Porphyromonas gingivalis possesses three major proteolytic activities with a) trypsin-like, b) collagenolytic, and c) glycylprolyl peptidase activities. Numerous proteins with thiol-dependent trypsin-like cleavage specificity can be isolated from cells and culture supernatants, but biochemical and genetic analyses indicate that many of these are derived by proteolytic processing of a larger, cell-associated, primary gene product. Structurally, the proteases contain a propeptide sequence, an N-terminal active site and a C-terminal putative adhesin domain with repeat regions. Recent studies indicate that P. gingivalis possesses a family of genes which contains part of the protease sequences. These proteases also possess hemagglutinating activity and share extensive DNA sequence homology with hemagglutinin genes hagA and D.

The black pigmentation of P. gingivalis is due to the accumulated hemin used as an iron source for growth. The organism appears to lack known siderophore activities and must use alternate mechanisms to sequester and transport exogenous iron. The expression of several outer membrane proteins is induced or repressed by heme, and a heme-repressible outer surface protein, which is translocated to the outer surface under heme-limiting conditions, is able to bind hemin. It was  showed that hemin binding was induced by growth in hemin, a discrepancy which is attributed to differences in bacterial growth and assay conditions. P. gingivalis hemolytic activity is associated with the cell surface and outer membrane vesicles and two hemolysin genes have been cloned. Hoover and Yoshimura  reported the isolation of pigment-deficient (non-heme accumulating) transposon mutants also defective in trypsin-like protease activity. These data suggest that these virulence functions may be genetically linked. Using a similar screen, Genco et al. observed that non-pigmented mutants had increased proteolytic activity.

Cell Structure and Metabolism

 

 

Porphyromonas gingivalis.

 

Porphyromonas are Gram-negative, nonsporeforming, anaerobic, rod-shaped bacteria that produce porphyrin pigments (dark brown/black pigments). Like Bacteroides, Porphyromonas are more closely related to Gram-positive bacteria than other Gram-negative bacteria. Also like Bacteroides, Porphyromonas have an outer membrane, a peptidoglycan layer, and a cytoplasmic membrane.

The black pigmentation of P. gingivalis is from the accumulation of hemin used as an iron source for bacterial growth. This may be a reason that people with higher metal intakes, such as iron, have more of a risk for getting gingivitis and periodontitis. Also, cell surface adhesion molecules on the surface of Porphyromonas, which interact with other bacteria, epithelial cells, and extracellular matrix proteins, assist the bacteria in living in their human host. The mouth generally has a consistant flow sugars and other simple carbohydrates, so it is likely that P. gingivalis living in peridontal tissue receive their energy from these materials. However, another common idea is that the main source of P. gingivalis energy and cell materials come mainly from peptides instead of single amino acids. However, due to the complicated amino acid composition of peptides or proteins, the amino acid metabolic pathway of P. gingivalis has been difficult to determine. Also, some enzymes involved in amino acid metabolism in these bacteria are known to be oxygen labile (changes in the presence of oxygen), which further complicates the detection and analysis of P. gingivalis metabolic enzymes.

Ecology

P. gingivalis may be one of the natural bacterial flora in the oral cavity that is comprised of over 400 different species of microorganisms. However, isolating it in a health oral cavity has proven difficult. It constitutes approximately 5% of the bacterial flora in an oral cavity with gingivitis and more than 5% in a mouth with advanced periodontitis. P. endodontalis also does not appear in a healthy mouth but can be detected in a diseased mouth. Porphyromonas also favor a slightly alkaline environmental pH. Like other bacteria that live in the human mouth, Porphyromonas favor an average temperature of around 95 degrees and a 100% humidity. It has been reported that anywhere from 1,000 to 1 billion bacteria can live on each tooth surface. P. asaccharolytica has been isolated from many nonoral sites such as the cervix, ear, intestine, genitalia, and from many infections throughout the body (only limited reports of P. asaccharolytica in the mouth). Other strains have been found in samples of blood, amniotic fluid, umbilical cord, empyema, peritoneal and pelvic abscesses, endometritis, and infections.

Pathology

Cell surface adhesion molecules on the surface of Porphyromonas interact with other bacteria, epithelial cells, and extracellular matrix proteins; they are currently being studied for their pathogenic potential. P. gingivalis is thought to spread through tissue, destroy tissue, and evade host defenses by the use of secreted cell-bound proteases, immunoactive cellular compounds, and toxins. P. gingivalis cytotoxic metabolic end products, which include butyrate, propionate, have low molecular weights which allows them to easily penetrate periodontal tissue and disrupt the host cell activity.

In the past, more research papers have been devoted to P. gingivalis that to any other dental pathogens. This is due to the high frequency in which P. gingivalis is associated with peridontal lesions, infections, and periodontitis. Projects such as The Forsyth Institute and TIGR’s Porphyromonas gingivalis genome project hope to switch the method of treating periodontal diseases by surgery and tooth scaling to antibiotic or vaccine therapies.

 

Diagram of gingivitis.

Gingivitis, which is inflamation of the gums that causes bleeding and exposes the base of the teeth, allows Porphyromonas gingivalis to infect the areas near the root of the teeth causing tooth decay and infection. the most common type of gingivitis is brought on by the accumlation of microbial plaques in people who do not take proper care of their mouth. Pockets form around the teeth, lesions can form, bacterial infections occur, and, eventually, peridontal ligaments break down and destruction of the local aveolar bone occurs. The teeth then loosen and fall out or can be broken off. Once bacterial infections occur, the gingivitis takes on a new, infectous form called acute necrotizing ulcerative gingivitis (ANUG). ANUG can cause an accelerated destruction of affected tissues as well as local or systemic spread of infection. When ANUG spread beyond the gingiva (gums) and invades the local tissues of the mouth and face, the syndrome is called noma (cancrum oris).

fusobacteria

Fusobacterium necrophorum is part of anaerobic normal throat flora  has a predisposition to abscess formation (termed ‘necrobacillus’ – this is very rare – affecting one per million of population) platelet aggregation and virulent toxin production results in internal jugular venous thrombosis (Lemierre’s syndrome)  cavitating pulmonary lesions and haemoptysis occur as a result of septic embolisation other possible features include empyema, septic arthritis, and abscesses in the liver, spleen and muscles if fusibacteria isolated on a throat swab consult local microbiologist for guidance re: treatment some strains are beta-lactimase producers so there may be advantages of prescribing a beta-lactimase inhibitor such as co-amoxiclav .

The bacterial species Fusobacterium nucleatum is one of the most frequently detected cultivable organisms in subgingival dental plaque from both inactive and active gigivitis and periodontitis sites. F. nucleatum is the most frequent cause of gingival inflammation that initiates periodontal disease and that it is the most common predominant pathogen in subsequent periodontal destruction.4 Moreover, it is found in a number of extra-oral sites where, together with other organisms, it causes polymicrobial infections.

Despite some confusion surrounding their validity, the heterogeneous collection of bacteria characterized as F. nucleatum has been divided into a number of subspecies; namely, subspecies n u c l e at u m , vincentii,  polymorphum, fusiforme and animalis, the first two of which are believed to be associated with sites of periodontal disease. In an attempt to explain differences in pathogenicity, studies in this laboratory have accordingly focused on various aspects of the physiology and metabolism of the Type strain and a clinical isolate from within each of the putative sub-species.

The growth and metabolism of Fusobacterium nucleatum

The growth and nutritional aspects of the metabolism of the various strains of F. Nucleatum were studied by growing them under continuous culture conditions in a chemostat using methods described previously. Briefly, the growth medium was a filter-sterilized chemically-defined medium (CDM). It contained a range of amino acids, a

number of vitamins, nucleotides, salts, trace elements and a fermentable carbohydrate such as glucose or fructose.10 Tween 80 was added to aid cell dispersion, as was thioglycollic acid to maintain a low redox potential. The growth temperature was 37°C, the pH controlled by the automatic addition of either 2 mol/L KOH or 2 mol/L HCl and anaerobic conditions maintained by gassing both the culture vessel and medium reservoir with a N₂/CO₂ (90:10)

mixture. Under varying conditions of growth rate and pH, growth parameters such as biomass – as measured by cell dry mass and protein content – and metabolic end-products were determined . All strains showed similar physiological and metabolic properties. For example, they grew well in various CDMs, with or without added carbohydrate, over a pH range of about 6 to 8, the optimum being between 7.0 and 8.0. In the absence of fermentable carbohydrate – most likely to occur in the subgingival environment – energy and carbon were obtained

from the fermentation of the amino acids glutamate (Glu), histidine (His), lysine (Lys) and serine (Ser). Most strains appeared to be auxotrophic for His and some for Lys. The end-products of fermentation were  acetate:butyrate: formate (3:2:1), irrespective of growth rate. It is worth noting here that butyrate was shown to be a potent in vitro inhibitor of gingival fibroblast proliferation and could, therefore, be a virulence factor. In terms of interbacterial nutritional co-operation – a key to explaining the coexistence of dental plaque bacteria – the formate produced by F. nucleatum could act as an electron donor for Wolinella recta with acetate performing the same function for Eubacterium species.

The breakdown and utilization of peptides. The low levels of free amino acids usually present in the oral environment would probably be insufficient to sustain the growth of Gram-negative anaerobes, including F. nucleatum, that obtain energy from the fermentation of amino acids. Since it lacks endopeptidase activities, F. nucleatum will not grow on proteins such as casein or albumin, but organisms such as Porphyromonas gingivalis, which is often found together with F. nucleatum in active disease sites, does possess such activities. Provided that they contained the appropriate residues, resultant peptides would thus be potential energy sources for

F. nucleatum. Accordingly, the ability of resting cells of F. nucleatum to attack unsubstituted peptides containing the appropriate residues – present C- or N – terminally or buried in the peptides – was investigated. The ability of growing cells to utilizean essentially amino acid-free peptide fraction prepared from a commercial peptone was also studied.

The pathogenic potential of Fusobacterium nucleatum and its significance in development of periodontal diseases, as well as in infections in other organs, have gained new interest for several reasons. First, this bacterium has the potential to be pathogenic because of its number and frequency in periodontal lesions, its production of tissue irritants, its synergism with other bacteria in mixed infections, and its ability to form aggregates with other suspected pathogens in periodontal disease and thus act as a bridge between early and late colonizers on the tooth surface. Second, of the microbial species that are statistically associated with periodontal disease, F. nucleatum is the most common in clinical infections of other body sites. Third, during the past few years, cloning and sequencing and the application of new techniques such as PCR have made it possible to obtain more information about F. nucleatum on the level, thereby also gaining better knowledge of the structure and functions of the outer membrane proteins (OMPs). OMPs are of great interest with respect to coaggregation, cell nutrition, and antibiotic susceptibility. Several studies have shown that OMPs are involved in the pathogenicity of gram-negative bacteria.

F. nucleatum is the type species of the genus Fusobacterium, which belongs to the family Bacteroidaceae. The name Fusobacterium has its origin in fusus, a spindle; and bacterion, a small rod: thus, a small spindle-shaped rod. The term nucleatum originates from the nucleated appearance frequently seen in light and electron microscope preparations owing to the presence of intracellular granules  F. nucleatum is nonsporeforming, nonmotile, and gram negative, with a G1C content of 27 to 28 mol% and a genome size of about 2.4 3 106 bp (34). Most cells are 5 to 10 mm long and have rather sharply pointed ends. Colony morphology is not a consistent parameter of the fusobacteria and is not sufficient for species identification. The bacterium is anaerobic but grows in the presence of up to 6% oxygen (204). The production of butyric acid as a major product of the fermentation of glucose and peptone, together with characteristic lipid constituents, differentiates Fusobacterium species from other anaerobic, gram-negative, nonsporeforming rods. F. nucleatum has no sialidase activity.

The species F. nucleatum is considered to be rather heterogeneous. On the bases of electrophoretic patterns of whole-cell proteins and DNA homology, there were proposed dividing F. nucleatum into three (or four) different subspecies: subspecies nucleatum, polymorphum, and vincentii.

Occurrence and Role in Periodontal Diseases. F. nucleatum is one of the most common species in human infections and can be found in body cavities of humans and other animals.

Of the periodontal species that are statistically associated with periodontal disease, it is the most common in clinical infections of other body sites. It has been isolated from several parts of the body  and from infections such as tropical skin ulcers, peritonsillar abscesses, pyomyositis and septic arthritis, bacteremia and liver abscesses, intrauterine infections, bacterial vaginosis, urinary tract infections, pericarditis and endocarditis, and lung and pleuropulmonary infections. The origin of F. nucleatum in infection has been dental in several cases. Fusobacteria, including F. nucleatum, are recovered from a variety of infections in children. Studies of the predominant cultivable oral microflora reveal that only a small number of the over 300 species found in human subgingival plaque are associated with periodontal disease. Collective microbiological studies implicate the gram-negative species Porphyromonas gingivalis,

Prevotella intermedia, Bacteroides forsythus, F. nucleatum, Capnocytophaga rectus, Eikenella corrodens, Capnocytophaga spp., certain spirochetes, and the gram-positive Eubacterium spp. in adult periodontitis. Actinobacillus actinomycetemcomitans seems to be the prime candidate in the etiology of juvenile periodontitis.

The role of F. nucleatum in the development of periodontal diseases has lately attracted new interest. Of over

51,000 isolates examined by Moore and Moore (208), F. Nucleatum and Actinomyces naeslundii were the most commonly occurring species in the human gingival crevice. From the early to the late stages of plaque formation, there is a shift from a gram-positive to a gram-negative microflora in which, among others, F. nucleatum increases in proportion as plaque forms. From studies on the bacteriology of experimental gingivitis in children (4 to 6 years) and young adults (22 to 31 years), F. nucleatum appeared to be one of the nonspirochetal organisms most closely correlated with gingivitis, and it appeared to be more common in young adults. This also seems to be the case in naturally occurring gingivitis. F. nucleatum has been detected less frequently in the first 6 months of life compared with older age groups, ranging from 25% of children below 6 months to 67% of children by 2 years, but of total anaerobic CFU, the proportion of F. Nucleatum was generally low (85, 173). In children 5 to 7 years of age, F. nucleatum is found commonly in plaque, being isolated from 60 to 70% of children examined (86). Even in juvenile periodontitis lesions, F. nucleatum has been reported in large amounts at active sites of inflammation. F. nucleatum is detected more commonly in dental plaque than on the tongue or in saliva, but these sites are a more common habitat of the organism than are the tonsils.

It has been suggested that certain combinations of bacterial species (clusters) present at the same time in the periodontal pocket are more prone to elicit periodontitis than other bacterial clusters. In experimentally induced infections in mice, strains of F. nucleatum were pathogenic when administered in pure culture; however, a mixed culture of F. nucleatum with either P. gingivalis or Prevotella intermedia was significantly more pathogenic than F. Nucleatum in pure culture. Positive correlations for disease production between F. nucleatum, C. rectus, Prevotella intermedia, and Peptostreptococcus micros have been found in periodontal as well as endodontal lesions. Recently, it was demonstrated positive associations between F. nucleatum, P. gingivalis, Prevotella intermedia, and B. Forsythus in subgingival plaque samples from untreated Sudanese patients with periodontitis. The most important finding was the effect exerted by F. nucleatum on the colonization of Prevotella intermedia; Prevotella intermedia was never detected in a site unless F. nucleatum also was present. Combinations of F. nucleatum, B. forsythus, and C. rectus or of P. gingivalis, Prevotella intermedia, and Streptococcus intermedius in sites that had the most attachment loss and the deepest pockets have been reported. F. nucleatum was also present in the majority of instances when B. forsythus was detected

 However, F. nucleatum is rather widespread in periodontal pockets in general, and F. nucleatum and C. rectus were the most frequently recovered species in an analysis of the subgingival flora of randomly selected subjects; 80 to 81% of the subjects were found positive for these microorganisms. F. nucleatum has been isolated from both active and inactive sites of disease, and it has been suggested that different subgroups may vary in pathogenesis and be related to different levels of disease activity. The most common subspecies in the gingival crevice is F.  nucleatum subsp. vincentii (this is also the case for other body sites), with F. nucleatum subsp. nucleatum and F. nucleatum subsp. Polymorphum following in a ratio of 7:3:2.

Growth and Metabolism

Fusobacteria require rich media for growth and usually grow well in media containing Trypticase, peptone, or yeast extract. Much attention has been paid to the utilization of amino acids and peptides by F. Nucleatum. F. nucleatum seems to be one of the few nonsporulating anaerobic species that uses amino acid catabolism to provide energy, and some strains of F. nucleatum utilize and apparently need peptides for growth. ATCC 10953 did not use any peptides to a noticeable extent (16), whereas all other strains examined utilized peptides containing glutamate and aspartate. All strains used amino acids, and glutamate, histidine, and aspartate utilization was common to all strains. The glutamate and histidine pools were characteristically depleted before the other amino acids were attacked, and at that time all strains except ATCC 10953 started to utilize peptides at a noticeable rate.

The utilization of peptides by these species is in accordance with available substrates in the environmental niches that these bacteria colonize. In the gingival crevice, the saccharolytic bacteria utilize the available carbohydrates. Peptides are generated by the hydrolytic activity of P. gingivalis, and therefore the levels of protein and ammonium ions are high and probably available to F. nucleatum. Carbohydrate metabolism and uptake by F. nucleatum have been the focus of interest for several studies.

F. nucleatum utilizes glucose to a low extent compared with other species, and F. nucleatum does not grow with sugars as the main energy source. Available data on fusobacterial species indicate that glucose is used for the biosynthesis of intracellular molecules and not energy metabolism. The ability of F. nucleatum to metabolize its storage glycopolymers before utilizing amino acids has recently been demonstrated (254). F. nucleatum possesses an amino aciddependent (only glutamine, lysine, and histidine are effective) carbohydrate transport system for glucose, galactose, and fructose that operates exclusively under anaerobic conditions and results in the production of polysaccharides inside the cell. Catabolism of these polysaccharides is controlled by the same amino acids, and the polymer can be degraded to yield butyric, lactic, formic, and acetic acids. Addition of glutamine, lysine, or histidine to the anaerobic cell suspension inhibits polymer degradation. Polymer catabolism is resumed when specific enzymes required for amino acid fermentation are inactivated by exposure of the cells to air. The energy necessary for active transport of the sugars (acetylphosphate and ATP) is derived from the anaerobic fermentation of glutamine, lysine, and histidine, and these compounds must provide the energy for glucose and galactose accumulation by a three-stage process involving membrane translocation, intracellular phosphorylation, and polymer synthesis. The capacity of F. nucleatum to form intracellular polymers from glucose, galactose, and fructose under conditions of amino acid excess and to ferment this sugar reserve under conditions of aminoacid deprivation may contribute to the survival of F. nucleatum in the environment of the oral cavity and to the persistence of this organism in periodontal disease. Certain strains of F. nucleatum can catabolize dextrans, and the dextran hydrolase is found to be cell associated. Since dental plaque bacteria can synthesize and partly utilize dextran, it is suggested that this polysaccharide can act as a carbohydrate storage compound.

The major product from metabolism of peptone or carbohydrate by fusobacteria is butyrate without any iso-acids but often with acetate and lactate and lesser amounts of propionate, succinate, formate, and short-chained alcohols. F. Nucleatum produces propionate from threonine but not from lactate; it does not hydrolyze esculin, but it produces indole. Butyrate, propionate, and ammonium ions inhibit proliferation of human gingival fibroblasts (21), may have the ability to penetrate the gingival epithelium (, and are present in elevated levels in plaque associated with periodontitis. Because of this, they may have an etiological role in periodontal disease.

Although the effect of the metabolites is not sufficient to cause cell death, inhibition of fibroblast proliferation is serious because the potential for rapid wound healing is compromised. Proteases from pathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes, the bacterial proteases may participate in periodontal destruction. F. nucleatum is capable of desulfuration of cysteine and methionine, resulting in the formation of ammonia, hydrogen sulfide, butyric acid, and methyl mercaptan. Hydrogen sulfide and methyl mercaptan account for 90% of the total content of volatile sulfur compounds in mouth air. A biotin-dependent sodium ion pump from F. nucleatum, glutaconyl-coenzyme A decarboxylase, has been characterized.

From a nutritional point of view, the organization of different bacterial species, for example, saccharolytic and asaccharolytic species, aerobic and anaerobic species, and clusters of bacteria in the tooth environment, is fascinating and logical. There exists a symbiotic life in the periodontal pocket that apparently several species make use of. This is best illustrated by the coexistence of different bacterial species in clusters and by coaggregation of F. nucleatum and P. gingivalis in intimate contact, which probably supplies each with essential metabolites. The saccharolytic aerobic bacteria found mostly in supragingival plaque convert carbohydrates into short-chain organic acids, lowering the pH in the local environment. The asaccharolytic bacteria are nearly always anaerobic and generally found subgingivally, where they utilize nitrogenous substances for energy, are usually weakly fermentative, and tend to raise the local pH. More than 90% of the carbohydrates utilized by bacteria in dental plaque are used for energy production, but carbohydrates are also utilized by asaccharolytic species like F. nucleatum in which, e.g., glucose is used for biosynthesis of intracellular macromolecules and not energy metabolism (. Most of the carbohydrate utilized by the subgingival microflora is probably derived from the carbohydrate side chains of glycoproteins. Removal of the carbohydrate residues leaves the protein core available for further hydrolysis by the asaccharolytic species.

The fusobacteria are susceptible to many of the most commonly used antibiotics, but they have reduced susceptibility or may be resistant to vancomycin, neomycin, erythromycin, amoxicillin, ampicillin, and phenoxymethylpenicillin.  Penicillinase-producing strains of F.nucleatum have been isolated, and isolation of beta-lactamase-producing strains of fusobacteria is increasing. As beta-lactamase production and beta-lactam resistance have been increasingly found in gram-negative bacteria, including F. nucleatum, the susceptibility of different bacteria to new agents has been tested. Biapenem, imipenem, the penem WY-49605, and trospectomycin were active against F. Nucleatum in vitro, as were the commonly used agents chloramphenicol and metronidazole.

Antimicrobial agents have been used in periodontal treatment either alone or preferentially in combination with conventional treatment to eliminate putative periodontal pathogens. The most extensively used antimicrobial agents as an adjunct in the treatment of periodontal disease have been the broad-spectrum bacteriostatic tetracyclines, which inhibit protein synthesis in the bacterial cells. Tetracycline, doxycycline, and minocycline concentrate in gingival crevicular fluid at concentrations up to five times those found in serum. As many as 75% of the bacteria in the subgingival pocket may be resistant to tetracycline after long-term, low-dose treatment. Besides systemic administration, antibiotics can be delivered locally to the periodontal pocket. Examples of antibiotics and antibiotic vehicles used for sustained release subgingivally are tetracycline-impregnated fibers and metronidazole gel. Tetracycline-resistant F. nucleatum strains have been found in subgingival plaque samples from patients with periodontal disease.

 

Eikenella corrodens

Eikenella corrodens was first described in 1948 as a slow-growing, anaerobic, Gram-negative rod.  A distinguishing feature of this organism is the ability to pit or corrode the agar in plated culture.  The colonies grow in the little grooves and for this reason it was called a corroding bacillus.  It was classified as Bacteroides corrodens.  Further studies proved that the classification had been applied to two organisms.  The major difference between the two being that one was a facultative anaerobe and the other was an obligate anaerobe.  The facultative anaerobe was renamed Eikenella corrodens.

 

 

E. corrodens inhabits the mucous membrane surfaces of humans, most commonly the respiratory tract.  E. corrodens can cause infections in humans when their immune system is weak.  Once an infection has occurred it can travel to other parts of the body.  E. corrodens is usually found with other bacteria in infections, commonly streptococci. E. corrodens is also responsible for about a quarter of human hand-bite wound infections and clenched-fist injuries.  It is also a putative periodontal pathogen, found at high levels in humans with periodontitis. E. corrodens infections can be treated with antibiotics such as penicillin, ampicillin and tetracycline.

E. corrodens does not grow on selective media.  When it is incubated aerobically it requires hemin.  However, when it is incubated anaerobically it does not require hemin.  Plate growth may be stimulated in a 3-10% CO₂ enviroment, even though CO₂ is not required.  E. corrodens grows so slowly that sometimes it is hidden by other faster-growing bacteria.  However, adding 5 ug/ml of clindamycin increases recovery.

E. corrodens must be incubated for 2-3 days before the colonies grow to a size sufficient for counting.  When plated the organism is dry, flat and has a yellow-pigmented colony.  The colony growth has three zones.  There is a clear and moist center, a highly visible ring that appears like droplets, and an outer growth ring.  The organism can produce either a musty smell or a bleach smell.

E. corrodens is small, straight, nonsporeforming, nonencapsulated and nonmotile.  It is biochemically inactive for most biochemical tests.  It does not produce catalase, urease, indole or H₂S₂.  They are oxidase-positive, catalase-negative, urease-negative, indole-negative and reduce nitrate to nitrite.

Medical importance

E. corrodens is a commensal of the human mouth and upper respiratory tract. It is an unusual cause of infection and when it is cultured, it is most usually found mixed with other organisms. Infections most commonly occur in patients with cancers of the head and neck, but it is also the common in human bite infections, especially “reverse bite” or “clenched fist injuries“. It has also causes infections in insulin-dependent diabetics and intravenous drug users who lick their needles. It is one of the HACEK group of infections which are a cause of culture-negative endocarditis.

E. corrodens infections are typically indolent (the infection does not become clinically evident until a week or more after the injury). They also mimic anaerobic infection in being extremely foul-smelling.

Treatment

E. corrodens can be treated with penicillins, cephalosporins or tetracyclines. It is innately resistant to macrolides (e.g., erythromycin), clindamycin and metronidazole. It is susceptible to fluoroquinolones (e.g., ciprofloxacin) in vitro but there is no clinical evidence available to advocate its use in these infections.

 

Students Practical activities:

1. To carry out indirect hemagglutination test for serologic diagnosis of plague.

 

 

Test results are assessed after complete erythrocyte sedimentation in control (6 well) – markedly localized erythrocytes sediment (“rouleaus”), In the experimental wells rapid erythrocytes agglutination with starlike, marginally festooned sediment (“umbrella”) on the bottom are observed. The titer of serum is its maximum dilution, which causes hemagglutination. Diagnostic titre –   1:40.

 

 

Causative agents of zoonose diseases.  laboratory  diagnosis of plague, other yersiniosis.  laboratory diagnosis of brucellosis,  tularaemia, and anthrax.

 

Yersinia

Yersinia pestis. Genus Yersinia includes the following three bacterial species: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. They were separated from genus Pasteurella on the basis of certain properties and included in family Enterobacteriaceae.

The causative agent of plague, Yersinia pestis, was discovered by the French microbiologist A. Yersin in Hong Kong in 1894. Russian scientists D. Samoilovich, D. Zabolotny, N. Klodnitsky, I. Deminsky, N. Gamaleya and others contributed greatly to the study of the mechanisms of its transmission.

The French microbiologists G. Girard and T. Robic obtained a live vaccine from the attenuated EV strain. R. Karamchamdani and K. Rao of India introduced the extremely effective antibiotic streptomycin into practice for the treatment of all forms of plague.

Bubonic plague, caused by Y. pestis, is an ancient disease that has killed millions of people over the centuries. For example, it is believed to have killed more than 100 million persons in an epidemic in the sixth century. Another epidemic in the 14th century killed one fourth of the European population, and the London plague in 1665 killed more than 70,000 persons. In 1893, an epidemic began in Hong Kong and spread to India where more than 10 million individuals died over a 20-year period. This epidemic eventually reached San Francisco in about1900, and the disease is firmly established in the south-western United States (eg, in prairie dogs, ground squirrels, wood rats, chipmunks, and mice), as well as in many other areas of the world.

Morphology. The plague bacillus, as seen in tissue smears, is ovoid-shaped, 1-2 mcm in length and 0.3-0.7 mcm in breadth (Fig.). It is non-motile, forms no spores, and on solid media cultures is elongated in form. In preparations from tissues and cultures Y. pestis is found to have a delicate capsule. The organism stains with ordinary aniline dyes and gives a bipolar appearance, its ends staining more intensively. It is Gram-negative.

 

Figure.  Yersinia pestis recovered from a bubo and colonies of Y. pestis on meat-peptone agar.

 

Y. pestis

 

 

Y. pestis in blood

 

Y. pestis is characterized by marked individual variability (pleomorphism). In smears from organs and in young cultures it has an ovoid shape, while in cultures on solid media it is elongated and sometimes thread-like. If common salt is added to agar the bacillus shows various forms: ovoid, club-shaped, thread-like, and granular. These forms are usually known as involution forms. The occurrence of filterable types of Y. pestis has also been demonstrated. The G+C content in DNA ranges between 45.8 and 46.8 per cent.

A capsule, a three-layer cell wall, and a three-layer cytoplasmic membrane are demonstrated on ultrathin sections, the cytoplasm is filled with ribosomes and small-granular inclusions and the nucleoid occupies the central part of the cell.

Cultivation. Y. pestis is a facultative anaerobe but can also grow under anaerobic conditions. It is cultivated on ordinary media with pH 6.9-7.0. The optimum temperature for cultivation is 25-30° C The pathogen can also grow at temperatures ranging from 0° to 45° C and at pH from 5.8 to 8.0.

On agar slants the culture forms a viscid translucent mucilaginous mass. On agar plates it forms colonies with turbid white centres and scalloped borders (see fig. 1) resembling lace or crumpled lace handkerchiefs.

In meat broth the cultures form a pellicle on the surface with thread-like growth resembling stalactites and a flocculent precipitate. Sodium sulphite, fresh haemolytic blood, sarcinic extract, and live sarcina (“feeders”) are used as growth stimulators. They are of special value when the seeded material contains a small number of organisms.

 

 

Y. pestis colonies

 

Y. pestis possesses intraspecies variability. It changes quite easily from die virulent R-forms to avirulent S-forms through the O-forms. Resistant S-forms develop in the presence of bacteriophage. They closely resemble the Y. pseudotuberculosis of rodents. Vaccine strains of the plague bacillus are of great practical value and are used for preparing live vaccines.

Fermentative properties. Y. pestis does not liquefy gelatin, nor does it produce indole. It reduces nitrates to nitrites, ferments glucose levulose, maltose, galactose, xylose, mannitol and, occasionally, arabinose with acid formation. Some strains ferment glycerin, while others do not. The differential diagnosis of Y pestis and Y. pseudotuberculosis is very difficult. Unlike Y. pseudotuberculosis of rodents, Y. pestis does not break down adonitol and rarely ferments rhamnose and lactose (Table 1).

Table 1

Differentiation of Yersinia Species

 

Toxin production. Y. pestis is very virulent for humans. The important virulence factors of Y. pestis seem to be directed toward two goals for the organisms: (1) invasion and proliferation within host cells, and (2) resistance to killing by the host. The incredibly high fatality rate of bubonic plague is probably primarily because of septic shock resulting from the bacteremia occurring in the disease.

Y. pestis produces a variety of different virulence factors, but only a few have been characterized sufficiently well to postulate their role in production of disease: (1) a capsular antigen, designated Fraction 1 (Fl), is antiphagocytic, and antibodies to Fl seem to be protective; (2) V/W antigens, consisting of a protein (V) and a lipoprotein (W), are produced together, but their role as virulence factors is unclear, although there are data that V-antigen suppresses the production of interferon-gamma and tumor necrosis factor-alpha, thus suppressing granuloma formation with the resulting delayed-type hypersensitivity to the organisms; and (3) an intracellular murine toxin that is lethal for the mouse (LD₅₀< 1 mcg) and rat but is essentially inert in other hosts. Also described has been a bacteriocin, Pesticin I, that has a lethal effect on Y. pseudotuberculosis and some E coli strains through an N-acetylglucosammidase-incdiated hydrolysis of peptidoglycan. It is always produced coincidentally with coagulase and fibrinolysin. Mutants unable to synthesize fibrinolysin have a million-fold higher LD₅₀ than wild-type strains if the mutant is injected subcutaneously, but not if the organisms are injected into deeper tissues. This postulated that the ability of fibrinolysin to convert plasminogen to plasmin is essential for the invasion of deeper tissues after the superficial inoculation of the host by an infected flea bite. Interestingly, these factors are encoded on a small plasmid not found in the other pathogenic yersiniae. Other determinants that are associated with virulence include the ability to synthesize purines and the property to absorb and store hemin and certain basic aromatic dyes to form coloured colonies. It is not possible to associate these characteristics with any specific virulence determinant, but mutants unable to synthesize purines or store iron to form coloured colonies display a decreased virulence for experimental animals.

Probably the most confusing virulence factors are encoded on a plasmid termed the low calcium response(Lcr) plasmid. This plasmid is expressed only at 37°C (not at 26 °C) and in the presence of low Ca²⁺ levels as would be present in mammalian intracellular fluid. Expression of the plasmid results in (1) a reduced adenylate charge; (2) shutoff of stable RNA synthesis; (3) synthesis of V and W antigens; and (4) synthesis of a number of new outer membrane proteins, which have been termed YOPs. The importance of the Lcr plasmid in the pathogenesis of infection with  Y. pestis is apparent in that Lcr-negative mutants are avirulent.

Some of the YOPs have been shown to be virulence factors. YOP E disrupts actin filaments and may be involved in the intenalization of the bacteria into epithelial cells. YOP H is a protein tyrosine phosphatase, and it has been proposed that this protein may be secreted into the cytoplasm of the infected cell where it could cause dephosphorylation of protein tyrosines that are important in signal transduction pathways. YOP H also has been shown to inhibit phagocytosis. Mutants lacking YOPs K and L were rapidly cleared from organs, and it is assumed that these YOPs are able to inhibit cell-mediated immune reactions. YOP M has been shown to inhibit the aggregation of platelets.

The virulence of Y. pestis is closely linked with a number of another factors: the formation of a brown pigment in a culture on hemin agar, synthesis of purines, dehydrase, catalase, the requirement for calcium, strontium and zinc ions, the sensitivity to glucose and survival and growth in macrophages. The continental strains produce a toxic substance urease.The mouse toxin has been obtained in pure form as an active preparation.

Table 2 summarizes those virulence factors that seem to be important in human disease.

Table 2

Yersinia Virulence Factors in Human Disease

 

Antigen structure. The causative agent of plague contains several antigens among which D, F1, T, W, and V-antigens have been studied.

Virulent Y. pestis cells contain thermostable somatic antigen highly toxic for mice and rats which changes to anatoxin under the effect of formalin, as well as haemolysins and other toxic substances. The agar precipitation technique demonstrated antigens in the causative agent of plague, which were also common to Y. pseudotuberculosis, bacteria of the enteric fever and dysentery groups, and human group O erythrocytes.

Resistance. The plague bacillus can withstand low temperatures. At 0° C it lives for 6 months. It survives on clothes for 5-6 months; in sterile soil and in milk for 90 days; in grain and on cadaver for 40 days; in water for 30 days; in bubo pus for 20-30 days; in sputum for 10 days; in vegetables and fruits for 6-11 days; and in bread for 4 days.

Y. pestis is very sensitive to drying and high temperatures. Boiling kills the organism within 1 minute, and when heated to 60°C it is destroyed in 1 hour. In a 5 per cent phenol solution it is killed in 5-10 minutes and in a 5 per cent solution of lysol in 2-10 minutes.

Pathogenicity for animals. Rodents, among them black rats, grey rats, mice, susliks, midday gerbils, tumarisks, and marmots (tarbagans) are susceptible to plague. More than 300 rodent species may spontaneously contract the disease. In addition, 19 rodent species are susceptible to laboratory infection with plague. Camels died in the Astrakhan steppes m 1911, and humans who ate camel meat contracted plague. Pigs, sheep, goats, donkeys, mules, dogs, cats, monkeys, and certain carnivores are susceptible to the disease iatural environments. However, little epidemiological importance is attached to them.

Guinea pigs, white mice, white rats, and rabbits are the experimental animals which easily  acquire the infection. Animals experimentally inoculated with plague display sepsis, necrosis at the site of injection, enlargement of lymph nodes and spleen, haemorrhages in the skin and mucous membranes (haemorrhagic septicaemia).

 

Pathogenesis and disease in man.

Plague is normally a disease of rodents that exists in two kinds of epidemic centres: the permanent but relatively resistant rat population, where the organisms reside in interepidemic periods; and the temporary but susceptible rodents, particularly the domestic rat population (Fig. 2).

Rats are the primary reservoir: they usually die acutely, with a high-grade bacteremia but occasionally develop amore chronic form of infection. The disease is transmitted by the bites of fleas (e.g., Xenopsylla cheopis, the rat flea) which have previously sucked blood from an infected animal. The ingested bacilli proliferate in the intestinal tract of the flea and eventually block the lumen of the proventriculus. The hungry flea, upon biting another rodent, regurgitates into the wound a mixture of plague bacilli and aspirated blood. If its host dies the flea leaves promptly, seeking a replacement. If another rodent is not available it will accept, a human host, an accidental intruder in the rat-flea-rat transmission cycle.

Thus. the spread of plague to humans is a function of the relative balance between resistant and susceptible species of rats. When the domestic rat population overlaps into the wild rat population, the domestic rats become infected by the bite of an infected rat flea. The domestic rat fleas then become infected and, when biting other rats, regurgitate the plague bacilli into the new host, instituting a new epidemic. As the domestic rats continue to die, the rat fleas will bite humans –intruders in the normal rat-rat flea cycle. Because epidemics of plague usually occur in crowded areas with poor sanitation, it has been proposed that, once started, human-to-human spread also occurs by human fleas.

 

 

Sporadic cases of plague also occur world-wide as a result of human  contact with infected animals. In the United States, this occurs primarily in rural residents of the south-west and, to a lesser extent, in hikers and campers in that region. Infection in such cases results from being bitten by an infected rodent flea or by handling an infected animal. Of 40 cases of human plague reported in 1983,occurring primarily in New Mexico and Arizona, 6 were fatal. Only four cases, occurred during 1989 and none in 1993.

In Africa, it has been shown that camels and goats can contract plague under natural conditions through contamination of their forage areas by plague-infected rodents. A number of human cases have resulted from contact with carcasses of diseased animals.

A small pustule may be present at the portal of entry in the skin but more often there is no discernible lesion. The bacilli introduced by the flea bite enter the dermal lymphatics and are transported to the regional lymph nodes, usually in the groin, where they cause the formation of enlarged, tender buboes. In severe bubonic plague the regional lymph nodes fail to filter out all the multiplying bacilli; organisms that gain entrance to the efferent lymphatics disseminate via the circulation (septicemic plague) to the spleen, liver, lungs, and sometimes the meninges. The parenchymatous lesions produced are hemorrhagic; disseminated intravascular coagulation may occur. In the terminal stages bacteremia is often intense.

After the flea bite, Y. pestisis transported in the human body to the regional lymph nodes (usually in the groin),causing them to become enlarged, tender nodes called buboes. Oddly, the organisms entering from the flea bite possess neither the Fl capsule nor the antiphagocytic V/W antigens (V/W antigens are not formed at 28°C, the normal flea temperature). As a result, the bacilli that undergo phagocytosis by polymorphonuclear neutrophils are destroyed. However, those that undergo phagocytosis by macrophages are not killed and are alive at 37°C in a Ca²⁺-deficient intracellular environment; the response to this low Ca²⁺ concentration is a turn-off in chromosome replication and the expression of the virulence determinants V/W and Fl. The bacilli can then leave the macro-phage as fully virulent organisms resistant to phagocytosis and, because plasma and other body fluids provide sufficient Ca²⁺ for Ca²⁺ -dependent growth to occur, Y. pestisis able to replicate rapidly.

After leaving the regional lymph nodes, Y. pestis disseminates by way of the bloodstream to the spleen, liver, lungs, and other organs and may cause subcutaneous hemorrhages, which gave bubonic plague the name black death. However, from 1980 to 1984, 25% of the cases of plague occurring in New Mexico (18 of 71 cases)were characterized by positive blood culture results in the absence of a bubo or lymph- node involvement. Such cases are difficult to diagnose because they are similar to septicemia caused by other gram-negative organisms. If lung involvement occurs, the resulting pneumonia also can be spread by respiratory discharges.

The incubation period of bubonic plague in man varies from 1 to 6 days, depending upon the infecting dose. Onset is usually abrupt with high fever, tachycardia, malaise, and aching of the extremities and back. If the disease progresses to the fulminant bacteremic stage, it causes prostration, shock, and delirium; death usually occurs within 3 to 5days of the first symptoms. The course of plague pneumonia is even more fulminant; untreated patients rarely survive longer than3 days. Pulmonary signs may be totally lacking until the final day of illness, making early diagnosis particularly difficult. Late in the disease copious bloody, frothy sputum is produced.

The occurrence of asymptomatic cases is suggested by serological studies on persons living in areas where the disease is endemic and by the finding in Vietnam of a pharyngeal carrier rate of about 10% in family members of plague patients.

Less than 10 organisms of a fully virulent strain of Y. pestis injected into a mouse is lethal. The mechanism by which the plague bacillus produces death is not entirely clear but probably results from septic shock because of the release of tumor necrosis factor-alpha and interleukin-1. It is also noteworthy that a superantigen analogous to TSST-1 and the streptococcal pyrogenic toxins has been reported in Y. pseudotuberculosis, and it seems that such an antigen also may be formed by Y. pestis.

In the pneumonic form of the disease the plague bacilli are spread in the air with sputum expelled by the patient when he coughs or talks. Quite ofteo inflammation is seen at the site of microbe entry. Depending on the location of the pathogen, reactivity of the infected body, virulence of the microbe, and extent of cellular and humoral activity, human plague may be of the following forms: cutaneous, bubonic, cutaneous-bubonic, primary septicaemia, secondary septicaemic (primary pneumonic), secondary pneumonic, and intestinal.

As a rule, plague develops quickly without a prodromal stage. It is characterized by a violent chill, severe headache, and dizzinesss. The face is pale and bluish, with an expression of suffering (horror-stricken), and is known as fades pestica. Each form of plague shows characteristic clinical symptoms. Before the use of streptomycin the mortality rate was very high, i.e. from 40 to 100 per cent. Post-mortem examination reveals centres of inflammation in the lymph nodes, haemorrhages, and haemorrhagic periadenitis. A large number of Y. pestis organisms are present in the lymph nodes and cellular tissue. Phagocytosis is inhibited. There is marked disintegration and necrosis of the buboes. Small haemorrhages form in the skin. The liver is enlarged, showing haemorrhages and necrosis. The spleen is enlarged and dark-red in colour.

The pneumonic foci fuse and assume the form of lobar pneumonia. The lungs are distended, violet-red or grey-red in colour, hyperaemic, oedematous, and contain a large number of Y. pestis organisms.

Immunity. After recovery from the disease a stable immunity of long duration is acquired. Realizing this, in ancient times people living in countries invaded by plague made use of convalescents for nursing plague patients and burying corpses.

Postinfection and postvaccinal immunities are predominantly due to the phagocytic activity of the cells of the lymphoid-macrophage system. An important role is played by the protective capsular antigen which serves as the basis in the preparation of chemical antiplague vaccines.

Laboratory diagnosis. Examination is carried out in special laboratories and in antiplague protective clothing. A strict work regimen must be observed. Depending on the clinical form of the disease and the location of the causative agent, test specimens are collected from bubo content (in bubonic plague), ulcer secretions (in cutaneous plague), mucus from the pharynx and sputum (in pneumonic plague), and blood (in septicaemic plague). Test matter is also recovered from necropsy material (organs, blood, lungs, contents of lymph nodes), rodent cadavers, fleas, foodstuff’s, water, air, etc. Examination is performed in the following stages.

1. Microscopy of smears, fixed in Nikiforov’s mixture and stained by the Gram method or with methylene blue by Loeffler’s method.

2. Inoculation of the test material into nutrient media, isolation of a pure culture and its identification. To inhibit the growth of the accompanying microflora, 1 ml of a 2.5 per cent sodium sulphite solution and 1 ml of a concentrated alcohol solution of gentian violet, diluted in distilled water in a ratio of 1 :100, are added to 100 ml of meat-peptone agar. Prior to inoculation 0.1 ml of antiphage serum is added to the culture to render the plague bacteriophage harmless.

3. Biological tests of the isolated pure culture and of material from which isolation of the organism is difficult are conducted on guinea pigs. In the latter case a thick emulsion prepared from the test material is rubbed into a shaven area of skin on the abdomen. If plague bacilli are present the animals die on the fifth-seventh day. To hasten diagnosis the infected guinea pigs are killed on the second-third day and the plague bacillus is isolated from their organs.

Y. pestis is identified by determining the morphological, cultural, fermentative, phagocytolytic, and agglutinative properties of the isolated culture. The growth is differentiated from the causative agent of rodent pseudotuberculosis (see Table 1). The biological test is decisive in the diagnosis of plague.

Decomposed rodent cadavers are examined by the thermoprecipitin test.

The importance of prompt diagnosis of plague has led 10 the elaboration of accelerated diagnostic methods in recent times.

Treatment At present streptomycin is used for treatment of plague, the drug being very effective and curing even pneumonic plague in a high per cent of cases. Good results have been obtained from a combination of streptomycin with chloromycetin or tetracycline with antiplague serum. Antiplague gamma-globulin and a specific bacteriophage are also used for treatment of plague patients. Penicillin, chlortetracycline, and sulphonamides are recommended in cases with complications.

Prophylaxis. General prophylaxis comprises the following measures:

(1) early diagnosis of plague, particularly the first cases;

(2) immediate isolation and hospitalization of patients and enforcement of quarantine; individuals who have been in contact with patients are placed under quarantine for 6 days and prescribed prophylactic streptomycin treatment;

(3) observation (i. e. isolation of individuals or groups of people suspected of having been in contact with infected material, daily inspection from house to house, temperature measurement twice a day, and observation during the possible incubation period);

(4) thorough disinfection and extermination of rats in disease foci;

(5) individual protection of medical personnel and prophylactic treatment with streptomycin and vaccination;

(6) prophylactic measures and systematic observation carried out by plague control laboratories, stations, and institutes in endemic areas;

(7) observance of international plague control conventions (extermination of rats and disinfection of ships, aircraft, trains, and harbours and, if necessary, compulsory quarantine for passengers);

(8) security measures from plague invasion at frontiers. Specific prophylaxis is accomplished with live EV vaccine. It is produced in dry form and administered subcutaneously, intracutaneously or percutaneously once or twice. Immunity lasts for no longer than one year. Depending on the epidemiological condition, revaccination is conducted in six or 12.months. The efficacy of the vaccination is not high. According to WHO data, a total of 2.5 million of persons with this disease were registered in all the countries of the world in the period between 1921 and 1965 and 12140 in 1970-1976. The incidence of plague has sharply reduced in the recent years and it has become a sporadic disease.

Yersinia enterocolitica. Yersinia enterocolitica is a motile, gram-negative rod that is involved primarily in animal diseases. It can be distinguished from Y. pestis on the basis of serologic reactions, biochemical reactions, and pathogenicity in animals. Thirty-four serotypes and five biotypes have been reported.

 

Yersinia enterocolitica.

 

Epidemiology and pathogenesis of Y. enterocolitica infections. Yersinia enterocolitica has been isolated from a variety of rodents and domestic farm animals, including cats and dogs. It has also been isolated from lakes and well water, and food-borne transmission (particularly via contaminated milk) is known to occur. Humans frequently acquire the infection from eating raw or undercooked pork (particularly in Europe).

Y. enterocolitica is second only to the Salmonella as a cause of pediatric enterocolitis that is characterized by fever, diarrhea, and abdominal pain. Frequently, considerable swelling of the mesenteric lymph nodes occurs, which, with the abdominal pain, mimics appendicitis. Septicemia is rare but can occur, resulting in lesions through-out the internal organs. Probably the most frequent complication of infections with Y. enterocolitica is a severe arthritis that occurs 1 to 14 days after acute enteritis. The mechanism of the arthritis is unclear, but arthritic patients have been shown to express a multiclonal T-cell response to Y. enterocolitica antigens and to have high titers of IgA antibodies that are not found in patients who do not develop arthritis. Moreover, Yersinia antigen has been demonstrated within synovial fluid mononuclear cells and in synovial membrane cells of patients with Yersinia-induced arthritis. In most cases, the arthritis is self-limiting but, in about 10% of affected patients, it may become chronic. Interestingly, most cases of enterocolitis occur in children whereas most cases of reactive arthritis occur in adults.

Rare cases of Y. enterocolitica bacteremia and endotoxin shock have occurred that were acquired from blood transfusions (7 of 10 patients died). In these cases, the organisms were isolated from residual red blood cells, and at least three of the blood donors gave a history of having diarrhea within 30 days of donating their blood. It is concluded that such persons had an asymptomatic bacteremia at the time of the blood donation and that the organisms grew to large numbers under the conditions of cold storage of the blood. As a result, it has been proposed that blood that is over 25 days old be checked for endotoxin.

 

Y. enterocolitica colonies

 

As with Y. pestis, the precise nature of the virulence determinants of Y. enterocolitica is complex. Virulent strains possess a low calcium response plasmid that is probably identical to that described for Y. pestis. Thus, both V and W antigens are produced as well as a large array of YOPs Y. enterocolitica also produces an outer membrane protein called YadA. YadA confers adherence to epithelial cells by binding collagen fibers and fibronectin. It also is responsible for resistance to the bactencidal activity of normal serum by inhibiting the formation of the membrane attack complex of complement. In addition, Y. enterocolitica possesses a chromosomally encoded gene called inv, which encodes for a surface protein termed invasin. Invasin interacts with eucaryotic transmembrane proteins belonging to the beta1 integrin family. Integrins normally tend to various basal membrane proteins such as fibronectin, collagen, and laminin, but when bound to invasin, they induce the internalization of the bacterium into the host cell. A second gene, termed ail, also appears to be involved in cell invasion by Y. enterocolitica, but this gene facilitates entry into only a limited number of host cell types by an unknown mechanism. Cloning of either inv or ail confers an invasive phenotype ooninvasive strains of E. coli. The product of these genes permits the organisms to gain access to the reticuloendothelial system and eventually to disseminate through out the body. Thus, for complete virulence, both plasmid and chromosomally encoded products are required.

Interestingly, Y. enterocolitica produces no siderophores, even though there are membrane receptors for them. This fact has prompted the proposal that the organism’s low virulence in humans may be because of its inability to obtain iron readily.

The mechanism by which this organism causes diarrhea seems to result from the production of a heat-stable enterotoxin, YST, that is similar, or identical, to the ST of E coli. Like ST, the Y. enterocolitica heat-stable enterotoxin manifests its toxicity by stimulation of guanyl cyclase activity, resulting in an increased cyclic guanosine 3′,5′-monophosphate synthesis.

Laboratory diagnosis and treatmrent of Y. enterocolitica infections. Isolation of the organisms from feces, blood, or lymph node biopsy specimens provides the best definitive diagnosis of this infection. Isolation of the organisms from fecal suspensions is facilitated by suspending the fecal specimens in phosphate-buffered saline and holding them at refrigerator temperatures for 2 to 4 weeks before streaking them on enteric media.

 

Treatment of the enteritis is of little value because patients recover from these infections spontaneously after a few days. Septicemia, however, carries a high mortality and should be treated with gentamicin or chloramphenicol

 

Yersinia pseudotuberculosis. The disease was first revealed in Vladivostok, and then in Leningrad and other places of the Soviet Union. It is encountered in 30 countries of the world among adults and children in the form of epidemic out-breaks or sporadic cases.

Yersinia pseudotuberculosis is primarily an animal pathogen, infecting both wild and domestic animals. The disease in animals is characterized by necrotic lesions occuring in the liver, spleen, and lymph nodes. Humans acquire the infection by way of an oral route after contact with infected animals.

The most common manifestation of human infection is the painful swelling of the mesenteric lymph nodes, resulting in an appendicitis-like syndrome, although diarrhea and fever also are usual. Rarely does Y. pseudotuberculosis  invade the bloodstream, but such infections are associated with a high mortality rate, resulting in clinical symptoms that are similar to toxic shock syndrome. The disease is marked by headache, malaise, vomiting, abdominal pain, fever (38-40°C), eruption of lesions of various morphology, and hyperaemia of the mouth and throat. A report that Y. pseudotuberculosis produces a superantigen that activates human T cells in the presence of macro-phages bearing MHC class II molecules supports this observation. Late complications may include arthritis, uveitis, and nephritis.

Virulence determinants for Y. pseudotuberculosis are essentially identical to those described for Y. enterocolitica.

Laboratory diagnosis consists in isolating the causative agent from the patient’s stool and identifying it by means of the agglutination reaction, phagolysis, and biochemical properties and in demonstrating the antibodies by the reaction of agglutination and indirect agglutination. Chloramphenicol and pathogenic measures are used for the treatment of patients. Prophylaxis comprises the extermination of rats, protection of foodstuff’s and water against rodents, the observance of sanitary and hygienic regimens at catering establishments, food storehouses, etc.

Ampicillin, streptomycin, or tetracycline are effective for treatment of bloodstream infections by Y. pseudotuberculosis. Antibiotic therapy probably is not necessary for the mesenteric lymph node infection inasmuch as such infections spontaneously resolve after several days.

 

Diagnosis of diseases in detail

The causal organism of plague, which is an acute, particularly dangerous zoonotic infection, is Yersinia pestis. The nature of the material obtained from the patient depends on the clinical form of plague: a puncture sample from a bubo (bubonic plague), exudate of an ulcer (cutaneous form), sputum, a smear from the fauces (pulmo­nary form), or blood (septic form). Section material (blood, internal organs, lymph nodes) is also examined. Corpses of rodents, fleas, water, foodstuffs, air, and other objects are also tested. The material is collected with special precautions by the operator wearing a type 1 antiplague suit. Bacterioscopic, bacteriological, serological, and biological exami­nation is carried out with a diagnostic purpose.

Bacterioscopic examination. Make several smears from the mate­rial collected and fix them in Nikiforov’s mixture. The smears are stained by the Gram technique and simultaneously with methylene blue, using the Loeffler method. If bacterioscopic examination reveals Gram-negative ovoid bacteria surrounded by a tender capsule and stained bipolarly with the Loeffler technique, a preliminary report with the description of the morphology of the bacteria recovered is issued. The smears may also be treated with labelled luminescent antiserum against Y. pestis (direct immunofluorescence).

Bacteriological examination. The material to be tested is inocu­lated into appropriate nutrient media: blood (5-10 ml), in 100 ml of meat-peptone broth (in vials); puncture sample from a bubo, exudates from an ulcer, sputum, and other materials, onto plates with meat-peptone agar (pH 7.2-7.3). To stimulate the growth of the plague bacteria, 0.1 per cent of rabbit or horse blood is added to the meat-peptone agar. To inactivate the plague phage, 0.1 ml of antiphage serum is placed and uniformly spread on the medium surface. To suppress the growth of extraneous decaying microflora in the tested material, to 100 ml of meat-peptone agar add 1 ml of 2.5 per cent sodium sulphite and 1 ml of saturated alcohol solution of gentian violet diluted 1:100 with distilled water. Place the inocu­lated cultures in the incubator at 25-28 °C. Examination of the plates in 10-12 hrs may demonstrate R-shaped colonies with a compact centre and fringed periphery. At 24 hrs, a loose film with descending threads is formed in the broth.

Pure culture of the plague bacteria is isolated in the usual manner and then identified by morphological, cultural, fermentative, agglu­tinating, phagolytic, and biological properties.

From a fermentative standpoint, the causative agents of plague are active: they split (with the formation of acid) glucose, fructose, galactose, xylose, mannitol, and occasionally arabinose. Some strains may also ferment glycerol. On the other hand, Y. pestis does not split adonite, does not liquify gelatin, does not form indol, and reduces nitrates to nitrites.

For the agglutination test diagnostic sera against somatic and capsular antigens are employed. Sera containing O-antibodies (somatic) give a group reaction with the  causative agent of pseudo-tuberculosis. Sera against capsular antigens are distinguished by great specificity.

To identify the causative agent of plague, one can use the aggluti­nation test with lyophilized erythrocyte diagnoіticum loaded with antibodies of antiplague serum.

Investigation of the above attributes does not always allow differ­entiation of the plague causative organism from similar bacteria of pseudotuberculosis (Yersinia pseudotuberculosis). To distinguish be­tween the se microorganisms, consideration of a number of other signs is helpful (Tabl. 4).

Table 4

Some diagnostic signs differentiating the bacteria of plague

from those of pseudotuberculosis in rodents

 

Study of the isolated culture of the plague bacteria for their phago-lysis is carried out in the  conventional manner on a meat-peptone agar and by adding the plague phage (in the amount of one-tenth of the culture volume) to a 3-hour broth culture in liquid media. The phage is diluted to the titre and various dilutions are tested.

Biological examination is conducted together with bacterioscopic and bacteriological one. Guinea pigs and mice are infected by various techniques: epicutaneously, subcutaneously, and intraperitoneally. Inoculation with the post-mortem material is done in the following way: shave a definite area of the animal skin and scarify it, place on the skin 2-3 drops of the test liquid, and rub it in until the skin is dry with a flat part of the lancet. The blood of the patient and suspension of the isolated pure culture are injected intraperitoneally; the sputum and bubonic puncture specimen are introduced sub­cutaneously in the internal surface of the hip.

There should be no ectoparasites on the animals to be tested. Infected guinea pigs are kept in tall glass jars closed tightly by two layers of gauze soaked in lysol solution. Mice are also kept in jars which are placed into metallic net bags with iron lids.

Animals inoculated epicutaneously die on the 5th-7th day. To reduce the time of examination, one of the infected animals is sacri­ficed in 2-3 days. Isolate the culture from the post-mortem blood and internal organs and identify it, using the above described tech­nique.

The following patho-anatomical alterations found during autopsy indicate the presence of plague: multiple haemorrhages oil mucosal and serous membranes, inflammation of lymph nodes, presence of necrotic foci in the spleen, and less commonly in the liver. Exami­nation of the abdominal cavity reveals viscous exudate and degener­ation of the parenchymatous organs. The presence of a large number of Gram-negative bipolarly stained bacteria in smears from the internal organs, blood, and exudate also suggests the positive diag­nosis.

When decayed cadavers of rodents are examined, use the precipi­tation test since isolation of pure bacterial culture in this case is difficult.

Prior to autopsy, immerse the carcasses of the rodents for several minutes in kerosene to kill the insects, then proceed with an autopsy looking for patho-anatomical alterations, make impression smears from the internal organs, and inoculate for pure culture. The per­formance of the biological test is mandatory. If the post-mortem examination involves a large number of rodents, the group biological test is employed, i.e., a guinea pig is inoculated with suspension from the lymph nodes or spleens of 15-20 cadavers to be examined.

Serological examination is carried out for a retrospective diagno­sis as well as during epizootic examinations iatural foci of plague.

Indirect haemagglutination with erythrocytes, which have ad­sorbed the capsular antigen of the causative agent of plague, is per­formed. The result is considered positive if haemagglutination occurs at a 1:40 dilution of the serum obtained from sick or dead animals.

Along with the above techniques of recovery of the causative agent of plague, one can employ variable methods including phago-diagnosis, the immunofluorescence test, the method of a rapid growth of the responsible pathogen on enriched and elective media, etc. According to the latter method, the material (0.2-0.3 ml) is inocu­lated into four test tubes with semi-solid agar containing blood and gentian violet, with 0.2-0.3 ml of the bacteriophage being added to one of the tubes. Simultaneously, 0.1 ml of the material is streaked onto plates with agar of the same composition. The inoculated cul­tures are incubated at 28 “C. From test tubes in which macroscopic inspection conducted 3 hours later elucidates growth, transfer 2-3 drops onto two glass slides, prepare smears, fix them in Nikiforov’s mixture, stain with methylene blue and by the Gram technique.

A positive result is recognized by the appearance of chains of Gram-negative bipolar rods in smears, while the tube with the phage shows no growth. Aspirate 0.4-ml portions of the material from the tube with a visible growth and administer intraperitoneally to several albino mice. In 8-10 hrs, examine the plates with the agar for growth of the causative agent of plague.

Some 10-12 hrs after the inoculation, kill the mice and introduce pieces of organs and exudate of the abdominal cavity into test tubes with a semi-solid agar containing blood and gentian violet. Simulta­neously, streak onto plates with agar. Inspect the test tubes in 4 hrs and the plates in 8 hrs.

Hence, a preliminary answer may he given 4 hrs after the onset of the examination and the final one, in 18-20 hrs.

 

Brucella and Francisella

 

Causative Agent of Tularaemia. The specific cause of tularaemia (Francisella tularensis} was discovered in 1912 by G. McCou and C. Chapin in Tulare (California) and studied in detail by E. Francis.

Morphology. The tularaemia bacteria are short coccal-shaped or rod-like cocci (Fig.) measuring 0.2-0.7 mcm. In old ‘cultures the organisms retain the coccal form. They are non-motile, polychromatophilic, and Gram-negative. In the animal body they are sometimes surrounded by a fine capsule.

 

Figure.  Causative agent of tularaemia from the blood of water rat (left) and colonies on fish-yeast agar (right)

                       Tularaemia bacteria

 

Tularaemia bacteria are pleomorphous. They may assume a club-like structure or the form of very small cocci (0.1-0.2 mcm) which pass through filters. Average-sized cocci, very large spherical forms, or spherical forms with kidney-like protrusions are also to be found. Smears demonstrate rod-like and thread-like forms of the organism, which may reach 8 mсm in length. In animal organs the tularaemia bacteria are seen mainly as cochlea bacteria or as rod-like forms, while in cultures cochlea forms are more often observed. Cultures of low virulence (vaccine strains) are cochlea-shaped, larger than those of the virulent strain, and, as a rule, do not have a capsule.

Submicroscopic structure. The cell cytoplasm is granular, the nucleoid is in the central part of the cell. During division the cytoplasmic membrane grows into the cell and sporulation occurs.

Cultivation. The tularaemia organism is an aerobe which does not grow on ordinary media, but grows well at 37° C on media rich in vitamins, e. g. yolk medium which consists of 60 per cent of yolk and 40 per cent of a 0.85 per cent sodium chloride solution with pH 6.7-7A. The organisms are cultured in a thermostat for 2-14 days.

Tularaemia bacteria multiply on cystine agar, containing 0.05-0.1 per cent cystine and 1 per cent glucose. The mixture is boiled for several minutes, cooled to 40-50 °C, after which defibrinated blood is added. The latter should constitute 5-10 per cent of the nutrient medium. Milk-white colonies are formed. The bacteria can be cultivated on media containing brain, spleen, and liver tissues, heart extracts, brewer’s yeast, and fish flour. Such media contain vitamins necessary for growth of the tularaemia organisms. Culture collections on solid media may be well preserved for 2-6 months. Tularaemia bacteria grow well in the yolk sac of a 12-day-old chick embryo.

 

Tularaemia bacteria colonies     Tularaemia bacteria growth

 

 

When cultivated in the laboratory, tularaemia organisms lose the K-antigen with which their virulence and immunogenic properties are associated.

Fermentative properties. Tularaemia bacteria break down proteins with the elimination of hydrogen sulphide, and do not produce indole. They ferment glucose, levulose, mannose, and maltose, with acid formation. Dextrin, saccharose, and glycerin fermentation is not a stable property. Biochemical properties are unstable and liable to comparatively rapid changes. This is due not only to the properties of the bacteria themselves but also to the nutrient media and to the ability of the tularaemia organism to break down proteins. The products of this process mask the production of acids which occurs simultaneously.

Toxin production. The existence of a soluble toxin in tularaemia bacteria has not been demonstrated. The organism’s virulence is associated with its K-antigen. The tularaemia bacterium grows poorly in liquid media, and for this reason it is difficult to isolate any toxin.

Antigenic structure. Agglutination test and precipitin reaction are highly specific. Tularaemia bacteria have been shown to possess thermostable specific haptens. The common character of the antigens of tularaemia and brucellosis agents in the agglutination reaction has been ascertained. This fact must be taken into account in serological diagnosis of these diseases.

The R-form cultures containing only the O-antigen are avirulent and possess no immunogenic properties. The S-form which contains K- and 0-antigens is more common. The intermediate SR-forms from which live vaccines are prepared contain the O-antigen and, in a smaller number, the K-antigen. With prolonged growth on laboratory media all cultures transform to the R-form.

Since complete avirulence is accompanied by the loss of immunogenic properties, a certain degree of virulence for white mice is preserved (residual virulence) in tularaemia strains from which the vaccine is prepared.

Classification. Two varieties of tularaemia organisms can be distinguished: the American type which is highly pathogenic for rabbits and ferments glycerin, and the European type which is non-pathogenic for rabbits and does not ferment glycerin. The former variety is also more virulent for human beings, causing death in 5-6 per cent of tularaemia cases. The second variety was responsible for a low death rate among humans, mortality being 0.5 per cent.

Resistance. The tularaemia organism lives in glycerin for 240 days, in grain for 130, in water and rodent cadavers for 90, in baked bread for 20, and in soil for 10 days. It survives for long periods (over 3 months) at low temperatures. It is killed in several minutes by 3 per cent solutions of lysol, cresol, saponaceous cresol, formalin, and alcohol. When heated to 60° C it is destroyed in 10-15 minutes, while exposure to direct rays destroys it in 30 minutes.

Pathogenicity for animals. The organism is pathogenic for water rats, field voles, grey rats, common field mice and house mice, hares, susliks, chipmunks, hamsters, muskrats, gerbils, moles, shrews, and other animals. Among the domestic animals camels, sheep, cats, dogs, and pigs are susceptible to the disease, and among laboratory animals, guinea pigs and white mice.

Guinea pigs are inoculated intraperitoneally, subcutaneously, intracutaneously, and by rubbing the infective material into the skin. The inoculated animals develop a fever and lassitude, and lose weight. The spleen, liver, and inguinal lymph nodes become enlarged and inflamed. Microscopic studies reveal the presence of the causative agent in the spleen, liver, bone marrow, lymph nodes and in the blood recovered from the heart.

Wide adaptivity is characteristic of the tularaemia bacterium. It has adapted itself to more than 140 species of vertebrates and 110 species of arthropods which are capable of transmitting the disease. However, water rats, field voles, mice, muskrats, and, among domestic animals, sheep, and, among the vectors, horseflies, ticks, mosquitoes, and sandflies have the most epidemiological importance.

Pathogenesis and disease in man. Tularaemia is a zoonotic disease. Humans are infected with air-borne dust. The pathogen may also gain entrance into the body through the integuments and mucous membranes as a result of bites by arthropods and insects (ticks, horseflies, mosquitoes, etc.).

Contingent on the route of entry, the bacteria invade the skin, mucous membranes, lymph nodes, respiratory and gastro-intestinal tracts, and other organs, causing the respective clinical form of the disease (bubonic, ulcerative-bubonic, ocular, anginose-bubonic, abdominal or intestinal, pneumonic, and generalized or primary septicaemia). The lymph nodes are affected in all forms of the disease. In the generalized form all tissues and ‘organs are involved as a result of bacteraemia.

Tularaemia may be acute, lingering, or relapsing, depending on the duration of the disease, and may be mild, severe, or mildly severe. During tularaemia allergy develops and remains for years and sometimes for life. In recent years the death rate is low and most patients recover owing to the wide use of antibiotics.

According to the mode of spread and route of infection, the following types of tularaemia epidemics are known: trapping outbreaks associated with water rat and muskrat trapping; agricultural outbreaks associated with thrashing stacks inhabited by mice rodents; water-borne outbreaks due to diseases caused by drinking infected water; alimentary outbreaks due to the use of contaminated foodstuffs; transmissive outbreaks spread by the bites of bloodsuckers (ticks, stable-flies, mosquitoes, etc.).

Immunity. Following recovery, a stable immunity of long duration develops, being of the cell and humoral type.

Laboratory diagnosis. The differential diagnosis of plague, anthrax, enteric fever, typhus fever, influenza, malaria, and brucellosis is difficult because these diseases have common symptoms. For differentiation of tularaemia from other diseases laboratory tests are the most effective. Those peculiarities of the disease which can be revealed easily and quickly by laboratory methods are taken into account.

1. Allergy develops on the third-fifth day of the disease. For this reason, intracutaneous and cutaneous tests with tularine are made for early diagnosis. In tularaemia patients the test gives a positive reaction 6-12 hours after inoculation of tularine. In distinguishing tularaemia from other infections one must bear in mind that allergic tests may show positive reactions in convalescents and vaccinated individuals.

2. In the second week of the disease agglutinins begin to accumulate in the blood. They are detected by carrying out the agglutination reaction by the blood-drop and volume methods.

 

Blood-drop agglutination reaction

 

In some cases this test may give a positive reaction with material containing brucella organisms, since they possess antigens common to tularaemia bacteria.

3. The tularaemia culture is isolated by the biological method as it is impossible to recover the pathogen directly from a tularaemia patient. For this purpose white mice or guinea pigs are infected by material obtained from people suffering from the disease (bubo punctate, scrapings from ulcers, conjunctiva! discharge, throat films, sputum, and blood). Biological tests are conducted in special laboratories where a standard regimen is observed. The laboratory animals die in 4-12 days if tularaemia bacteria are present in the test material. Autopsy is performed, smears from organs are made and organ specimens are inoculated onto coagulated egg medium for culture isolation. Microscopic, microbiological, and biological studies of the cultured organisms are made. If no culture can be isolated from the first infected guinea pig, an emulsion, obtained from the latter’s organs, is inoculated into a second guinea pig, etc.

4. Laboratory diagnosis of rodent tularaemia is made by microscopy of smears from organs, precipitin ring reaction (thermoprecipitation), and biological tests.

Water, foodstuffs, and blood-sucking arthropods are examined by biological tests.

Treatment. Streptomycin, tetracycline, chloramphenicol, and a vaccine prepared from killed tularaemia bacteria are prescribed.

Prophylaxis comprises the following measures:

(1) systematic observation, absolute and relative registration of rodent invasion, and extermination of rats;

(2) prevention of mass reproduction of the rodents;

(3) protective measures in agricultural enterprises against contamination by tularaemia-infected rodents;

(4) protection of foodstuffs and water from rodents;

(5) control of ticks, horseflies, stable-flies, mosquitoes, and protection from these insects;

(6) specific prophylaxis with a live vaccine.

The vaccine is prepared in a dry form. A single application is made by rubbing it into the skin and it produces immunity for a period of 3-6 years.

Due to the application of a complex of measures on a wide scale (immunization of people living in a zone of natural foci) and the marked improvement in agricultural management tularaemia morbidity reduced to one thousandth within a period of 20 years (from 1945 to 1965) and has become a sporadic disease.

 

Brucellae. In 1886 on the Island of Malta D. Bruce, an English bacteriologist, demonstrated the presence of the causative agent of Malta fever in the spleen of a deceased patient and in 1887 isolated the organism in pure culture.

In 1896 the Danish scientist B. Bang established the aetiology of contagious abortion of cattle. In 1914 the American investigator G. Traum isolated from pigs the organism responsible for contagious abortion among these animals. Other Brucellae species were discovered in 1953, 1957, and 1966.

A more detailed study of these organisms was made in 1918 by the American scientist A. Evans. She came to the conclusion that, according to their main features, they were all closely related. She grouped them in one genus which she named after D. Bruce, the discoverer of the causative agent of brucellosis. All the former names of the disease (Malta fever. Mediterranean fever, undulant fever. Bang’s disease, contagious abortion of pigs, etc.) were substituted by the general name of brucellosis. In 1920 K. Mayer and M. Feseaux verified these data.

Morphology. Brucellae are small, coccal, ovoid-shaped micro-organisms 0.5-0.7 mcm in size (Fig.). Elongated forms are b.6-1.5 mem in length and 0.4 mcm in breadth. Under the electron microscope Brucella organisms of cattle, sheep and goats appear as coccal and coccobacilary forms, while those of pigs are rod-shaped. They are Gram-negative, non-motile, and do not form spores or capsules (in some strains capsules are sometimes present). DNA contains 56 to 58 per cent of G+C.

 

Figure. Brucella melitensis, a pure culture and colonies

 

 

Cultivation. The organisms are aerobic. When cultivated from material recovered from patients, they grow slowly, over a period of 8-15 days; in some cases, however, this period is reduced to 3 days, while in others it is prolonged to 30 days. In laboratory subcultures growth becomes visible in 24-48 hours. The pH of medium for these organisms is 6.8-7.2. The optimum temperature for growth is 37 °C, the limits of growth temperatures being 6 and 45 °C.

Brucella organisms may be cultivated on ordinary media, but they grow best on liver-extract agar and liver-extract broth. On liver-extract agar the organisms form round, smooth colonies (see Fig. 2) with a white or pearly hue. In liver-extract broth they produce a turbidity, and subsequently a mucilaginous precipitate settles at the bottom of the tubes Brucella organisms grow well on unfertilized eggs and on the yolk sac of a 10-12-day-old chick embryo.

The brucellae of bovine origin (Brucella abortus) only grow in an atmosphere of 10 per cent carbon dioxide, which serves as a growth factor.

Selective media containing definite dyes and antibiotics (polymixin B, bacitracin, and others) are used for isolating Brucellae.

Brucella growth

 

Dissociation of the organisms from the S-form to the R-form has been demonstrated. L-forms have also been observed. These forms possess manifest adaptability. They adapt relatively easily to cultivation outrient media on which the first generation shows no growth; e. g. the first generation of brucellae of bovine cattle grow well only in the presence of carbon dioxide, while in subcultures they grow without it.

Prolonged cultivation of the organisms outrient media leads to a significant weakening of their virulence and to the loss of the Vi-antigen.

Fermentative properties. Brucellae do not liquefy gelatin and do not produce indole. Some strains produce hydrogen sulphide, break down urea and asparagin, reduce nitrates to nitrites, and hydrolize proteins, peptones and amino acids, with release of ammonia and hydrogen sulphide. No carbohydrates are fermented, although a small number of strains ferment glucose and arabinose.

 

Biochemical properties

 

Toxin production. Brucellae do not produce soluble toxins. An endotoxin is produced as a result of disintegration of the bacterial cells. This endotoxin possesses characteristic properties and may be used in allergic skin tests

Antigenic structure. The organism contains four antigens: A, M, G and R. The M-antigen is predominant among brucellae of sheep and goats, and the A-antigen, in the other species. Substances of polysaccharide character, with no type specificity, have been extracted from brucellae of cattle, sheep and goats. At present it is known that all three Brucella species contain 7 antigens (1, 2, 3, 4, 5, 6, and 7) which are arranged in the form of a mosaic on. the cell surface. Brucellae have an antigen common with tularaemia bacteria.

In recent years it has been discovered that brucellae, in addition to the O-antigen, possess a thermolabile Vi-antigen. Experiments haw confirmed that separate immunization with Vi- and O-antigens does not produce complete immunity to the disease in animals, while simultaneous immunization with all the antigens gives good results

Classification. Brucellae are subdivided into some species: (1) brucellae of goats and sheep (Brucella melitensis); (2) brucellae of cattle (Brucella bovis); (3) brucellae of pigs (Brucella suis); (4) brucellae of forest rats (Brucella neotomae); (5) the causative agents of epididymitis and abortion in sheep (Brucella ovis). The first three species produce cross immunity. In Canada, Siberia, and Alaska, B. rengiferi (fourth type of B suis) has been discovered: it affects northern reindeer from which humans are contaminated. B. canis was isolated from hounds in 1966 Brucellae are differentiated according to the characteristics given in table 5.

 

Table 5.

Distinguishing Characteristics of Various Brucella Species and Their Biotypes

Species	Biotype	Requirements for CO2	H2S production	Growth on media with dyes		Aglutination in monospecific sera AM	Lysis under effect of T-b phage in TD	Metabolic tests
				thionin	basic fuchsin			glutami-nic acid	arginine	ribose	lysine
				abc	bc
B. melitensis	1	–	–	– + +	+ +	– +	–	+	–	–	–
	2	–	–	– + +	+ +	+ –	–	+	–	–	–
	3	–	–	– + +	+ +	+ +	–	+	–	–	–
B. abortus	1	+(–)	+	– – –	+ +	+ –	+	+	–	+	–
	2	+(–)	+	– – –	– –	+ –	+	+	–	+	–
	3	+(–)	+	+ + +	+ +	+ –	+	+	–	+	–
	4	+(–)	+	– – –	+ +	– +	+	+	–	+	–
	5	–	–	– + +	+ +	+ –	+	+	–	+	–
	6	–	– +	– + +	+ +	+ –	+	+	–	+	–
	7	–	+ –	– + +	+ – +	+ +	+	+	–	+	–
	8	+	–	– + +	+ +	– +	+	+	–	+	–
	9	–	+	– + +	+ +	– +	+	+	–	+	+
B. suis	1	–	+	+ + +	– –	+ –	–	–	+	+	–
	2	–	–	– + +	– –	+ –	–	+	+	+	+
	3	–	–	+ + +	+ +	+ –	–	+	+	+	+
	4	–	–	+ + +	+ +	+ +	–	+	+	+
B. neotomae	1	–	+	– – +	– –	+ –	–	+	–	±	–
B. ovis		+	–	+ + +		– –	–	+	–	–	–
B. canis	–	–	–	+ +	–	–	–	–	+	+	+

 

 

Brucella susceptibility to fuchsin and thionin

 

Biotypes have been described within three brucella species (B melitensis, B. bovis, B. suis). Brucellae can also be differentiated by the agglutinin adsorption method. This method defines the species of the organisms as well as their genetic interrelationships.

When identifying Brucella organisms, the variations in their antigen structure should be considered. Variants that cannot be identified by the usual methods are differentiated by Vi-agglutinating sera and Brucella bacteriophage. The most important criteria are the peculiarities of metabolism, the urease activity, the ability to form hydrogen sulphide, the antibiograms, and the relation to the phage.

Resistance. Brucellae are characterized by high resistance and viability. They survive for a long time at low temperatures. The organisms live for 18 weeks m winter soil, urine, animal faeces, manure, hay dust and bran, up to 16 weeks in ice, snow, butter and sheep’s milk cheese, from 12 to 16 weeks m sheep’s wool, for 30 days in dust, for 20 days in meat, and for 7 days in milk.

The organisms are sensitive to high temperatures and disinfectants. At 60 °C are destroyed in 30 minutes, at 70 °C in 10 minutes, at 80-95 °C in 5 minutes, and at boiling point in a few seconds. They are very sensitive to phenol, creolin, formalin, chloramine, and other disinfectants. The best results are obtained with a 1 per cent solution of hydrochloric acid in combination with an 8 per cent sodium chloride solution.

Pathogenity for animals. Goats, sheep, cattle, pigs, horses, camels, deer, dogs, cats, and rodents (rats, mice, susliks, hamsters, rabbits, field- voles, water rats, and other animals) are all susceptible to infection by brucellae. The high concentration of brucellae in the placenta of cattle is explained by the presence in this tissue of the growth stimulator erythrol.

The disease assumes an acute or a clinically latent course in animals. In sheep and goats abortions and delivery of non-viable foetus are the most typical  symptoms. Affected cows have miscarriages, yield less milk, and lose flesh. In the weakest and emaciated animals the disease is sometimes fatal. Apart from miscarriages the organism produces arthritis, bursitis, orchitis, epididymitis, and other complications in pigs. In horses and camels the disease is usually of a latent form, abortions are rare, but emaciation, lassitude and numerous abscesses of long duration occur.

The excretions of sick animals (urine, faeces, amniotic fluid, and uterine mucus), and their milk, particularly that of goats and sheep, and milk products are sources of infection.

The migration of Brucella organisms from the usual hosts to animals of other species has been shown. This fact is of great epidemiological importance and must be taken into account in the laboratory diagnosis of brucellosis and when prophylactic measures are applied. Cows infected with B. melitensis may transmit virulent types of Brucella organisms to humans.

Among experimental animals guinea pigs are susceptible to brucellosis. The disease lasts for 3 months and the animals die showing lesions in the bones, joints, cartilages and eyes. During the disease they become emaciated, their skin atrophies, the hair falls out, and orchitis develops. In mice the disease produces septicaemia and the bacteria are recovered from the liver and spleen.

Pathogenesis and disease in man. Brucellosis is a zoonotic infection. Man contracts it from animals (goats, sheep, cows, pigs). From the epidemiological standpoint goats and sheep are the most important. At present there is a sufficient amount of cases to prove that healthy individuals may acquire the disease from humans infected by B. melitensis. The causative agents of brucellosis may be transmitted through wild animals (rodents and herbivores), ticks, and other blood-sucking insects; the infection may also be air-born.

Man contracts brucellosis very often through the milk of goats, sheep, and cows, and dairy products made from such milk also carry infection. The organism may likewise gain entry through abrasions in the skin and mucous membranes. For the most part veterinary and zootechnical staff, shepherds, workers of dairy farms and farms where sheep’s milk cheese is produced, workers of stockyards, etc., are attacked by the disease. On farms brucellosis prevails in certain seasons when sheep and goats bear their young (March-May).

From the site of primary location brucellae enter the lymphatic apparatus where they multiply. Then they enter the blood and cause protracted bacteraemia (from 4 to 12 months or longer). Via the blood-stream the bacteria invade the whole body and give rise to orchitis, ostitis, periostitis, arthritis, etc.

Allergy, which develops from the onset of the disease, lasts throughout the disease and remains long after recovery, is characteristic of brucellosis in humans and animals. A sensitized body becomes extremely sensitive to exposure to the specific action of the brucellosis antigen and to various non-specific factors, such as cooling, secondary infection, trauma, etc.

In human beings brucellosis is characterized by undulant fever with atypical and polymorphous symptoms. The disease may assume an acute septic or a chronic metastatic course. The structural and motor systems, haemopoietic, hepatolienal, nervous and genital systems are often involved. Pregnant women may have miscarriages. Often brucellosis recurs, continuing for months and years. The death rate is 1-3 per cent. The diagnosis of mild, asymptomatic forms presents difficulties and is based on laboratory tests.

Brucellosis in humans has many clinical symptoms common to other diseases (malaria, tuberculosis, rheumatic fever, enteric fever, typhus fever, Q fever, and various septic processes of other aetiology). For this reason, the differential diagnosis of brucellosis is of great importance. It is made with regard to the peculiarities of the disease and of other infections which have a similar course.

Immunity. A characteristic immunity is acquired following brucellosis, the patient becoming insusceptible to repeated infection. The activity of the T-lymphocyte system forms the basis for infectious and postinfectious immunity in brucellosis; phagocytosis plays a particularly important role. At the onset the immunity is non-sterile and infectious, but later it becomes sterile, although labile and of a low grade. All Brucella species produce cross immunity in the body.

The humoral factors, i. e. the production of opsonins, agglutinins, complement-fixing substance, and incomplete antibodies which block the causative agents of brucellosis, play a definite role in rendering the brucellae harmless. The phage, being a powerful factor in bacterial variation, plays an important role in producing insusceptibility to brucellosis. Nearly all patients show marked improvement with the clinical course of the disease and on recovery a higher titre and lytic activity of the bacteriophage are displayed.

Laboratory diagnosis. The patient’s blood and urine (for isolation of the pathogen), serum (for detection of agglutinins), milk and dairy products (for detection of brucellae or agglutinins in milk) are examined. The microbe is isolated in special laboratories.

1. Culture isolation. Since brucellosis is often accompanied by bacteraemia, blood is examined during the first days of the disease (preferably when the patient has a high temperature). For this purpose, 5-10 ml of blood is collected and transferred into two or three flasks (2-5 ml per flask) containing 100 ml of liver-extract or ascitic-fluid broth (pH 6.8). The cultures are grown for 3-4 weeks or more. Five to ten per cent of carbon dioxide is introduced into one of the flasks (for growth of the 23 bovine species of the bacteria). Inoculations on agar slants are made every 4-5 days for isolation and identification ‘of the pure culture.

An antiphage serum is introduced into the cultures for neutralization of the phage which inhibits the growth of brucellae. The best results are obtained when the blood is inoculated into the yolk of an unfertilized egg or the yolk sac of a chick embryo. For this, 0.1-0.2 ml of the tested blood diluted in citrate broth in a ratio of 1 : 3 is introduced into each egg. The infected eggs are placed in an incubation chamber for 5 days, after which 0.3-0.5 ml of their contents is inoculated into the liquid nutrient media. Growth is examined every 2-3 days.

If the blood culture produces a negative result bone marrow obtained by sternum puncture is inoculated onto solid and liquid media for isolation of myelocultures.

The urine is also examined. It is obtained with a catheter, centrifuged, and 0.1 ml of the precipitate is seeded onto agar plates containing 1 :200000 gentian violet. In some cases faeces, cow’s and human milk, and amniotic fluid of sick humans and animals are examined for the presence of Brucella organisms.

 

Brucella susceptibility to phages

 

Brucella cultures may be isolated by the biological method. For this purpose healthy guinea pigs or white mice are injected with 0.5 or 3 ml of the test material. A month later the guinea pigs’ blood is tested for agglutinins, the allergic test is carried out, and the pure culture is isolated. White mice are tested bacteriologically every three weeks.

2. Serological test. From the tenth-twelfth day of the disease onwards, the agglutinins accumulate in the blood in an amount sufficient for their detection by the agglutination tests. The Wright (in test tubes) and Huddleson (on glass) reactions are carried out. The Wright reaction is valued highly positive in a 1 800 serum dilution, positive in a 1 :400-1 :200 dilution, weakly positive in 1 :100 dilution, and doubtful at a titre of 1 :50.

The Huddleson reaction is used mainly in mass examinations for brucellosis.

However, there is a disadvantage of this reaction in that it sometimes shows positive results with sera of healthy individuals who have normal antibodies in their blood.

3. Skin allergic test. To determine allergy, Burne’s test is made beginning from the fifteenth-twentieth day of the disease. A 0.1 ml sample of the filtrate of a 3- or 4-week-old broth culture (brucellin) is injected intracutaneously into the forearm. The test is considered positive if a painful red swelling 4 by 6 cm in size appears within 24 hours (Plate III).

4. Opsono-phagocytic test. This test detects changes in the phagocytic reaction. The index of healthy individuals averages 0-1 and occasionally 3-5. In sick persons the reaction is considered high if the index is 50-70, mild, if it is 25-49, and low, if it is 10-24.

For detecting brucellae in the external environment the reaction for demonstrating a rise in bacteriophage titre is carried out. 5. In some cases the complement-fixation test, the indirect haemagglutination reaction, and the immunofluorescence reaction are used.

Treatment. Patients suffering from brucellosis are treated with antibiotics (amphenicol, tetracycline, etc.). Chronic cases are best treated by vaccine therapy. X-ray therapy, blood transfusions, electropyrexia, and balneotherapy, hormonotherapy. Injection of antibrucellosis gammaglobulin is recommended for the prevention of recurrences.

Prophylaxis comprises a complex of general and specific measures carried out in conjunction with veterinary services. This includes:

(1) early recognition of brucellosis, hospitalization of sick individuals, exposure of the sources of the disease;

(2) sanitary treatment of cattle-breeding farms, identification, examination and isolation of sick animals, immunization with live vaccine;

(3) systematic disinfection of discharges of sick humans and animals, prophylactic disinfection of hands of shepherds and persons engaged in the care of sick animals;

(4) observance of hygienic measures during consumption of milk (pasteurization or boiling) and dairy products in districts where there are cases of brucellosis;

(5) protection of cattle-breeding farms, control of cattle driven from farm to farm, the enforcement of quarantine for new cattle, and isolation of young livestock from sick animals;

(6) sanitary education among the population. Immunization with the live or killed vaccine is an additional measure in districts where there are cases of goat-sheep brucellosis.

 

Laboratory diagnosis in details

Tularemia. The causative agent of tularaemia, an acute zoonotic disease largely affecting the lymph nodes, is Francisella tularensis. The laboratory diagnosis of this illness is based on bacteriological, biological, and serological methods of investigation, as well as on allergy tests.

Allergy and serological tests are performed in usual hospital conditions and microbiological laboratories. The recovery and isola­tion of the tularaemia bacteria are carried out in special laboratories.

Bacteriological and biological examination. Attempts to isolate a culture of the causative agent from the patient by direct inocu­lation of the material onto media almost invariably end in failure. The biological method is more promising. Using the material obtained from patients, which may vary depending on the clinical form of tularaemia (blood, puncture sample from a bubo, scraping from an ulcer, conjunctival exudate, faucial deposit, sputum, etc.), the total of 3-5 white mice or 2 guinea pigs are infected. The material collected before the antibiotic treatment is administered subdermally, epicutaneously, intraperitoneally, and by mouth. The infected animals are kept under special conditions (as in the diagnosis of plague) and observed for 6-14 days.

If the experimental animals fail to die within 7-15 days, they are sacrificed on the 15th-20th day and subjected to autopsy. Tularaemia is recognized by pathoanatomical changes in the form of a produc­tive process with necrosis and without marked haemorrhagic com­ponent. The blood, bone marrow, and pieces of the internal organs and lymph nodes from the animal corpse are inoculated by rubbing them into the surface of one of the media such as yolk, glucose-cystine agar with blood, Emelyanova’s medium, etc.

Yolk medium consists of three parts of egg yolks and two parts of isotonic sodium chloride solution (pH 7.0-7.4). It is dispensed into sterile test tubes by 5-ml portions and coagulated for 60 min at 75 °C. On the following day the medium is heated for 30min at 82-85 “C. To check for sterility, the medium is placed in an incubator for 48 hrs. After the inoculation, the test tubes are left in almost horizontal position to fix the micro-organisms on the medium surface to prevent evaporation of the medium, the test tubes are plugged with rubber caps (the presence of a condense is a must) and incubated at 37 °C On the 3rd-5th day, occasionally on the 20th day and later, tender small colonies can be noted.

Glucose-cystine blood agar. Supplement melted meat-peptone agar with U.05-0.1 per cent of cystine and 1 per cent of glucose (pH 7.2–7.4), then boil it for several minutes, cool to 45–50 °C, and add 5–10 per cent of defibrinated rabbit blood. Colonies of the tularaemia growth on a glucose-cystine medium are moist and milk-white in colour.

Emelyanova’s medium contains 2.5 ml of yeast autolysate, 10 ml of gelatin hydrolysate, 20 ml of fish Hour hydrolysate, 1 g of glucose, 0.1 g of cystine 0.5 g of sodium chloride, 2 g of agar, 100 ml of distilled water (pH 7.2–7.4).

The causative agent of tularaemia demonstrates good propagation in the yolk sac of a 12-day chick embryo and poor growth in liquid nutrient media.

Simultaneously with culturing, the same material is used to make impression smears to be stained by the Romanowsky-Giemsa tech­nique. The tularaemia causative agents are small (0.2–0.7 pan), coccal, and rod-shaped bacteria. In smears from the organs they are arranged intracellularly, and in the form of clusters, forming a tender capsule. The isolated pure culture is identified by morpholog­ical, antigenic, and biological attributes.

Freshly isolated pathogenic tularaemia bacteria show weak bio­chemical activity. They may ferment glucose and release hydrogen sulphide; with further cultivation they acquire a capacity to ferment maltose, mannose, and occasionally other carbohydrates. To identify the species of the isolated culture, agglutination with a specific agglutinating serum is performed. The pathogenicity of the culture is measured by inoculation of sensitive animals.

To isolate a culture of the tularaemia bacteria from water, sen­sitive animals are infected with a sediment obtained upon the centrifugation or filtration of water.

In examining foodstuffs, they are washed with meat-peptone broth, centrifuged, and the deposit is administered to animals.

In enzootic foci, planned systemic studies are carried out to isolate the causative agent of tularaemia from rodents (using the above described method).

Serological diagnosis. A standard agglutination test with pa­tients’ serum is performed in test tubes, while a blood drop agglutin­ation reaction (rapid) is carried out on glass slides.

To carry out a standard agglutination test, on the second week of the disease (the 10th–15th day) withdraw 2-3 ml of blood from the patient’s cubital vein. Dilute successively the obtained serum (Table) with isotonic saline in titres from 1:25 to 1:400.

Table

Schematic Representation of the Standard Agglutination Test

Ingredient	Number of test the tubes
	1	2	3	4	5	6 serum control	7 diagnosti­cum control
Isotonic  sodium chloride solution, ml	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Patient’s serum in dilution 1:25, ml	0.5→	0.5→	0.5→	0.5→	0.5­	0.5	–
Diagnosticum, ml	0.5	0.5	0.5	0.5	0.5	–	0.5
Obtained serum      dilu­tion	1:50	1:100	1:200	1:400	1:800	1:50	–

 

Introduce 0.5 ml of the diluted serum into each test tube, add 0.5 ml of tula­raemia diagnosticum to obtain the final dilutions of the serum from 1:50 to 1:800. Simultaneously, check the adequacy of the serum and diagnosticum by the ordinary method.

The test tubes are placed in an incubator for 2 hrs. The prelimin­ary results of the test are read after removal of the test tubes from the incubator, the final ones after the test tubes have been allowed to stay at room temperature for 18–20 hrs.

A positive agglutination reaction in dilution 1:100 and higher is diagnostically significant. It is recommended that the sera be re-examined in the course of the disease (paired sera), the second examination being conducted 7–10 days after the first one. An in­crease in the titre of antibodies by four-fold or more is of a delimit & diagnostic significance.

A blood drop agglutination reaction is used in mass screening. Blood withdrawn from a finger is placed on a glass slide in the form of a thick drop to which an equal amount of distilled water is added to induce haemolysis. Put a drop of the tularaemia diagnosticum near the blood drop and mix both drops with a glass rod. If the serum contains any antibodies, the agglutination reaction occurs immediately.

The blood serum from patients with tularaemia may give a positive agglutination reaction with brucellosis diagnosticum since tula­raemia bacteria and Brucella have common antigens. Individuals with a history of tularaemia show agglutinins in their blood for a long time.

A fairly sensitive method of serological diagnosis of tularaemia is the indirect haemagglutination test. It may he positive already at the end of the first, or the beginning of the second, week of the disease. Tularaemia erythrocyte diagnosticum consisting of a sus­pension of formalin-preserved sheep red blood cells sensitized by the tularaemia antigen is employed as an antigen. Haemagglutinating litres of sera in patients with tularaemia rise to 1:1280–1:2560 and even higher.

The complement-fixation test in the cold is utilized for the early diagnosis of tularaemia. The patient’s blood is obtained during the first week of the disease and a diagnosticum is used as an antigen.

Allergy tests are also used for the early diagnosis of tula­raemia (from the fifth day of the disease onset). Two types of tularin and, respectively, two methods of their administration (epicutaneous and intracutaneous) are utilized. Since the concentration of the allergen in both types of tularin is different, one should not use epicutaneous tularin for the intracutaneous test and vice versa.

The results of the allergic reaction are read 24–36–48 hours after allergen administration.  The presence of a marked infiltrate and hyperaemia of 0.5 cm in diameter is considered a positive reac­tion. Some patients may present the formation of a pustula and even skiecrosis at the place of drug administration, as well as lymphangitis and regional lymphadenitis with elevation of the body tem­perature up to 37.5–38.0°C, which persists for 24–48 hours.

The allergy tests may remain positive (anamnestic reaction) for several years in vaccinated subjects or those with a history of tularaemia.

 

Brucellosis. The causative agents of brucellosis (a chronic zoonotic infection) belong to the genus Brucella which includes three main types: B. melitensis, B. abortus, B. suis. Other types comprise B. neotomae, inducing disease in common field mice, B. ovis, affecting birds, B. canis, causing infection in dogs, and B. rangiferi which is patho­genic to deer.

In diagnosing brucellosis, one uses bacteriological, serological, and biological examination, as well as allergy tests. Serologi­cal and allergy tests for the diagnosis of brucellosis are conduct­ed in common microbiological laboratories. The pure culture of Brucella organisms is isolated and identified in special laboratories.

Bacteriological examination. To isolate a pure culture of Bru­cella, blood, puncture sample of the bone marrow, urine, milk, and pieces of the organs are studied.

To isolate a haemoculture, blood obtained from a feverish patient is inoculated during the first days of the disease; if the results are negative, the examination is performed several times (bacteraemia persists for a long time, for 12 months and more).

No less than 10 ml of blood is withdrawn from the cubital vein and inoculated (in 5 ml-portions) into two vials containing 100 ml of liver or ascitic broth (pH 7.1–7.2) or meat-peptone broth with addition of 1 per cent of glucose, 2 per cent of solution of glycerol, and antiphage serum for phage suppression. To prevent blood co­agulation, 0.2 per cent of sodium citrate is added to the sera. One vial is kept under the usual aerobic conditions, the other, in a sealed vessel containing 10 per cent of carbon dioxide which enters the vessel from a gas cylinder via a gasometre. The necessary concentra­tion of carbon dioxide can also be achieved in the following way:

Insert a burning cotton plug deep into the mouth of the vial with the inoculated culture, additionally close the vial with a rubber plug, and then seal it with liquid paraffin. Taking into consideration a slow growth of Brucella, keep the cultures in a 37 °C incubator for up to one month. Subinoculate to a liver agar slant and “D” agar every 4–5 days. Dry nutrient “D” agar made up of nutrient broth, yeast autolysate, and sodium chloride (pH 7.2–7.4) enhances the rate of Brucella culturing. If tiny isolated colonies have formed on a solid medium, determine purity of the culture and identify the type of Brucella organism (see below).

The puncture specimen of the bone marrow for isolating a, myelo-culture is inoculated onto the same media. It has been established that myelocultures in patients with brucellosis may be isolated 1.5–2 times as often as haemocultures.

Urine for isolating a urinoculture is collected aseptically at the end of the feverish period with the help of a catheter and then centrifuged. The deposit is streaked onto plates with liver infusion agar containing gentian violet in 1:200 000 dilution to retard the growth of Gram-positive microflora. Urinocultures are obtained in 9–14 per cent of cases.

Milk of animals (goats, sheep, and cows) is centrifuged and 0.1–0.2 ml of cream is placed into plates with liver agar containing gentian violet.

The best Brucella growth occurs when the test material is inocu­lated into a yolk of a fresh chicken egg or the yolk sac of the chicken embryo. The patient’s blood is diluted 1 to 3 with citrate broth and introduced into several eggs in 0.1–0.2 ml amounts. The infected eggs are placed into a 37 °C incubator for five days, and then 0.3–0.5 ml of the contents of eggs is streaked onto solid and liquid nutrient media. The inoculated cultures are inspected every 2–3 days.

Prior to infecting eggs with the material tested (faeces, urine, sputum, pus, etc.) containing extraneous microflora, it is subjected to centrifugation. The obtained sediment is diluted with gentian violet solution (1:200000) by 3–5-fold and inoculated in 0.2 ml portions into 3–5 eggs with subsequent subculturing (live days later) onto nutrient media.

Biological examination. Guinea pigs and white mice are the most sensitive animals with any method of inoculation. The patients’ blood is injected to animals intraperitoneally (2–3 ml to guinea pigs, 0.5–1 ml to white mice), urine sediment and cream centrifuged from the milk of animals are administered subcutaneously.

Some 20–30 days following the inoculation withdraw some blood from a guinea pig (or a mouse) and carry out the agglutination test. Then sacrifice the animals, dissect them, and culture the heart blood and suspension from the internal organs and lymph nodes.

Growth of Brucella (of any type) in the broth is manifested by the appearance of turbidity with the subsequent formation of 1 mucilaginous sediment. In plates with liver infusion agar Brucella organisms form tiny smooth, whitish-opaque colonies with a pearly hue. When smears are stained by Kozlovsky’s technique, Brucella are pink.

The smear is flame fixed, stained with 3 per cent aqueous solution of safranine with heating it up until the appearance of fumes, washed with water, and counter stained for 1–1.5 min with 1 per cent aqueous solution of methylene blue, then washed in water once again and dried.

Brucella organisms do not ferment carbohydrates, do not coagulate milk, and do not liquify gelatine. Various types of Brucella contain different antigens so they have to be identified by means of the agglutination reaction with monospecific sera.

Brucella organisms can also be differentiated by their ability to grow in the absence of elevated levels of carbon dioxide, by produc­tion of hydrogen sulphide, sensitivity to the specific bacteriophage, and a bacteriostatic action of aniline dyes (Table 14).

Serological examination in brucellosis includes the agglutination test with the patient’s serum (Wright’s reaction), accelerated plate reaction on a glass slide (Huddleson’s test), an opsonocytophagic test, and the complement-fixation test.

Wright’s reaction is performed at the beginning of the second week of the disease. Suspension of killed Brucella organisms stained with gentian violet or methylene violet and containing 1 mird of brucellae (per ml) killed with formalin or phenol is used as a diagnosticum. Currently a uniform colour brucellar diagnosticum for Wright’s and Huddleson’s tests is commercially available.

The procedure of performing the Wright test is similar to that used in other agglutination tests, with the exception of the fact that the serum is diluted in a multiple order with isotonic NaCI solution containing 0.5 per cent of phenol. The test tubes are placed in the incubator for 18–20 hrs and then left at room temper­ature for 2 hrs. The results of agglutination are denoted with pluses. The reaction is considered positive if the titre is 1:200 and doubtful if it is 1:50. Wright’s reaction may be positive in vaccinated sub­jects and patients with brucellosis. So, it is to be repeated in the course of the disease to look for increase in the antibody litre.

The plate agglutination test proposed by Huddleson (Table) is most frequently used in brucellosis foci (in field conditions) since it is simple to perform.

Table 

Schematic Representation of Huddleson’s Agglutination Test

 

Using a wax pencil, divide a glass plate into six squares, then pipette on them undiluted serum to be studied in 0.08, 0.04, 0.02, 0.01, and 0.02-ml portions, and add 0.03 ml of the same diagnosticum that is used in Wright’s reaction to each dose of the serum but the last (the fifth square) into which 0.03 ml of isotonic sodium chloride solution (control of the serum) is placed. In the sixth square, place 0.03 ml of the antigen and 0.03 ml of iso­tonic saline (control of the diagnosticum). Mix drops of the serum and antigen with a glass rod and after slight rocking of the plate read the results of the reaction which is manifested already in the first minutes by the appearance of microgranular stained agglutinate. The absence of agglutination in all doses of the serum is assessed as a negative reaction, the presence of agglutination in the first dose (0.08 ml of the serum) as a doubtful one, in the second dose (0.04 ml), as a weakly positive one, in the third or fourth doses (0.02–0.01 ml), as a positive one; the agglutination reaction expressed by 4 pluses in all doses is evaluated as a drastically positive one. The value of Huddleson’s agglutination test is diminished by the fact that not infrequently it produces positive results with healthy people’s sera due to the presence of normal antibodies. Hence, sera showing posi­tive Huddleson’s reaction should be subjected to the tube aggluti­nation test.

For the rapid diagnosis of brucellosis one can use an agglutination reaction of Bengal pink with an acid antigen. The antigen is prepared from dense suspension of heat-killed (85 °C for 1.5 hrs) Brucellae with the use of buffer (pH 3.6–3.8) solvent and Bengal pink. Put 0.03 ml aliquots of patient’s whole serum and of the antigen of Bengal pink on plates and then mix them by circular movements.

Read the results of the reaction in 4 min. The presence of macro- or microgranular agglutinate indicates a positive test.

The opsonocytophagic test is performed beginning with the 15–20th day of the disease with four milliard broth suspension of killed Brucellae. It is considered that the parameter varying from 10 to 24 is characteristic of a weakly positive reaction; from 25 to 49, of a definitely expressed one; and from 50 to 75, of a dramatically positive one. Iormal subjects, this parameter usually equals 0, or 1, rarely 3–5.

Complement fixation is a fairly specific test with regard to brucel­losis. A positive result is registered from the 20th–25th day of the disease and persists for a long time.

When the IHA test is made in patients with acute and subacute brucellosis, agglutinins are detected in 91–100 per cent of cases.

In acute, subacute, and chronic forms of brucellosis, antibodies in patients’ sera may be recovered by the indirect IF test that is more sensitive than the agglutination reaction. It is recommended that one should determine the composition of serum immunoglobulins at different stages of the disease. At early stages after inoculation, IgM can be demonstrated, later IgG are observed. These immunoglobulins are differentiated with the help of the cystine test.

Burnet’s intracutaneous allergy test is used for detecting an allergic status in patients with an active disease or a history of brucellosis. It becomes positive from the 15th–20th day of the dis­ease. A total of 0.1 ml of brucellin is injected intracutaneously into the middle area of the upper arm. The reaction is con­sidered positive if 24 hrs after the injection there is tenderness, hyperaemia, and infiltration of the skin some 3 X 3 cm in size, weakly positive if the area of hyperaemia and infiltration is 1 X 1 cm in size, and sharply positive if the area of hyperaemia and infiltration is 6 X 6 cm in size. Allergic reaction remains positive for many years after the disease and in inoculated persons.

 

Bacillus anthracis

The agent responsible for anthrax (B. anthracis) was described by A. Pollander (Germany) in 1849, by K. Davaine (France) in 1850, and by F. Brauell (Russian) in 1854. Studies of anthrax were originated by R. Koch (1876), L. Pasteur (1881) and L. Tsenkovsky (1883). Its isolationb y German bacteriologist Robert Koch in 1877 marked the beginning of modern medical microbiology, because it was, in part, Koch’s work with this organism that was responsible for the development of the well-known Koch’s postulates

B. anthracis belongs to the family Bacillaceae.

Morphology. Anthrax bacilli are large organisms, measuring 3-5 mcm in length and 1-1.2 mcm in breadth. In the body of animals and man they occur in pairs or in short chains, while iutrient media they form long chains (Fig.). In stained preparations the ends of the bacilli appear either to be sharply cut across or slightly concave, resembling bamboo canes with elbow-shaped articulations. The G+C content in DNA is 32 to 62 per cent.

 

Figure. Anthrax bacilli: 1 — smear from sporogenous culture; 2 — structure of the periphery of colony; 3 —  smear from cadaver specimens

 

The bacilli are non-motile. Outside the host’s body they produce oval-shaped central spores which are smaller in diameter than the bacillus. Spores are best produced in the presence of oxygen at 30-40° C. No spores are produced in the body of man or animal or at temperatures above 43 and below 15°C.

It has been ascertained that spores may germinate into vegetative forms during the warm months under favourable conditions, and transform again into spores in the autumn.

In the bodies of man and animals the bacilli produce capsules which surround a single organism or are continuous over the whole chain (Fig.).

 

Anthrax bacilli in tissue

 

Capsules are also produced outrient media which contain blood, serum, egg yolk, or brain tissue. The capsule which contains specific proteins provides a defence mechanism and determines the virulence of the organism. The anthrax bacillus readily stains with all aniline dyes and is Gram-positive.

It can be seen on ultrathin sections that the cell wall is 360 to 400 nm thick, the cytoplasmatic membrane has three layers, the cytoplasm is granular and the vacuoles large, and that the nucleoid is in the centre of the cell. Division begins before the cells have separated after the previous division, which leads to the formation of streptobacilli.

Cultivation. The bacillus is aerobic and facultatively anaerobic. The optimum growth temperature is 37° C, and the organism does not grow below 35° and above 43° C. It grows well on all ordinary media at pH7.2-7.6. On meat-peptone agar the bacilli form rough colonies (R) which have uneven edges and resemble the head of a medusa (Fig.).The edges of the colonies have the appearance of locks of hair or a lion’s mane. The smooth S-forms possess low virulence or are completely avirulent and non-capsulated in the body of the host.

 

Bacillus anthracis colonies

 

Broth cultures of the anthrax bacillus produce flocculent growth resembling cotton wool which sinks to the bottom of the tube or flask, leaving the broth clear. Spore production is inhibited by adding a 1 per cent calcium chloride solution to the medium, and is stimulated by the presence of neutral sodium oxalate.

The anthrax bacillus undergoes a morphological change when it transforms from the R-form to the S-form. It loses its ability to form chains in smears and occurs in coccal and diplobacillary forms or the cells are arranged in groups. Incubation at 42.5° C produces thread-like, non-sporeforming organisms of low virulence. On meat-peptone agar containing penicillin the bacilli break up into globules which are arranged in the form of a necklace (a pearl necklace). The anthrax bacilli transform usually from the R-form (typical, with rough colonies, and virulent) to the S-form (atypical, with smooth even-edged colonies, and avirulent) through the intermediate 0-form (with mucilaginous, pigmented, and patterned colonies).

Fermentative properties. The anthrax bacilli possess great biochemical activity. They contain the enzymes — dehydrogenase, lipase, diastase, peroxidase, and catalase. In gelatin stab-cultures growth resembles an inverted fir tree (Fig.), the gelatin being liquefied in layers. The organisms cause late liquefaction of coagulated serum and produce ammonia and hydrogen sulphide. They slowly reduce nitrates to nitrites and coagulate and peptonize milk. The organisms ferment glucose, levulose, saccharose, maltose, trehalose, and dextrin with acid production.

 

Figure. Growth of B. anthracis on gelatin stab

 

Toxin production. When growing on semisynthetic medium, B. anthracis discharges an exotoxin (oedema factor) into the culture fluid. The capsular substance is very toxic, it contains Bayle’s aggressins. Loss of the capsule results in loss of virulence.

It has been established that some strains of B. anthracis produce in the animal’s body a lethal toxin (mouse factor), which on addition of the oedema factor or the protective antigen causes death of the animal.

The serum of guinea pigs who died from anthrax possesses the property of causing death of albino mice and guinea pigs on being injected intravenously in small doses.

Antigenic structure. There is only one antigenic type of B anthracis, and it contains three major types of cell antigens. The anthrax bacillus contains a protein (P) and a polysaccharide (C) antigen. The polysaccharide antigen is present in the bacterial cell, while the protein antigen is in the capsule.

The polysaccharide antigen consists of N-acetylglucosamine and d-galactose and is thermoresistant. It survives for long periods in tissues obtained from corpses. Ascoli’s thermoprecipitin test is based on this principle. A boiled B. anthracis extract contains a polysaccharide fraction (thermoresistant) which yields a precipitin reaction with the precipitant serum.

The capsule contains a protein-like substance, a polypeptide, which. is a polymer of gamma-d-glutamic acid

In animal bodies and on media containing tissue extracts and plasma, B. anthracis produces a peculiar antigen (a protective antigen). This antigen is a non-toxic thermolabile protein which possesses marked immunogenic properties. It gives rise to the formation of protective (incomplete) antibodies which neutralize the aggressive enzyme of the anthrax bacillus. The capsule is unusual in that it contains only the D (or unnatural) isomer of the amino acid, furthermore, the virulence of the organism depends, in part, on the antiphagocytic activity of this capsular material.

Classification (Table).

Table 

Differential-Diagnostic Signs of B. anthracis, Antracoides, and Soil Bacilli

 

Resistance. B. anthracis survives in meat-broth cultures in hermetically sealed ampoules for 40 years and the spores survive for 58 to 65years. Spores remain viable in soil for decades and when dried — for as long as 28 years. They are more resistant to disinfectants than the vegetative cells. The vegetative cells are killed in 40 minutes at 55° C, in 15 minutes at 60° C, and in 1-2 minutes at 100 ºC. The spores are thermoresistant, and withstand boiling for 15-20 minutes and autoclaving at 110° C for 5-10 minutes. They are destroyed by 1 per cent formalin and10 per cent sodium hydrate solutions in 2 hours. The capsule is moreresistant than the microbial cell. Post-mortem examination of animal bodies which had previously been exposed to putrefactive microflora reveals quite frequently empty microbial capsules (shadows), devoid of cytoplasm.

Pathogenicity for animals. Sheep, cows, horses, deer, camels, and pigs are susceptible to anthrax infection. The animals are usually infected through the mouth, ingesting the spores in the fodder. The organisms localise in the intestine. Blood-sucking insects (gadflies and stable flies) may be vehicles of infection in some cases.

The disease is characterized by lassitude, cyanosis, and sanguineous discharge from the intestine, nostrils, and mouth. Septicaemia sets in preceding death which occurs in 2 or 3 days. In horses the infection is less severe, affecting the glandular tissues and causing the development of anthrax carbuncles.

Among the laboratory animals, most susceptible are white mice, then guinea pigs, and rabbits which die 2-4 days after infection. A swelling and haemorrhages appear at the site of injection. The internal organs become congested and enlarged (particularly the spleen), and septicaemia develops. The blood of the animals does not clot after their death because of the anticoagulative effect of the anthrax bacilli, and it is thick and black-red in colour (Gk. anthrax coal).

Pathogenesis and diseases in man. Anthrax is a typical zoonosis. Anthrax is primarily a disease of sheep, goats, cattle, and, to a lesser extent, other herbivorous animals. Although in the United States it is found only in a few areas (Louisiana, Texas, California, Nebraska, and South Dakota), it is a world-wide problem, especially in parts of Europe, Asia, and Africa Once the disease is established in an area, bacterial endospores from infected or dead animals are able to contaminate the soil and, because of the resistant endospores, the pasture areas remain infectious for other animals for many years In most animal infections, the spores enter the body by way of abrasions in the oral or intestinal mucosa, and after entering the bloodstream, they germinate and multiply to tremendous numbers, causing death in 2 to 3 days.

Humans acquire the disease from sick animals or articles and clothes manufactured from contaminated raw materials: sheepskin coats, fur mittens, collars, hats, shaving-brushes, etc. In summer the infection may be transmitted by blood-sucking insects. Anthrax occurs in three main clinical forms: cutaneous, respiratory, and intestinal.

In the cutaneous form the causative agent enters the body most frequently through injured integuments, mainly those parts of the body which are not covered with clothes (face, neck, hands, and forearms). Cutaneous infection is an occupational hazard for persons who handle livestock or work with items derived from contaminated wool or hides. An anthrax carbuncle (malignant pustule) develops at the site of bacilli localization. An initial lesion occuiring at the site of entry soon develops into a black necrotic area (Fig. 3). If the lesion is not treated, the organisms may invade the regional lymph nodes and bloodstream, causing death.

FIGURE. Lesion of cutaneous anthrax (eighth day of illness) on the arm of a woman who had been a carder in a wool factory.

 

The pulmonary form of the disease occurs less frequently than the cutaneous form but is more serious and carries a higher mortality rate. This form is acquired through the air when working with material contaminated with bacillary spores. The disease assumes the form of a severe bronchopneumonia. The bacilli are discharged in the sputum.

The intestinal form is due to ingestion of meat of sick animals. It is characterized by grave lesions in the intestinal mucosa with haemorrhages and necrotic foci The bacilli are excreted in the faeces. It is considered by a number of authors that the intestinal form of the disease is caused by the bacilli invading the intestine through the blood.

A seemingly rare source of anthrax infection came to the force in 1979 when an epidemic of anthrax occurred in Sverdlovsk in the Soviet Union This epidemic, which is believed to have killed almost 100 people, supposedly originated as gastric anthrax caused by eating anthrax infected meat. In such cases, it is assumed that the organisms enter the bloodstream from the intestine. Although gastric anthrax is rare in the western world, Soviet textbooks have described previous epidemics of gastric anthrax that resulted in 100% mortality.

At present the cutaneous form of anthrax occurs sporadically, while the intestinal form is very rare. Anthrax septicaemia may develop as a complication in any of the clinical forms or in debilitated and emaciated patients.

Until 1955, it was believed that anthrax caused death by its ability to resist phagocytosis, allowing it to grow to tremendous numbers of bacilli that clogged capillaries. This explanation had been termed the “log jam” theory. It is known that this concept is incorrect and that B. anthracis secretes a highly toxic exotoxin.

The toxin consists of a complex of proteins that has been isolated as three components named deem factor (EF), lethal factor (LF), and protective antigen (PA)None of these factors has any biologic activity by itself. An insight into the mechanism of action of this toxin complex was provided by Stephen Leppla when he discovered that EF is an inactive form of adenylate cyclase, which is activated by eucaryotic calmodulin, and that PA is required to facilitate the entry of EF into cells. Thus, it is similar to other A-B toxins in that PA is analogous to what is termed the B subunit, with EF acting as the A subunit of this toxin. It differs in that unlike the A-B toxins already discussed, PA and EF are secreted as separate components. The end result is an increase in cAMP in polymorphonuclear neutrophils, inhibiting their ability to carry out normal phagocytosis. Thus, as described for Bordetella pertussis, the phagocyte impotence seen in anthrax infections results from the secretion of an extracellular adenylate cyclase and its effect on host leukocytes. Table summarizes the effects of these components.

Table

Components of Anthrax Toxin

EF, edema factor, LF, lethal factor, PA, protective antigen

Interestingly, PA also is required to show a biologic effect of LF, indicating that PA acts as a common binding subunit for both EF and LF. No enzymatic activity is known for LF, but when injected together with PA, LF causes severe pulmonary edema and death in Fisher 344rats. PA and LF together are also rapidly cytolytic for macrophages.

When PA binds to a specific receptor on a host cell, a cell-surface protease cleaves off a peptide of about 20kd to generate a cell-bound 63-kd PA, which possesses a new binding site to which LF and EF bind with high affinity. The resulting complexes of PA63-LF and PA63-EF are then internalized by receptor-mediated endocytosis. Mutants lacking LF produced edema when injected subcutaneously but were not lethal. Mutants missing EF but still producing LF were lethal when injected into susceptible animals.

Notice that the virulence of. B. anthracis is dependent on two separate plasmids, one of which codes for the D-glutamic acid capsule, and the other for the three separate genes of the secreted toxin.

Immunity following anthrax is antimicrobial and depends on the presence of protective antigens. Phagocytosis plays no defensive role in the disease. The protective antigen does not give rise to the production of complete antibodies, but it stimulates the formation of protective (in-complete) antibodies which cause the destruction of the virulent anthrax bacilli.

Serum of individuals who have recovered from anthrax is found to contain substances which are capable of destroying capsular matter and neutralizing aggressins and toxins (lethal factor).

Laboratory diagnosis. In cases of cutaneous anthrax the malignant pustular exudate is examined; it is obtained from the deep layers of the oedematous area where it borders with the healthy tissues. Sputum is examined in cases of the respiratory form, faeces and urine, in intestinal form, and blood is examined in cases of septicaemia.

1. The specimens are examined under the microscope, the smears are Gram-stained, or stained by the Romanowsky-Giemsa method. The presence of morphologically characteristic capsulated bacilli, arranged in chains, allows a preliminary diagnosis.

2. For isolation of the pure culture the specimens are inoculated into meat-peptone agar and meat-peptone broth. The isolated culture is differentiated from other morphologically similar bacteria by its morphological and biochemical properties.

3. Laboratory animals (white mice, guinea pigs and rabbits) are inoculated with the pathological material and with the pure culture de-rived from it. B. anthracis causes the death of white mice in 24-48 hours and of guinea pigs in 2-3 days following inoculation. Microscopic examination of smears made from blood and internal organs reveals anthrax bacilli which are surrounded by a capsule.

A rapid biological test is also employed. The culture obtained which has to be identified is introduced intraperitoneally into white mice. Several hours after inoculation smears are prepared from the peritoneal contents. Detection of typical capsulated bacilli gives a basis for con-firming the final result of the biological test.

The allergic test with anthracin (a purified anthrax allergen) is employed when a retrospective diagnosis is required in cases which have yielded negative results with microscopical and bacteriological examination.

Postmortem material as well as leather and fur used as raw materials are examined serologically by the thermoprecipitin reaction (Ascoli’s test) since isolation of the bacilli is a matter of difficulty in such cases.

As can be seen in Fig. 3, the result in the first test tube (containing the test material) may be either positive or negative, in the second test tube (control) it must be only positive, and in the third, fourth, fifth, and sixth control test tubes the results must always be negative.

 

Figure. Positive thermoprecipitation reaction (Ascoli’s test)

 

When employing laboratory diagnosis of anthrax, one must bear in mind the possibility of the presence of bacteria identical with B. anthracis in their biological properties (see Table 1). These sporing aerobes are widely distributed iature and are normally sporeforming saprophytes. They include B. cereus, B. subtilis, B. megaterium, etc.

The anthrax bacilli may be differentiated from anthracoids (false anthrax organisms) and other similar sporing aerobes by phagodiagnosis. The specific bacteriophage only causes lysis of the B. anthracis culture.

Treatment comprises timely intramuscular injection of antianthraxglobulin, and the use of antibiotics (penicillin, tetracycline, and streptomycin).

Prophylaxis. General measures of anthrax control are carried out in joint action with veterinary workers. These measures are aimed at timely recognition, isolation, and treatment of sick animals. They also include thorough disinfection of premises for live-stock, territory an dall objects found on it, and ploughing over of pastures. Carcasses of animals which have died of anthrax are burnt or buried on specially assigned territory, not less than 2 meters deep, and covered with lime chloride.

The veterinary authorities also enforce regulations banning the use of contaminated meat for food and introduce thorough control of manufactured articles from animal hide and fur which are to be marketed.

Ever since Pasteur’s celebrated field trial, in which animals were successfully immunized with a living attenuated suspension of B. anthracis, efforts have been directed toward the production of effective vaccines that possess little or no toxicity. Because killed vaccines are of little value, two different approaches for the stimulation of artificial immunity have been undertaken: (1) the isolation and use of the protective antigenic component of the anthrax toxin, and (2) the use of attenuated living bacteria to induce antitoxic immunity.

At present, a vaccine, prepared from non-capsulated anthrax bacilli and consisting of a suspension of live spores of vaccine strains, is used. It is employed for immunization of man and domestic animals.

The vaccine is completely harmless, producing immunity quite rapidly (in 48 hours) and for a period of over a year. It is inoculated in a single dose.

Vaccination is carried out among people who work at raw-material processing factories (processing of animal hide and hair), at meat-packing factories, and at farms where anthrax is encountered. Reinoculation is performed after a period of 12 months.

However, all effective living vaccines possess some toxicity, and they have not been used in the United States for humans. The vaccine licensed in the United States for use in humans is an aluminum hydroxide-adsorbed supernatant material from ferment or cultures of a toxigenic, but nonencapsulated strain of B. anthracis. Unfortunately, it induces a short-lived immunity and re-quires annual boosters.

Individuals who have been in contact with material contaminated with anthrax organisms (when dressing infected carcasses or using such meat for food) are given intramuscular injections of 20-25 ml of antianthrax globulin together with penicillin.

PA seems to be the only effective component of the vaccine because neither EF nor LF induced protection. It has been cloned in Bacillus subtilis, and guinea pigs immunized by the intramuscular injection of the B subtilis cell suspension were protected against challenge by B. anthracis. PA also has been cloned in vaccinia virus, where it induced at least partial immunity to challenge in guinea pigs and mice. It is, therefore, possible that a cloned source of PA will be used in future anthrax vaccines for humans. The vaccine used in domestic animals consists of a living spore culture, which is designated the Sterne strain. It still carries the plasmid encoding for PA, EF, and LF, but its avirulence is attributed to the loss of the plasmid encoding the antiphagocytic capsule.

A chemical anthrax vaccine consisting of a protective antigen (a filtrate of non-capsulated non-proteolytic anthrax strains which had been cultivated on synthetic and semisynthetic media) is used in England and the USA, it is as effective as the living vaccine.

Control of anthrax in humans is, therefore, not a simple matter, particularly for those who are occupationally exposed, but sterilization of wool, hair, and other animal materials capable of transmitting the disease does prevent the spread of the bacilli to persons not normally exposed to infected hides. However, not all such products are sterilized, as exemplified by the person who contracted anthrax from contaminated goat hair fringe on a souvenir drum purchased in Haiti. Other Haitian products using goat hair have been found contaminated with anthrax spores, and the Centres for Disease Control in Atlanta has recommended that all such items be considered potentially contaminated and that they be given to local health authorities for disposal.

However, due to the prophylactic measures conducted regularly by the veterinary and public health services, mortality has decreased 130-fold while the disease is a very rare occurrence.

 

BACILLUS CEREUS. Bacillus cereus is an aerobic, spore-forming, gram-positive rod that is the etiologic agent of a food borne intoxication.

Pathogenesis of B cereus Intoxication. Bacillus cereus produces two distinct clinical forms of food poisoning (1) an illness with an incubation period of 10 to 12 hours, which is characterized by abdominal pain, profuse diarrhea, and nausea, lasting 12 to 24 hours, and (2) an illness with an incubation period of 1 to 6 hours characterized by vomiting, with or without a mild diarrhea.

These two clinical entities result from the production of two different enterotoxins by B cereus. The first, like cholera enterotoxin and LT from Eschenchia coli, stimulates the adenylate cyclase-cyclic AMP system in intestinal epithelial cells. When fed to rhesus monkeys, this heatlabile toxin causes only diarrhea. The second enterotoxin does not stimulate the synthesis of cAMP, and this heatstable toxin causes only vomiting when fed to rhesus monkeys

Epidemiology and Control of B. cereus  Intoxication. Bacillus cereus is readily found in soil and on raw and dried foods, including uncooked rice, a major source of B. cereus food poisonings. The spores may not be killed during cooking, and will germinate when the boiled rice is left unrefrigerated (to avoid clumping of grains). Brief warming or flash frying does not always destroy the elaborated enterotoxins, particularly the heat stable toxin. Meat or meat products, as well as cream or pudding preparations, also have been sources of B. cereus food poisoning. A diagnosis usually is based on finding 10^sorganisms per gram of the incriminated food.

Prevention is best accomplished by the prompt refrigeration of boiled rice and other dried foods that have been cooked. Because the symptoms are mediated by preformed enterotoxins, antibiotic therapy is of no value.

 

Laboratory diagnosis in details

The material for examination, depending on the clinical form of the disease (skin, septic), is contents of vesicles, carbuncles, secretion of ulcers, scabs, sputum, faeces, blood, and cerebrospinal fluid. Autopsy material usually studied is blood and pieces of the internal organs.

The material is collected into sterile test tubes or vials. When autopsy material has to be transported to the laboratory, blood, a puncture sample of the spleen, and bone marrow are placed in a thick layer on glass slides (this ensures the formation from vegetative forms of spores with a high rate of survival). After drying, the slides are wrapped with paper, put into a box, sealed, and sent to the labo­ratory with a courier. In case of epidemiological indications various objects of the environment, as well as the fur and bristle of animals, are examined.

All examinations related to the identification of the culture of anthrax bacilli are carried out in special laboratories.

Bacterioscopic examination. Clinical and autopsy material is examined microscopically. The presence in smears of large Gram-positive bacilli, which form chains surrounded by a capsule), makes it possible to make a preliminary diagnosis of anthrax. Staining of capsules of anthrax bacilli is made by Gins’ method or some other technique. The capsular form of the causative agent is usually detected in blood in a septic form of the disease.

Luminescent microscopy is used as a supplementary method of the diagnosis of anthrax. In this kind of examination anthrax bacilli treated with specific fluorescent serum appear as rods with a rim, which give off green light.

Bacteriological and biological examination. To confirm the diag­nosis, inoculation into nutrient media and animals is carried out. The material is introduced into plates with a meat-peptone agar and test tubes with a meat-peptone broth, which are placed into a 37 °C incubator. Simultaneously, two white mice are infected subcutaneously.

In 20-24 hrs the inoculated cultures are examined and dead mice are autopsied (death occurs 24-48 hrs after inoculation). Characteris­tic rough colonies (“medusa head”) with curly edges are noticed on plates with a meat-peptone agar. A growth resembling a piece of cotton wool forms on the bottom of a test tube with broth; the medium remains transparent. A smear and a hanging-drop prepa­ration are made from the sediment. Acapsular Gram-positive bacilli appearing as long chains are found in the smear. The causative agent of anthrax, unlike mobile soil bacilli, is immobile. To isolate a pure culture, typical colonies are subcultured onto an agar slant.

Of great diagnostic significance is detection of capsule formation outrient media. For this purpose a medium containing Hanks’ solu­tion (or Hottinger’s broth) and 40 per cent of sterile cattle serum, Bouze’s medium (3 per cent hunger agar, pH 7.4, to which 15 per cent of defibrinated sheep blood is added), and other media are employed. The inoculated cultures are incubated for 18-24 hrs at 37 °C in a microanaerostatic jar with a 20-40 per cent concentration of carbon dioxide gas. Capsule formation may also be demonstrated in vivo. Sacrify infected animals in 1-2 hrs; make smears from the peritoneal fluid and heart blood and impression smears from the spleen, kidneys, and lungs. Then stain the prepared smears.

Dissect the corpses of the mice in 24-48 hrs and look for signs characteristic of anthrax, namely: oedema at the site of material introduction, darkened uncoagulated blood, haemorrhages into fat tissue, loose consistence of the spleen, and a compact red liver. Typical bacilli surrounded with a capsule are found in smears from the internal organs and blood. On the basis of morphological, tincto­rial, cultural, and biological data a conclusion is drawn on the detec­tion of B. anthracis.

The isolated culture is identified by inoculating it into gelatin and blood-containing- agar and into animals. B. anthracis induce a characteristic growth in the gelatin column which resembles a rod whose processes go downward in the form of an inverted fir-tree. Later on the gelatin liquefies. There is no haemolysis on a blood-agar.

Study of biological properties of the isolated culture makes it possible to differentiate between B. anthracis and soil bacilli (Table 1).

The penicillin test also allows for the differentiation between the causative agent of anthrax and soil bacilli. It is conducted in the following manner. A three-hour-broth culture of the isolated micro­organism is streaked onto plates with a meat-peptone agar contain­ing 0-5 and 0.05 U/ml of penicillin. After 3 hrs of inoculation smears are made and examined microscopically for the appearance of “a pearl necklace”, i.e., disintegration of anthrax bacilli with the formation of balls. Soil bacilli retain the form of chain-arranged rods.

Bacilli of anthrax, in contrast to similar non-pathogenic bacilli are lysed by a specific anthrax phage.

The presence of the anthrax antigen in the decayed carcass of the animal, in the skin (fresh, dry, and tanned) and products from it. as well as in pelts and fur is determined using Ascoli’s thermoprecipitation reaction. The material studied is comminuted, immersed with 10-20-fold volume of isotonic saline and boiled for 10-45 min or kept for 16-20 hrs at 6-14 °C. Then, the fluid is filtered and the ob­tained transparent extract is layered on the precipitating serum. At the place where the two fluids converge a white ring appears for 1-5 min, which is considered as a sign of a positive reaction }.

The reaction is considered non-specific if a precipitate forms in 10 min. Simultaneously, control reactions with an extract and pre­cipitating serum are run.

 

 

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Serological diagnosis is resorted to in those cases where the caus­ative agent cannot be detected from the material studied. To demon­strate antibodies in the patient’s blood serum, the highly sensitive latex agglutination and passive agglutination reactions with a protec­tive anthrax antigen are employed.

The allergy test is used for detecting immunological changes fol­lowing a disease and vaccination. The allergen anthraxin is injected intracutaneously in a dose of 0.1 ml. The results are read in 24-48 hrs. The test is considered positive if the patient develops hyperaemia over 16 mm in diameter and an infiltrate.