Theme 16

Theme 16. Causetive agents of bacterial intestinal diseases: dysentery and cholera. Microbiological diagnosis and prophylaxis of them.

Theme17. Corynebacteria. Microbiological diagnosis of diphtheria

 

SHIGELLAE

The causative agent of dysentery was described in 1888 by A. Chantemesse and in 1891 by A. Grigoryev and F. Widal. In 1898 this organism was studied in detail by K. Shiga in Japan and in 1900-1901 by V. Kruse in Germany (Shiga bacillus). In 1900 S. Flexner and R. Strong in the Philippines isolated dysentery organisms (Flexner bacillus) which possessed properties different to those of the above-mentioned bacillus. In 1904 P. Hiss and F. Russel described a dysentery bacterium which became to be known as the Hiss-Russel bacillus. At present this organism is included in the Flexner species. In 1904 K. Duval, in 1907 V. Kruse and others, and in 1915 K. Sonne recognized a dysentery bacillus which ferments lactose. In 1917 M. Stutzer in Russia and K. Schmitz in Rumania simultaneously isolated another species of dysentery bacilli (Stutzeri-Schmitzii bacillus). Later other bacilli causing dysentery were discovered. According to the current International Nomenclature, all dysentery bacilli are grouped together in one genus known as Shigella.

Morphology. Morphologically dysentery bacilli correspond to the organisms of the family Enterobacteriaceae. Dysentery bacilli have no flagella and this is one of the differential characters between these organisms and bacteria of the coli-typhoid-paratyphoid group. Some strains of Flexner bacilli are found to possess cilia.

Cultivation. Dysentery bacilli are facultatively aerobic and grow readily on common media at pH 6.7-7.2, the optimum temperature for growth being 37° C, they do not grow at 45° C. On solid media they form small (1-1.5 mm in diameter), fragile, semitransparent colonies which are similar to those of the typhoid bacteria. In meat broth dysentery bacilli produce a diffuse turbidity.

 

 

Fermentative properties. None of the species of dysentery bacilli liquefy gelatior produce hydrogen sulphide. They ferment glucose, with acid formation, with the exception of the Newcastle subspecies which sometimes produce both acid and gas during this reaction. With the exception of the Sonne bacilli, none of them ferment lactose.

Table

Shigellae Biochemical Properties

Note: “+”, fermentation of carbohydrates, formation of undol and catalase; “–”, the absence of carbohydrate fermentation and indol and ornithine formation; “±”,  weak for­mation of indol and ornithine and weak carbohydrate fermentation.

 

Toxin production. S. dysenteriae produce thermolabile exotoxin which displays marked tropism to the nervous system and intestinal mucous membrane. This toxin may be found in old meat broth cultures, lysates of a 24-hour-old agar culture, and in desiccated bacterial cells.

An intravenous injection of small doses of the exotoxin is fatal to rabbits and white mice. Such an injection produces diarrhoea, paralysis of the hind limbs, and collapse.

The dysentery exotoxin causes the production of a corresponding antitoxin. The remaining types of dysentery bacilli produce no soluble toxins. They contain endotoxins, which are of a gluco-lipo-proteiature, and occur in the smooth but not in the rough variants.

Thermolabile substances exerting a neurotropic effect were revealed in some S. sonnei strains. They were extracted from old cultures by treating the latter with trichloracetic acid.

Antigenic structure. Dysentery bacilli are subdivided into 4 subgroups within which serovars may be distinguished. The antigenic structure of shigellae is associated with somatic 0-antigens and surface K-antigens.

Classification. Dysentery bacilli are differentiated on the basis of the whole complex of antigenic (Table) and biochemical (Table 2) properties. S. sonnei have four fermentative types which differ in the activity of ramnose and xylose and in sensitivity to phages and colicins.

Table

International Classification of Shigellae

 

 

Resistance. Dysentery bacilli live in the external environment for a period of 5-10 days (in soil, foodstuffs and water, and on objects, plates and dishes). Direct sunlight and a 1 per cent phenol solution destroy the organisms in 30 minutes and at a temperature of 60° C the organisms perish in 10 minutes. The bacilli are easily killed by treatment with chloramine and calcium chloride solutions. The Shiga bacilli are most sensitive to physical and chemical factors, while the Sonne bacilli are relatively resistant to them. Dysentery bacilli may acquire resistance to drugs (sulphonamides, antibiotics) and to ionizing radiation.

Pathogenicity for animals. Monkeys are susceptible to dysentery bacilli. They contract the infection from sick people or carriers in the nurseries. In some cases they become sources of contamination of personnel iurseries and zoological gardens.

Parenteral infection causes fatal toxicosis in rabbits. An intravenous injection of a S. dysenteriae culture exerts a highly toxic effect. The resulting infection constitutes diarrhoea, paresis or paralysis of the limbs, followed by collapse and death. Autopsy reveals hyperaemia of the intestinal mucous membrane, haemorrhages, necrosis, and ulcerations. Infected white mice die within the first four days.

When cultures of virulent dysentery bacilli are introduced into the respiratory tract of white mice, the organisms multiply. However, attempts to reproduce dysentery in white mice are of no success. Kittens and puppies are more susceptible. Guinea pigs display low susceptibility to dysentery bacilli, but infection through the eye conjunctiva results in keratoconjunctivitis which is assumed to be a specific lesion.

Epidemiology and Pathogenesis of Shigellosis. Humans seem to be the only natural hosts for the shigellae, becoming infected after the ingestion of contaminated food or water. Unlike Salmonella, the shigellae remain localized in the intestinal epithelial cells, and the debilitating effects of shigellosis are mostly attributed to the loss of fluids, electrolytes, and nutrients and to the ulceration that occurs in the colon wall.

It has been known for many years that Shigella dysenteriae type 1 secreted one or more exotoxins (called Shiga toxins), which would cause death when injected into experimental animals and fluid accumulation when placed in ligated segments of rabbit ileum. These toxins are essentially identical to the Shiga-like toxins produced by the EIEC and the EHEC. Thus, Shiga toxin consists of one A subunit and five B subunits and seems to kill an intestinal epithelial cell by inactivating the 60S ribosomal subunit, halting all protein synthesis. Moreover, although all virulent species of Shigella produce Shiga toxins, there seems to be a wide variation in the amount of toxin formed.

The mechanism whereby Shiga toxin causes fluid secretion is thought to occur by blocking fluid absorption in the intestine. In this model, Shiga toxin kills absorptive epithelial cells, and the diarrhea results from an inhibition of absorption rather than from active secretion.

Of note is that, like the EHEC, Shigella species can cause HUS. Moreover, Shiga-like toxins have been detected in certain strains of Vibrio cholerae and Vibrio parahaemolyticus that were associated with HUS, indicating an important role of Shiga toxin in this malady. There has also been a report indicating that tumor necrosisfactor-alpha acts synergistically with Shiga toxin to induce HUS.

To cause intestinal disease, shigellae must invade the epithelial cells lining of the intestine. After escaping from the phagocytic vacuole, they multiply within the epithelial cells in a manner similar to that described for EIEC strains. Thus, Shigella virulence requires that the organisms invade epithelial cells, multiply intracellularly, and spread from cell to cell by way of finger-like projections to expand the focus of infection, leading to ulceration and destruction of the epithelial layer of the colon. Interestingly, for Shigella to be fully invasive, both plasmid and chromosomally encoded products seem to be required. The invasion plasmids is identical for the Shigella and the EIEC and contains at least four genes, IpaA, IpaB, IpaC, and IpaD that encode for a series of proteins termed invasion-plasmid antigens, which arc involved in the virulence of these organisms. Interestingly, IpaB acts both as an invasin that triggers phagocytosis of the bacterium and as a cytolysin that allows escape from the phagocytic vacuole. The elaboration of toxic products causes a severe local inflammatory response involving both polymorphonuclear leukocytes and macrophages, resulting in a bloody, mucopurulent diarrhea.

During 1990, over 27,000 cases of shigellosis were reported to the Centres for Disease Control (CDC) and, of these, the most prevalent species in the United States was S sonnei. The disease produced by this species is transmitted by a fecal-oral route, and most of patients are preschool-age children, particularly those in day-care centres.

Immunity. Immunity acquired after dysentery is specific and type-specific but relatively weak and of a short duration. For this reason the disease may recur many times and, in some cases, may become chronic. This is probably explained by the fact that Shigella organisms share an antigen with human tissues.

Laboratory diagnosis. Reliable results of laboratory examination depend, to a large extent, on correct sampling of stool specimens and its immediate inoculation onto a selective differential medium. The procedure should be carried out at the patient’s bedside, and the plate sent to the laboratory.

In hospital conditions the stool is collected on a paper plate or napkin, placed into a bedpan. The latter should be washed previously with running water or, better still, with boiling water, be dry, and should contaio disinfectants. It is best to collect the faeces directly from the rectum by means of a rectal tube or rectal swab. The specimen should be sown in the isolation department immediately after collection. Portions of the stool, containing pus and mucus, are picked out with a swab and plated on Ploskirev’s medium. The plates are incubated at 37°C for 24 hours The isolated pure culture is identified by its biochemical and serological properties.

An accelerated method of dysentery diagnosis is employed to shorten the examination period. In some cases an agglutination reaction, similar to the Widal reaction, is used. This test is relevant to retrospective diagnosis.

The nature of the isolated culture may be determined m some cases by its lysis by a polyvalent dysentery phage and by the reaction of passive haemagglutmation as well as by the method of immunofluorescence. This method is used for demonstrating antigens of Shigella organisms in smears from faeces or in colonies by means of specific sera treated with fluorochromes.

An allergic test consisting in intracutaneous injection of 0.1 ml of dysenterin is applied in the diagnosis of dysentery in adults and children. Hyperaemia and a papule 2 to 3.5 cm in diameter develop at the site of the injection in 24 hours in a person who has dysentery. The test is strictly specific.

Treatment and Control of Shigellosis. Intravenous replacement of fluids and electrolytes plus antibiotic therapy are used for severe cases of shigellosis. Ampicillin frequently is not effective, and alternative therapies include sulfamethoxazole/trimethoprim and, with increasing sulfamethoxazole/trimethoprim resistance, the quinolone antibiotics such as nalidixic acid and ciprofloxacin. In the Far East, India, and Brazil where shigellosis is more common than in the United States, multiple antibiotic resistance because of the acquisition of plasmids has become common. Shigellosis also is common in Latin America.

Efforts to control the disease usually are directed toward sanitary measures designed to prevent the spread of organisms. This is particularly difficult in view of the fact that many persons remain asymptomatic carriers after recovery from an overt infection. Such individuals provide a major reservoir for the spread of the shigellae.

The injection of killed vaccines is worthless, because humoral IgG does not seem to be involved in immunity to the localized intestinal infection. Live vaccines that cannot grow in the absence of streptomycin (ie, streptomycin-dependent vaccines) have been developed and used in clinical trials, but success has been equivocal. It seems that the organisms must invade and colonize the intestine to induce a local immunity. An engineered vaccine designed to induce this type of immunity used an avirulent E coli K12 into which was transferred a 140-megadalton plasmid obtained from a virulent strain of Shigella flexneri. The transfected plasmid endowed the E. coli K12 strain with the ability to invade intestinal epithelial cells, and its use as an oral vaccine in monkeys conferred significant protection against oral challenge with virulent S flexneri. Acquired immunity seems to result from both a cell-mediated immune response and an IgA antibody production.

Interestingly, human milk contains a globotriaosylceramide that binds to Shiga and Shiga-like toxins. This suggests that human milk could contribute to a protective effect by preventing these toxins from binding to their intestinal target receptors.

Thus, dysentery control is ensured by a complex of general and specific measures; (1) early and a completely effective clinical, epidemiological, and laboratory diagnosis; (2) hospitalization of patients or their isolation at home with observance of the required regimen; (3) thorough disinfection of sources of the disease; (4) adequate treatment of patients with highly effective antibiotics and use of chemotherapy and immunotherapy; (5) control of disease centres with employment of prophylaxis measures; (6) surveillance over foci and the application of prophylactic measures there; (7) treatment with a phage of all persons who were in contact with the sick individuals; (8) observance of sanitary and hygienic regimens in children’s institutions, at home and at places of work, in food industry establishments, at catering establishments, in food stores.

 

Additional materials for diagnosis

Dysentery is an infectious disease with the predominant involve­ment of the large intestine and general intoxication caused by bacteria of the genus Shigella: S. dysenteriae, S. sonnei, S. flexneri, S. boydii.

Material used for isolating the causal organism of dysentery in­cludes faeces of patients, convalescents, and carriers, less frequently, vomited matter and waters from stomach and intestine lavage. Shigellae may be recovered in washings off hands, cutlery and crockery, and various other objects (toys, door handles, etc.) as well as in milk and other foodstuffs. The  results of laboratory examina­tion depend to a large degree on the correct procedure of material collection. The following rules should be strictly adhered to: (1) carry out bacteriological examination of faeces before aetiotropic therapy has been initiated; (2) collect faecal samples (mucus, mucosal admixtures) from the bedpan and with swabs (loops) directly from the rectum (the presence in the bedpan of even the traces of disin­fectants affects the results of examination); (3) inoculate without delay the collected material onto enrichment media, place them into an incubator or store them in preserving medium in the cold; (4) send the material to the laboratory as soon as possible.

Bacteriological examination. Faecal samples are streaked onto plates with Ploskirev’s medium and onto a selenite medium con­taining phenol derivatives, beta-galactosides, which retard the growth of the attendant flora, in particular E. coli. The inoculated cultures are placed into a 37 °C incubator for 1S-24 hrs. The nature of tile colonies is examined on the second day.

Colourless lactose-negative colonies are subcultured to Olkenitsky’s medium or to an agar slant to enrich for pure cultures. On the third day, examine the nature of the growth on Olkenitsky’s medium for changes in the colour of the medium column without gas formation. Subculture the material to Hiss’ media with malonate, arabinose, rhamnose, xylose, dulcite, salicine, and phenylalanine. Read the re­sults indicative of biochemical activity on the following day. Shigel­lae ferment carbohydrates with the formation of acid (Table 2).

For serological identification the agglutination test is performed first with a mixture of sera containing those species, and variants of Shigellae that are prevalent in a given area, and then the slide ag­glutination test with monoreceptor species sera.

To determine the species of Shigellae, one can employ the following tests:

1. Direct and indirect immunofluorescence test.

2. The coagglutination test which allows to determine the specificity of the causative agent by a positive reaction with protein A of staphylococci coated with specific antibodies. On a suspected colony put a drop of specific sensitized protein A of Staphylococcus aureus, then rock the dish and 15 min later examine it microscopically for the appearance of the agglutinate (these tests may also be carried out on the second day of the investigation with the material from lactose-negative colonies).

3. Another test, which is highly specific for dysentery, is ELISA. For the epidemiological purpose the phagovar and colicinovar of Shigellae are also identified.

To determine whether the isolated cultures belong to the genus Shigella, perform the keratoconjunctival test on guinea pigs. In contrast to causal organisms of other intestinal infections, the dy­sentery Shigellae cause marked keratitis.

Depending on the findings obtained, the presence of Shigella bac­teria in the test material is either confirmed or ruled out.

For the serological diagnosis of dysentery the indirect haemagglutination (IHA) test with erythrocyte diagnosticums with the titre of 1:160 and higher is performed. The test. is repeated after at least seven days. Diagnostically important is a four-fold rise in the anti­body litre, which can be elicited from the 10th-12th day of the disease. To distinguish between patients with subclinical forms of the disease and Shigella carriers, identify immunoglobulins of the G class.

An allergy intracutaneous test with Tsuverkalov’s dysenterine is of supplementary significance. It becomes positive in dysentery patients beginning with the fourth day of the disease. The result is read in 24 hrs by the size of the formed papula. The test is consid­ered markedly positive in the presence of oedema and skin hyperaemia 35 mm or more in diameter, moderately positive if this diameter is 20-34 mm, doubtful if there is no papula and the diameter of skin hyperaemia measures 10-15 mm, and negative if the hyperaemic area is less than 10 mm.

Another technique that can be employed is determination of the indicator of neutrophil damage in the presence of dysenterine.

Examination of water, milk, and washings off various objects for Shigellae is conducted utilizing the above mentioned techniques. Of especial importance for examination of these objects is the test aimed at determining the increase in the phage litre, which is also employed for demonstration of Shigella bacteria in the patient’s faeces.

To carry out this test, the indicator phages and reference strains of Flexner’s and Sonne’s Shigella bacteria are used. A rise in the phage titre by 3-5 orders (4-) is considered as weak positive reaction, by 5-7 orders (++) and 7-10 orders (+++), positive, and by over 10 orders (++++), markedly positive.

The immunofluorescence test for Shigella recovery is employed in examining objects containing minor amounts of the causative agents and for rapid laboratory diagnosis of dysentery.

 

VIBRIO. LABORATORY DIAGNOSIS of CHOLERA. CAmpYlobacter, helicobacter and aeromonas infections

 

The Cholera Vibrio.  The causative agents of cholera are the classical Vibrio cholera biovars discovered by R. Koch in 1883 and the El Tor vibrio biovar isolated from the cadaver of a pilgrim on the Sinai peninsula by Gotschlich in 1906, RO- and O139 strains.

Vibrio cholerae biovar Proteus (N. Gamaleya, 1888) and Vibrio cholerae biovar albensis were discovered” later. V. cholerae was described by F. Pacini in 1854.

Morphology. Cholera vibrios are shaped like a comma or a curved rod measuring 1-5 mcm in length and 0.3 mcm in breadth (Fig.).

 

 

Figure. Vibrio cholerae:

 

They are very actively motile, monotrichous, nonsporeforming, noncapsulated, and Gram-negative.

 

 

The cholera vibrio is subject to individual variation when it is exposed to physical and chemical factors. On artificial media and in old cultures it occurs in the form of grains, globes, rods, threads, clubs or spirals. When it is re-inoculated into fresh media, the organism assumes its initial form.

Electron microscopy reveals flagella 25 nm thick and three-layer cell wall and cytoplasmatic membrane. Small vacuoles are seen  between the wall and the cytoplasmatic membrane. The formation of the vacuoles is assumed to be linked with exotoxin synthesis. The nucleoid is in the centre of the cell. No essential difference has beeoted between the structure of the classical cholera vibrion and that of the El  Tor vibrio. The G-C content in DNA ranges between 45 and 49 per cent.                            

 

Cultivation. Cholera vibrios are facultative (anaerobes). The optimum growth temperature is 37° C, and growth is arrested below 14 °C and above 42° C. The organisms grow readily on alkaline media at pH 6.0-9.0, and on solid media the colonies are transparent with a light-blue hue, forming domes with smooth edges. On gelatin the organisms produce transparent granular colonies which, when examined under a microscope, resemble broken glass. In 48 hours the medium surrounding the colonies becomes liquefied and the colonies sink into this area. Six-hour-old cultures on alkaline meat broth and peptone water produce a pellicle, which consists of cholera vibrios.

 

 

 

The organism is also subjected to cultural changes. It dissociates from the S-form to the R-form, this process being accompanied by profound changes in antigenic structure.

Fermentative properties. The cholera vibrio liquefies coagulated serum and gelatin; it forms indole and ammonia, reduces nitrates to nitrites, breaks down urea, ferments glucose, levulose, galactose, maltose, saccharose, mannose, mannite, starch, and glycerine (slowly) with acid formation but does not ferment lactose in the first 48 hours, and always coagulates milk. The cholera vibrio possesses lysin and ornithine decarboxylases and oxidase activity. B. Heiberg differentiated vibrios into biochemical types according to their property of fermenting mannose, arabinose, and saccharose. Eight groups of vibrios are known to date; the cholera vibrios of the cholerae and El Tor biovar belong to biochemical variant 1.

The haemolytic activity and haemagglutinating properties of the cholera vibrios in relation to different erythrocytes (sheep, goat, chick, and others) as well as the ability for forming acetylmethylcarbinol are not stable characteristics and are taken into account as less important data in differentiating microbes of the genus Vibrio.

Toxin production. The cholera vibrio produces an exotoxin (cholerogen) which is marked by an enterotoxic effect and plays an important role in the pathogenesis of cholera; the endotoxin also exerts a powerful toxic effect. The cholera vibrios produce fibrinolysin, hyaluronidase, collagenase, mucinase, lecithinase, neuraminidase, and proteinases.

V cholerae produces diarrhea as a result of the secretion of an enterotoxin, choleratoxin, which acts identically to E coli LT to stimulate the activity of the enzyme adenylcyclase. This, in turn, converts ATP to cAMP, which stimulates the secretion of Cl^– and inhibits the absorption of NaCl. The copious fluid that is lost also contains large amounts of bicarbonate and K⁺. Thus, the patient has both a severe fluid loss and an electrolyte imbalance.

The enterotoxin has been shown to bind specifically to a membrane ganglioside designated GM₁. Interestingly, V cholerae produces a neuraminidase that is unable to remove the y-acetylneuraminic acid from GM₁, but it is able to convert other gangliosides to GM₁, thus synthesizing even more receptor sites to which its enterotoxin can bind. Like the LT of E coli, choleragen is composed of five B subunits that react with the cell receptor, an Ai-active subunit that enters the cell and, together with a cellular ADP-ribosylating factor, carries out the ADP-ribosylation of the GTP-binding protein, and a small A; subunit that seems to link the Ai subunit to the B subunit. Interestingly, unlike LT, the DNA en-coding choleragen is not plasmid mediated but is on the chromosome of V cholerae.

CT (as well as the LT produced by E coli) can be quantitated by a number of in vivo, cell culture, or immunologic assay units. In one method, a segment of rabbit small intestine is tied to form a loop. Enterotoxin is serially diluted, and an aliquot of each dilution is injected into a loop. The highest dilution that stimulates fluid accumulation in the loop is recorded as the titer of the enterotoxin. A second method takes advantage of the fact that cAMP causes a morphologic response in cultured Chinese hamster ovary cells, and that enterotoxin will induce such cells to produce cAMP. To quantitate enterotoxin using this assay, a standard, curve is established (with purified enterotoxin) that can be used subsequently to assay an unknown enterotoxin from E coli or V. cholerae

FIGURE. A standard curve to equate Escherichia coli enterotoxin with purified cholera toxin. The percentage of Chinese hamster ovary cells that have elongated after growing 24 hours in the presence of cholera toxin in 1% fetal calf serum is plotted against the concentration of cholera toxin present in the culture. As shown, heated toxin or toxin preincubated with antitoxin (anti-CT) have no effect on the morphologic features of the cells.

 

As is true with essentially all diarrhea-producing bacteria, V cholerae must specifically colonize the intestinal epithelial cells to produce disease. In this case, however, the pili binding the bacteria to the host cells seem to be under the same regulator as choleragen production and, as a result, are termed toxin-coregulated pili. Mutants unable to bind to intestinal cells are avirulent in spite of their ability to produce choleragen. Moreover, antibody directed to toxin-coregulated pili are protective.

Remember that non-01 and non-0139 strains of V.cholerae also cause a wide spectrum of infections, ranging from mild diarrhea to one indistinguishable from classic cholera. Some of these serotypes are known to produce a choleratoxin that is identical to that of the classic biotypes, whereas other products a heat-stable enterotoxin analogous to the ST of E. coli.

Antigenic structure. The cholera vibrios have thermostable O-antigens (somatic) and thermolabile H-antigens (flagellar). The O-antigen possesses species and type specificity, the H-antigen is common for the genus Vibrio. According to the O-antigen content, the vibrios are separated into subgroups of which there are more than 140. The cholerae vibrios, El Tor biovars and biovars cholera belong to the O-1 subgroup. In the 0-1 subgroup there are three O-antigens (A, B, and C) according to the combination of which three serological variants, Ogawa (AB), Inaba (AC) and an intermediate variant Hikojima (ABC), are distinguished.

Classification. Vibrio cholerae belongs to family Vibrionaceae, genus Vibrio consisting of 5 species. The species Vibrio cholerae is subdivided into four biological variants: biovar cholerae, biovar El Tor, biovar Proteus, and biovar albensis.

Biovar cholerae and biovar El Tor of Vibrio cholerae are the causative agents of human cholera. Biovar Proteus of Vibrio cholerae causes diarrhoea in birds and gastroenteritis in humans; biovar albensis of Vibrio cholerae was revealed in fresh water and in human faeces and bile.

Resistance. The cholera vibrio survives for a long time at low temperatures. It lives in faeces for up to a month, in oysters, crabs, on the surface offish and in their intestines from 1 to 40 days, in water for several days, on foodstuffs from 1 to 10 days, and in the intestines of flies from 4 to 5 days. .

The El Tor vibrio is marked by high resistance. It lives more than four weeks in sea and river water, 1-10 days on foodstuff’s, and 4-5 days in the guts of flies. It is possible that under favourable conditions El Tor vibrio may reproduce in various water reservoirs.

The organism shows a low resistance to sunlight, X-rays, desiccation, and high temperatures. It is destroyed instantly at 100°C, and in 5 minutes at 80° C. Cholera vibrios are highly sensitive to disinfectants, particularly to acids (e. g. a 1 :10000 solution of hydrochloric acid kills them within one minute). The organism is also very sensitive to the action of gastric juice.

Pathogenicity for animals. Iature animals are not attacked by cholera, but an intraperitoneal injection of the culture into rabbits and guinea pigs gives rise to general toxicosis and peritonitis which is followed by death.

In his experiments with rabbit-sucklings E. Metchnikoff produced a disease similar to human cholera by oral infection. R. Koch reproduced the disease in guinea pigs previously alkalizing the gastric juice and introducing opium. An intravenous vibrio injection into rabbits and dogs gives rise to lethal toxaemia.

Pathogenesis and diseases in man.  Cholera is undoubtedly the most dramatic of the water-borne diseases. As far as is known, cholera was confined to India for the almost 2000 years between its first description by Hindu physicians in 400 b c and its spread to Arabia, Persia, Turkey, and Southern Russia in the early1800s. There were six major pandemics of cholera during the 1800s covering the entire world, killing millions wherever it struck. During one such outbreak in London during 1849, the famous physician, John Snow, traced the spread of the disease to a Broad Street pump from which area residents obtained their water. The spread of cholera in this area was stopped when Snow recommended that the handle of the pump be removed. This is particularly remarkable when one remembers that the germ-theory of disease had not yet been formulated.

The cholera vibrios are transmitted from sick persons and carriers by food, water, flies, and contaminated hands. Via the mouth the organisms gain entrance into the small intestine, where the alkaline medium and an abundance of products of protein catabolism furnish favourable conditions for their multiplication. When the cholera vibrios perish, a large amount of toxin is released. This toxin invades the patient’s blood owing to necrosis of the intestinal    epithelium and the resulting condition promotes disturbance of the intestinal vegetative nerve fibres, dehydration of the body, and a development of intoxication.

Cholera is characterized by a short incubation period of several hours to up to 6 days (in a disease caused by the El Tor vibrio it lasts three to five days), and the disease symptoms include  general weakness, vomiting, and a frequent loose stool. The stools resemble rice-water and contain enormous numbers of torn-off intestinal epithelial cells and cholera vibrios. The major symptom of cholera is a severe diarrhea in which a patient may lose as much as 10 to 20 L or more of liquid per day. Death, which may occur in as many as 60% of untreated patients, results from severe dehydration and loss of electrolytes.

Three phases can be distinguished in the development of the disease.  1. Cholera enteritis (choleric diarrhoea) which lasts 1 or 2 days. In some cases the infectious process terminates in this period and the patient recovers. 2. Cholera gastroenteritis is the second phase of the disease. Profuse diarrhoea and continuous vomiting lead to dehydration of the patient’s body and this results in lowering of body temperature, decrease in the amount of urine excreted, drastic decrease in the number of mineral and protein substance, and the appearance of convulsions. The presence of cholera vibrios is revealed guite frequently in the vomit and particularly in the stools which have the appearance of rice water. 3.  Cholera algid which is characterized by severe symptoms. The skin becomes wrinkled due to the loss of water, cyanosis appears, and the voice becomes husky and is sometimes lost completely. The body temperature falls to 35.5-34° C. As a result of blood concentration cardiac activity is drastically weakened and urination is suppressed.

In severe cases the algid period is followed by the asphyctic phase characterized by cyanosis, dyspnoea, uraemia, azotaemia, and unconsciousness (cholera coma), which lead to prostration and death. Effective treatment and proper nursing care may induce a change of the algid period to the reactive phase during which urination becomes normal, intoxication decreases, and the patient recovers. Fulminate forms of cholera (dry cholera or cholera sicca) may occur in a number of cases.These forms are characterized by the absence of diarrhoea and vomiting and result in death due to severe intoxication. Atypical and latent forms of cholera are exhibited quite frequently, particularly in children, resembling mild cases of gastroenteritis.

Non-specific complications in cholera include pneumonia, erysipelas, phlegmons, abscesses, occasionally sepsis, etc. Among the specific complications cholera typhoid is the most menacing. It is accompanied by a rise in body temperature to 38-39° C, eruptions on the skin, vomiting and fetid loose stools. This condition causes a mortality rate of 80-90 per cent.

Erased and mild forms are observed in 80 to 90 per cent of cases caused by El Tor vibrio. Severe forms with a fatal outcome are encountered in individuals whose condition is aggravated by various somatic diseases which reduce the general body resistance, in those with hypoacidic gastric function, and in elderly persons.

Post-mortem examination of cholera cases reveals distinct hyperaemia of the peritoneum and serosa of the small intestine, which are covered with a sticky exudate. The mucous membrane of the small intestine is congested, peach-coloured, the intestinal epithelium is frequently desquamated, and there are haemorrhages in the submucosa. The vibrios are present in great abundance in the intestinal wall, particularly in Lieberkuhn’s glands, and, not infrequently, in the gall-bladder.

Cholera mortality was quite high in the past (50 to 60 per cent), but has markedly decreased with the application of aetiotropic and pathogenetic therapy. According to WHO, in 1969-1971 it was 17.7 per cent.

 

 

Immunity acquired after cholera is high-grade but of short duration and is of an anti-infectious (antibacterial and antitoxic) character. It is associated mainly with the presence of antibodies (lysins, agglutinins, and opsonins). The cholera vibrios rapidly undergo lysis under the influence of immune sera which contain bacteriolysins.

E. Metchnikoff attributed definite significance to phagocytosis following immunity. The normal activity of the stomach, whose contents are bactericidal to the cholera vibrio, plays an essential role in the natural defence mechanism.

Laboratory diagnosis. A strict regimen is established in the laboratory. Examinations are carried out in accordance with the general rules observed for particularly hazardous diseases.

 

Test specimens are collected from stools, vomit, organs obtained at autopsy, water, objects contaminated by patient’s stools, and, in some cases, from foodstuffs. Certain rules are observed when the material is collected and transported to the laboratory, and examination is made in the following stages.

1. Stool smears stained by a water solution of fuchsin are examined microscopically. In the smears, the cholera vibrios occur in groups similar to shoals of fish.

2. A stool sample is inoculated into 1 per cent peptone water and alkaline agar. After 6 hours incubation at 37°C the cholera vibrios form a thin pellicle in the peptone water, which adheres to the glass. The pellicle smears are Gram stained, and the culture is examined for motility. A slide agglutination reaction is performed with specific agglutinating 0-serum diluted in a ratio of 1 in  100.

The organisms are then transferred from the peptone water onto alkaline agar for isolation of the pure culture. If the first generation of the vibrios in peptone water is not visible, a drop taken from the surface layer is re-inoculated into another tube of peptone water. In some cases with such re-inoculations, an increase in the number of vibrios is achieved.

The vibrio culture grown on solid media is examined for motility and agglutinable properties. Then it is subcultured on an agar slant to obtain the pure culture.

3. The organism is identified finally by its agglutination reaction with specific 0-serum, determination of its fermentative properties (fermentation of mannose, saccharose, and arabinose), and its susceptibility to phagolysis (Table ).

Table

Differentiation of Biovars of Cholera vibrio

 

Note   “A” – carbohydrate fermentation with acid production; “+””– positive result; “–” – negative result; “+” – negative or positive result is not always observed.

 

The following procedures are undertaken for rapid diagnosis: (1) dark field microscopy of the stool; (2) stool culture by the method of  tampons incubated for 16-18 hours in an enrichment medium with repeated dark field microscopy; (3) agglutination reaction by the method of fluorescent antibodies;

 

 (4) bacterial diagnosis by isolation of cholera vibrios (the faecal mass is seeded as a thin layer into a dish containing non-inhibiting nutrient agar and grown for 4-5 hours, the vibrio colonies are detected with a stereoscopic microscope, and the culture is tested by the agglutination reaction with O-serum on glass; (5) since neuraminidase is discharged by the cholera vibrios and enters the intestine, a test for this enzyme is considered expedient as a means of early diagnosis (it is demonstrated in 66-76 per cent of patients, in 50-68 per cent of vibrio carriers, and occasionally in healthy individuals).

Treatment. The mortality rate of cholera can be reduced to less than1% by the adequate replacement of fluids and electrolytes. Antibiotics of the tetracycline group (tetracycline, sigmamycin), amphenicol, and streptomycin are prescribed at first intravenously and then by mouth.

Pathogenetic therapy is very important: control of dehydration, hypoproteinaemia, metabolic disorders, and the consequences of toxicosis, acidosis in particular, by infusion of saline (sodium and potassium) solutions, infusion of plasma or dry serum, glucose, the use of warm bath, administration of drugs which improve the tone of the heart and vessels.

Prophylaxis. Cholera patients and vibrio carriers are the source of the disease. Individuals remain carriers of the El Tor vibrio for a lengthy period of time, for several years. Vibrios of this biotype are widely distributed in countries with a low sanitary level. They survive in water reservoirs for a long time and have been found in the bodies of frogs and oysters. Infection may occur from bathing in contaminated water and fishing for and eating shrimps, oysters, and fish infected with El Tor vibrio.The following measures are applied in a cholera focus:

 

(1) detection of the first cases with cholera, careful registration of all sick individuals, immediate information of health protection organs;

(2) isolation and hospitalization, according to special rules, of all sick individuals and carriers, observation and laboratory testing of all contacts;

(3) concurrent and final disinfection in departments for cholera patients and in the focus;

(4) protection of sources of water supply, stricter sanitary control over catering establishments, control of flies; in view of the possibility of El Tor vibrio reproducing in water reservoirs under favourable conditions (temperature, the presence of nutrient substrates) systematic bacteriological control over water reservoirs has become necessary, especially in places of mass rest and recreation of the population in the summer;

(5) strict observance of individual hygiene; boiling or proper chlorination of water, decontamination of dishes, hand washing;

(6) specific prophylaxis: immunization with the cholera monovaccine containing 8 thousand million microbial bodies per 1 ml or with the cholera anatoxin. Chemoprophylaxis with oral tetracycline is conducted for persons who were in contact with the sick individual or for patients with suspected cholera.

 

Immunization with heat-killed cholera organisms seems to give some protection, and recovery from the disease imparts immunity of an unknown degree or duration. Killed whole cells of V cholerae given orally along with purified B subunits of the toxin induced immunity in about 85% of persons who received it. Another experimental engineered oral vaccine consists of a live attenuated V. cholerae El Tor Ogawa strain. This mutant no longer expresses the A subunit of the toxin but does produce B subunits. It seemed to provide good immunity in volunteers but it has not been used in large-scale field trials.

A experimental vaccine that induces toxin-neutralizing antibodies in mice uses. an ingenious technique in which a 45-base-pair oligonucleotide encoding an epitope of the B subunit of CT is inserted into the flagellin gene of an avirulent Salmonella. This 15-amino acid insert was expressed at the flagellar surface without abolishing flagellar function. The concept of placing an immunogen in a prominently displayed position on the bacterial surface could be used as a cholera vaccine as well as for inserting a number of other epitopes from both bacteria and viruses.

Remember, however, that none of these vaccines offer any protection against the newly described 0139 strain of V. cholera, and it is necessary to develop new vaccines for these organisms.

General epidemic measures play the principal role in cholera prophylaxis, whereas immunization is regarded as an auxiliary measure.

Cholera dates back to the most ancient times. Its endemic focus is India (Lower Bengal, and the deltas of the Ganges and Brahmaputra rivers).

There were six cholera pandemics between 1817 and 1926: in 1817-1823, 1826-1837, 1846-1862, 1864-1875, 1883-1896, and in 1900-1926. In 1961-1963 the seventh outbreak of cholera pandemic occurred, it was caused by the El Tor vibrio.

According to WHO, 668650 cholera cases were recorded between 1953 and 1961 in the countries of Asia and Africa and 348752 cases between 1961 and 1966.

Beginning with 1966, over 50 per cent of cholera cases in the countries of Asia were caused by the El Tor vibrio. According to WHO, however, the incidence of cholera induced by the classical Vibrio cholerae has doubled. There were cases with cholera in the southern regions of the Soviet Union (Astrakhan, Odessa, Kerch) in 1970. More than 464307 people sick with cholera were recorded in all countries in the period between 1970 and 1976. Cholera epidemic with high morbidity and
mortality rates occurred in the second half of 1977 in the Near East (Syria, Saudi Arabia, and other countries).

 

Additional materials for diagnosis

CHOLERA

Cholera is a particularly dangerous infectious disease, caused by Vibrio cholerae and Vibrio El Tor biovars, which runs as gastroenteritis associated with dehydration.

The main method in the laboratory diagnosis of cholera is bacterio­logical examination. Some 10-20 ml of faeces and vomited matter from patients with suspected cholera are collected with a sterile metallic or wooden spoon, transferred into a sterile wide-mouthed vessel, and tightly stoppered with a-glass or cork plug.

The second portion of faeces and vomit (1-2 ml) is inoculated into 1 per cent peptone water (50 ml) at the patient’s bedside. Both vessels are sealed and immediately sent to the laboratory.

If the patient has no bowel movements at the moment of material collection, cut off soiled samples of the bed linen or underclothes and collect the contents of the rectum with a sterile wire loop inserted 5-8 cm deep. Following removal, put the loop with a faecal sample into a flask with a nutrient medium. At autopsy the material to be tested is obtained in the following manners mark off three sites in the area of the upper, middle and lower portions of the small in­testine and a site of the rectum some 10-15 cm long; then from each end of the marked section express the contents of the intestine side-wise, apply two ligatures, and cut an intestine between them. The gallbladder is removed with a part of the liver. The water (1 L) and foodstuffs (no less than 200 g) should also be examined.

In examining convalescents, individuals who have contacted with patients or carriers, it is recommended that a purgative or a cholagogue (25-30 g of magnesium sulphate, etc.) be preliminary given to them to obtain liquid faeces from the upper part of the intestines and the contents of the gallbladder.

The material to be studied is collected, packed, and sent to the laboratory with special measures of precaution. The glassware should not contain any traces of disinfectants, particularly of acids; it is sterilized or boiled for 15 min.

Jars and test tubes should be closed with glass or rubber stoppers. When cork plugs are used, cellulosic film is placed under them. After the  material has been collected, the plugs are sealed with paraffin and wrapped with double cel­lulosic film.

On each vessel stick on a slip of paper with the name and age of the patient, his or her home and office address, diagnosis, the dates of the onset of the disease and hospitalization, as well as the date and exact time of material collection, and also the name of the person who has sent in the analysis.

The material should be brought to the laboratory no later than six hours after its collection. If the delivery within this period is impossible, the samples are inoculated into 1 per cent peptone water with potassium tellurite and onto plates with alkaline agar. If the laboratory is a long way off, jars and test tubes with the specimens to be tested are put, packing them with saw dust, into a metallic container which, in turn, is packed into a wooden box. The latter is wrapped, sealed, signed “Top, fragile”, and is sent with a cou­rier.

The material should be exam­ined in a special laboratory. Yet, if no such laboratory is available, the samples are sent to any bacte­riological laboratory which may provide an isolated room with a separate entrance and exit. No other analyses are taken in this case and stricter measures of precau­tion are introduced. Personnel with special training only is allowed to do this kind of investigation. No operators on a fasting stomach should be allowed in the laboratory. The examination is carried out around the clock since the results should be available no later than 30-36 hrs later.

Bacteriological examination. Stage I. Using the material col­lected, prepare smears, dry them in the air, fix with alcohol or Nikiforov’s mixture, stain by the Gram technique, and examine under the microscope. Later on, if laboratory findings confirm the diagnosis of cholera in at least one case, stain the smears with Pfeiffer’s fuchsine only. Cholera vibrios appear as thin curved Gram-negative rods (Fig. 15). Because of great polymorphism the smear may, along with typical cells, contain coccal, rod-shaped, and spiral forms, which diminishes the value of this method.

The first preliminary answer is given after the microscopic exam­ination of the smear. It refers to the presence of vibrios and the nature of their Gram-staining.

At this stage of bacteriological examination, one can also perform the immunoftuorescence test, using specific labelled 0-cholera sera. Moreover, the cholera vibrio may be recovered by the immune Indian ink method. In the latter case smears fixed on a glass slide are treated for 2 min in a humid chamber with Indian ink mixed with immune serum, then washed with water and examined with a microscope. The vibrio is stained black by Indian ink: the walls of the cell are black-brown, the centre is slightly greyish. If the bacteria are few, they are preliminarily cultivated for 3-5 hrs in peptone water.

Simultaneously with bacterioscopy, the material tested is inoculat­ed onto liquid and solid nutrient media. Enrichment liquid media that are usually recommended for use include alkaline 1 per cent peptone water, 1 per cent peptone water with potassium tellurite in a ratio of 1 to 100 000, and alkaline taurocholate-tellurite-peptone medium (Monsur’s liquid medium), etc.

Solid nutrient media usually employed are alkaline meat-peptone agar and one of the selective nutrient media: Aronson’s medium, Monsur’s  alkaline  taurocholate-lellurite-gelatine-agar medium, TCBS, etc.

To isolate the vibrio from carriers or patients with subclinical forms of cholera, use media which improve the growth of vibrios and suppress the attendant flora (predominantly E. coli). All inoculat­ed cultures are placed in an incubator at  37 °C.

Aronson’s medium consists of 2-3 per cent of meat-peptone agar to which sucrose and destained fuchsine are added.

Monsur’s alkaline taurocholate-tellurite-gelatine-agar medium contains 10 g of trypticase, l0 g of sodium chloride, 50 g of sodium taurocholate, 30g of sod­ium carbonate, 1 g of gelatin, 15 g of agar-agar, and 1 L of distilled water.

TCBS (thiosulphate-citrate-bromthymol sucrose) is manufactured in the form ready for use; 69 g of the dry medium is taken per 1 L of distilled water.

Stage II. Some 5-6 hours after inoculation examine the film on the peptone water. To do it, tilt the test tube or the vial so that a delicate bluish film is attached to the wall. Prepare smears from the  film or the surface of the medium, stain them by the Gram method, evaluate motility, and conduct presumptive slide agglutination test with 0-cholera (0-1) serum diluted 1:100 or the reaction of cholera vibrio immobilization with 0-cholera serum. The results of the latter are estimated by phase-contrast microscopy. Inhibition of vibrios motility and the formation of agglutinate occur within 1-2 min.

On the basis of the results obtained give a second preliminary result referring to the motility of the vibrio and its relation to the agglutinating serum.

Subculture the material from the film onto plates with alkaline agar or selective medium and simultaneously onto the second peptone water and look for changes in 5-6 hrs.

Stage III. Some 10-16 hrs after inoculation, examine the growth in the second enrichment medium (peptone water) and on the plates with the culture of the native material. The film formed on the pep­tone water is examined as described above.

On an alkaline agar the cholera vibrio grows with the formation of round, smooth, flat, bluish, homogeneous colonies which are 1–2 mm in diameter, transparent in the transmitted light and have smooth edges. They are oily in consistence, are readily removed and emulsified. Examination of the material from convalescents, bac­teria carriers, and individuals treated with antibiotics may reveal atypical colonies.

On Aronson’s medium colonies of cholera vibrios are scarlet in the centre and pale-pink or colourless at the periphery. On Monsur’s medium colonies are transparent or semitransparent, or they may be of a greyish-black colour with turbid edges. On the TCBS medium they appear as flat and yellow against a bluish-grey background.

The selected colonies are introduced into test tubes with Oikenitsky’s medium or onto an agar slant for enrichment of pure culture and placed in an incubator.

Preliminary identification of cholera vibrios grown on plates with solid media is based on the study of cultural and morphological characteristics and on a presumptive slide agglutination test with 0-cholera serum diluted 1:100 and with Ogawa’s and Inaba’s sera in a 1 to 60 dilution, which is carried out to determine the serovar.

If the examination demonstrates signs typical of the cholera vib­rio, a third preliminary answer about the positive result of the investigation is issued. Some material from the typical colonies may be transferred to a broth; then, using a 3-4-hour old culture, perform a standard agglutination test, check fermentation of carbo­hydrates, and determine whether the isolated culture belongs to Group I according to Heiberg and whether it is liable to phagolysis by cholera phages C and El Tor 2. If the results are positive, an answer concerning the isolation of the causative agent is given within 18-24 hrs from the beginning of the study.

Stage IV. After the results of the standard agglutination test and the reaction of phagolysis and fermentation of carbohydrates by 3-4-hour broth culture have been analysed, a preliminary conclusion about the isolation of the cholera vibrio is made. Plates with the inoculated culture on the second peptone water are examined, using the scheme which is employed in examining the plates with the culture of the native material. On Oikenitsky’s medium the vibrio breaks down sucrose without gas formation and does not ferment lactose (reddening of the medium in the column without gas for­mation).

To distinguish vibrios from homogeneous species of microorganisms (Aeromonas, Pseudomonas, Plesiomonas), a number of tests may be employed: the oxidase test, glucose oxidation-fermentation reac­tion, the “strand” test (Table ).

Table

Differential-Diagnostic Signs of Vibrios and Related Types of Bacteria

 

The oxidase test consists of placing a solution of paraaminodimethyl-aniline and alpha-naphthol onto the culture in a Petri dish or onto a meat-peptone agar slant.

To carry out the oxidation-fermentation test, medium with the following composition (per 100 ml) is prepared: 2.0 g of peptone; 5.0 g of sodium chloride;   0.3 g of potassium hydrophosphate; 3.0 g of agar-agar; bromthymol blue (1 per- cent aqueous solution).

Dispense the medium in 3-4-ml portions into 13 X 100 mm test tubes and sterilize for 15 min at 120 °C. After that, add to the tubes 10 per cent glucose solution sterilized by nitration to adjust to the final concentration of 1 per cent. Inoculate the test culture into two tubes with the above mentioned medium- Into one of the tubes pour a layer (1.5-2 cm) of sterile petrolatum oil. Incubate the test tubes for four days and note acid and gas formation. Darkening of the medium in the open test tube indicates oxidation and in the tube with the oil, fermentation. Gas formation is sometimes observed.

The “strand” test. Onto a glass slide, place a drop of 0.5 per cent solution of sodium desoxycholate in buffer isotonic saline. Into this drop, introduce a loop-ful of the tested culture of vibrios grown on a solid nutrient medium and mix. If the result is positive, the mixture becomes transparent, acquires mucilaginous consistency, and trails the loop in the form of a strand in the first minutes after its preparation.

To differentiate between the classical cholera vibrio and the El Tor vibrio, utilize tests determining the sensitivity of cholera vibrios toward phage’s and polymixin and the ability of vibrios to agglutinate chick erythrocytes.

Sensitivity to diagnostic phages is determined by streaking onto a plate with a culture of whole cholera phages C and EI Tor 2, with ten-fold dilutions of the above. Phage C is active only toward the classic cholera vibrio, while the El Tor 2 phage is active toward El Tor biovar. The presence of lysis in the form of one “sterile” spot or a group of small spots in the place of phage introduction is assessed as a positive result.

Sensitivity to polymixin is determined by inoculating the isolated culture onto Petri dishes with nutrient agar containing 50 U of polymixin M or B in 1 ml of nutrient medium. El Tor vibrios are insensitive to antibiotics and show good growth on the dishes, un­like the classic cholera vibrios.

Haemagglutination of chicken erythrocytes is performed on a glass slide. In a drop of isotonic sodium chloride solution, comminute a loopful of 18-hour culture of the vibrio and add a drop of 2.5 per cent suspension of chicken erythrocytes. The cholera El Tor vibrio agglutinates the red blood cells within 1-3 min, whereas the classical biovar fails to induce any clumping.

Haemolysis of sheep erythrocytes (Greig’s test) occurs after their 2-hour incubation with broth culture of cholera El Tor vibrios at 37 °C. Yet, this sign is not stable and some strains of the El Tor biovar, similar to the classical cholera vibrio, display no haemolytic effect.

The Voges-Proskauer test is based on the ability of El Tor vibrios to form acetylmethylcarbinol, which is recognized by the fact that Clark’s glucose-phosphate broth becomes pink or ruby-red, following 1-3 day incubation of the inoculated cultures with addition of alpha-naphthol.

The hexamine test is performed with 24-hour broth culture of the vibrio a loopful of which is streaked onto 1 ml of a glucose-hexamine medium. Following incubation at 37 °C for 6-24 hrs, the El Tor vibrio alters the colour of the medium from green to yellow. The classical cholera vibrio induces no changes in the medium colour over this time.

Production of enterotoxin by the cholera vibrio is determined by means of a specific reaction of passive immune haemolysis. To carry out this reaction, re-suspend in 0.08 M phosphate buffer the erythrocytes from defibrinated sheep blood (after their triple washing). Prepare 10 per cent suspension of red blood cells in 0.02 M solution of the buffer. With a micropipette introduce 0.025 ml portions of 0.02 M phosphate buffer into agglutinating plates, then add two­fold dilutions of the antigen and 1 per cent suspension of erythrocytes. Cover the plates and place them into a 37 °C incubator for 30 min, add to each well (1.025 ml of antitoxic serum diluted 1:50 and 0.02 per cent bovine serum al­bumin, reincubate the culture for 30 min, then add 0.025 ml of complement and replace the culture into the incubator for 90 min. .Haemolysis is evaluated after 30 min of keeping the plates at room temperature. Simultaneously, one monitors non-immune haemolysis and the ingredients of the serum, antigen, and complement.

Isolation of non-agglutinating vibrios brings about the  necessity of studying their biochemical properties, namely: liquefaction of gelatine, splitting of 5tarch (Cadamot’s test), formation of indol from triptophane, reduction of nitrates into nitrites, as well as oxidase and decarboxylase activity. It is also necessary to classify the culture with one of the biochemical groups according to Heiberg.

Demonstration of cholera vibrios in water is of great importance for identifying the factors of infection transmission and conducting anti-epidemic measures. Using a saturated solution of sodium hydrocar-bonate, alkalize the water (900 ml) delivered to the laboratory to pH of 7.8-8.0, add 100 ml of basic peptone, pH 8.0 (peptone, 100 g, sodium chloride, 50 g, potassium nitrate, 1 g, sodium hydrocarbon-ate, 20 g, distilled water, 1000 ml), and dispense it into flasks or vials in 100-200-ml portions. Incubate the inoculated cultures at 37 °C for 5-8 hrs and then inspect them in the manner employed for studying other inoculated cultures in peptone water (vomited matter and faeces). The results are more reliable when the water tested is filtered through membrane filters. Large amounts of water (1.5-2.5 1) are examined and the deposit from the filters is transferred to peptone water (pH 8.0) and alkaline agar.

Rapid detection of cholera vibrios in drinking water. If water con­tamination with cholera vibrios is heavy (at least 100 vibrios per 1 ml), the agglutination reaction is utilized for their recovery. To the water to be assayed add weakly alkaline concentrated solution of peptone in a quantity sufficient to produce 1 per cent solution. With this mixture dilute the 0-cholera agglutinating serum from 1:100 to its titre. Use a mixture free of the serum as a control. Place the test tubes into an incubator and read the results of the test in 6 hrs. The reaction is considered positive it flocculation is observed upon serum dilution to half the litre or the litre.

Rapid recovery of the cholera vibrio in water may also be based on increase in the phage titre.

Rapid method of wide-scale screening for carriers. During an out­break of cholera wide-scale screening for carriers of the cholera vibrio is performed. When a large number of analyses is to be made in the laboratory, faeces from ten subjects are examined simultaneously. Faeces are collected with wire loops and placed into one flask con­taining 200 ml of peptone water and 0-cholera agglutinating serum which is diluted to half the titre. The flask is placed into a 37 “C incubator. In 3-4 hrs the multiplied cholera vibrios begin to agglu­tinate and fall to the bottom in the form of flakes. If this is the case, faecal material is taken from each of the ten individuals, and the examination is repeated with each sample.

Serological diagnosis of cholera is supportive and relies on de­tecting agglutinins and vibriocidal antibodies in the patient’s serum. It is recommended that paired sera obtained from the patients at a 6-8 day interval be used for these reactions. Titres of agglutinins and vibriocidal antibodies usually tend to increase simultaneously. The most sensitive test is demonstration of vibriocidal antibodies. The presence of agglutinating antibodies in the titre of 1:80-1:320 and vibriocidal ones in the titre of 1:1000 is considered diagnostically positive.

 

 

Corynebacteria

Causative Agent of Diphtheria. Extensive clinical, pathoanatomical, epidemiological, and experimental investigations preceded the discovery of the agent responsible for diphtheria. They paved the way for the discovery of the organism (E.Klebs, 1883), its isolation in pure culture (F. Loeffler, 1884), separation of the toxin (E. Roux and A. Yersin, 1888), antitoxin (E. Behring and S.Kitasato, 1890) and diphtheria toxoid (G. Ramon, 1923).

 

Morphology. Corynebacterium diphtheriae (L. coryna club) is a straight or slightly curved rod, 1-8 mcm in length and 0.3-0.8 mcm in breadth. The organism is pleomorphous and stains more intensely at its ends (Fig. 1) which contain volutin granules (Babes-Ernst granules, metachromatin). C. diphtheriae frequently display terminal club-shaped swellings. Branched forms as well as short, almost coccal, forms sometimes occur. In smears the organisms are arranged at an angle and resemble spread-out fingers. They are Gram-positive and produce no spores, capsules, or flagella.

 

Figure 1. Corynebacterium diphtheriae

C. diphtheriae may change into cone-shaped, thread-like, fungi-like, and coccal forms. In old cultures the cytoplasm of the organisms acquires a zebra-like appearance with unequally stained stripes. On ultrathin sections the cell wall has two layers, an inner osmiophilic layer and an outer layer forming a microcapsule The cytoplasmatic membrane is composed of three layers. During maximum exotoxin liberation membrane structures are seen as ‘organelles’, ovals, and rings. The cytoplasm is granular. The nucleoid is filled with fine osmiophilic fibrils. The metachromatic granules appear as dense granular structures surrounded by a membrane. A correlation has been revealed between the development of the membrane and the production of exotoxin. The G^-C content in DNA ranges from 51.8 to 60 per cent.

Cultivation. The causative agent of diphtheria is an aerobe or a facultative aerobe. The optimal temperature for growth is 37° C and the organism does not grow at temperatures Below 15 and above 40° C. The pH of medium is 7.2-7.6 The organism grows readily on media which contain protein (coagulated serum, blood agar, and serum agar) and on sugar broth. On Roux’s (coagulated horse serum) and Loeffler’s (three parts of ox serum and one part of sugar broth) media the organisms produce growth in 16-18 hours The growth resembles shagreen leather, and the colonies do not merge together.

According to cultural and biological properties, three varieties of C.diphtheriae can be distinguished, gravis, mitis, and intermedius, which differ in a number of properties (fig. 2).

Corynebacteria of the gravis biovar produce large, rough (R-forms), rosette-like black or grey colonies (Fig. 2, 1) on tellurite agar which contains defibrinated blood and potassium tellurite. The organisms ferment dextrin, starch, and glycogen and produce a pellicle and a granular deposit in meat broth. They are usually highly toxic with very marked invasive properties.

The colonies produced by corynebacteria of the mitis biovar on tellurite agar are dark, smooth (S-forms), and shining (Fig. 2, 2). Starch and glycogen are not fermented, and dextrin fermentation is not a constant property. The organisms cause haemolysis of all animal erythrocytes and produce diffuse turbidity in meat broth. Cultures of this biovar are usually less toxic and invasive than those of the gravis biovar.

Organisms of the intermedius biovar are intermediate strains. They produce small (RS-forms) black colonies on tellurite agar. Starch and glycogen are not fermented. Growth in meat broth produces turbidity and a granular deposit.

   

 

Growth of different biovars of C. diphtheriae

Fermentative properties. All three biovars of C. diphtheriae do not coagulate milk, do not break down urea, produce no indole, and slowly produce hydrogen sulphide. They reduce nitrates to nitrites. Potassium tellurite is also reduced, and for this reason C. diphtheriae colonies grown on tellurite agar turn black or grey. Glucose and levulose are fermented whereas galactose, maltose, starch, dextrin, and glycerin fermentation is variable. Exposure to factors in the external environment renders the organisms incapable of carbohydrate fermentation.

Toxin production. In broth cultures C. diphtheriae produce potent exotoxins (histotoxin, dermonecrotoxin, haemolysin). The toxigenicity of these organisms is linked with lysogeny (the presence of moderate phages-prophages in the toxigenic strains). The classical International standard strain, Park-Williams 8 exotoxin-producing strain, is also lysogenic and has retained the property of toxin production for over 85 years. The genetic determinants of toxigenicity (tox⁺ genes) are located in the genome of the prophage, which is integrated with the C. diphtheriae nucleoid.

In the commercial production of diphtheria toxin for vaccine, the amount of iron present in the growth medium is critical. Good toxin production is obtained only at low concentrations of iron (2 mcmol/L). At concentrations aslow as 10 mcmol/L, toxin production becomes negligible. Evidence suggests that, normally, the bacterium forms are presser which prevents the expression of the phage tox⁺ gene, and that this represser is an iron-containing protein.Thus, when the concentration of iron is abnormally low, the complete represser is not formed, and the tox⁺ geneis transcribed, ultimately yielding toxin.

The diphtheria exotoxin is a complex of more than 20 antigens. It has been obtained in a crystalline form. C. diphtheriae also contain bacteriocines (corynecines) which provide these organisms with certain selective advantages.

The diphtheria toxin contains large amounts of amino-nitrogen and catalyses chemical reaction in the body. The toxigenic strains of C. diphtheriae are characterized by marked dehydrogenase activity, while the non-toxigenic strains do not possess such activity.

Diphtheria toxin is excreted from the bacterium as a single polypeptide chain of about 61,000 daltons with two disulfide bridges. Although highly toxic for cells or animals, the pure, intact toxin is inert in cell-free protein systems, even when NAD is present. Thus, the secreted toxin is actually a proenzyme which, in cell-free systems, must be activated before it can function as an enzyme. This activation, as shown in is accomplished in two steps: (1) treatment with trypsm hydrolyzes a peptide bond between the disulfide-linked amino acids; and (2)reduction of the disulfides to sulfhydryl groups using a reducing agent such as mercaptoethanol yields two smaller peptides, which have been designated fragment A (21,150 daltons) and fragment B (40,000 daltons).

 

Fragment A is active in cleaving the nicotinamide moiety from NAD and in catalyzing the transfer of ADP-ribose from NAD to EF-2 when added to cell-free, protein-synthesizing systems, but it has no effect when given to animals or to intact HeLa cells. Thus, although fragment A is the activated enzyme (and hence contains allthe toxic properties), it cannot get into intact cells.

Fragment B, on the other hand, has no enzymatic activity, but it is needed for attachment of the toxin tospecific receptor sites on cells. Cells possess specific glycoprotein receptor sites for the diphtheria toxin, as suggested by the following observation: Rats and mice areover 1000 times more resistant to the intact toxin thanare other susceptible animals, but their cell-free protein-synthesizing system is equally sensitive to the enzymaticaction of fragment A. Moreover, toxin that is defectivein its A fragment (and is, therefore, nontoxic) but retains a normal B fragment, will competitively inhibit the actionof normal toxin on HeLa cells.

The question of whether the phage genome itself codes for the toxin or merely derepresses a bacterial gene, which could then synthesize the toxin, originally was solved using a series of mutant phages that induced the synthesis of mutant toxins. Moreover, the tox gene has been completely sequenced and unequivocally shown to exist in the phage genome.

 

Also, different toxigcnic strains of C diphtheriae vary considerably in the amount of toxin produced under identical conditions. This is, in part, because of subtle differences in the regulation of the tox gene expression, but amore obvious explanation for this observation was shown by Rino Rappuoli and his colleagues. Using specific DNA probes, they conclusively demonstrated that high-toxin-producing strains had two or even three tox genes inserted into their genome. Thus, the quantity of toxin produced was correlated to the amount of tox DNA within thetoxin-producing strain of C diphtherias.

In summary, the usual series of events leading totoxin action is as follows: (1) the toxin binds to specificreceptor sites on susceptible cells; (2) the toxin enters the cell (perhaps through a phagocytic vesicle that can then fuse with a lysosome), and lysosomal proteases hydrolyze the toxin into fragments A and B; and (3) reduction ofthe disulfide bridges (perhaps by glutathione) releases fragment A from fragment B; and (4) fragment A canthen enzymatically inactivate EF-2.

The diphtheria toxin is unstable, and is destroyed easily by exposure to heat, light, and oxygen of the air, but is relatively resistant to super-sonic vibrations. The toxin is transformed into the toxoid by mixture with 0.3-0.4 per cent formalin and maintenance at 38-40° C for a period of 3 or 4 weeks. The toxoid is more resistant to physical and chemical factors than the toxin.

Because diphtheria toxin is effective against many cells, the use of tissue cultures provides a model for studying its mode of action. Early studies reported that, although toxin had no effect on the respiration of HeLa cells (human cervical carcinoma tissue culture cells), all protein synthesis stopped about 1 to 1.5 hours after the additionof the toxin. Surprisingly, dialyzed, cell-free, protein-synthesizing systems were entirely insensitive to the action of the toxin, unless oxidized nicotinamide-adenine dinu-cleotide (NAD) was added to the reaction.

Subsequent research has shown that the toxin possesses enzymatic activity that cleaves nicotmamide from NAD and then catalyzes the ADP-ribosylation of elongation factor 2 (EF-2). EF-2 is required for the translocasc reaction of polypcptide synthesis, in which the ribosome is moved to the next codon on the mRNA after the peptide bond is formed to the most recent aminoacid to be added to the chain. When EF-2 is inactivated by the addition of ADP-ribose, the ribosome is frozen, and protein synthesis stops. Insofar as is known, EF-2 from all eucaryotic cells (those studied include vertebrate, invertebrate, wheat, and yeast) is inactivated in the presence of diphtheria toxin and NAD, whereas the corresponding factor, EF-G (which occurs in bacteria), or the analogous factor from mitochondria, is not affected. The ADP-ribose is transferred to a histidine modified residue on the EF-2 molecule. This modified ammo acid (commonly called diphtheramide) does not exist in bacterialor mitochondnal elongation factors.

Antigenic structure. Eleven serovars of C. diphtheriae have been deter-mined on the basis of the agglutination reaction. They all produce toxins which do not differ from each other and are neutralized completely by the standard diphtheria antitoxin. A number of authors have confirmed the presence of type-specific thermolabile surface protein antigens (K-antigens) and group-specific thermostable somatic polysaccharide antigens (O-antigens) in the diphtheria corynebacteria.

Classification. The genus Corynebacterium comprises a species pathogenic for human beings and several species which are non-pathogenic for man and conditionally designated as diphtheroids. The majority of diphtheroids occurs in the external environment (water, soil, air), some of them are present as commensals in the human body. There are 19 phage types among C. diphtheriae, by means of which the source of the infection is identified The phage types are also taken into account in identificaition of isolated cultures.

 

Resistance. C. diphtheriae are relatively resistant to harmful environmental factors. They survive for one year on coagulated serum, for two months at room temperature, and for several days on children’s toys. Corynebacteria remain viable in the membranes of diphtheria patients for long periods, particularly when the membranes are not exposed to light. The organisms are killed by a temperature of 60° C and by a 1 percent phenol solution in 10 minutes.

Pathogenicity for animals. Animals do not naturally acquire diphtheria. Although, virulent diphtheria organisms were found to be pre-sent in horses, cows, and dogs, the epidemiological significance of animals in diphtheria is negligible.

Among the laboratory animals, guinea pigs and rabbits are most susceptible to the disease. Inoculation of these animals with a culture or toxin gives rise to typical manifestations of a toxinfection and the appearance of inflammation, oedema, and necrosis at the site of inoculation. The internal organs become conjested, particularly the adrenals in which haemorrhages occur.

Pathogenesis and disease in man. Patients suffering from the disease and carriers are the sources of infection in diphtheria. The disease is transmitted by an air-droplet route, and sometimes with dust particles. Transmission by various objects (toys, dishes, books, towels, handkerchiefs, etc.) and foodstuffs (milk, cold dishes, etc.) contaminated with C. diphtheriae is also possible.

Carriers play an essential part in the epidemiology of diphtheria. The carrier state averages from 3 to 5 per cent among convalescents and healthy individuals.

Diphtheria is most prevalent in autumn. This is due to the fact that children are more crowded in the autumn months and that body resistance is reduced by a drop in temperature.

Histotoxin plays the principal role in the pathogenesis of diphtheria. It blocks protein synthesis in the cells of mammals and inactivates transferase, the enzyme responsible for the formation of the polypeptide chain.

C. diphtheriae penetrate into the blood and tissues of sick humans and infected animals. The diffusion factor due to which these organisms are capable of invasion is formed of a complex of K-antigen and lipids of the wall of bacterial cells. The lipids contain corynemicolic and corynemicolenic acids, the cord factor (trehalose dimicolate), and mannose and inositol phosphatides. The cord factor causes the death of mice, destroys mitochondria, and disturbs the processes of respiration and phosphorylation. The necrotic factor, alpha-glutaric acid, and haemolysin are considered to be factors of invasiveness.

Clinical studies and experiments on animals have provided evidence of the influence of pathogenic staphylococci and streptococci, on the development of diphtheria, the infection becoming more severe in the presence of these organisms. Hypersensitivity to C. diphtheriae and to the products of their metabolism is of definite significance in the pathogenesis of diphtheria.

 

 

 

 

 

In man, membranes containing a large number of C. diphtheriae and other bacteria are formed at the site of entry of the causative agent(pharynx, nose, trachea, eye conjunctiva, skin, vulva, vagina, and wounds). The toxin produces diphtheria! inflammation and necrosis in the mucous membranes or skin. On being absorbed, the toxin affects the nerve cells, cardiac muscle, and parenchymatous organs and causes severe toxaemia.

Deep changes take place in the cardiac muscle, vessels, adrenals, and in the central and peripheral nervous systems.

According to the site of the lesion, faucial diphtheria and diphtheritic croup occur most frequently, and nasal diphtheria somewhat less frequently. The incidence of diphtheria of the eyes, ears, genital organs, and skin is relatively rare. Faucial diphtheria constitutes more than 90per cent of all the diphtherial cases, and nasal diphtheria takes the second place.

Immunity following diphtheria depends mainly on the antitoxin con-tent m the blood However, a definite role of the antibacterial component, associated with phagocytosis and the presence of opsonins, agglutinins, precipitins, and complement-fixing substances cannot be ruled out. Therefore, immunity produced by diphtheria is anti-infectious (anti-toxic and antibacterial) in character.

Schick test. This test is used for detecting the presence of antitoxin in children’s blood. The toxin is injected intracutaneously into the forearm in a 0 2 ml volume which is equivalent to 1/40 DLM for guinea pigs. A positive reaction, which indicates susceptibility to the disease, is manifested by an erythematous swelling measuring 2 cm in diameter which appears at the site of injection in 24-48 hours. The Schick test is positive when the blood contains either no antitoxin or not more than0.005 units per millilitre of blood serum. A negative Schick reaction indicates, to a certain degree, insusceptibility to diphtheria.

In view of the fact that the diphtheria exotoxin produces a state of sensitization and causes the development of severe reaction in many children, it is advisable to restrict the application of the Schick test and conduct it with great care.

Children from 1 to 4 years old are most susceptible to diphtheria. A relative increase of the incidence of the disease among individuals 15years of age and older has beeoted in recent years.

Diphtheria leaves a less stable immunity than do other children’s diseases (measles, whooping cough). Diphtheria reinfection occurs in 6-7per cent of the cases.

Laboratory diagnosis. Discharges from the pharynx, nose, and, some-times, from the vulva, eyes, and skin are collected with a sterile cotton-wool swab for examination.

The material under test is seeded on special media, e. g. coagulated serum, Clauberg’s II medium, blood-tellurite agar, serum-tellurite agar, etc. Smears are examined under the microscope after 12-24-48 hours’ growth, and preliminary diagnosis is made on the basis of microscopic findings.

C. diphtheriae does not always occur m its typical form. Short rods arranged not at a particular angle but in disorder and containing few granules are found in a number of cases. Diagnostic errors are made most frequently when investigations are confined to microscopical examination. Other bacterial species and non-pathogenic corynebacteria which are morphologically identical with the diphtheria organisms maybe mistaken for the diphtheria corynebacteria (Plate VIII). It must also be borne in mind that formation of volutin granules is variable, and therefore, this is not an absolute property. For this reason, contemporary laboratory diagnosis comprises isolation of the pure culture and its identification by cultural, biochemical, serological and toxigenic properties.

The toxigenic and non-toxigenic strains of diphtheria corynebacteria are differentiated either by subcutaneous or intracutaneous infection of guinea pigs, or by the agar precipitation method, the latter being relatively simple and may be carried out in any laboratory. It is based on the ability of the diphtheria toxin to react with the antitoxin and produce a precipitate resembling arrow-tendrils.

The agglutination reaction with patient’s sera (similar to the Widal reaction) is employed as an auxiliary and retrospective method. It is performed with 5 serovars of C. diphtheriae; the reaction is considered positive beginning from 1 :50-1 :100 dilutions of serum.

To detect the sources of infection, the isolated cultures are subject to phagotyping. There are 19 known phage types.

Treatment. According to the physician’s prescriptions, patients are given antitoxin in doses ranging from 5000 to 15000 units in mildly severe cases, and from 30 000 to 50 000 units in severe cases of the disease. Penicillin, streptomycin, tetracycline, erythromycin, sulphonamides, and cardiac drugs are also employed. Diphtheria toxoid is recommended in definite doses for improving the immunobiological state of the body, i.e for stimulating antitoxin production.

Carriers are treated with antibiotics. Tetracycline, erythromycin, and oxytetracycline in combination with vitamin C are very effective.

Prophylaxis. General control measures comprise early diagnosis, prompt hospitalization, thorough disinfection of premises and objects, recognition of carriers, and systematic health education.

Specific prophylaxis is afforded by active immunization. A number of preparations are used: the pertussis-diphtheria vaccine, purified adsorbed toxoid, pertussis-diphtheria-tetanus vaccine All preparations are used according to instructions and directions.

Reports show that only antibodies to the fragment B portion of the toxin molecule are capable of neutralizing the toxin, supposedly by preventing the attachment of toxin to the specific receptor sites on the cell surface. Treatment of the toxin with formalin, however, both detoxifies the toxin and protects fragment B from the action of proteolytic enzymes, resulting in better protective antibody production than that obtained by using untreated fragment B or defective toxins possessing anormal fragment B

It should be noted that not all immunized children acquire resistance to diphtheria. An average of 5-10 per cent of them remain susceptible or refractory (not capable of producing antibodies after immunization).Such a condition is considered to be the result of tolerance, agamma-globulmaemia, or hypoagammaglobulinaemia.

Haifa century ago diphtheria was a menacing disease of children. In Russia every year more than 250 000 persons contracted the disease in 1886-1912. The death rate was very high (12 to 30 per cent).With the introduction of compulsory immunization against diphtheria great success has been gained in the control of this disease.

Other Corynebacteria. Many species of Corynebacterium exist in the soil; a few cause animal diseases, and a large number are plant pathogens. Such species, however, are only rare causes of human diseases. Interestingly, both Corynebacterium ulcerans and Corynebacterium pseudotuberculosis are known to cause occasional diphthena-like illnesses. Moreover, selected isolates of these species have been shown to produce a toxin that is indistinguishable from that of C. diphtheriae. The fact that human disease by these speciesis both rare and mild suggests that even though toxigemc, they may lack some virulence factor possessed by C. diphtheriae.

 

Additional materials about laboratory Diagnosis

Diphtheria is an acute infectious disease with the predominant localization of the causative organism in the mucosa of the fauces and upper respiratory pathways. The causative agent of the disease is Corynebacterium diphtheriae.

The material tested is diphtheritic films or secretions of the involved mucosal membrane of the fauces, nose, and occasionally of the external genitalia and conjunctiva. From carriers, secretions of the faucial and nasal mucosa are examined. At the requirement of the epidemiologist foodstuffs (milk, ice-cream) and washings from various objects (toys, etc.) are examined.

 

 

 

 

 

 

 

 

Secretions of the faucial mucosa should be taken on a fasting stomach or two hours after meal. It is recommended that no disin­fectants or antibiotics be used before this procedure. Material from the fauces and nose is taken with two sterile tampons which are placed into test tubes and sent to the laboratory without delay. If transportation of specimens is to take over 3-4 hrs, use tampons soaked with a 5 per cent glycerol solution in isotonic saline.

Bacterioscopic examination of the material obtained from the patient is carried out only if the physician considers it advisable. In such cases the secretion of the mucosa or film is removed with two swabs: one of them is used for culturing. the other, for preparing smears. Smears may be stained with Gram’s dye, acetic-acidic methyl violet, Loeffler’s blue, toluidine blue, and Neisser’s stain. In smears prepared from the film corynebacteria of diphtheria appear as single rods arranged at an angle to each other (V-like arrangement), less commonly they form clusters. They are Gram-positive; staining with acetic-acidic methyl violet or Loeffler’s blue reveals intensely stained volutin granules. False diphtheria bacteria and diphlheroids are arranged in parallel (“a fence-like arrangement”) and are ordinarily deprived of volutin granules. Volulin granules may be detected with the help of luminescent microscopy. For this purpose the preparation is stained with coryphosphine. Microscopic findings are yellow-green bodies of bacteria with orange-red volutin granules against a dark background. Upon the detection of typical corynehacteria. the laboratory immediately issues a preliminary result which reads “Diphtheritic corynebacteria have been detected, proceed with examination”.

 

 

 

Bacteriological examination. The material is introduced onto one of the elective media: into test tubes with coagulated serum and in a Petri dish with telluric blood agar, cystine-tellurite-serum me­dium (Tinsdal-Sadykova), Buchin’s quinosol medium, etc. It is recommended that one of the above media should be constantly used for the corynebacteria isolation as this practice makes it possible to obtain more clear-cut and comparable results.

 

Telluric blood agar. To 100 ml of melted 2-3 per cent agar cooled to 50 °C, add 5-10 per cent of defibrinated blood and 1 ml of a 2 per cent solution of potas­sium tellurite. Thoroughly mix the mixture and pour into sterile plates.

Cystine-tellurite-serum medium (Tinsdal-Sadykova medium). To 100 ml of melt­ed meat-peptone agar cooled to 60 °C, add consecutively the following compo­nents: (1) 1 per cent solution of cystine in 0.1 N sodium hydroxide solution (12 ml); (2) 0.1 N solution of hydrochloric acid (12 ml); (3) 2 per cent solution of potassium tellurite (1.5-1.8 ml); (4) 2.5 per cent of sodium hyposulphite solu­tion (1.8 ml); (5) normal horse or bovine serum (20 ml). The mixture is stirred and dispensed into sterile Petri dishes.

Buchin’s quinosol medium is prepared from powder, according to the label instructions. It is boiled for 2-3 min and cooled to 50 °C after which 511 ml of defibrinated blood (rabbit or human) is added. The prepared medium is dark blue.

During inoculation the material is rubbed with a swab into the medium surface. The growth of diphtheria corynebacteria on co­agulated serum is faster than that of other microorganisms, their colonies are small and separate. After 8-12 hrs of incubation smears are made; if the result is negative, microscopic examination is re­peated in 18-24 hrs. If diphtheria corynebacteria cannot be found, the inoculated cultures are kept in the  incubator for 48 hrs after which a negative result can be issued. If typical corynebacteria are demonstrated, a pure culture is isolated and identified by fermentative and toxigenic properties (Table 2 ).

Table 2

Differential-Diagnostic Signs of Diphtheria and Non-Pathogenic

 

Urease production

 

Study the colonies in dishes in 24-48 hrs. On media with potas­sium tellurite diphtheria corynebacteria of the gravis type form relatively large, greyish-black, flat, rough colonies with radial lines and a wavy margin; colonies of the mitis type are small, protuberant, lustrous, black, with a smooth surface and an even margin. Diphtheroids grow in the form of protuberant moist colonies of a grey or brown colour. False diphtheria bacteria form dry. small mucoid colonies of a grey colour. On the Tinsdal-Sadykova medium colonies of diphtheria corynebacteria are surrounded with a dark brown halo, on Buchin’s medium they are blue. while diphtheroids on the same medium form colourless colonies and false diphtheria bacteria form bluish colonies.

To obtain a pure culture and assess toxigenicity, suspicious colonies are examined microscopically, subcultured to a serum medium and onto a plate with a phosphate-peptone agar. Pure cultures are in­troduced into Hiss’s media (glucose, sucrose, starch), cystine medium (cystinase test), and into a medium with urea (urease test).

Medium for cystinase determination. To 90 ml of melted 2 per cent meat-pep­tone agar (pH 7.6) add 2 ml of cystine solution (1 percent cystine solution with 0.1 M solution of sodium hydroxide), mix thoroughly, and add 2 ml of 0. 1 N solution of sulphuric acid. Sterilize the medium at 112 °C for 30 mill. To the melted medium cooled to 50 °C add 1 ml of 10 per cent solution of acetic-acidic lead (after its double sterilization with flowing steam), stir the mixture, and add 9 ml of normal horse serum. Decant the medium in 2-ml quantities into small test tubes under sterile conditions. When diphtheria corynebacteria are inoculated by injection, they induce blackening of the medium (combination of lead with hydrogen sulphide) along the course of the injection and around it in the form of a cloud.

Medium for demonstrating the urease enzyme. To 100 ml of a meat-peptone broth or Hottinger’s broth (pH 7.0) add 1 g of urea and 0.2 ml of cresol red (1.6 per cent alcohol solution). Pour the resultant medium (in 2-3-ml aliquots) into sterile test tubes and sterilize with flowing steam for 10 min. Reddening of the medium observed 20-24 hrs after the inoculation of the diphtheroid culture into the urea broth witnesses the presence of the urease enzyme. Diphtheria coryne­bacteria do not alter the colour of the medium.

Simultaneously, the agglutination test is performed on a slide with monospecific diphtheria sera of the first-fourth serovars. Agglu­tinating sera are diluted 1:25 in advance. Using this reaction, 11 serological types or variants of the diphtheria causative organism have been established; in the USSR the second serovar is the most common one. In corynebacteria of diphtheria 12 phagovars have been iden­tified, with the help of which the sources of the infection are estab­lished.

Upon the isolation of toxigenic strains of diphtheria corynebacte­ria the final answer may be issued in 48 hrs. It specifies a biological (gravis or mitis) and serological variants of the causative agent, a phagovar of the isolated microorganism, and its toxigenicity.

Determination of toxigenicity of cultures in vitro. For this purpose 12 ml of melted phosphate-peptone agar cooled to 50 °C are poured into a Petri dish.

Phosphate-peptone agar. 1. Preparation of marten peptone. Minced pieces of the pig stomach (250 g) are immersed with 1 l of 1 per cent aqueous solution of chemically pure hydrochloric acid and placed into a 37 °C incubator for 18-20 hrs. Following digestion, the peptone is heat­ed at 80 °C for 10 min and allowed to settle down for 8-10 days in a cool place, then it is filtered, heated to 80 °C, alkalized with a 10 per cent solution of sodium hydroxide to pH 8-8.2, boiled for 10 min, filtered, dispensed into bottles, and, after being supplemented with 1 per cent. of chloroform, stored in a cool place.

2. Preparation of a phosphate agar. Per 11 of distilled water take 40 got agar-agar, 125 g of sodium hydrophosphate, and 3.75 g of potassium dihydrophosphate. Place the mixture into a sterilizer and allow it to stand there for 20 min at flowing steam and for 10 min at 115 °C. Leave the mixture in the sterilizer for 2 hrs to allow sedimentation to take place, then filter it and sterilize at 115 °C for 30 min.

To obtain a phosphate-peptone agar, mix 50 ml of heated peptone and 50 ml of a phosphate agar. Bring the pH to 7.8-8.0 by adding 0.5 per cent of sodium acetate and 0.3 per cent of maltose, dispense the mixture in 10-ml volumes and sterilize them with flowing steam for 3D min.

After the nutrient medium has solidified, on the middle of the plate place a strip of sterile filter paper (2.5 X 8 cm) soaked with an anti-toxic serum con­taining 500 AU per ml or with a specific gamma-globulin. The plate is dried for 15-20 min in an incubator, then the  culture examined is streaked with strokes perpendicular to the filter paper or with patches 1 cm in diameter at a dis­tance of 1 cm from the edge of the strip. From 3-4 to 10 cultures can be streaked onto one plate (one of the cultures, the control, is known to be toxigenic). Inoc­ulated cultures are put into an incubator. The results are read in 24, 48, and 72 hrs. If the culture is toxigenic, lines of precipitation form at some distance from the paper strip, which coincide with the lines of the precipitate of the con­trol culture. They are readily seen in transmitted light (Fig. 4)

 

Figure  4. Determination of the in vitro toxigenicity of Corynebacterium diphtheriae

 

 

Biological examination is conducted to determine the toxigenicity of isolated cultures in vivo.

Intracutaneous method. The day before the examination clip off hair from the sides of two guinea pigs (preferably with white hair). On the day of the examination prepare 100-200-million suspension of the culture to be studied and inject intracutaneously  0.2-ml por­tions of each suspension into two prepared guinea pigs. In 4 hrs administer intraperitoneally 100 IU of the antitoxic diphtheria serum to the control infected guinea pig. If the culture is toxigenic. the test guinea pig develops reddening, oedema, and theecrosis at the site of injection. The final assessment of the results is made in 72 hrs. Control animals present no alterations. The intracutaneous method of toxigenicity determination makes it possible to test 6 cultures in one guinea pig.

Guinea pigs weighing 240-300 g are used for the subcutaneous administration, of the material. One day before the test administer 1000 IU of the antitoxic serum to the control animal. On the exami­nation day inject subcutaneously 0.5 ml of suspension of the culture tested (500 min and 1 mlrd of microorganisms per ml) to both test and control guinea pigs. If the examined strain of diphtheria is toxigenic, the test guinea pigs die on the 2nd-5th day. Post-mortem findings include oedema at the site of the culture administration, exudate in the peritoneal and thoracic cavity, hyperemia of the adrenal cortex. The control guinea pig remains alive.

Serological examination remains supplementary in the diagnosis of diphtheria. Sera of patients or convalescents are diluted with sodium chloride solution in ratios 1:-100, 1:200, 1:400, 1:800, 1:1600, etc. Add a specially prepared diagnosticum (diphtheria culture washed off with saline and killed with 0.2 per cent formalin solution) to the serum dilutions. The reaction is considered positive if the dilution of the serum is no less than 1:100. Agglutinins against diphtheria corynebacteria usually appear within the first days of the disease and disappear in 12-15 days. The usually employed test is IHA with an erythrocyte bacterial diagnosticum: a 1:8 or greater titre during the second week of the disease is considered diagnostically significant.

The current employment of Schick’s test is limited. It is intended for detecting antitoxic immunity. For this purpose utilize diluted diphtherial toxin 0.2 ml of which contains 1/40 Dim for a guinea pig. The toxin is injected intracutaneously into a median internal surface of the upper arm. If 1 ml of the blood serum contains 1/30 IU of antitoxin or over, Schick’s reaction is negative. If antitoxins are absent, redness and infiltrate develop at the site of toxin adminis­tration in 48-96 hrs.

 

References: 

1.                  Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002, P. 223-225, 235-241, 

2.                  Essential of Medical Microbiology /Wesley A. Volk and al. / Lippincott-Raven Publishers, Philadelphia-Ney-York, 1995, 725 p.

3.                  Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed. by Yu.S. Krivoshein, 1989, P. 96-105.