Microbiological diagnosis of plague, other yersiniosis and tularaemia. Microbiological diagnosis of brucellosis and anthrax.
Yersinia
Yersinia pestis. Genus Yersinia includes the following three bacterial species: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. They were separated from genus Pasteurella on the basis of certain properties and included in family Enterobacteriaceae.
The causative agent of plague, Yersinia pestis, was discovered by the French microbiologist A. Yersin in Hong Kong in 1894. Russian scientists D. Samoilovich, D. Zabolotny, N. Klodnitsky, I. Deminsky, N. Gamaleya and others contributed greatly to the study of the mechanisms of its transmission.
The French microbiologists G. Girard and T. Robic obtained a live vaccine from the attenuated EV strain. R. Karamchamdani and K. Rao of India introduced the extremely effective antibiotic streptomycin into practice for the treatment of all forms of plague.
The genus Yersinia includes 11 species, three of which are important human pathogens: Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The yersinioses are zoonotic infections of domestic and wild animals; humans are considered incidental hosts that do not contribute to the natural disease cycle.
Y. pestis causes plague and is transmitted by fleas. The most common clinical manifestation is acute febrile lymphadenitis, called bubonic plague. Less common forms include septicemia, pneumonia, pharyngeal, and meningeal plague.
The clinical features, diagnosis, and treatment of plague will be reviewed here. The epidemiology, microbiology, and pathogenesis of Yersinia pestis are discussed separately. (See “Epidemiology, microbiology and pathogenesis of plague (Yersinia pestis infection)”.)
Key microbiological characteristics of Yersinia pestis include the following (ASM 2013, Sneath 1986):
· Pleomorphic gram-negative bacillus (1.0 to 2.0 mcm x 0.5 mcm); single cells or short chains in direct smears
· Bipolar (“closed safety pin”) staining with Giemsa, Wright’s, or Wayson stains (may not be visible on Gram stain)
· Facultative anaerobe
· Nonmotile, nonsporulating
· Non–lactose fermenter
· Slow-growing in culture (colonies are pinpoint after 24 hours on sheep blood agar [SBA] and much smaller than other Enterobacteriaceae growing for 24 hours on SBA; colonies may not be visible on MacConkey or eosin methylene blue agar at 24 hours)
· Catalase-positive, oxidase- and urease-negative (rarely, strains may be urease-positive)
· Optimal growth at 28°C
· “Stalactite pattern” in broth culture with clumps of cells from the side of the tube settling to the bottom if disturbed
· At 48 to 72 hours of incubation on solid media, colonies have a raised, irregular, “fried egg” appearance under 4X enlargement, which becomes more pronounced as the culture ages; colonies also have been described as having a “hammered copper” shiny surface
· Alkaline slant/acid butt (K/A) on triple sugar iron agar (TSI) without gas or H2S
· Generally susceptible to tetracyclines, chloramphenicol, aminoglycosides, sulfonamides (with or without trimethoprim), and fluoroquinolone antibiotics
Y pestis is divided into three classic biovariants (biovars) (Dennis 1997).
· Biovar antiqua (Africa, southeastern Russia, central Asia)
· Biovar medievalis (Caspian Sea)
· Biovar orientalis (Asia, Western Hemisphere)
Previously, the three biovars were thought to be responsible for the first, second, and third pandemics, respectively. Limited archeologic evidence suggests that all three pandemics were caused by the orientalis biovar (Drancourt 2007).
Other classification and diversity information includes:
· A nonvirulent strain, microtus, has been proposed as a fourth biovar (Zhou 2004: Genetics of metabolic variations between Yersinia pestis biovars and the proposal of a new biovar, microtus).
· Y pestis is thought to have evolved from Yersinia pseudotuberculosis 1,500 to 20,000 years ago, and the two species remain closely related (Achtman 1999). Whole-genome sequence comparisons have identified 32 chromosomal genes and 2 plasmids in Y pestis but not Y pseudotuberculosis (Chain 2004).
· The complete genomes of several strains have been sequenced and are available online (National Center for Biotechnology Information, Parkhill 2001, Song 2004, Zhou 2002).
Virulence Factors
Virulence factors for Y pestis are primarily encoded on the chromosome and on three plasmids (the Pst plasmid, the Lcr plasmid, and the pFra plasmid) (Dennis 1997).
The major virulence factors for Y pestis are responsible for the following activities (Dennis 1997, McGovern 1997, Perry 1997, Titball 2003):
· The ability of Y pestis organisms to adhere to cell surfaces is a key step in pathogenesis. Irreversible binding to host cell receptors via adhesins allows the organisms to then penetrate the cell surfaces (Zhou 2006).
· The F1 antigen is antiphagocytic, elicits a humoral response, and is a target for immunologic-based diagnostic tests. Most pathogenic Y pestis strains isolated from humans contain the F1 antigen.
· Plasminogen activator (Pla) is a protease that appears to degrade fibrin and other extracellular proteins and to facilitate systemic spread from the inoculation site. Expression of Pla allows Y pestis to replicate rapidly in the airways. Pla is essential for Y pestis to cause primary pneumonic plague but is less important for dissemination during pneumonic than bubonic plague (Lathem 2007).
· The V and W antigens (produced at 37°C) cause the organisms to be resistant to phagocytosis; the V antigen is important for survival of Y pestis in macrophages.
· Yersinia outer proteins (Yops) have a variety of activities, including inhibiting phagocytosis, inhibiting platelet aggregation, and preventing an effective inflammatory response.
· The outer membrane protein A (OmpA) enhances intracellular survival of Y pestis (Bartra 2012).
· Lipopolysaccharide (LPS) endotoxin (encoded on the chromosome) causes the classic features of endotoxic shock. LPS consists of three domains: the hydrophobic membrane anchor (lipid A), the surface-exposed O-antigen polysaccharide, and the core sugar region connecting the other two. Most of the effects of LPS are caused by lipid A (LPS-lipid A) (Zhou 2006).
· Effectors secreted by the type 3 secretion system enable immunosuppression in the lungs, allowing for rapid, uninhibited growth of Y pestis during the early preinflammatory phase of infection. These effectors alone, however, are not sufficient for this immune suppression to occur (Price 2012).
· Phospholipase D (PLD) allows the bacilli to survive in the flea midgut.
· Yersinia murine toxin (Ymt) is one of the factors required for maintaining Y pestis in fleas. Ymt is highly toxic for mice and rats but less active in other animals (Zhou 2006).
Bubonic Plague
- After a flea initially ingests Y pestis, the organisms elaborate a coagulase that clots ingested blood in the proventriculus (an organ between the esophagus and stomach) of the flea, thus blocking passage of the next blood meal into the flea’s stomach. Fleas with this blockage regurgitate Y pestis into the bite wound while attempting to feed (Perry 1997).
- From 25,000 to 100,000 Y pestis organisms are inoculated into the skin via the bite of an infected flea (Reed 1970).
- As few as 1 to 10 organisms are sufficient to cause infection via the subcutaneous, intradermal, oral, or intravenous routes (Worsham 2007).
- A papule, vesicle, pustule, or furuncle may occur at the site of the fleabite but is noted in less than 10% of patients (Dennis 1997).
- The organisms migrate through the cutaneous lymphatics to regional lymph nodes. Comparative studies in mice reveal that Y pestis virulence is associated with a distinct ability to massively infiltrate the draining lymph node without inducing an organized polymorphonuclear cell reaction (Guinet 2008).
- Once in the lymph nodes, they are phagocytized by polymorphonuclear neutrophils (PMNs) and mononuclear phagocytes. Organisms that are phagocytized by PMNs generally are destroyed, whereas those phagocytized by mononuclear cells proliferate intracellularly and develop resistance to further phagocytosis (Perry 1997). These organisms are released when cell lysis occurs.
- Initially, a thick, proteinaceous exudate that includes plague bacilli, PMNs, lymphocytes, and fewer macrophages can be found in affected nodes (Dennis 1997).
- Subsequently, the exudative pattern gives way to lakes of hemorrhagic necrosis, which obliterate the underlying lymph node architecture. A ground-glass amphophilic material that represents masses of bacilli may be present (CDC 2004).
- The inflammatory process creates swollen painful buboes and surrounding edematous tissues that are characteristic of bubonic plague. Bubo location is a function of the site of inoculation of plague bacilli by the infected flea (Worsham 2007).
- The organisms often enter the bloodstream, causing hemorrhagic lesions in other lymph nodes and in organs throughout the body (initially the liver and spleen). Findings from a study using a mouse model suggest that the organisms replicate in splenic macrophages during the later stages of infection (Lukaszewski 2005).
- Septicemia, disseminated intravascular coagulation (DIC), and shock can ensue.
- Unless treated promptly with appropriate antibiotic therapy, death usually results from overwhelming sepsis.
- A bioluminescence imaging study in mice found that Y pestis bacteria initially multiply at the site of inoculation before colonizing the draining inguinal lymph nodes and the associated ipsilateral axillary lymph node. A mild bacteremia then develops and is cleared by the liver and spleen. Once the liver and spleen reach their filtering capacity, terminal septicemia develops. The study also showed that the primary location of multiplication of Y pestis is the secondary lymph nodes and that disease progression after colonization of the secondary lymph nodes is rapid (Nham 2012).
Septicemic Plague
- Primary septicemic plague is defined as systemic toxicity caused by Y pestis infection but without apparent preceding lymph node involvement. Secondary septicemic plague occurs commonly with either bubonic or primary pneumonic plague.
- In primary septicemic plague, Y pestis organisms can disseminate from a fleabitesite through thelymphatic system (but without clinically apparent involvement of the lymph nodes), directly through the circulatory system, orboth (Sebbane 2006).
- Septicemic plague causes sepsis syndrome with multiorgan involvement, DIC, and shock. In the late stages of infection, high-density bacteremia often occurs, leading to identification of organisms on peripheral blood smears (Butler 1991).
- The spleen, liver, kidneys, skin, and brain are the most commonly affected organs. Meningitis can occur and is characterized by a thick, yellow, fibrinous-purulent exudate. Foci of necrosis with hemorrhage are common, as are characteristic lesions of DIC (such as fibrin thrombi in glomerular capillaries or purpuric skin lesions) (Dennis 1997).
Pneumonic Plague
- Y pestis can enter the lungs either through direct inhalation (primary pneumonic plague) or through hematogenous spread as a complication of bubonic or septicemic plague (secondary pneumonic plague).
- Primary pneumonic plague is acquired naturally by inhaling respiratory droplets from infected humans or animals (such as cats).
- The infectious dose by inhalation is estimated to be 100 to 500 organisms (Franz 1997).
- Marked intra-alveolar edema and congestion of the lungs are common (CDC 2004). Pulmonary lesions include areas of central exudate with peripheral congestion. This pattern initially is lobular, but usually progresses to lobar consolidation (Dennis 1997).
- Distinguishing primary pneumonic plague from secondary hematogenous spread to the lungs can be difficult. Features that occur more commonly with primary pneumonic plague include the following (Dennis 1997):
- Tracheal and bronchial mucosal hemorrhages
- Fibrinous pleuritis and subpleural hemorrhages overlying areas of exudative pneumonia
- Less inflammation and necrosis and more exudation in lobular foci of the parenchyma
- Foci of pneumonia along medium and large bronchi
- More involvement of hilar lymph nodes
- Less severe evidence of disease in organs other than the lungs, if such evidence is present
- In primary pneumonic plague, as with bubonic plague, organisms often enter the bloodstream and cause multiorgan involvement, DIC, and shock.
- In the absence of early antibiotic therapy (ie, within the first 24 hours), death occurs from overwhelming sepsis (usually within several days after illness onset). Without therapy, mortality approaches 100%.
Y pestis may persist iecrotic tissues after antibiotic treatment despite negative blood cultures. Presumably, Y pestis becomes trapped in hypoperfused tissues and is able to persist because of: (1) inadequate delivery of antibiotics to affected areas and (2) the ability of the organisms to overcome local host defenses (Guarner 2005).
Plague still poses a significant threat to human health, and interest has been renewed recently in the possible use of Yersinia pestis as a biological weapon by terrorists. The septicaemic and pneumonic forms are always lethal if untreated. Attempts to treat this deadly disease date back to the era of global pandemics, when various methods were explored. The successful isolation of the plague pathogen led to the beginning of more scientific approaches to the treatment and cure of plague. This subsequently led to specific antibiotic prophylaxis and therapy for Y. pestis. The use of antibiotics such as tetracycline and streptomycin for the treatment of plague has been embraced by the World Health Organization Expert Committee on Plague as the ‘gold standard’ treatment. However, concerns regarding the development of antibiotic-resistant Y. pestis strains have led to the exploration of alternatives to antibiotics. Several investigators have looked into the use of alternatives, such as immunotherapy, non-pathogen-specific immunomodulatory therapy, phage therapy, bacteriocin therapy, and treatment with inhibitors of virulence factors. The alternative therapies reported in this review should be further investigated by comprehensive studies of their clinical application for the treatment of plague.
As a Gram-negative bacterium, the causative agent of plague, Yersinia pestis (Yersin, 1894), has been the cause of three pandemics (Achtman et al., 1999; Drancourt et al., 2004; Guiyoule et al., 1994; Raoult et al., 2000), and has led to the deaths of millions of people, the devastation of cities and villages, and the collapse of governments and civilizations (Zietz & Dunkelberg, 2004). At the present time, the circulation of Y. pestis has been detected within the populations of more than 200 species of wild rodent inhabiting natural plague foci on all continents, except for Australia and Antarctica, and the transmission of plague is provided for by a minimum of 80 species of flea. Plague epizootics, during which the pathogen spreads through new territories, alternate with a decrease in epizootic activity. Wheatural foci of infection are investigated during periods between epizootics, no antibodies to Y. pestis are detected in the animals, and the plague microbe is not detected by bacteriological and biological methods. Morbidity in humans is noted, as a rule, when epizootics become acute, and is a consequence of bites of fleas, direct contact with infected animal tissues, the consumption of insufficiently cooked meat products, or the inhalation of aerosolized respiratory excreta of animals or patients with the pneumonic form of infection (Anisimov, 2002a, b; Anisimov et al., 2004; Aparin & Golubinskii, 1989; Brubaker, 1991; Butler, 1983; Dennis et al., 1999; Domaradskii, 1993, 1998; Gage & Kosoy, 2005; Hinnebusch, 2003; Inglesby et al., 2000; Lien-Teh et al., 1936; Naumov & Samoilova, 1992; Nikolaev, 1972; Perry & Fetherston, 1997; Pollitzer, 1954).
Plague remains a serious problem for international public health. Small outbreaks of plague continue to occur throughout the world, and at least 2000 cases of plague are reported annually (Gage & Kosoy, 2005). Plague has recently been recognized as a reemerging disease, and Y. pestis, if used by the aerosol route of exposure as a bioterrorism agent, could cause mass casualties (Gage & Kosoy, 2005; Inglesby et al., 2000). According to World Health Organization (WHO) data, if 50 kg of the plague pathogen was released as an aerosol over a city with a population of 5 million, 150 000 people might fall ill with pneumonic plague, 36 000 of whom would die (WHO, 1970).
Immunization against plague is one of the major approaches being pursued to deal with potential infection. Commercially available human plague vaccines are based on either a live, attenuated strain or a killed, whole-cell preparation. The live, attenuated vaccine is produced in the former Soviet Union, and is based on Y. pestis strain EV, line NIIEG. This strain is attenuated due to deletion of the 102 kb pgm locus that includes the hms locus responsible for the ability to store haemin, and a cluster of genes needed for production of the siderophore-based yersiniabactin biosynthetic/transport systems. The parental wild-type strain was isolated in Madagascar. The second vaccine is a formalin-fixed virulent Y. pestis strain 195/P (originally isolated in India) that was developed in the USA, although at present it is manufactured only in Australia. Both these types of vaccines have unacceptable side effects when used (Meyer, 1970; Naumov et al., 1992). A number of subunit-based vaccines are currently under development and/or at different stages of preclinical/clinical trials. These offer the advantage of employing a defined antigen that possesses the ability to elicit high-level protection, and they are also less reactogenic than the currently used whole-cell vaccines (Anisimov et al., 2004; Williamson, 2001; Williamson et al., 2005). The main limitation of the prevention of plague by vaccination is that protection is delayed for at least 1 week after immunization, and this time may be crucial with respect to the lethal outcome of the disease (Butler, 1983; Dennis et al., 1999; Domaradskii, 1993, 1998; Inglesby et al., 2000; Lien-Teh et al., 1936; Naumov & Samoilova, 1992; Nikolaev, 1972; Perry & Fetherston, 1997; Pollitzer, 1954; Rudnev, 1940). As a consequence, antibiotics are employed for the early initiation of prophylaxis and therapy of plague.
The overwhelming majority of Y. pestis natural isolates are susceptible in vitro and in vivo to antimicrobials such as tetracycline, doxycycline, ciprofloxacin, streptomycin, gentamicin, chloramphenicol, fluoroquinolones, sulphonamides, and trimethoprim-sulfamethoxazole (Domaradskii, 1993; Frean et al., 2003; Inglesby et al., 2000). The infrequent recovery of natural drug-resistant isolates of Y. pestis has been explained by the relative rarity of cases of human plague at the present time and by the acute nature of the disease (Domaradskii, 1993). However, a ‘natural’ strain with resistance to multiple antibiotics, including all of those recommended for plague prophylaxis and treatment, was isolated in 1995 in the Ambalavao district of Madagascar from a 16-year-old boy. The Y. pestis strain 17/95 was resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, spectinomycin, sulfonamides, tetracycline and minocycline. The resistance genes were carried by a plasmid that could conjugate with high frequency to other Y. pestis strains in vitro (Galimand et al., 1997) or in fleas (Hinnebusch et al., 2002). The possibility of the rise of such multidrug-resistant strains in the natural environment, the ease of generation of such strains under laboratory conditions (Hinnebusch et al., 2002; Hurtle et al., 2003), the potential use of such strains for bioterrorist attack, together with the rapidity and high lethality of the disease (Butler, 1983; Dennis et al., 1999; Domaradskii, 1993, 1998; Inglesby et al., 2000; Lien-Teh et al., 1936; Naumov & Samoilova, 1992; Nikolaev, 1972; Perry & Fetherston, 1997; Pollitzer, 1954; Rudnev, 1940), are evidence of the necessity for a search for novel antimicrobial alternatives to antibiotics. Here, we will briefly review the existing methods, as well as promising current strategies under investigation for the treatment of plague.
Pathogenesis and virulence factors
For constant circulation in natural foci, the plague pathogen must penetrate into the host organism, counteract the protective bactericidal systems of the rodent, and reproduce to ensure bacteraemia, essential for further transmission of the infection by fleas to a new host. In the case of a human pneumonic plague outbreak, the bacteria must also overcome host innate immunity to develop pneumonia with abundant exhalation of Y. pestis, causing plague pneumonia iaïve persons. Each of these stages in the cyclic existence of Y. pestis is supported by numerous factors of the plague pathogen, including pathogenicity factors and housekeeping genes (Table 1⇓), which may exert an influence, jointly or individually, upon various stages of the infectious process or transmission. The removal of any one of these components may or may not render the organism avirulent. However, only in aggregate do these factors ensure survival of Y. pestis in the host organism, no matter how significant or insignificant their individual effect might be (Anisimov et al., 2004; Brubaker, 1991; Perry & Fetherston, 1997).
The main pathogenicity factor of Yersinia resides in the complex traits encoded by the plasmid pCad (also termed pCD, pVW, pYV or pLcr), the presence of which in the cell is essential for the manifestation of virulence (Portnoy & Falkow, 1981). It is considered as the Yop virulon: the system that permits extracellular bacteria to disarm the cells involved in the host immune response, disrupt their binding, and induce their apoptosis by injection of bacterial effector proteins. This system consists of the Yop proteins and the apparatus of their type III secretion, called Ysc. The Ysc apparatus consists of 25 proteins, while most of the Yops can be divided into two groups according to their functions. Some of them are intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, YopT), whereas others (YopB, YopD, LcrV) form the translocation apparatus, which is unfolded on the surface of the bacterium to deliver the effectors into eukaryotic cells through the plasma membrane. The secretion of Yops proteins is triggered by contact with eukaryotic cells. In the absence of attachment, yersiniae do not release Yops from the bacterial cell. The adhesion mechanism is not yet known, but it seems that close contact with eukaryotic membrane lipids alone might be enough to trigger secretion. The secretion channel may be blocked at different levels by proteins of the virulon, including YopN, TyeA and LcrG, which cover the bacterial secretory channel, presumably in the form of a trap door. The precise functioning of the system also requires several chaperones, called Syc proteins, that assist Yops to be secreted by the injectisome. Each of these chaperones serves only one Yop, and they do not leave the bacterial cytosol. Transcription of the genes is controlled by the temperature and activity of the secretion apparatus (Cornelis, 2002; Viboud & Bliska, 2005).
Infective doses of 10 bacteria or less are sufficient to cause lethal infection iaïve rodents and primates via the intradermal, subcutaneous and intravenous routes (Anisimov et al., 2004; Brubaker, 1991; Perry & Fetherston, 1997). LD50 values in the case of the respiratory and oral routes of infection increase to 102–104 c.f.u. (Anisimov, 2002a; Anisimov et al., 2004; Ehrenkranz & Meyer, 1955) and 105–109 c.f.u. (Anisimov, 2002a), respectively.
Y. pestis is primarily a rodent pathogen, and is usually transmitted to another mammalian host by fleas, following the death of the rodent. After being introduced into the host, the bacterium is initially susceptible to phagocytosis and killing by neutrophils (Cavanaugh & Randall, 1959) and CD11c+ cells (Bosio et al., 2005), but it may survive and multiply within macrophages. Released bacteria are resistant to capture by neutrophils and begin an unchecked propagation (Cavanaugh & Randall, 1959). The bacteria then spread from the initial flea-bite site to the lymph nodes, where local replication causes a suppurative lymphadenitis (bubo). If the lymphatic system becomes overwhelmed, septicaemia results, with Y. pestis spreading to other organs. The LPS of Y. pestis causes the development of endotoxic shock peculiar to other Gram-negative infections (Butler, 1983; Dmitrovskii, 1994; Van Amersfoort et al., 2003). After the development of highly contagious plague pneumonia, the disease spreads in the air in droplets (Lien-Teh, 1926; Lien-Teh et al., 1936; Rudnev, 1940).
Clinical manifestations
There are three main primary forms of human plague, bubonic, septicaemic and pneumonic. Complications such as secondary septicaemic plague, secondary pneumonic plague, plague meningitis, plague endophthalmitis and multiple lymph node involvement result from bacteraemic dissemination of the pathogen (Butler, 1983; Dennis et al., 1999; Lien-Teh et al., 1936; Naumov & Samoilova, 1992; Nikolaev, 1972; Pollitzer, 1954; Rudnev, 1940). Septicaemia may be followed by digital gangrene (Kuberski et al., 2003).
Irrespective of clinical form, the mean incubation period is 3–6 days, but may decrease to 1–2 days in patients with primary septicaemic or pneumonic plague, and increase in vaccinated people. All of the clinical forms may be characterized by sudden onset of illness, malaise, chills and temperature increase up to 39–40 °C, headache, myalgia, insomnia, indistinct speech, wobbly walk, and sometimes vomiting. In the case of serious illness, patients are delirious, and violent and aggressive. Attempts to make an escape from a patient care institution may be considered as a sign of developing meningitis. In extremely severe cases, a profound prostration and cyanosis can be observed.
Fatality for untreated bubonic plague varies from 40 to 60 %, while untreated septicaemic and pneumonic forms of the disease are always lethal (Albizo & Surgalla, 1970; Butler, 1983; Dennis et al., 1999; Dmitrovskii, 1994; Kool, 2005; Krishna & Chitkara, 2003; Lien-Teh, 1926; Lien-Teh et al., 1936; Naumov & Samoilova, 1992; Nikolaev, 1972; Pollitzer, 1954; Rudnev, 1940).
Bubonic plague
Bubonic plague results from a flea bite or direct contact of an open skin lesion with plague-infected material. Sometimes, a vesicle, pustule or ulcer develops at the inoculation site. Y. pestis spreads via the lymphatic vessels to the regional lymph nodes, causing inflammation and swelling in one or several nodes: buboes (‘bubo’ is derived from the Greek ‘boubon’, groin). Buboes are usually no greater than 5 cm in diameter, extremely tender, erythematous, and surrounded by a boggy haemorrhagic area. Buboes typically arise in the inguinal and femoral regions, but also occur in other regional lymph node sites, including popliteal, axillary, supraclavicular, cervical, post-auricular, pharyngeal and other sites. Deeper nodes (such as intrabdominal or intrathoracic nodes) may also be involved through the lymphatic or haematogenous spread of Y. pestis. Initial symptoms include malaise, high fever, and one or more painful lymph nodes. Pulse rate is increased to 110–140 min−1. Blood pressure is low, usually about 100/60 mm Hg, due to extreme vasodilatation. Circulatory collapse, haemorrhage and peripheral thrombosis are the terminal events (Butler, 1983; Dennis et al., 1999; Lien-Teh et al., 1936; Naumov & Samoilova, 1992; Nikolaev, 1972; Pollitzer, 1954; Rudnev, 1940).
Septicaemic plague
Septicaemic plague may occur primarily as a result of infection with massive doses of the pathogen, or when the bacillus is deposited in the vasculature, bypassing the lymphatics. In this case, bacteria proliferate in the body without producing a bubo. Secondary septicaemic plague is a complication of haematogenous dissemination of bubonic plague. The presenting signs and symptoms of primary septicaemic plague are essentially the same as those of any Gram-negative septicaemia: fever, chills, nausea, vomiting and diarrhoea. Later, purpura, disseminated intravascular coagulation, and acral cyanosis and necrosis may be seen. Other symptoms include abdominal pain and digital gangrene (Albizo & Surgalla, 1970; Butler, 1983; Dennis et al., 1999; Dmitrovskii, 1994; Lien-Teh et al., 1936; Naumov & Samoilova, 1992; Nikolaev, 1972; Pollitzer, 1954; Rudnev, 1940; Van Amersfoort et al., 2003).
Pneumonic plague
Pneumonic plague is rare, but is the most dangerous and fatal form of the disease, and is spread via respiratory droplets. It can develop as a secondary complication of septicaemic plague or result from the inhalation of infectious respiratory excreta of humans or animals with the pneumonic form of infection. The onset of pneumonic plague is acute and fulminant, with high fever, chills, headache, malaise, myalgia and productive cough (with sputum that may be clear, bloody or purulent) within 24 h of the onset of symptoms. Buboes on the neck are rare, but are a symptom of pneumonic plague. Nausea, diarrhoea, vomiting and abdominal pain may also accompany the disease. The pneumonia progresses rapidly, resulting in dyspnoea, stridor and cyanosis. The disease rapidly engulfs the lungs and haemorrhages develop, filling them with fluid (a haemorrhagic pneumonia). The terminal events are respiratory failure, circulatory collapse, and bleeding diathesis (Albizo & Surgalla, 1970; Butler, 1983; Dennis et al., 1999; Dmitrovskii, 1994; Kool, 2005; Krishna & Chitkara, 2003; Lien-Teh, 1926; Lien-Teh et al., 1936; Naumov & Samoilova, 1992; Nikolaev, 1972; Pollitzer, 1954; Rudnev, 1940).
Methods of plague treatment
Until the nineteenth century, the treatment of plague was based on mysticism and superstition. Such ‘remedies’ as magic and talismans, mixtures of bird and animal blood, tablets made from rattlesnake meat, and even material squeezed from fresh horse dung, were widely used (Afanas’ev & Vaks, 1903). Later, methods such as phlebotomy, emetics, purgatives and diaphoretics were also applied to plague treatment (Rudnev, 1940). The isolation of the plague pathogen, Y. pestis, in 1894 (Yersin, 1894) made possible scientific approaches to the cure of plague-infected patients. The local application of antiseptics such as iodine, mercuric chloride, carbolic acid or quinine, together with the incision or even searing of bubos, were promising in some cases, as were attempts to surmount severe systemic disease by the use of the same remedies, specific bacteriophages or animal hyperimmune sera (Lien-Teh et al., 1936; Rudnev, 1940); however, real success in plague therapy was observed when sulfanilamides (Carman, 1938) and then streptomycin (Hornibrook, 1946) became available.
Antibiotic prophylaxis and therapy
Currently, antibiotics are the cornerstone of plague treatment. The antibiotic prophylaxis and therapy for Y. pestis infection, as suggested by the WHO Expert Committee on Plague (1970), focuses on the use of tetracycline, streptomycin, and chloramphenicol for eradication of the organism. More recently, the US Working Group on Civilian Biodefence has added gentamicin, doxycycline and ciprofloxacin to this list (Inglesby et al., 2000). Chloramphenicol is optional for the treatment of plague meningitis (Becker et al., 1987). Aminoglycosides (streptomycin, kanamycin, tobramycin, gentamicin and amikacin) and cephalosporins (ceftriaxone and ceftazidim) are recommended for the treatment of plague caused by F1-negative Y. pestis strains that are resistant to doxycycline, ampicillin and cefoperazone in vivo. Studies in mice suggest that an increase in the daily doses of less-efficient drugs such as cefotaxime, cefoperazone, sulbactam/ampicillin, azthreonam, ciprofloxacin and rifampicin, along with prolongation of the treatment course for up to 7 days, make it possible to increase the protective effects to 80–100 % (Anisimov et al., 2004; Ryzhko et al., 1998). Furthermore, studies using ciprofloxacin, doxycycline and the newer fluoroquinolones gatifloxacin and moxifloxacin have shown an increase in the survival of mice presenting with pneumonic plague (Byrne et al., 1998; Russell et al., 1998; Steward et al., 2004).
It is recommended that antibiotic therapy be started as early as possible. A delay in antibiotic therapy will result in an increase in bacterial biomass, and in a more harmful subsequent inflammatory response caused by endotoxin release induced by both lytic and non-lytic antimicrobials (Nau & Eiffert, 2002). During the first few hours of antibiotic treatment, patients should be monitored closely, because shock may develop after the start of antibiotic application due to bacteriolysis, with the subsequent release of large amounts of endotoxin (Jacobs et al., 1990). Those in contact with pneumonic plague patients and persons who have been exposed to aerosols should receive doxycycline, ciprofloxacin or chloramphenicol as post-exposure prophylaxis (Inglesby et al., 2000). The recent increase in multidrug-resistant strains (Galimand et al., 1997; Wong et al., 2000) has led to renewed efforts to find alternatives to antibiotics.
Immunotherapy
Serum therapy.
A. Yersin and others were the first to use the serum of vaccinated rabbits to cure infected animals (Yersin et al., 1895). In 1896, Yersin was able to cure several patients in Asia with a horse serum (Yersin, 1897). Howevфer, the experiences of other researchers with the use of plague serum were conflicting. The horse, mule or bull sera were produced in different laboratories with the use of different immunization schemes involving killed or live bacteria of different strains. In the course of treatment, patients were injected subcutaneously or intravenously with up to 1000 ml immune serum (200–500 ml for one subcutaneous or intramuscular injection, and 50–100 ml for one intravenous injection). In the case of bubonic plague, the reported death rate in treated (13 %) and untreated (64 %) patients differed significantly. In patients with septicaemic or pneumonic plague, treatment did not cause a detectable decrease in mortality. The sooner treatment was started the better the result. Even in patients that succumbed to infection, an up to twofold increase in time to death was usually reported. The dangers of serum sickness and anaphylactic shock frequently induced by serum therapy were believed to be less significant than that of septic shock (Lien-Teh et al., 1936; Pollitzer, 1954; Rudnev, 1940). In 1970, the WHO Expert Committee on Plague recommended the continuation of further development of plague antitoxic sera that might be used for the treatment of plague patients with severe toxicosis (WHO, 1970).
Antibody therapy.
Beginning in the 1960s, separate antibody preparations were introduced into clinical practice for preventing and treating infectious diseases. In contrast to older remedies based on animal sera, these specific immunoglobulins contained antibodies derived from immunized human donors, and so had minimal side effects. Although human-specific globulins lack the toxicities associated with animal sera, they have several limitations, such as high cost, low availability, and the potential to transmit infectious disease (Buchwald & Pirofski, 2003; Casadevall, 2002; 2005; Keller & Stiehm, 2000).
Hybridoma technology, introduced in 1975 (Kohler & Milstein, 1975), provides a method for the unlimited production of homogeneous mAbs. The construction of mouse–human chimeric or humanized mAbs (Morrison, 1992), completely human mAbs (Kang et al., 1991) from hybridomas or combinatorial libraries, and recombinant human polyclonal antibodies (Bregenholt & Haurum, 2004), makes it possible to reduce the immunogenicity of rodent mAbs in humans. These approaches provide the possibility of raising relevant antibodies against protective antigens only, and even against protective epitopes of protective antigens.
In the case of Y. pestis, several antigens have been shown to be able to produce protective immunity. Among them are F1 capsular antigen (Meyer et al., 1974; Simpson et al., 1990), LcrV or simply V antigen (Leary et al., 1995; Motin et al., 1994; Une & Brubaker, 1984), YopD (Andrews et al., 1999), and type III secretion system needle complex protein, YscF (Matson et al., 2005). Immunodominant epitopes have been found for the F1 (Sabhnani & Rao, 2000; Sabhnani et al., 2003; Zav’yalov et al., 1995) and V (Hill et al., 1997) antigens. Passive administration of antibodies against target antigens protects macrophages from Y. pestis-induced cell death, promotes phagocytosis (Cowan et al., 2005; Philipovskiy et al., 2005; Weeks et al., 2002), and protects animals against both bubonic and pneumonic plague (Anderson et al., 1997; Friedlander et al., 1995; Green et al., 1999; Hill et al., 2006; Motin et al., 1994; Roggenkamp et al., 1997; Une & Brubaker, 1984; Williamson et al., 2005). Importantly, therapy based on a single antibody against a single antigen or epitope will be ineffective in the case of infection with a virulent strain lacking the antigen or expressing a different serological variant of the antigen (Anisimov et al., 2004; Friedlander et al., 1995; Roggenkamp et al., 1997).
Non-pathogen-specific immunomodulatory therapy.
Y. pestis is known to efficiently overcome the innate immune system of many mammals. However, it has been shown that neutrophils (Cavanaugh & Randall, 1959) and CD11c+ cells (Bosio et al., 2005), which represent the initial line of host defence against invading pathogens, play an important role in suppressing the initial replication and dissemination of inhaled Y. pestis. Recent studies (Liles, 2001) conducted in vitro and in vivo have shown that granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor and interferon-γ can augment the functional antimicrobial activities of neutrophils. Studies conducted in animal models have shown the potential use of each of these cytokines for the treatment of bacterial infections. We can speculate that such adjunctive treatment may be useful for plague therapy.
Y. pestis mediates septic shock, which contributes significantly to host death (Butler, 1983; Dmitrovskii, 1994). Sepsis and the systemic inflammatory response syndrome are accompanied by the inability to regulate the inflammatory response (Van Amersfoort et al., 2003). The most potent class of cholesterol-lowering drugs, statins, has been shown to also possess cholesterol-independent effects, including diverse immunomodulatory and anti-inflammatory properties (Almog, 2003). Recent studies have demonstrated that simvastatin (Merx et al., 2004) and cerivastatin (Ando et al., 2000) pretreatment profoundly improves survival in a murine model of sepsis. Another retrospective study has shown that statin therapy reduces both overall and attributable mortality in patients with bacteraemia (Liappis et al., 2001). The lipid profiles of the statin-pretreated patients were considerably higher than those of the no-statin group, and it has been suggested that the medicinal efficacy is a result of the attenuation of the intensity of the inflammatory response due to binding of endotoxins by increased amounts of lipids (Almog et al., 2004).
Several other immune response modifiers that target different stages of innate immunity and might be useful for plague therapy have recently been reviewed (Amlie-Lefond et al., 2005; Masihi, 2000), but only one, glutoxim, a sulfur-containing hexapeptide with an immunomodulatory effect on lymphocytes (Fimiani et al., 2002), has been used in an animal model of plague (Zhemchugov, 2004). When injected into mice (12.5 mg per animal) 6 h before challenge with 10 LD50 of the virulent Y. pestis strain 231, it protected half of the animals from death. Glutoxim has also been shown to protect mice from tularaemia and melioidosis. However, the data thus far are inconclusive, as they were obtained in isolated experiments that employed a minimal number of laboratory animals.
Phage therapy
Bacteriophages seem to be good candidates for antibacterial therapy: they often possess high species specificity, they are non-toxic to eukaryotes, and they kill the target bacteria that they infect and within which they multiply. After the discovery of bacteriophages by Twort in 1915, and independently by d’Herelle in 1917, a number of researchers and physicians were overenthusiastic in the use of bacteriophages for the therapy of bacterial disease (Skurnik & Strauch, 2006; Sulakvelidze, 2005; Summers, 1999, 2001). With respect to plague, in 1925, d’Herelle used a highly virulent anti-plague phage that had been isolated in 1920 in Indo-China from rat faeces to treat four cases of bubonic plague as the first bacteriophage therapy (d’Herelle, 1925). All the patients had laboratory-diagnosed bubonic plague with very serious clinical signs. d’Herelle treated all four with anti-plague phage preparations by single/double direct injection of phage (0.5–1 ml) into the buboes. Within several hours of injection, the patients felt better, a 2 °C average fall in temperature was recorded, and severe pain in the buboes decreased. All four patients recovered in what was considered a remarkable fashion. On the basis of this work, a number of attempts to confirm the efficacy of the phage therapy of plague both in animal models and in clinical trials were performed (d’Herelle, 1925; Flu, 1929; Fonquernie, 1932; Lien-Teh et al., 1936; Naidu & Avari, 1932; Rudnev 1940). However, a poor understanding of the mechanisms of phage–bacterial interactions, including lysogeny and phage DNA restriction, and poorly designed and executed experiments and clinical trials, together with the use of undefined phages in the form of non-purified phage preparations, led to conflicting results (Skurnik & Strauch, 2006; Sulakvelidze, 2005; Summers, 1999, 2001).
Bacteriophages have received renewed attention as possible agents against bacterial pathogens. Evidence from several recent trials designed and executed in accordance with good laboratory practice (GLP) regulations indicates that phage therapy can be effective, although the efficacy of the phage treatment depends greatly on the route (oral, intramuscular, aerosol spray, etc.) and/or frequency of phage application (Skurnik & Strauch, 2006; Sulakvelidze, 2005; Summers, 1999, 2001). Numerous obstacles to the use of phage as antimicrobials for clinical practice remain. Among them are the issue of phage resistance, and the possibility of phage-mediated transfer of undesirable genetic material to bacterial hosts. Therapy based on the simultaneous use of several phages with known genetic sequences and targeting different bacterial receptors can help in overcoming these obstacles. Currently, the genomes of several Y. pestis-specific phages have been sequenced (Filippov et al., 2005; Garcia et al., 2003), and they could be candidates for the revival of plague phage therapy.
A phage-encoded peptidoglycan-degrading activity is responsible for the cell-wall-hydrolysing action of bacteriophages. At least four types of these enzymes, endolysins, are responsible for this activity in different phages (Young, 1992). Taking into account that if a sufficiently high phage dose is given, phages are able to control infection through a non-proliferative lytic mechanism (Berchieri et al., 1991; Goode et al., 2003), the therapeutic use of endolysins in a controlled fashion seems advantageous in comparison with the uncontrollable increase of the live active agent.
Bacteriocin therapy
Bacteriocins, bacterially produced antimicrobial peptides, range from the well-studied, narrow spectrum, high-molecular-mass colicins produced by Escherichia coli and the short polypeptide lantibiotics of lactic acid bacteria to the relatively unknown halocins produced almost universally by halobacteria. Purified bacteriocins could be used for the reduction or elimination of certain pathogens (Braude & Siemienski, 1965; Riley & Wertz, 2002). It has been shown in assays for potency that purified colicins V and K have similar inhibitory activities on a per weight basis to those of the therapeutic antibiotics kanamycin, streptomycin and oxytetracycline (McGeachie, 1970). Bacteriocins nisin A (produced by Lactococcus lactis) and mutacin B-Ny266 (produced by Streptococcus mutans) are as active as vancomycin and oxacillin against most strains tested (Bacillus, Enterococcus, Lactococcus, Listeria, Mycobacterium, Pediococcus, Staphylococcus, Bordetella, Clostridium, Peptostreptococcus, Propionibacterium, Streptococcus and Micrococcus). Furthermore, mutacin B-Ny266 remains active against strains that are resistant to nisin A, oxacillin and vancomycin (Mota-Meira et al., 2000).
Intraperitoneal injections of mutacin B-Ny266 have been used for the treatment of mice infected with Staphylococcus aureus. While there was 100 % mortality in the control group of mice, no mortality was observed in the mice injected with vancomycin or mutacin B-Ny266 (Mota-Meira et al., 2005). Lacticin 3147, a broad-spectrum bacteriocin produced by the food-grade organism L. lactis, reduced the incidence of mastitis after experimental challenge with Streptococcus dysgalactiae ion-lactating dairy cows (Ryan et al., 1999). Local injections of staphylococcin A-1262a were used to treat 50 patients with a variety of staphylococcal lesions. Complete recovery was observed in 42 of the patients (Lachowicz, 1965). The streptococcal bacteriocin tomicide was used for protection of white mice from staphylococcal infection. Tomicide, administered in the maximum dose admissible for mice, ensured the protection of up to two-thirds of the total number of mice. A single oral administration of the preparation immediately after infection protected one-third of the surviving mice from local staphylococcal infection (Blinkova et al., 2003). The pronounced prophylactic effect of tomicide, manifested by a reliable decrease of group A streptococcal carrier state, as well as by a decrease in morbidity in respiratory streptococcal infection among children in a test group in comparison with controls, has been reported (Briko & Zhuravlev, 2004). Enterocoliticin, a phage-tail-like bacteriocin, has been administered as an antimicrobial compound by the oral route for the treatment of BALB/c mice infected with Yersinia enterocolitica, the nearest relative of Y. pestis. The increase in the number of Y. enterocolitica c.f.u. in animals was retarded at time points shortly after the application of enterocoliticin, indicating that the bacteriocin was effective during the early phase of infection (Damasko et al., 2005). A purified class IIa bacteriocin, secreted by Paenibacillus polymyxa NRRL-B-30509, has been incorporated into chicken feed, and dramatically reduced both intestinal levels and the frequency of chicken colonization by Campylobacter jejuni (Stern et al., 2005). The same bacteriocin has been shown to inhibit the growth of Y. pestis in vitro (E. A. Svetoch and B. V. Eruslanov, personal communication) in doses similar to those recommended by the WHO Expert Committee on Plague (WHO, 1970) for antibiotics. The above studies suggest that there is a potential therapeutic effect associated with bacteriocins. When contemplating the clinical use of bacteriocins, one important consideration is their possible pathological effects. In early studies with partially purified bacteriocins (Montgomerie et al., 1973; Tagg & McGiven, 1972; Turnowsky et al., 1973) or strain pairs differing in their expression (Brubaker et al., 1965; Burrows, 1965; Smith, 1974), the data showed their potential toxicity, and also a relationship between the carriage of bacteriocinogenic factors and the virulence of certain strains. However, studies with purified substances have in many cases failed to confirm toxicity (Braude & Siemienski, 1965; Tagg et al., 1976). Although intraperitoneal injection of 10 mg mutacin B-Ny266 kg−1 did not apparently affect the health of mice, these results will have to be confirmed with more relevant toxicity tests (Mota-Meira et al., 2005). The investigation of the in vitro cytotoxicities of two bacteriocins, gallidermin (Staphylococcus gallinarum) and nisin A, in comparison with those of antimicrobial peptides of eukaryotic origin, magainin I, magainin II and melittin, indicated that gallidermin was the least cytotoxic antimicrobial peptide, followed by nisin A, magainin I, magainin II and melittin. Nisin caused haemolysis, but at concentrations which were 1000-fold higher than those required for antimicrobial activity. Gallidermin shows the most promise as a therapeutic agent, with relatively low cytotoxicity and potent antimicrobial activities (Maher & McClean, 2006). In considering the potential toxicity of bacteriocins, the well-known toxicity of antibiotics such as quinolones, doxycycline, streptomycin and gentamicin, and the possible problems associated with massive use of antimicrobial agents for prophylaxis or therapy during a bioterrorist attack, should be noted (Navas, 2002).
Inhibitors of virulence factors
Virulence refers to the ability of an organism to establish an infection and cause disease. Many steps are involved in the infection process, including adherence, invasion and the evasion of host defences (Finlay & Falkow, 1997). Microbial factors involved in the process of virulence are unique, in that their inhibition, by definition, should interfere with the process of infection rather than with bacterial viability. Inhibitors of such targets would be unlikely to affect host cells, to be cross-resistant to existing therapies, or to induce resistance themselves. Bacterial virulence may therefore offer unique opportunities to inhibit the establishment of infection or alter its course as a method of antimicrobial chemotherapy (Alksne, 2002; Y. M. Lee et al., 2003; Marra, 2004).
A necessary step in the successful colonization and, ultimately, production of disease by microbial pathogens is the ability to adhere to host surfaces (Finlay & Falkow, 1997), in many cases involving oligosaccharides located on the host cell surface and bacterial adhesins. The terminal di- or trisaccharide units of these oligosaccharides may be used to inhibit these interactions, preventing attachment and therefore disease. The validity of this approach has been unequivocally demonstrated in experiments performed in a wide variety of animals, from mice to monkeys, and also in humans (Ofek et al., 2003).
Y. pestis expresses a range of adhesins, including pH 6 (PsaA), F1 (Caf1), Pla and S-layer protein (Anisimov, 2002b; Kienle et al., 1992; Parkhill et al., 2001; Payne et al., 1998). pH 6 antigen binds to several human immunoglobulin G subclasses by acting as a bacterial Fc receptor (Zav’yalov et al., 1996). It is also able to bind to gangliotetraosylceramide, gangliotriaosylceramide and lactosylceramide, and also to attach to hydroxylated galactosylceramide. Recombinant pH 6 antigen, present on the surface of intact E. coli cells, possesses the same specificity as the purified antigen, with the exception of binding to non-hydroxylated galactosylceramide. The observed binding patterns indicate that the presence of β1-linked galactosyl residues in glycosphingolipids is the minimum requirement for binding of the pH 6 antigen. The glycosphingolipids recognized by the pH 6 antigen are common and may be found on a range of host cell types (Payne et al., 1998). In fact, pH 6 antigen preparations are able to agglutinate human, rabbit, guinea pig and murine erythrocytes (Bichowsky-Slomnicki & Ben-Efraim, 1963). Studies that are currently in progress in the Laboratory for Plague Microbiology, Department of Infectious Diseases, State Research Center for Applied Microbiology and Biotechnology, indicate that pH 6 antigen is capable of promoting the adhesion of macrophage-like eukaryotic cells to each other and to plastic surfaces, and the formation of cell monolayers. The high-molecular-weight capsular antigen F1 possesses haemagglutinating activity on account of its ability to bind specifically to d-galactosamine-HCl and glucuronic acid (Anisimov, 2002b). An analysis of the complete genome of Y. pestis has revealed the presence of eight more gene clusters similar to the operons psa and caf1, each of which is potentially capable of promoting the expression of the pilus adhesins (Parkhill et al., 2001). Transfer of the pla locus, which encodes the production of the plasminogen activator Pla, to E. coli cells imparted adhesive activity to the latter with respect to a number of eukaryotic cells (Kienle et al., 1992), on account of the ability of Pla to bind to the mammalian extracellular matrix (Lähteenmäki et al., 1998). Y. pestis S-layer protein at a concentration of 8–16 μg ml−1 agglutinated rabbit erythrocytes; this agglutination was inhibited by 0.1 M sugars: rhamnose (fourfold) and dulcitol (eightfold) (Diatlov & Antonova, 1999). Until recently it was customarily considered that the plague pathogen had lost the ability to synthesize adhesin/invasin Ail on account of the insertion of IS285 (Brubaker, 2004); however, in the genome of the Y. pestis virulent strain CO92, this gene is intact (Parkhill et al., 2001). More recently, the Y. pestis genome has been shown to possess two homologues to the Erwinia HecA adhesin (Rojas et al., 2002). Y. pestis lipopolysaccharide is also reported to be an adhesin (Straley, 1993).
Y. pestis, as a species associated with pneumonic infections, adheres more efficiently to an alveolar epithelial A549 cell line than enteric bacteria, including enteric yersiniae. The plague pathogen demonstrates a restricted tropism for oligosaccharides compared with environmental and opportunistic bacteria. It has been shown that the compound with the greatest anti-adhesion activity toward A549 cells is p-nitrophenol (Thomas & Brooks, 2004). As an alternative to antibiotics, the inhibition of attachment can be mediated through the use of oligosaccharide receptor mimics. In a more recent report, Thomas & Brooks (2006) have demonstrated that the attachment of Y. pestis strain GB to the murine monocyte cell line J774A.1 and a range of human respiratory epithelial cell lines is reduced by 55–65 % after pre-treatment of the cell lines with tunicamycin (an inhibitor of the biosynthesis and processing of N-linked oligosaccharides, produced by Streptomyces lysosuperificus). This further demonstrates the potential of oligosaccharides as anti-adhesion therapeutics. Other generic attachment inhibitors include polymeric saccharides (dextran and heparin), GalNAcβ1-4Gal, GalNAcβ1-3Gal, Galβ1-4GlcNAc and Galβ1-3GlcNAc (Thomas & Brooks, 2004). It is possible that mixtures of such compounds may serve as a novel class of therapeutics for respiratory tract infections, including pneumonic plague.
The Ysc-Yop-‘type III’ weaponry delivering effector proteins into the cytosol of the eukaryotic target cell seems to be the main pathogenicity factor of Yersinia. Inhibitors specifically targeting type III secretion are attracting attention (Chen et al., 2003; K. Lee et al.; 2003, 2005; Kauppi et al., 2003a,b; Nordfelth et al., 2005; Xie et al., 2004). The majority of them target YopH, protein tyrosine phosphatase (Chen et al., 2003; K. Lee et al., 2003, 2005; Xie et al., 2004) or YopE, a GTPase-activating protein (Kauppi et al., 2003b). Among such inhibitors are compounds that belong to a class of acylated hydrazones of different salicylaldehydes (Nordfelth et al., 2005), monoanionic squaric acids (Xie et al., 2004), peptidic α-ketocarboxylic acids, and sulfonamides (Chen et al., 2003). A eukaryotic cell model that mimics in vivo conditions has shown that some of the inhibitors attenuate the pathogen to the advantage of the host cell.
Iron is a necessary nutrient for all pro- and eukaryotic cells, and at physiological pH values, iron salts (Fe3+) form an almost-insoluble ferric hydroxide, Fe(OH)3. To assimilate the negligible portion of the iron that is still present in the dissolved state, both in the mammalian organism and in micro-organisms, special iron-binding molecules, siderophores, are synthesized. Bacterial siderophores have a critical role in the competition between parasite and host for iron acquisition (Braun, 2001; Finlay & Falkow, 1997). Therefore, the Y. pestis siderophore yersiniabactin, as well as its receptor Psn (Perry & Fetherston, 1997), are promising targets for the development of new antimicrobial inhibitors to treat plague. An inhibitor of the domain salicylation enzymes required for siderophore biosynthesis in Y. pestis, the arylic acyl adenylate analogue 5′-O-[N-(salicyl)-sulfamoyl] adenosine, has recently been designed, synthesized, and shown to inhibit yersiniabactin biosynthesis and the growth of Y. pestis under iron-limiting conditions (Ferreras et al., 2005). More recently, a set of newly synthesized aryl sulfamoyl adenosine derivatives has also been shown to inhibit yersiniabactin biosynthesis in vitro (Miethke et al., 2006).
Other possible targets for inhibiting Y. pestis virulence are quorum sensing (Suga & Smith, 2003), the two-component regulatory systems (Oyston et al., 2000; Winfield et al., 2005) that govern virulence, and/or the enzymes for the biosynthesis of the LPS that is believed to determine antimicrobial-cationic-peptide (Anisimov et al., 2005; Bengoechea et al., 1998) and serum resistance (Anisimov et al., 2005; Porat et al., 1995).
Symptomatic treatment
Severe cases of plague are characterized by shock and extensive diffuse intravascular coagulation (DIC), two interrelated processes that have common causal mechanisms and reinforce each other (Hardaway, 1982). The standard treatment of the pathophysiological changes accompanying endotoxic shock and DIC consists of administration of fluid and vasopressors to restore blood pressure and organ blood flow, and oxygenation (Cohen & Glauser, 1991; Naumov & Samoilova, 1992; Rackow & Astiz, 1991; Wheeler & Bernard, 1999).
Very important to the management of shock is early fluid resuscitation and the immediate start of mechanical ventilation (Carcillo et al., 1991). In addition to extensive capillary leakage, severe plague cases are characterized by severe cardiac depression (Albizo & Surgalla, 1970; Butler, 1983; Dennis et al., 1999; Dmitrovskii, 1994; Lien-Teh et al., 1936; Naumov & Samoilova, 1992; Nikolaev, 1972; Pollitzer, 1954; Rudnev, 1940; Van Amersfoort et al., 2003). Consequently, pulmonary congestion may develop early, and this limits the amount of fluid that can be administered. In general, inotropic and vasopressive support is needed from an early stage. Dobutamine is preferred for its beneficial effects on cardiac function and peripheral oxygenation (Vincent et al., 1990; Winslow et al., 1973). As for the anti-DIC therapy, currently the only undoubtedly successful treatment is anti-shock therapy. Bleeding due to DIC should probably be treated with replacement therapy: platelets for thrombocytopenia, cryoprecipitate for hypofibrinogenaemia and fresh frozen plasma for decreased coagulation factors (Jacobson & Young, 1986). DIC may cause severe distal necrosis (Welty et al., 1985), and if gangrene has completely demarcated then plastic surgery or amputation may be needed (Kuberski et al., 2003). In the case of evolving gangrene, in a patient with good peripheral blood supply and no bleeding diathesis, sympathetic blockade to preserve peripheral feet tissues has been shown to be successful (Kuberski et al., 2003).
Concluding remarks
The treatment dynamics of plague are of critical interest because of the high human mortality rate of the disease, and current threat for use in bioterrorism. Even though antibiotics such as ciprofloxacin, doxycycline and the newer fluoroquinolones have shown an increase in survival in mice presenting with pneumonic plague, there has recently been a worrying increase in multidrug-resistant Y. pestis strains. This has necessitated the need to look at alternatives to antibiotics as treatment regimens for plague. This review has highlighted some of the promising non-antibiotic therapeutic strategies that can be explored further. Thus, a novel treatment mechanism of pneumonic plague infection may be via inhibiting adhesion of Y. pestis to the respiratory tract. Recent progress in the use of short-chain oligosaccharides as potential anti-adhesion therapeutics, and also a set of newly synthesized aryl sulfamoyl adenosine derivatives as inhibitors of yersiniabactin biosynthesis, make these receptor mimics potential alternatives to antibiotics. Although hopes remain high that these alternative strategies of plague treatment, alone or as part of combination therapies, will provide a valuable new line of defence against multdrug-resistant Y. pestis strains, they remain a long way from clinical practice. The alternative therapies reported in this review should be investigated by comprehensive studies of their clinical applications as a form of preparedness against any possible bioterrorist attack involving the use of antibiotic-resistant Y. pestis.
Acknowledgments
This work was partially funded by US Civilian Research and Development Foundation (CRDF) grant RBO-11012-MO-01 (DOE/LAB agreement 325786-A-K6) and by the International Science and Technology Center (ISTC) grant 2927.
Bubonic plague, caused by Y. pestis, is an ancient disease that has killed millions of people over the centuries. For example, it is believed to have killed more than 100 million persons in an epidemic in the sixth century. Another epidemic in the 14th century killed one fourth of the European population, and the London plague in 1665 killed more than 70,000 persons. In 1893, an epidemic began in Hong Kong and spread to India where more than 10 million individuals died over a 20-year period. This epidemic eventually reached San Francisco in about1900, and the disease is firmly established in the south-western United States (eg, in prairie dogs, ground squirrels, wood rats, chipmunks, and mice), as well as in many other areas of the world.
Morphology. The plague bacillus, as seen in tissue smears, is ovoid-shaped, 1-2 mcm in length and 0.3-0.7 mcm in breadth (Fig.). It is non-motile, forms no spores, and on solid media cultures is elongated in form. In preparations from tissues and cultures Y. pestis is found to have a delicate capsule. The organism stains with ordinary aniline dyes and gives a bipolar appearance, its ends staining more intensively. It is Gram-negative.
Figure. Yersinia pestis recovered from a bubo and colonies of Y. pestis on meat-peptone agar.
Y. pestis
Y. pestis in blood
Y. pestis is characterized by marked individual variability (pleomorphism). In smears from organs and in young cultures it has an ovoid shape, while in cultures on solid media it is elongated and sometimes thread-like. If common salt is added to agar the bacillus shows various forms: ovoid, club-shaped, thread-like, and granular. These forms are usually known as involution forms. The occurrence of filterable types of Y. pestis has also been demonstrated. The G+C content in DNA ranges between 45.8 and 46.8 per cent.
A capsule, a three-layer cell wall, and a three-layer cytoplasmic membrane are demonstrated on ultrathin sections, the cytoplasm is filled with ribosomes and small-granular inclusions and the nucleoid occupies the central part of the cell.
Cultivation. Y. pestis is a facultative anaerobe but can also grow under anaerobic conditions. It is cultivated on ordinary media with pH 6.9-7.0. The optimum temperature for cultivation is 25-30° C The pathogen can also grow at temperatures ranging from 0° to 45° C and at pH from 5.8 to 8.0.
On agar slants the culture forms a viscid translucent mucilaginous mass. On agar plates it forms colonies with turbid white centres and scalloped borders (see fig. 1) resembling lace or crumpled lace handkerchiefs.
In meat broth the cultures form a pellicle on the surface with thread-like growth resembling stalactites and a flocculent precipitate. Sodium sulphite, fresh haemolytic blood, sarcinic extract, and live sarcina (“feeders”) are used as growth stimulators. They are of special value when the seeded material contains a small number of organisms.
Y. pestis colonies
Y. pestis possesses intraspecies variability. It changes quite easily from die virulent R-forms to avirulent S-forms through the O-forms. Resistant S-forms develop in the presence of bacteriophage. They closely resemble the Y. pseudotuberculosis of rodents. Vaccine strains of the plague bacillus are of great practical value and are used for preparing live vaccines.
Fermentative properties. Y. pestis does not liquefy gelatin, nor does it produce indole. It reduces nitrates to nitrites, ferments glucose levulose, maltose, galactose, xylose, mannitol and, occasionally, arabinose with acid formation. Some strains ferment glycerin, while others do not. The differential diagnosis of Y pestis and Y. pseudotuberculosis is very difficult. Unlike Y. pseudotuberculosis of rodents, Y. pestis does not break down adonitol and rarely ferments rhamnose and lactose (Table 1).
Table 1
Differentiation of Yersinia Species
|
Species
|
Carbohydrate fermentation |
Production of Hydrogen
|
|||||
|
adonitol
|
arabinose |
arabitol |
arbutin |
sorbitol |
xylose |
||
|
Y pestis |
– |
+ |
– |
– |
+ |
+ |
+ |
|
Y pseudotuberculosis |
+ |
+ |
– |
+ |
– |
– |
– |
|
Y. enterocolitica |
– |
+ |
– |
+ |
+ |
+ |
+ |
Toxin production. Y. pestis is very virulent for humans. The important virulence factors of Y. pestis seem to be directed toward two goals for the organisms: (1) invasion and proliferation within host cells, and (2) resistance to killing by the host. The incredibly high fatality rate of bubonic plague is probably primarily because of septic shock resulting from the bacteremia occurring in the disease.
Y. pestis produces a variety of different virulence factors, but only a few have been characterized sufficiently well to postulate their role in production of disease: (1) a capsular antigen, designated Fraction 1 (Fl), is antiphagocytic, and antibodies to Fl seem to be protective; (2) V/W antigens, consisting of a protein (V) and a lipoprotein (W), are produced together, but their role as virulence factors is unclear, although there are data that V-antigen suppresses the production of interferon-gamma and tumor necrosis factor-alpha, thus suppressing granuloma formation with the resulting delayed-type hypersensitivity to the organisms; and (3) an intracellular murine toxin that is lethal for the mouse (LD50< 1 mcg) and rat but is essentially inert in other hosts. Also described has been a bacteriocin, Pesticin I, that has a lethal effect on Y. pseudotuberculosis and some E coli strains through an N-acetylglucosammidase-incdiated hydrolysis of peptidoglycan. It is always produced coincidentally with coagulase and fibrinolysin. Mutants unable to synthesize fibrinolysin have a million-fold higher LD50 than wild-type strains if the mutant is injected subcutaneously, but not if the organisms are injected into deeper tissues. This postulated that the ability of fibrinolysin to convert plasminogen to plasmin is essential for the invasion of deeper tissues after the superficial inoculation of the host by an infected flea bite. Interestingly, these factors are encoded on a small plasmid not found in the other pathogenic yersiniae. Other determinants that are associated with virulence include the ability to synthesize purines and the property to absorb and store hemin and certain basic aromatic dyes to form coloured colonies. It is not possible to associate these characteristics with any specific virulence determinant, but mutants unable to synthesize purines or store iron to form coloured colonies display a decreased virulence for experimental animals.
Probably the most confusing virulence factors are encoded on a plasmid termed the low calcium response(Lcr) plasmid. This plasmid is expressed only at
Some of the YOPs have been shown to be virulence factors. YOP E disrupts actin filaments and may be involved in the intenalization of the bacteria into epithelial cells. YOP H is a protein tyrosine phosphatase, and it has been proposed that this protein may be secreted into the cytoplasm of the infected cell where it could cause dephosphorylation of protein tyrosines that are important in signal transduction pathways. YOP H also has been shown to inhibit phagocytosis. Mutants lacking YOPs K and L were rapidly cleared from organs, and it is assumed that these YOPs are able to inhibit cell-mediated immune reactions. YOP M has been shown to inhibit the aggregation of platelets.
The virulence of Y. pestis is closely linked with a number of another factors: the formation of a brown pigment in a culture on hemin agar, synthesis of purines, dehydrase, catalase, the requirement for calcium, strontium and zinc ions, the sensitivity to glucose and survival and growth in macrophages. The continental strains produce a toxic substance urease.The mouse toxin has been obtained in pure form as an active preparation.
Table 2 summarizes those virulence factors that seem to be important in human disease.
Table 2
Yersinia Virulence Factors in Human Disease
|
Factor |
Apparent Function |
|
Fraction 1 capsule |
Antiphagocytic |
|
V/W antigens |
Suppress granuloma formation |
|
Fibrinolysin |
Tissue invasion |
|
Low Ca2+ response gene |
YOP synthesis |
|
YOP H |
Protein tyrosine phosphatase |
|
YOP K & L |
Inhibit cell-mediated immune response |
Antigen structure. The causative agent of plague contains several antigens among which D, F1, T, W, and V-antigens have been studied.
Virulent Y. pestis cells contain thermostable somatic antigen highly toxic for mice and rats which changes to anatoxin under the effect of formalin, as well as haemolysins and other toxic substances. The agar precipitation technique demonstrated antigens in the causative agent of plague, which were also common to Y. pseudotuberculosis, bacteria of the enteric fever and dysentery groups, and human group O erythrocytes.
Resistance. The plague bacillus can withstand low temperatures. At 0° C it lives for 6 months. It survives on clothes for 5-6 months; in sterile soil and in milk for 90 days; in grain and on cadaver for 40 days; in water for 30 days; in bubo pus for 20-30 days; in sputum for 10 days; in vegetables and fruits for 6-11 days; and in bread for 4 days.
Y. pestis is very sensitive to drying and high temperatures. Boiling kills the organism within 1 minute, and when heated to
Pathogenicity for animals. Rodents, among them black rats, grey rats, mice, susliks, midday gerbils, tumarisks, and marmots (tarbagans) are susceptible to plague. More than 300 rodent species may spontaneously contract the disease. In addition, 19 rodent species are susceptible to laboratory infection with plague. Camels died in the Astrakhan steppes m 1911, and humans who ate camel meat contracted plague. Pigs, sheep, goats, donkeys, mules, dogs, cats, monkeys, and certain carnivores are susceptible to the disease iatural environments. However, little epidemiological importance is attached to them.
Guinea pigs, white mice, white rats, and rabbits are the experimental animals which easily acquire the infection. Animals experimentally inoculated with plague display sepsis, necrosis at the site of injection, enlargement of lymph nodes and spleen, haemorrhages in the skin and mucous membranes (haemorrhagic septicaemia).
Pathogenesis and disease in man.
Plague is normally a disease of rodents that exists in two kinds of epidemic centres: the permanent but relatively resistant rat population, where the organisms reside in interepidemic periods; and the temporary but susceptible rodents, particularly the domestic rat population (Fig. 2).
Rats are the primary reservoir: they usually die acutely, with a high-grade bacteremia but occasionally develop amore chronic form of infection. The disease is transmitted by the bites of fleas (e.g., Xenopsylla cheopis, the rat flea) which have previously sucked blood from an infected animal. The ingested bacilli proliferate in the intestinal tract of the flea and eventually block the lumen of the proventriculus. The hungry flea, upon biting another rodent, regurgitates into the wound a mixture of plague bacilli and aspirated blood. If its host dies the flea leaves promptly, seeking a replacement. If another rodent is not available it will accept, a human host, an accidental intruder in the rat-flea-rat transmission cycle.
Thus. the spread of plague to humans is a function of the relative balance between resistant and susceptible species of rats. When the domestic rat population overlaps into the wild rat population, the domestic rats become infected by the bite of an infected rat flea. The domestic rat fleas then become infected and, when biting other rats, regurgitate the plague bacilli into the new host, instituting a new epidemic. As the domestic rats continue to die, the rat fleas will bite humans –intruders in the normal rat-rat flea cycle. Because epidemics of plague usually occur in crowded areas with poor sanitation, it has been proposed that, once started, human-to-human spread also occurs by human fleas.
|
1. Reservoir: Fleas and wild rodents (ground squirrel, gerbil, marmot, cavy) |
1. Reservoir: Rodents in contact with man (house rat, sewer rat) |
|
2. Vectors: Fleas on infected rodents, flea i rodent burrows |
2. Vectors: Fleas |
|
3. Rodent – Flea – Rodent |
Bubonic palgue: Rat – Xenopsilla – Man Man – Pulex irritans – Man Hangling infected rodents |
|
4. Wild Rodent to man – Sporadic human plague. Usually direct contact |
4. Sepicemic plague: Primary – entry throgh mucose. Secondary – complication of bubonic |
|
5. Wild rodent – flea – domestic rodent 6. Domestic rodent – flea – man: human plague |
5. Pneumonic plague: Secondary – complication of bubonic Primary – droplet infection man – to – man |
Sporadic cases of plague also occur world-wide as a result of human contact with infected animals. In the United States, this occurs primarily in rural residents of the south-west and, to a lesser extent, in hikers and campers in that region. Infection in such cases results from being bitten by an infected rodent flea or by handling an infected animal. Of 40 cases of human plague reported in 1983,occurring primarily in New Mexico and Arizona, 6 were fatal. Only four cases, occurred during 1989 and none in 1993.
In Africa, it has been shown that camels and goats can contract plague under natural conditions through contamination of their forage areas by plague-infected rodents. A number of human cases have resulted from contact with carcasses of diseased animals.
A small pustule may be present at the portal of entry in the skin but more often there is no discernible lesion. The bacilli introduced by the flea bite enter the dermal lymphatics and are transported to the regional lymph nodes, usually in the groin, where they cause the formation of enlarged, tender buboes. In severe bubonic plague the regional lymph nodes fail to filter out all the multiplying bacilli; organisms that gain entrance to the efferent lymphatics disseminate via the circulation (septicemic plague) to the spleen, liver, lungs, and sometimes the meninges. The parenchymatous lesions produced are hemorrhagic; disseminated intravascular coagulation may occur. In the terminal stages bacteremia is often intense.
After the flea bite, Y. pestisis transported in the human body to the regional lymph nodes (usually in the groin),causing them to become enlarged, tender nodes called buboes. Oddly, the organisms entering from the flea bite possess neither the Fl capsule nor the antiphagocytic V/W antigens (V/W antigens are not formed at
After leaving the regional lymph nodes, Y. pestis disseminates by way of the bloodstream to the spleen, liver, lungs, and other organs and may cause subcutaneous hemorrhages, which gave bubonic plague the name black death. However, from 1980 to 1984, 25% of the cases of plague occurring in New Mexico (18 of 71 cases)were characterized by positive blood culture results in the absence of a bubo or lymph- node involvement. Such cases are difficult to diagnose because they are similar to septicemia caused by other gram-negative organisms. If lung involvement occurs, the resulting pneumonia also can be spread by respiratory discharges.
The incubation period of bubonic plague in man varies from 1 to 6 days, depending upon the infecting dose. Onset is usually abrupt with high fever, tachycardia, malaise, and aching of the extremities and back. If the disease progresses to the fulminant bacteremic stage, it causes prostration, shock, and delirium; death usually occurs within 3 to 5days of the first symptoms. The course of plague pneumonia is even more fulminant; untreated patients rarely survive longer than3 days. Pulmonary signs may be totally lacking until the final day of illness, making early diagnosis particularly difficult. Late in the disease copious bloody, frothy sputum is produced.
The occurrence of asymptomatic cases is suggested by serological studies on persons living in areas where the disease is endemic and by the finding in Vietnam of a pharyngeal carrier rate of about 10% in family members of plague patients.
Less than 10 organisms of a fully virulent strain of Y. pestis injected into a mouse is lethal. The mechanism by which the plague bacillus produces death is not entirely clear but probably results from septic shock because of the release of tumor necrosis factor-alpha and interleukin-1. It is also noteworthy that a superantigen analogous to TSST-1 and the streptococcal pyrogenic toxins has been reported in Y. pseudotuberculosis, and it seems that such an antigen also may be formed by Y. pestis.
In the pneumonic form of the disease the plague bacilli are spread in the air with sputum expelled by the patient when he coughs or talks. Quite ofteo inflammation is seen at the site of microbe entry. Depending on the location of the pathogen, reactivity of the infected body, virulence of the microbe, and extent of cellular and humoral activity, human plague may be of the following forms: cutaneous, bubonic, cutaneous-bubonic, primary septicaemia, secondary septicaemic (primary pneumonic), secondary pneumonic, and intestinal.
As a rule, plague develops quickly without a prodromal stage. It is characterized by a violent chill, severe headache, and dizzinesss. The face is pale and bluish, with an expression of suffering (horror-stricken), and is known as fades pestica. Each form of plague shows characteristic clinical symptoms. Before the use of streptomycin the mortality rate was very high, i.e. from 40 to 100 per cent. Post-mortem examination reveals centres of inflammation in the lymph nodes, haemorrhages, and haemorrhagic periadenitis. A large number of Y. pestis organisms are present in the lymph nodes and cellular tissue. Phagocytosis is inhibited. There is marked disintegration and necrosis of the buboes. Small haemorrhages form in the skin. The liver is enlarged, showing haemorrhages and necrosis. The spleen is enlarged and dark-red in colour.
The pneumonic foci fuse and assume the form of lobar pneumonia. The lungs are distended, violet-red or grey-red in colour, hyperaemic, oedematous, and contain a large number of Y. pestis organisms.
Immunity. After recovery from the disease a stable immunity of long duration is acquired. Realizing this, in ancient times people living in countries invaded by plague made use of convalescents for nursing plague patients and burying corpses.
Postinfection and postvaccinal immunities are predominantly due to the phagocytic activity of the cells of the lymphoid-macrophage system. An important role is played by the protective capsular antigen which serves as the basis in the preparation of chemical antiplague vaccines.
Laboratory diagnosis. Examination is carried out in special laboratories and in antiplague protective clothing. A strict work regimen must be observed. Depending on the clinical form of the disease and the location of the causative agent, test specimens are collected from bubo content (in bubonic plague), ulcer secretions (in cutaneous plague), mucus from the pharynx and sputum (in pneumonic plague), and blood (in septicaemic plague). Test matter is also recovered from necropsy material (organs, blood, lungs, contents of lymph nodes), rodent cadavers, fleas, foodstuff’s, water, air, etc. Examination is performed in the following stages.
1. Microscopy of smears, fixed in Nikiforov’s mixture and stained by the Gram method or with methylene blue by Loeffler’s method.
2. Inoculation of the test material into nutrient media, isolation of a pure culture and its identification. To inhibit the growth of the accompanying microflora, 1 ml of a 2.5 per cent sodium sulphite solution and 1 ml of a concentrated alcohol solution of gentian violet, diluted in distilled water in a ratio of 1 :100, are added to 100 ml of meat-peptone agar. Prior to inoculation 0.1 ml of antiphage serum is added to the culture to render the plague bacteriophage harmless.
3. Biological tests of the isolated pure culture and of material from which isolation of the organism is difficult are conducted on guinea pigs. In the latter case a thick emulsion prepared from the test material is rubbed into a shaven area of skin on the abdomen. If plague bacilli are present the animals die on the fifth-seventh day. To hasten diagnosis the infected guinea pigs are killed on the second-third day and the plague bacillus is isolated from their organs.
Y. pestis is identified by determining the morphological, cultural, fermentative, phagocytolytic, and agglutinative properties of the isolated culture. The growth is differentiated from the causative agent of rodent pseudotuberculosis (see Table 1). The biological test is decisive in the diagnosis of plague.
Decomposed rodent cadavers are examined by the thermoprecipitin test.
The importance of prompt diagnosis of plague has led 10 the elaboration of accelerated diagnostic methods in recent times.
Treatment At present streptomycin is used for treatment of plague, the drug being very effective and curing even pneumonic plague in a high per cent of cases. Good results have been obtained from a combination of streptomycin with chloromycetin or tetracycline with antiplague serum. Antiplague gamma-globulin and a specific bacteriophage are also used for treatment of plague patients. Penicillin, chlortetracycline, and sulphonamides are recommended in cases with complications.
Prophylaxis. General prophylaxis comprises the following measures:
(1) early diagnosis of plague, particularly the first cases;
(2) immediate isolation and hospitalization of patients and enforcement of quarantine; individuals who have been in contact with patients are placed under quarantine for 6 days and prescribed prophylactic streptomycin treatment;
(3) observation (i. e. isolation of individuals or groups of people suspected of having been in contact with infected material, daily inspection from house to house, temperature measurement twice a day, and observation during the possible incubation period);
(4) thorough disinfection and extermination of rats in disease foci;
(5) individual protection of medical personnel and prophylactic treatment with streptomycin and vaccination;
(6) prophylactic measures and systematic observation carried out by plague control laboratories, stations, and institutes in endemic areas;
(7) observance of international plague control conventions (extermination of rats and disinfection of ships, aircraft, trains, and harbours and, if necessary, compulsory quarantine for passengers);
(8) security measures from plague invasion at frontiers. Specific prophylaxis is accomplished with live EV vaccine. It is produced in dry form and administered subcutaneously, intracutaneously or percutaneously once or twice. Immunity lasts for no longer than one year. Depending on the epidemiological condition, revaccination is conducted in six or 12.months. The efficacy of the vaccination is not high. According to WHO data, a total of 2.5 million of persons with this disease were registered in all the countries of the world in the period between 1921 and 1965 and
Yersinia enterocolitica. Yersinia enterocolitica is a motile, gram-negative rod that is involved primarily in animal diseases. It can be distinguished from Y. pestis on the basis of serologic reactions, biochemical reactions, and pathogenicity in animals. Thirty-four serotypes and five biotypes have been reported.
Yersinia enterocolitica.
Epidemiology and pathogenesis of Y. enterocolitica infections. Yersinia enterocolitica has been isolated from a variety of rodents and domestic farm animals, including cats and dogs. It has also been isolated from lakes and well water, and food-borne transmission (particularly via contaminated milk) is known to occur. Humans frequently acquire the infection from eating raw or undercooked pork (particularly in Europe).
Y. enterocolitica is second only to the Salmonella as a cause of pediatric enterocolitis that is characterized by fever, diarrhea, and abdominal pain. Frequently, considerable swelling of the mesenteric lymph nodes occurs, which, with the abdominal pain, mimics appendicitis. Septicemia is rare but can occur, resulting in lesions through-out the internal organs. Probably the most frequent complication of infections with Y. enterocolitica is a severe arthritis that occurs 1 to 14 days after acute enteritis. The mechanism of the arthritis is unclear, but arthritic patients have been shown to express a multiclonal T-cell response to Y. enterocolitica antigens and to have high titers of IgA antibodies that are not found in patients who do not develop arthritis. Moreover, Yersinia antigen has been demonstrated within synovial fluid mononuclear cells and in synovial membrane cells of patients with Yersinia-induced arthritis. In most cases, the arthritis is self-limiting but, in about 10% of affected patients, it may become chronic. Interestingly, most cases of enterocolitis occur in children whereas most cases of reactive arthritis occur in adults.
Rare cases of Y. enterocolitica bacteremia and endotoxin shock have occurred that were acquired from blood transfusions (7 of 10 patients died). In these cases, the organisms were isolated from residual red blood cells, and at least three of the blood donors gave a history of having diarrhea within 30 days of donating their blood. It is concluded that such persons had an asymptomatic bacteremia at the time of the blood donation and that the organisms grew to large numbers under the conditions of cold storage of the blood. As a result, it has been proposed that blood that is over 25 days old be checked for endotoxin.
Y. enterocolitica colonies
As with Y. pestis, the precise nature of the virulence determinants of Y. enterocolitica is complex. Virulent strains possess a low calcium response plasmid that is probably identical to that described for Y. pestis. Thus, both V and W antigens are produced as well as a large array of YOPs Y. enterocolitica also produces an outer membrane protein called YadA. YadA confers adherence to epithelial cells by binding collagen fibers and fibronectin. It also is responsible for resistance to the bactencidal activity of normal serum by inhibiting the formation of the membrane attack complex of complement. In addition, Y. enterocolitica possesses a chromosomally encoded gene called inv, which encodes for a surface protein termed invasin. Invasin interacts with eucaryotic transmembrane proteins belonging to the beta1 integrin family. Integrins normally tend to various basal membrane proteins such as fibronectin, collagen, and laminin, but when bound to invasin, they induce the internalization of the bacterium into the host cell. A second gene, termed ail, also appears to be involved in cell invasion by Y. enterocolitica, but this gene facilitates entry into only a limited number of host cell types by an unknown mechanism. Cloning of either inv or ail confers an invasive phenotype ooninvasive strains of E. coli. The product of these genes permits the organisms to gain access to the reticuloendothelial system and eventually to disseminate through out the body. Thus, for complete virulence, both plasmid and chromosomally encoded products are required.
Interestingly, Y. enterocolitica produces no siderophores, even though there are membrane receptors for them. This fact has prompted the proposal that the organism’s low virulence in humans may be because of its inability to obtain iron readily.
The mechanism by which this organism causes diarrhea seems to result from the production of a heat-stable enterotoxin, YST, that is similar, or identical, to the ST of E coli. Like ST, the Y. enterocolitica heat-stable enterotoxin manifests its toxicity by stimulation of guanyl cyclase activity, resulting in an increased cyclic guanosine 3′,5′-monophosphate synthesis.
Laboratory diagnosis and treatmrent of Y. enterocolitica infections. Isolation of the organisms from feces, blood, or lymph node biopsy specimens provides the best definitive diagnosis of this infection. Isolation of the organisms from fecal suspensions is facilitated by suspending the fecal specimens in phosphate-buffered saline and holding them at refrigerator temperatures for 2 to 4 weeks before streaking them on enteric media.
Treatment of the enteritis is of little value because patients recover from these infections spontaneously after a few days. Septicemia, however, carries a high mortality and should be treated with gentamicin or chloramphenicol
Yersinia pseudotuberculosis. The disease was first revealed in Vladivostok, and then in Leningrad and other places of the Soviet Union. It is encountered in 30 countries of the world among adults and children in the form of epidemic out-breaks or sporadic cases.
Yersinia pseudotuberculosis is primarily an animal pathogen, infecting both wild and domestic animals. The disease in animals is characterized by necrotic lesions occuring in the liver, spleen, and lymph nodes. Humans acquire the infection by way of an oral route after contact with infected animals.
The most common manifestation of human infection is the painful swelling of the mesenteric lymph nodes, resulting in an appendicitis-like syndrome, although diarrhea and fever also are usual. Rarely does Y. pseudotuberculosis invade the bloodstream, but such infections are associated with a high mortality rate, resulting in clinical symptoms that are similar to toxic shock syndrome. The disease is marked by headache, malaise, vomiting, abdominal pain, fever (38-
Virulence determinants for Y. pseudotuberculosis are essentially identical to those described for Y. enterocolitica.
Laboratory diagnosis consists in isolating the causative agent from the patient’s stool and identifying it by means of the agglutination reaction, phagolysis, and biochemical properties and in demonstrating the antibodies by the reaction of agglutination and indirect agglutination. Chloramphenicol and pathogenic measures are used for the treatment of patients. Prophylaxis comprises the extermination of rats, protection of foodstuff’s and water against rodents, the observance of sanitary and hygienic regimens at catering establishments, food storehouses, etc.
Ampicillin, streptomycin, or tetracycline are effective for treatment of bloodstream infections by Y. pseudotuberculosis. Antibiotic therapy probably is not necessary for the mesenteric lymph node infection inasmuch as such infections spontaneously resolve after several days.
Diagnosis of diseases in detail
The causal organism of plague, which is an acute, particularly dangerous zoonotic infection, is Yersinia pestis. The nature of the material obtained from the patient depends on the clinical form of plague: a puncture sample from a bubo (bubonic plague), exudate of an ulcer (cutaneous form), sputum, a smear from the fauces (pulmonary form), or blood (septic form). Section material (blood, internal organs, lymph nodes) is also examined. Corpses of rodents, fleas, water, foodstuffs, air, and other objects are also tested. The material is collected with special precautions by the operator wearing a type 1 antiplague suit. Bacterioscopic, bacteriological, serological, and biological examination is carried out with a diagnostic purpose.
Bacterioscopic examination. Make several smears from the material collected and fix them in Nikiforov’s mixture. The smears are stained by the Gram technique and simultaneously with methylene blue, using the Loeffler method. If bacterioscopic examination reveals Gram-negative ovoid bacteria surrounded by a tender capsule and stained bipolarly with the Loeffler technique, a preliminary report with the description of the morphology of the bacteria recovered is issued. The smears may also be treated with labelled luminescent antiserum against Y. pestis (direct immunofluorescence).
Bacteriological examination. The material to be tested is inoculated into appropriate nutrient media: blood (5-10 ml), in 100 ml of meat-peptone broth (in vials); puncture sample from a bubo, exudates from an ulcer, sputum, and other materials, onto plates with meat-peptone agar (pH 7.2-7.3). To stimulate the growth of the plague bacteria, 0.1 per cent of rabbit or horse blood is added to the meat-peptone agar. To inactivate the plague phage, 0.1 ml of antiphage serum is placed and uniformly spread on the medium surface. To suppress the growth of extraneous decaying microflora in the tested material, to 100 ml of meat-peptone agar add 1 ml of 2.5 per cent sodium sulphite and 1 ml of saturated alcohol solution of gentian violet diluted 1:100 with distilled water. Place the inoculated cultures in the incubator at 25-
Pure culture of the plague bacteria is isolated in the usual manner and then identified by morphological, cultural, fermentative, agglutinating, phagolytic, and biological properties.
From a fermentative standpoint, the causative agents of plague are active: they split (with the formation of acid) glucose, fructose, galactose, xylose, mannitol, and occasionally arabinose. Some strains may also ferment glycerol. On the other hand, Y. pestis does not split adonite, does not liquify gelatin, does not form indol, and reduces nitrates to nitrites.
For the agglutination test diagnostic sera against somatic and capsular antigens are employed. Sera containing O-antibodies (somatic) give a group reaction with the causative agent of pseudo-tuberculosis. Sera against capsular antigens are distinguished by great specificity.
To identify the causative agent of plague, one can use the agglutination test with lyophilized erythrocyte diagnoіticum loaded with antibodies of antiplague serum.
Investigation of the above attributes does not always allow differentiation of the plague causative organism from similar bacteria of pseudotuberculosis (Yersinia pseudotuberculosis). To distinguish between the se microorganisms, consideration of a number of other signs is helpful (Tabl. 4).
Table 4
Some diagnostic signs differentiating the bacteria of plague
from those of pseudotuberculosis in rodents
|
Yersinia pestis |
Yersinia pseudotuberculosis
|
|
1. Fresh strains do not usually ferment rhamnose |
1. Fresh strains usually ferment rhamnose |
|
2. Do not ferment adonite
|
2. Ferment adonite with the formation of acid |
|
3. Do not ferment urea |
3. Ferment urea |
|
4. On desoxycholic citrate agar they grow with the formation of red colonies |
4. On desoxycholic citrate agar they grow with the formation of yellow colonies |
|
5. Are lysed by the plague phage to the titre |
5. Do not undergo lysis |
Study of the isolated culture of the plague bacteria for their phago-lysis is carried out in the conventional manner on a meat-peptone agar and by adding the plague phage (in the amount of one-tenth of the culture volume) to a 3-hour broth culture in liquid media. The phage is diluted to the titre and various dilutions are tested.
Biological examination is conducted together with bacterioscopic and bacteriological one. Guinea pigs and mice are infected by various techniques: epicutaneously, subcutaneously, and intraperitoneally. Inoculation with the post-mortem material is done in the following way: shave a definite area of the animal skin and scarify it, place on the skin 2-3 drops of the test liquid, and rub it in until the skin is dry with a flat part of the lancet. The blood of the patient and suspension of the isolated pure culture are injected intraperitoneally; the sputum and bubonic puncture specimen are introduced subcutaneously in the internal surface of the hip.
There should be no ectoparasites on the animals to be tested. Infected guinea pigs are kept in tall glass jars closed tightly by two layers of gauze soaked in lysol solution. Mice are also kept in jars which are placed into metallic net bags with iron lids.
Animals inoculated epicutaneously die on the 5th-7th day. To reduce the time of examination, one of the infected animals is sacrificed in 2-3 days. Isolate the culture from the post-mortem blood and internal organs and identify it, using the above described technique.
The following patho-anatomical alterations found during autopsy indicate the presence of plague: multiple haemorrhages oil mucosal and serous membranes, inflammation of lymph nodes, presence of necrotic foci in the spleen, and less commonly in the liver. Examination of the abdominal cavity reveals viscous exudate and degeneration of the parenchymatous organs. The presence of a large number of Gram-negative bipolarly stained bacteria in smears from the internal organs, blood, and exudate also suggests the positive diagnosis.
When decayed cadavers of rodents are examined, use the precipitation test since isolation of pure bacterial culture in this case is difficult.
Prior to autopsy, immerse the carcasses of the rodents for several minutes in kerosene to kill the insects, then proceed with an autopsy looking for patho-anatomical alterations, make impression smears from the internal organs, and inoculate for pure culture. The performance of the biological test is mandatory. If the post-mortem examination involves a large number of rodents, the group biological test is employed, i.e., a guinea pig is inoculated with suspension from the lymph nodes or spleens of 15-20 cadavers to be examined.
Serological examination is carried out for a retrospective diagnosis as well as during epizootic examinations iatural foci of plague.
Indirect haemagglutination with erythrocytes, which have adsorbed the capsular antigen of the causative agent of plague, is performed. The result is considered positive if haemagglutination occurs at a 1:40 dilution of the serum obtained from sick or dead animals.
Along with the above techniques of recovery of the causative agent of plague, one can employ variable methods including phago-diagnosis, the immunofluorescence test, the method of a rapid growth of the responsible pathogen on enriched and elective media, etc. According to the latter method, the material (0.2-0.3 ml) is inoculated into four test tubes with semi-solid agar containing blood and gentian violet, with 0.2-0.3 ml of the bacteriophage being added to one of the tubes. Simultaneously, 0.1 ml of the material is streaked onto plates with agar of the same composition. The inoculated cultures are incubated at 28 “C. From test tubes in which macroscopic inspection conducted 3 hours later elucidates growth, transfer 2-3 drops onto two glass slides, prepare smears, fix them in Nikiforov’s mixture, stain with methylene blue and by the Gram technique.
A positive result is recognized by the appearance of chains of Gram-negative bipolar rods in smears, while the tube with the phage shows no growth. Aspirate 0.4-ml portions of the material from the tube with a visible growth and administer intraperitoneally to several albino mice. In 8-10 hrs, examine the plates with the agar for growth of the causative agent of plague.
Some 10-12 hrs after the inoculation, kill the mice and introduce pieces of organs and exudate of the abdominal cavity into test tubes with a semi-solid agar containing blood and gentian violet. Simultaneously, streak onto plates with agar. Inspect the test tubes in 4 hrs and the plates in 8 hrs.
Hence, a preliminary answer may he given 4 hrs after the onset of the examination and the final one, in 18-20 hrs.
Brucella and Francisella
Causative Agent of Tularaemia. The specific cause of tularaemia (Francisella tularensis} was discovered in 1912 by G. McCou and C. Chapin in Tulare (California) and studied in detail by E. Francis.
Morphology. The tularaemia bacteria are short coccal-shaped or rod-like cocci (Fig.) measuring 0.2-0.7 mcm. In old ‘cultures the organisms retain the coccal form. They are non-motile, polychromatophilic, and Gram-negative. In the animal body they are sometimes surrounded by a fine capsule.
Figure. Causative agent of tularaemia from the blood of water rat (left) and colonies on fish-yeast agar (right)
Tularaemia bacteria
Tularaemia bacteria are pleomorphous. They may assume a club-like structure or the form of very small cocci (0.1-0.2 mcm) which pass through filters. Average-sized cocci, very large spherical forms, or spherical forms with kidney-like protrusions are also to be found. Smears demonstrate rod-like and thread-like forms of the organism, which may reach 8 mсm in length. In animal organs the tularaemia bacteria are seen mainly as cochlea bacteria or as rod-like forms, while in cultures cochlea forms are more often observed. Cultures of low virulence (vaccine strains) are cochlea-shaped, larger than those of the virulent strain, and, as a rule, do not have a capsule.
Submicroscopic structure. The cell cytoplasm is granular, the nucleoid is in the central part of the cell. During division the cytoplasmic membrane grows into the cell and sporulation occurs.
Cultivation. The tularaemia organism is an aerobe which does not grow on ordinary media, but grows well at 37° C on media rich in vitamins, e. g. yolk medium which consists of 60 per cent of yolk and 40 per cent of a 0.85 per cent sodium chloride solution with pH 6.7-7A. The organisms are cultured in a thermostat for 2-14 days.
Tularaemia bacteria multiply on cystine agar, containing 0.05-0.1 per cent cystine and 1 per cent glucose. The mixture is boiled for several minutes, cooled to 40-
Tularaemia bacteria colonies Tularaemia bacteria growth
When cultivated in the laboratory, tularaemia organisms lose the K-antigen with which their virulence and immunogenic properties are associated.
Fermentative properties. Tularaemia bacteria break down proteins with the elimination of hydrogen sulphide, and do not produce indole. They ferment glucose, levulose, mannose, and maltose, with acid formation. Dextrin, saccharose, and glycerin fermentation is not a stable property. Biochemical properties are unstable and liable to comparatively rapid changes. This is due not only to the properties of the bacteria themselves but also to the nutrient media and to the ability of the tularaemia organism to break down proteins. The products of this process mask the production of acids which occurs simultaneously.
Toxin production. The existence of a soluble toxin in tularaemia bacteria has not been demonstrated. The organism’s virulence is associated with its K-antigen. The tularaemia bacterium grows poorly in liquid media, and for this reason it is difficult to isolate any toxin.
Antigenic structure. Agglutination test and precipitin reaction are highly specific. Tularaemia bacteria have been shown to possess thermostable specific haptens. The common character of the antigens of tularaemia and brucellosis agents in the agglutination reaction has been ascertained. This fact must be taken into account in serological diagnosis of these diseases.
The R-form cultures containing only the O-antigen are avirulent and possess no immunogenic properties. The S-form which contains K- and 0-antigens is more common. The intermediate SR-forms from which live vaccines are prepared contain the O-antigen and, in a smaller number, the K-antigen. With prolonged growth on laboratory media all cultures transform to the R-form.
Since complete avirulence is accompanied by the loss of immunogenic properties, a certain degree of virulence for white mice is preserved (residual virulence) in tularaemia strains from which the vaccine is prepared.
Classification. Two varieties of tularaemia organisms can be distinguished: the American type which is highly pathogenic for rabbits and ferments glycerin, and the European type which is non-pathogenic for rabbits and does not ferment glycerin. The former variety is also more virulent for human beings, causing death in 5-6 per cent of tularaemia cases. The second variety was responsible for a low death rate among humans, mortality being 0.5 per cent.
Resistance. The tularaemia organism lives in glycerin for 240 days, in grain for
Pathogenicity for animals. The organism is pathogenic for water rats, field voles, grey rats, common field mice and house mice, hares, susliks, chipmunks, hamsters, muskrats, gerbils, moles, shrews, and other animals. Among the domestic animals camels, sheep, cats, dogs, and pigs are susceptible to the disease, and among laboratory animals, guinea pigs and white mice.
Guinea pigs are inoculated intraperitoneally, subcutaneously, intracutaneously, and by rubbing the infective material into the skin. The inoculated animals develop a fever and lassitude, and lose weight. The spleen, liver, and inguinal lymph nodes become enlarged and inflamed. Microscopic studies reveal the presence of the causative agent in the spleen, liver, bone marrow, lymph nodes and in the blood recovered from the heart.
Wide adaptivity is characteristic of the tularaemia bacterium. It has adapted itself to more than 140 species of vertebrates and 110 species of arthropods which are capable of transmitting the disease. However, water rats, field voles, mice, muskrats, and, among domestic animals, sheep, and, among the vectors, horseflies, ticks, mosquitoes, and sandflies have the most epidemiological importance.
Pathogenesis and disease in man. Tularaemia is a zoonotic disease. Humans are infected with air-borne dust. The pathogen may also gain entrance into the body through the integuments and mucous membranes as a result of bites by arthropods and insects (ticks, horseflies, mosquitoes, etc.).
Contingent on the route of entry, the bacteria invade the skin, mucous membranes, lymph nodes, respiratory and gastro-intestinal tracts, and other organs, causing the respective clinical form of the disease (bubonic, ulcerative-bubonic, ocular, anginose-bubonic, abdominal or intestinal, pneumonic, and generalized or primary septicaemia). The lymph nodes are affected in all forms of the disease. In the generalized form all tissues and ‘organs are involved as a result of bacteraemia.
Tularaemia may be acute, lingering, or relapsing, depending on the duration of the disease, and may be mild, severe, or mildly severe. During tularaemia allergy develops and remains for years and sometimes for life. In recent years the death rate is low and most patients recover owing to the wide use of antibiotics.
According to the mode of spread and route of infection, the following types of tularaemia epidemics are known: trapping outbreaks associated with water rat and muskrat trapping; agricultural outbreaks associated with thrashing stacks inhabited by mice rodents; water-borne outbreaks due to diseases caused by drinking infected water; alimentary outbreaks due to the use of contaminated foodstuffs; transmissive outbreaks spread by the bites of bloodsuckers (ticks, stable-flies, mosquitoes, etc.).
Immunity. Following recovery, a stable immunity of long duration develops, being of the cell and humoral type.
Laboratory diagnosis. The differential diagnosis of plague, anthrax, enteric fever, typhus fever, influenza, malaria, and brucellosis is difficult because these diseases have common symptoms. For differentiation of tularaemia from other diseases laboratory tests are the most effective. Those peculiarities of the disease which can be revealed easily and quickly by laboratory methods are taken into account.
1. Allergy develops on the third-fifth day of the disease. For this reason, intracutaneous and cutaneous tests with tularine are made for early diagnosis. In tularaemia patients the test gives a positive reaction 6-12 hours after inoculation of tularine. In distinguishing tularaemia from other infections one must bear in mind that allergic tests may show positive reactions in convalescents and vaccinated individuals.
Blood-drop agglutination reaction
In some cases this test may give a positive reaction with material containing brucella organisms, since they possess antigens common to tularaemia bacteria.
3. The tularaemia culture is isolated by the biological method as it is impossible to recover the pathogen directly from a tularaemia patient. For this purpose white mice or guinea pigs are infected by material obtained from people suffering from the disease (bubo punctate, scrapings from ulcers, conjunctiva! discharge, throat films, sputum, and blood). Biological tests are conducted in special laboratories where a standard regimen is observed. The laboratory animals die in 4-12 days if tularaemia bacteria are present in the test material. Autopsy is performed, smears from organs are made and organ specimens are inoculated onto coagulated egg medium for culture isolation. Microscopic, microbiological, and biological studies of the cultured organisms are made. If no culture can be isolated from the first infected guinea pig, an emulsion, obtained from the latter’s organs, is inoculated into a second guinea pig, etc.
4. Laboratory diagnosis of rodent tularaemia is made by microscopy of smears from organs, precipitin ring reaction (thermoprecipitation), and biological tests.
Water, foodstuffs, and blood-sucking arthropods are examined by biological tests.
Treatment. Streptomycin, tetracycline, chloramphenicol, and a vaccine prepared from killed tularaemia bacteria are prescribed.
Prophylaxis comprises the following measures:
(1) systematic observation, absolute and relative registration of rodent invasion, and extermination of rats;
(2) prevention of mass reproduction of the rodents;
(3) protective measures in agricultural enterprises against contamination by tularaemia-infected rodents;
(4) protection of foodstuffs and water from rodents;
(5) control of ticks, horseflies, stable-flies, mosquitoes, and protection from these insects;
(6) specific prophylaxis with a live vaccine.
The vaccine is prepared in a dry form. A single application is made by rubbing it into the skin and it produces immunity for a period of 3-6 years.
Due to the application of a complex of measures on a wide scale (immunization of people living in a zone of natural foci) and the marked improvement in agricultural management tularaemia morbidity reduced to one thousandth within a period of 20 years (from 1945 to 1965) and has become a sporadic disease.
Brucellae. In 1886 on the Island of Malta D. Bruce, an English bacteriologist, demonstrated the presence of the causative agent of Malta fever in the spleen of a deceased patient and in 1887 isolated the organism in pure culture.
In 1896 the Danish scientist B. Bang established the aetiology of contagious abortion of cattle. In 1914 the American investigator G. Traum isolated from pigs the organism responsible for contagious abortion among these animals. Other Brucellae species were discovered in 1953, 1957, and 1966.
A more detailed study of these organisms was made in 1918 by the American scientist A. Evans. She came to the conclusion that, according to their main features, they were all closely related. She grouped them in one genus which she named after D. Bruce, the discoverer of the causative agent of brucellosis. All the former names of the disease (Malta fever. Mediterranean fever, undulant fever. Bang’s disease, contagious abortion of pigs, etc.) were substituted by the general name of brucellosis. In 1920 K. Mayer and M. Feseaux verified these data.
Morphology. Brucellae are small, coccal, ovoid-shaped micro-organisms 0.5-0.7 mcm in size (Fig.). Elongated forms are b.6-1.5 mem in length and 0.4 mcm in breadth. Under the electron microscope Brucella organisms of cattle, sheep and goats appear as coccal and coccobacilary forms, while those of pigs are rod-shaped. They are Gram-negative, non-motile, and do not form spores or capsules (in some strains capsules are sometimes present). DNA contains 56 to 58 per cent of G+C.
Figure. Brucella melitensis, a pure culture and colonies
Cultivation. The organisms are aerobic. When cultivated from material recovered from patients, they grow slowly, over a period of 8-15 days; in some cases, however, this period is reduced to 3 days, while in others it is prolonged to 30 days. In laboratory subcultures growth becomes visible in 24-48 hours. The pH of medium for these organisms is 6.8-7.2. The optimum temperature for growth is
Brucella organisms may be cultivated on ordinary media, but they grow best on liver-extract agar and liver-extract broth. On liver-extract agar the organisms form round, smooth colonies (see Fig. 2) with a white or pearly hue. In liver-extract broth they produce a turbidity, and subsequently a mucilaginous precipitate settles at the bottom of the tubes Brucella organisms grow well on unfertilized eggs and on the yolk sac of a 10-12-day-old chick embryo.
The brucellae of bovine origin (Brucella abortus) only grow in an atmosphere of 10 per cent carbon dioxide, which serves as a growth factor.
Selective media containing definite dyes and antibiotics (polymixin B, bacitracin, and others) are used for isolating Brucellae.
Brucella growth
Dissociation of the organisms from the S-form to the R-form has been demonstrated. L-forms have also been observed. These forms possess manifest adaptability. They adapt relatively easily to cultivation on nutrient media on which the first generation shows no growth; e. g. the first generation of brucellae of bovine cattle grow well only in the presence of carbon dioxide, while in subcultures they grow without it.
Prolonged cultivation of the organisms outrient media leads to a significant weakening of their virulence and to the loss of the Vi-antigen.
Fermentative properties. Brucellae do not liquefy gelatin and do not produce indole. Some strains produce hydrogen sulphide, break down urea and asparagin, reduce nitrates to nitrites, and hydrolize proteins, peptones and amino acids, with release of ammonia and hydrogen sulphide. No carbohydrates are fermented, although a small number of strains ferment glucose and arabinose.
Biochemical properties
Toxin production. Brucellae do not produce soluble toxins. An endotoxin is produced as a result of disintegration of the bacterial cells. This endotoxin possesses characteristic properties and may be used in allergic skin tests
Antigenic structure. The organism contains four antigens: A, M, G and R. The M-antigen is predominant among brucellae of sheep and goats, and the A-antigen, in the other species. Substances of polysaccharide character, with no type specificity, have been extracted from brucellae of cattle, sheep and goats. At present it is known that all three Brucella species contain 7 antigens (1, 2, 3, 4, 5, 6, and 7) which are arranged in the form of a mosaic on. the cell surface. Brucellae have an antigen common with tularaemia bacteria.
In recent years it has been discovered that brucellae, in addition to the O-antigen, possess a thermolabile Vi-antigen. Experiments haw confirmed that separate immunization with Vi- and O-antigens does not produce complete immunity to the disease in animals, while simultaneous immunization with all the antigens gives good results
Classification. Brucellae are subdivided into some species: (1) brucellae of goats and sheep (Brucella melitensis); (2) brucellae of cattle (Brucella bovis); (3) brucellae of pigs (Brucella suis); (4) brucellae of forest rats (Brucella neotomae); (5) the causative agents of epididymitis and abortion in sheep (Brucella ovis). The first three species produce cross immunity. In Canada, Siberia, and Alaska, B. rengiferi (fourth type of B suis) has been discovered: it affects northern reindeer from which humans are contaminated. B. canis was isolated from hounds in 1966
Brucella susceptibility to fuchsin and thionin
Biotypes have been described within three brucella species (B melitensis, B. bovis, B. suis). Brucellae can also be differentiated by the agglutinin adsorption method. This method defines the species of the organisms as well as their genetic interrelationships.
When identifying Brucella organisms, the variations in their antigen structure should be considered. Variants that cannot be identified by the usual methods are differentiated by Vi-agglutinating sera and Brucella bacteriophage. The most important criteria are the peculiarities of metabolism, the urease activity, the ability to form hydrogen sulphide, the antibiograms, and the relation to the phage.
Resistance. Brucellae are characterized by high resistance and viability. They survive for a long time at low temperatures. The organisms live for 18 weeks m winter soil, urine, animal faeces, manure, hay dust and bran, up to 16 weeks in ice, snow, butter and sheep’s milk cheese, from 12 to 16 weeks m sheep’s wool, for 30 days in dust, for 20 days in meat, and for 7 days in milk.
The organisms are sensitive to high temperatures and disinfectants. At
Pathogenity for animals. Goats, sheep, cattle, pigs, horses, camels, deer, dogs, cats, and rodents (rats, mice, susliks, hamsters, rabbits, field- voles, water rats, and other animals) are all susceptible to infection by brucellae. The high concentration of brucellae in the placenta of cattle is explained by the presence in this tissue of the growth stimulator erythrol.
The disease assumes an acute or a clinically latent course in animals. In sheep and goats abortions and delivery of non-viable foetus are the most typical symptoms. Affected cows have miscarriages, yield less milk, and lose flesh. In the weakest and emaciated animals the disease is sometimes fatal. Apart from miscarriages the organism produces arthritis, bursitis, orchitis, epididymitis, and other complications in pigs. In horses and camels the disease is usually of a latent form, abortions are rare, but emaciation, lassitude and numerous abscesses of long duration occur.
The excretions of sick animals (urine, faeces, amniotic fluid, and uterine mucus), and their milk, particularly that of goats and sheep, and milk products are sources of infection.
The migration of Brucella organisms from the usual hosts to animals of other species has been shown. This fact is of great epidemiological importance and must be taken into account in the laboratory diagnosis of brucellosis and when prophylactic measures are applied. Cows infected with B. melitensis may transmit virulent types of Brucella organisms to humans.
Among experimental animals guinea pigs are susceptible to brucellosis. The disease lasts for 3 months and the animals die showing lesions in the bones, joints, cartilages and eyes. During the disease they become emaciated, their skin atrophies, the hair falls out, and orchitis develops. In mice the disease produces septicaemia and the bacteria are recovered from the liver and spleen.
Pathogenesis and disease in man. Brucellosis is a zoonotic infection. Man contracts it from animals (goats, sheep, cows, pigs). From the epidemiological standpoint goats and sheep are the most important. At present there is a sufficient amount of cases to prove that healthy individuals may acquire the disease from humans infected by B. melitensis. The causative agents of brucellosis may be transmitted through wild animals (rodents and herbivores), ticks, and other blood-sucking insects; the infection may also be air-born.
Man contracts brucellosis very often through the milk of goats, sheep, and cows, and dairy products made from such milk also carry infection. The organism may likewise gain entry through abrasions in the skin and mucous membranes. For the most part veterinary and zootechnical staff, shepherds, workers of dairy farms and farms where sheep’s milk cheese is produced, workers of stockyards, etc., are attacked by the disease. On farms brucellosis prevails in certain seasons when sheep and goats bear their young (March-May).
From the site of primary location brucellae enter the lymphatic apparatus where they multiply. Then they enter the blood and cause protracted bacteraemia (from 4 to 12 months or longer). Via the blood-stream the bacteria invade the whole body and give rise to orchitis, ostitis, periostitis, arthritis, etc.
Allergy, which develops from the onset of the disease, lasts throughout the disease and remains long after recovery, is characteristic of brucellosis in humans and animals. A sensitized body becomes extremely sensitive to exposure to the specific action of the brucellosis antigen and to various non-specific factors, such as cooling, secondary infection, trauma, etc.
In human beings brucellosis is characterized by undulant fever with atypical and polymorphous symptoms. The disease may assume an acute septic or a chronic metastatic course. The structural and motor systems, haemopoietic, hepatolienal, nervous and genital systems are often involved. Pregnant women may have miscarriages. Often brucellosis recurs, continuing for months and years. The death rate is 1-3 per cent. The diagnosis of mild, asymptomatic forms presents difficulties and is based on laboratory tests.
Brucellosis in humans has many clinical symptoms common to other diseases (malaria, tuberculosis, rheumatic fever, enteric fever, typhus fever, Q fever, and various septic processes of other aetiology). For this reason, the differential diagnosis of brucellosis is of great importance. It is made with regard to the peculiarities of the disease and of other infections which have a similar course.
Immunity. A characteristic immunity is acquired following brucellosis, the patient becoming insusceptible to repeated infection. The activity of the T-lymphocyte system forms the basis for infectious and postinfectious immunity in brucellosis; phagocytosis plays a particularly important role. At the onset the immunity is non-sterile and infectious, but later it becomes sterile, although labile and of a low grade. All Brucella species produce cross immunity in the body.
The humoral factors, i. e. the production of opsonins, agglutinins, complement-fixing substance, and incomplete antibodies which block the causative agents of brucellosis, play a definite role in rendering the brucellae harmless. The phage, being a powerful factor in bacterial variation, plays an important role in producing insusceptibility to brucellosis. Nearly all patients show marked improvement with the clinical course of the disease and on recovery a higher titre and lytic activity of the bacteriophage are displayed.
Laboratory diagnosis. The patient’s blood and urine (for isolation of the pathogen), serum (for detection of agglutinins), milk and dairy products (for detection of brucellae or agglutinins in milk) are examined. The microbe is isolated in special laboratories.
1. Culture isolation. Since brucellosis is often accompanied by bacteraemia, blood is examined during the first days of the disease (preferably when the patient has a high temperature). For this purpose, 5-10 ml of blood is collected and transferred into two or three flasks (2-5 ml per flask) containing 100 ml of liver-extract or ascitic-fluid broth (pH 6.8). The cultures are grown for 3-4 weeks or more. Five to ten per cent of carbon dioxide is introduced into one of the flasks (for growth of the 23 bovine species of the bacteria). Inoculations on agar slants are made every 4-5 days for isolation and identification ‘of the pure culture.
An antiphage serum is introduced into the cultures for neutralization of the phage which inhibits the growth of brucellae. The best results are obtained when the blood is inoculated into the yolk of an unfertilized egg or the yolk sac of a chick embryo. For this, 0.1-0.2 ml of the tested blood diluted in citrate broth in a ratio of 1 : 3 is introduced into each egg. The infected eggs are placed in an incubation chamber for 5 days, after which 0.3-0.5 ml of their contents is inoculated into the liquid nutrient media. Growth is examined every 2-3 days.
If the blood culture produces a negative result bone marrow obtained by sternum puncture is inoculated onto solid and liquid media for isolation of myelocultures.
The urine is also examined. It is obtained with a catheter, centrifuged, and 0.1 ml of the precipitate is seeded onto agar plates containing 1 :200000 gentian violet. In some cases faeces, cow’s and human milk, and amniotic fluid of sick humans and animals are examined for the presence of Brucella organisms.
Brucella susceptibility to phages
Brucella cultures may be isolated by the biological method. For this purpose healthy guinea pigs or white mice are injected with 0.5 or 3 ml of the test material. A month later the guinea pigs’ blood is tested for agglutinins, the allergic test is carried out, and the pure culture is isolated. White mice are tested bacteriologically every three weeks.
2. Serological test. From the tenth-twelfth day of the disease onwards, the agglutinins accumulate in the blood in an amount sufficient for their detection by the agglutination tests. The Wright (in test tubes) and Huddleson (on glass) reactions are carried out. The Wright reaction is valued highly positive in a 1 800 serum dilution, positive in a 1 :400-1 :200 dilution, weakly positive in 1 :100 dilution, and doubtful at a titre of 1 :50.
The Huddleson reaction is used mainly in mass examinations for brucellosis.
However, there is a disadvantage of this reaction in that it sometimes shows positive results with sera of healthy individuals who have normal antibodies in their blood.
3. Skin allergic test. To determine allergy, Burne’s test is made beginning from the fifteenth-twentieth day of the disease. A 0.1 ml sample of the filtrate of a 3- or 4-week-old broth culture (brucellin) is injected intracutaneously into the forearm. The test is considered positive if a painful red swelling 4 by
4. Opsono-phagocytic test. This test detects changes in the phagocytic reaction. The index of healthy individuals averages 0-1 and occasionally 3-
For detecting brucellae in the external environment the reaction for demonstrating a rise in bacteriophage titre is carried out.
Treatment. Patients suffering from brucellosis are treated with antibiotics (amphenicol, tetracycline, etc.). Chronic cases are best treated by vaccine therapy. X-ray therapy, blood transfusions, electropyrexia, and balneotherapy, hormonotherapy. Injection of antibrucellosis gammaglobulin is recommended for the prevention of recurrences.
Prophylaxis comprises a complex of general and specific measures carried out in conjunction with veterinary services. This includes:
(1) early recognition of brucellosis, hospitalization of sick individuals, exposure of the sources of the disease;
(2) sanitary treatment of cattle-breeding farms, identification, examination and isolation of sick animals, immunization with live vaccine;
(3) systematic disinfection of discharges of sick humans and animals, prophylactic disinfection of hands of shepherds and persons engaged in the care of sick animals;
(4) observance of hygienic measures during consumption of milk (pasteurization or boiling) and dairy products in districts where there are cases of brucellosis;
(5) protection of cattle-breeding farms, control of cattle driven from farm to farm, the enforcement of quarantine for new cattle, and isolation of young livestock from sick animals;
(6) sanitary education among the population. Immunization with the live or killed vaccine is an additional measure in districts where there are cases of goat-sheep brucellosis.
Laboratory diagnosis in details
Tularemia. The causative agent of tularaemia, an acute zoonotic disease largely affecting the lymph nodes, is Francisella tularensis. The laboratory diagnosis of this illness is based on bacteriological, biological, and serological methods of investigation, as well as on allergy tests.
Allergy and serological tests are performed in usual hospital conditions and microbiological laboratories. The recovery and isolation of the tularaemia bacteria are carried out in special laboratories.
Bacteriological and biological examination. Attempts to isolate a culture of the causative agent from the patient by direct inoculation of the material onto media almost invariably end in failure. The biological method is more promising. Using the material obtained from patients, which may vary depending on the clinical form of tularaemia (blood, puncture sample from a bubo, scraping from an ulcer, conjunctival exudate, faucial deposit, sputum, etc.), the total of 3-5 white mice or 2 guinea pigs are infected. The material collected before the antibiotic treatment is administered subdermally, epicutaneously, intraperitoneally, and by mouth. The infected animals are kept under special conditions (as in the diagnosis of plague) and observed for 6-14 days.
If the experimental animals fail to die within 7-15 days, they are sacrificed on the 15th-20th day and subjected to autopsy. Tularaemia is recognized by pathoanatomical changes in the form of a productive process with necrosis and without marked haemorrhagic component. The blood, bone marrow, and pieces of the internal organs and lymph nodes from the animal corpse are inoculated by rubbing them into the surface of one of the media such as yolk, glucose-cystine agar with blood, Emelyanova’s medium, etc.
Yolk medium consists of three parts of egg yolks and two parts of isotonic sodium chloride solution (pH 7.0-7.4). It is dispensed into sterile test tubes by 5-ml portions and coagulated for 60 min at
Glucose-cystine blood agar. Supplement melted meat-peptone agar with U.05-0.1 per cent of cystine and 1 per cent of glucose (pH 7.2–7.4), then boil it for several minutes, cool to 45–50 °C, and add 5–10 per cent of defibrinated rabbit blood. Colonies of the tularaemia growth on a glucose-cystine medium are moist and milk-white in colour.
Emelyanova’s medium contains 2.5 ml of yeast autolysate, 10 ml of gelatin hydrolysate, 20 ml of fish Hour hydrolysate,
The causative agent of tularaemia demonstrates good propagation in the yolk sac of a 12-day chick embryo and poor growth in liquid nutrient media.
Simultaneously with culturing, the same material is used to make impression smears to be stained by the Romanowsky-Giemsa technique. The tularaemia causative agents are small (0.2–0.7 pan), coccal, and rod-shaped bacteria. In smears from the organs they are arranged intracellularly, and in the form of clusters, forming a tender capsule. The isolated pure culture is identified by morphological, antigenic, and biological attributes.
Freshly isolated pathogenic tularaemia bacteria show weak biochemical activity. They may ferment glucose and release hydrogen sulphide; with further cultivation they acquire a capacity to ferment maltose, mannose, and occasionally other carbohydrates. To identify the species of the isolated culture, agglutination with a specific agglutinating serum is performed. The pathogenicity of the culture is measured by inoculation of sensitive animals.
To isolate a culture of the tularaemia bacteria from water, sensitive animals are infected with a sediment obtained upon the centrifugation or filtration of water.
In examining foodstuffs, they are washed with meat-peptone broth, centrifuged, and the deposit is administered to animals.
In enzootic foci, planned systemic studies are carried out to isolate the causative agent of tularaemia from rodents (using the above described method).
Serological diagnosis. A standard agglutination test with patients’ serum is performed in test tubes, while a blood drop agglutination reaction (rapid) is carried out on glass slides.
To carry out a standard agglutination test, on the second week of the disease (the 10th–15th day) withdraw 2-3 ml of blood from the patient’s cubital vein. Dilute successively the obtained serum (Table) with isotonic saline in titres from 1:25 to 1:400.
Table
Schematic Representation of the Standard Agglutination Test
|
Ingredient |
Number of test the tubes |
||||||
|
1 |
2 |
3 |
4 |
5 |
6 serum control |
7 diagnosticum control |
|
|
Isotonic sodium chloride solution, ml |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
|
Patient’s serum in dilution 1:25, ml |
0.5→ |
0.5→ |
0.5→ |
0.5→ |
0.5 |
0.5 |
– |
|
Diagnosticum, ml |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
– |
0.5 |
|
Obtained serum dilution |
1:50 |
1:100 |
1:200 |
1:400 |
1:800 |
1:50 |
– |
Introduce 0.5 ml of the diluted serum into each test tube, add 0.5 ml of tularaemia diagnosticum to obtain the final dilutions of the serum from 1:50 to 1:800. Simultaneously, check the adequacy of the serum and diagnosticum by the ordinary method.
The test tubes are placed in an incubator for 2 hrs. The preliminary results of the test are read after removal of the test tubes from the incubator, the final ones after the test tubes have been allowed to stay at room temperature for 18–20 hrs.
A positive agglutination reaction in dilution 1:100 and higher is diagnostically significant. It is recommended that the sera be re-examined in the course of the disease (paired sera), the second examination being conducted 7–10 days after the first one. An increase in the titre of antibodies by four-fold or more is of a delimit & diagnostic significance.
A blood drop agglutination reaction is used in mass screening. Blood withdrawn from a finger is placed on a glass slide in the form of a thick drop to which an equal amount of distilled water is added to induce haemolysis. Put a drop of the tularaemia diagnosticum near the blood drop and mix both drops with a glass rod. If the serum contains any antibodies, the agglutination reaction occurs immediately.
The blood serum from patients with tularaemia may give a positive agglutination reaction with brucellosis diagnosticum since tularaemia bacteria and Brucella have common antigens. Individuals with a history of tularaemia show agglutinins in their blood for a long time.
A fairly sensitive method of serological diagnosis of tularaemia is the indirect haemagglutination test. It may he positive already at the end of the first, or the beginning of the second, week of the disease. Tularaemia erythrocyte diagnosticum consisting of a suspension of formalin-preserved sheep red blood cells sensitized by the tularaemia antigen is employed as an antigen. Haemagglutinating litres of sera in patients with tularaemia rise to 1:1280–1:2560 and even higher.
The complement-fixation test in the cold is utilized for the early diagnosis of tularaemia. The patient’s blood is obtained during the first week of the disease and a diagnosticum is used as an antigen.
Allergy tests are also used for the early diagnosis of tularaemia (from the fifth day of the disease onset). Two types of tularin and, respectively, two methods of their administration (epicutaneous and intracutaneous) are utilized. Since the concentration of the allergen in both types of tularin is different, one should not use epicutaneous tularin for the intracutaneous test and vice versa.
The results of the allergic reaction are read 24–36–48 hours after allergen administration. The presence of a marked infiltrate and hyperaemia of
The allergy tests may remain positive (anamnestic reaction) for several years in vaccinated subjects or those with a history of tularaemia.
Brucellosis. The causative agents of brucellosis (a chronic zoonotic infection) belong to the genus Brucella which includes three main types: B. melitensis, B. abortus, B. suis. Other types comprise B. neotomae, inducing disease in common field mice, B. ovis, affecting birds, B. canis, causing infection in dogs, and B. rangiferi which is pathogenic to deer.
In diagnosing brucellosis, one uses bacteriological, serological, and biological examination, as well as allergy tests. Serological and allergy tests for the diagnosis of brucellosis are conducted in common microbiological laboratories. The pure culture of Brucella organisms is isolated and identified in special laboratories.
Bacteriological examination. To isolate a pure culture of Brucella, blood, puncture sample of the bone marrow, urine, milk, and pieces of the organs are studied.
To isolate a haemoculture, blood obtained from a feverish patient is inoculated during the first days of the disease; if the results are negative, the examination is performed several times (bacteraemia persists for a long time, for 12 months and more).
No less than 10 ml of blood is withdrawn from the cubital vein and inoculated (in 5 ml-portions) into two vials containing 100 ml of liver or ascitic broth (pH 7.1–7.2) or meat-peptone broth with addition of 1 per cent of glucose, 2 per cent of solution of glycerol, and antiphage serum for phage suppression. To prevent blood coagulation, 0.2 per cent of sodium citrate is added to the sera. One vial is kept under the usual aerobic conditions, the other, in a sealed vessel containing 10 per cent of carbon dioxide which enters the vessel from a gas cylinder via a gasometre. The necessary concentration of carbon dioxide can also be achieved in the following way:
Insert a burning cotton plug deep into the mouth of the vial with the inoculated culture, additionally close the vial with a rubber plug, and then seal it with liquid paraffin. Taking into consideration a slow growth of Brucella, keep the cultures in a
The puncture specimen of the bone marrow for isolating a, myelo-culture is inoculated onto the same media. It has been established that myelocultures in patients with brucellosis may be isolated 1.5–2 times as often as haemocultures.
Urine for isolating a urinoculture is collected aseptically at the end of the feverish period with the help of a catheter and then centrifuged. The deposit is streaked onto plates with liver infusion agar containing gentian violet in 1:200 000 dilution to retard the growth of Gram-positive microflora. Urinocultures are obtained in 9–14 per cent of cases.
Milk of animals (goats, sheep, and cows) is centrifuged and 0.1–0.2 ml of cream is placed into plates with liver agar containing gentian violet.
The best Brucella growth occurs when the test material is inoculated into a yolk of a fresh chicken egg or the yolk sac of the chicken embryo. The patient’s blood is diluted 1 to 3 with citrate broth and introduced into several eggs in 0.1–0.2 ml amounts. The infected eggs are placed into a
Prior to infecting eggs with the material tested (faeces, urine, sputum, pus, etc.) containing extraneous microflora, it is subjected to centrifugation. The obtained sediment is diluted with gentian violet solution (1:200000) by 3–5-fold and inoculated in 0.2 ml portions into 3–5 eggs with subsequent subculturing (live days later) onto nutrient media.
Biological examination. Guinea pigs and white mice are the most sensitive animals with any method of inoculation. The patients’ blood is injected to animals intraperitoneally (2–3 ml to guinea pigs, 0.5–1 ml to white mice), urine sediment and cream centrifuged from the milk of animals are administered subcutaneously.
Some 20–30 days following the inoculation withdraw some blood from a guinea pig (or a mouse) and carry out the agglutination test. Then sacrifice the animals, dissect them, and culture the heart blood and suspension from the internal organs and lymph nodes.
Growth of Brucella (of any type) in the broth is manifested by the appearance of turbidity with the subsequent formation of 1 mucilaginous sediment. In plates with liver infusion agar Brucella organisms form tiny smooth, whitish-opaque colonies with a pearly hue. When smears are stained by Kozlovsky’s technique, Brucella are pink.
The smear is flame fixed, stained with 3 per cent aqueous solution of safranine with heating it up until the appearance of fumes, washed with water, and counter stained for 1–1.5 min with 1 per cent aqueous solution of methylene blue, then washed in water once again and dried.
Brucella organisms do not ferment carbohydrates, do not coagulate milk, and do not liquify gelatine. Various types of Brucella contain different antigens so they have to be identified by means of the agglutination reaction with monospecific sera.
Brucella organisms can also be differentiated by their ability to grow in the absence of elevated levels of carbon dioxide, by production of hydrogen sulphide, sensitivity to the specific bacteriophage, and a bacteriostatic action of aniline dyes (Table 14).
Serological examination in brucellosis includes the agglutination test with the patient’s serum (Wright’s reaction), accelerated plate reaction on a glass slide (Huddleson’s test), an opsonocytophagic test, and the complement-fixation test.
Wright’s reaction is performed at the beginning of the second week of the disease. Suspension of killed Brucella organisms stained with gentian violet or methylene violet and containing 1 mird of brucellae (per ml) killed with formalin or phenol is used as a diagnosticum. Currently a uniform colour brucellar diagnosticum for Wright’s and Huddleson’s tests is commercially available.
The procedure of performing the Wright test is similar to that used in other agglutination tests, with the exception of the fact that the serum is diluted in a multiple order with isotonic NaCI solution containing 0.5 per cent of phenol. The test tubes are placed in the incubator for 18–20 hrs and then left at room temperature for 2 hrs. The results of agglutination are denoted with pluses. The reaction is considered positive if the titre is 1:200 and doubtful if it is 1:50. Wright’s reaction may be positive in vaccinated subjects and patients with brucellosis. So, it is to be repeated in the course of the disease to look for increase in the antibody litre.
The plate agglutination test proposed by Huddleson (Table) is most frequently used in brucellosis foci (in field conditions) since it is simple to perform.
Table
Schematic Representation of Huddleson’s Agglutination Test
|
Ingredient, ml
|
Number of square |
Control |
||||
|
1 |
2 |
3 |
4 |
of serum |
of antigen |
|
|
5 |
6 |
|||||
|
Isotonic saline |
– |
– |
– |
– |
0.03 |
0.03 |
|
Patient’s serum |
0.08 |
0.04 |
0.02 |
0.01 |
0.02 |
– |
|
Diagnosticum |
0.03 |
0.03 |
0.03 |
0.03 |
– |
0.03 |
Using a wax pencil, divide a glass plate into six squares, then pipette on them undiluted serum to be studied in 0.08, 0.04, 0.02, 0.01, and 0.02-ml portions, and add 0.03 ml of the same diagnosticum that is used in Wright’s reaction to each dose of the serum but the last (the fifth square) into which 0.03 ml of isotonic sodium chloride solution (control of the serum) is placed. In the sixth square, place 0.03 ml of the antigen and 0.03 ml of isotonic saline (control of the diagnosticum). Mix drops of the serum and antigen with a glass rod and after slight rocking of the plate read the results of the reaction which is manifested already in the first minutes by the appearance of microgranular stained agglutinate. The absence of agglutination in all doses of the serum is assessed as a negative reaction, the presence of agglutination in the first dose (0.08 ml of the serum) as a doubtful one, in the second dose (0.04 ml), as a weakly positive one, in the third or fourth doses (0.02–0.01 ml), as a positive one; the agglutination reaction expressed by 4 pluses in all doses is evaluated as a drastically positive one. The value of Huddleson’s agglutination test is diminished by the fact that not infrequently it produces positive results with healthy people’s sera due to the presence of normal antibodies. Hence, sera showing positive Huddleson’s reaction should be subjected to the tube agglutination test.
For the rapid diagnosis of brucellosis one can use an agglutination reaction of Bengal pink with an acid antigen. The antigen is prepared from dense suspension of heat-killed (
Read the results of the reaction in 4 min. The presence of macro- or microgranular agglutinate indicates a positive test.
The opsonocytophagic test is performed beginning with the 15–20th day of the disease with four milliard broth suspension of killed Brucellae. It is considered that the parameter varying from 10 to 24 is characteristic of a weakly positive reaction; from 25 to 49, of a definitely expressed one; and from 50 to 75, of a dramatically positive one. Iormal subjects, this parameter usually equals 0, or 1, rarely 3–5.
Complement fixation is a fairly specific test with regard to brucellosis. A positive result is registered from the 20th–25th day of the disease and persists for a long time.
When the IHA test is made in patients with acute and subacute brucellosis, agglutinins are detected in 91–100 per cent of cases.
In acute, subacute, and chronic forms of brucellosis, antibodies in patients’ sera may be recovered by the indirect IF test that is more sensitive than the agglutination reaction. It is recommended that one should determine the composition of serum immunoglobulins at different stages of the disease. At early stages after inoculation, IgM can be demonstrated, later IgG are observed. These immunoglobulins are differentiated with the help of the cystine test.
Burnet’s intracutaneous allergy test is used for detecting an allergic status in patients with an active disease or a history of brucellosis. It becomes positive from the 15th–20th day of the disease. A total of 0.1 ml of brucellin is injected intracutaneously into the middle area of the upper arm. The reaction is considered positive if 24 hrs after the injection there is tenderness, hyperaemia, and infiltration of the skin some 3 X
Bacillus anthracis
The agent responsible for anthrax (B. anthracis) was described by A. Pollander (Germany) in 1849, by K. Davaine (France) in 1850, and by F. Brauell (Russian) in 1854. Studies of anthrax were originated by R. Koch (1876), L. Pasteur (1881) and L. Tsenkovsky (1883). Its isolationb y German bacteriologist Robert Koch in 1877 marked the beginning of modern medical microbiology, because it was, in part, Koch’s work with this organism that was responsible for the development of the well-known Koch’s postulates
B. anthracis belongs to the family Bacillaceae.
Morphology. Anthrax bacilli are large organisms, measuring 3-5 mcm in length and 1-1.2 mcm in breadth. In the body of animals and man they occur in pairs or in short chains, while iutrient media they form long chains (Fig.). In stained preparations the ends of the bacilli appear either to be sharply cut across or slightly concave, resembling bamboo canes with elbow-shaped articulations. The G+C content in DNA is 32 to 62 per cent.
Figure. Anthrax bacilli: 1 — smear from sporogenous culture; 2 — structure of the periphery of colony; 3 — smear from cadaver specimens
The bacilli are non-motile. Outside the host’s body they produce oval-shaped central spores which are smaller in diameter than the bacillus. Spores are best produced in the presence of oxygen at 30-40° C. No spores are produced in the body of man or animal or at temperatures above 43 and below
It has been ascertained that spores may germinate into vegetative forms during the warm months under favourable conditions, and transform again into spores in the autumn.
In the bodies of man and animals the bacilli produce capsules which surround a single organism or are continuous over the whole chain (Fig.).
Anthrax bacilli in tissue
Capsules are also produced outrient media which contain blood, serum, egg yolk, or brain tissue. The capsule which contains specific proteins provides a defence mechanism and determines the virulence of the organism. The anthrax bacillus readily stains with all aniline dyes and is Gram-positive.
It can be seen on ultrathin sections that the cell wall is 360 to 400 nm thick, the cytoplasmatic membrane has three layers, the cytoplasm is granular and the vacuoles large, and that the nucleoid is in the centre of the cell. Division begins before the cells have separated after the previous division, which leads to the formation of streptobacilli.
Cultivation. The bacillus is aerobic and facultatively anaerobic. The optimum growth temperature is 37° C, and the organism does not grow below 35° and above 43° C. It grows well on all ordinary media at pH7.2-7.6. On meat-peptone agar the bacilli form rough colonies (R) which have uneven edges and resemble the head of a medusa (Fig.).The edges of the colonies have the appearance of locks of hair or a lion’s mane. The smooth S-forms possess low virulence or are completely avirulent and non-capsulated in the body of the host.
Bacillus anthracis colonies
Broth cultures of the anthrax bacillus produce flocculent growth resembling cotton wool which sinks to the bottom of the tube or flask, leaving the broth clear. Spore production is inhibited by adding a 1 per cent calcium chloride solution to the medium, and is stimulated by the presence of neutral sodium oxalate.
The anthrax bacillus undergoes a morphological change when it transforms from the R-form to the S-form. It loses its ability to form chains in smears and occurs in coccal and diplobacillary forms or the cells are arranged in groups. Incubation at 42.5° C produces thread-like, non-sporeforming organisms of low virulence. On meat-peptone agar containing penicillin the bacilli break up into globules which are arranged in the form of a necklace (a pearl necklace). The anthrax bacilli transform usually from the R-form (typical, with rough colonies, and virulent) to the S-form (atypical, with smooth even-edged colonies, and avirulent) through the intermediate 0-form (with mucilaginous, pigmented, and patterned colonies).
Fermentative properties. The anthrax bacilli possess great biochemical activity. They contain the enzymes — dehydrogenase, lipase, diastase, peroxidase, and catalase. In gelatin stab-cultures growth resembles an inverted fir tree (Fig.), the gelatin being liquefied in layers. The organisms cause late liquefaction of coagulated serum and produce ammonia and hydrogen sulphide. They slowly reduce nitrates to nitrites and coagulate and peptonize milk. The organisms ferment glucose, levulose, saccharose, maltose, trehalose, and dextrin with acid production.
Figure. Growth of B. anthracis on gelatin stab
Toxin production. When growing on semisynthetic medium, B. anthracis discharges an exotoxin (oedema factor) into the culture fluid. The capsular substance is very toxic, it contains Bayle’s aggressins. Loss of the capsule results in loss of virulence.
It has been established that some strains of B. anthracis produce in the animal’s body a lethal toxin (mouse factor), which on addition of the oedema factor or the protective antigen causes death of the animal.
The serum of guinea pigs who died from anthrax possesses the property of causing death of albino mice and guinea pigs on being injected intravenously in small doses.
Antigenic structure. There is only one antigenic type of B anthracis, and it contains three major types of cell antigens. The anthrax bacillus contains a protein (P) and a polysaccharide (C) antigen. The polysaccharide antigen is present in the bacterial cell, while the protein antigen is in the capsule.
The polysaccharide antigen consists of N-acetylglucosamine and d-galactose and is thermoresistant. It survives for long periods in tissues obtained from corpses. Ascoli’s thermoprecipitin test is based on this principle. A boiled B. anthracis extract contains a polysaccharide fraction (thermoresistant) which yields a precipitin reaction with the precipitant serum.
The capsule contains a protein-like substance, a polypeptide, which. is a polymer of gamma-d-glutamic acid
In animal bodies and on media containing tissue extracts and plasma, B. anthracis produces a peculiar antigen (a protective antigen). This antigen is a non-toxic thermolabile protein which possesses marked immunogenic properties. It gives rise to the formation of protective (incomplete) antibodies which neutralize the aggressive enzyme of the anthrax bacillus. The capsule is unusual in that it contains only the D (or unnatural) isomer of the amino acid, furthermore, the virulence of the organism depends, in part, on the antiphagocytic activity of this capsular material.
Classification (Table).
Table
Differential-Diagnostic Signs of B. anthracis, Antracoides, and Soil Bacilli
|
Type of bacilli |
Motility |
Capsule |
Nature of growth of growth |
Pathogenicity for ogenicity for
|
|||
|
on blood |
in litmus |
mice |
guinea pigs |
rabbits |
|||
|
B. anthracis |
— |
+ |
No haemo- |
Reddening |
Die in 24 hrs |
Die in |
Die in |
|
B. anthracoides |
Weak |
— |
Haemolysis |
Medium |
Pathogenic when large amounts of |
Non-pathogenic |
Non-pathogenic |
|
B. subtilis |
Active |
— |
Haemolysis |
Medium |
Some strains are pathogenic when administered in large doses |
Non-patho |
|
|
B. megatherium |
Moderate |
— |
No haemo-lysis |
— |
|
Non-pathogenic |
Non-pathogenic |
|
B. cereus var. mycoides |
Weak |
— |
No haemo-lysis |
— |
Produce soluble toxin lethal for mice
|
Non-pathogenic |
Non-pathogenic |
Resistance. B. anthracis survives in meat-broth cultures in hermetically sealed ampoules for 40 years and the spores survive for 58 to 65years. Spores remain viable in soil for decades and when dried — for as long as 28 years. They are more resistant to disinfectants than the vegetative cells. The vegetative cells are killed in 40 minutes at 55° C, in 15 minutes at 60° C, and in 1-2 minutes at 100 ºC. The spores are thermoresistant, and withstand boiling for 15-20 minutes and autoclaving at 110° C for 5-10 minutes. They are destroyed by 1 per cent formalin and10 per cent sodium hydrate solutions in 2 hours. The capsule is moreresistant than the microbial cell. Post-mortem examination of animal bodies which had previously been exposed to putrefactive microflora reveals quite frequently empty microbial capsules (shadows), devoid of cytoplasm.
Pathogenicity for animals. Sheep, cows, horses, deer, camels, and pigs are susceptible to anthrax infection. The animals are usually infected through the mouth, ingesting the spores in the fodder. The organisms localise in the intestine. Blood-sucking insects (gadflies and stable flies) may be vehicles of infection in some cases.
The disease is characterized by lassitude, cyanosis, and sanguineous discharge from the intestine, nostrils, and mouth. Septicaemia sets in preceding death which occurs in 2 or 3 days. In horses the infection is less severe, affecting the glandular tissues and causing the development of anthrax carbuncles.
Among the laboratory animals, most susceptible are white mice, then guinea pigs, and rabbits which die 2-4 days after infection. A swelling and haemorrhages appear at the site of injection. The internal organs become congested and enlarged (particularly the spleen), and septicaemia develops. The blood of the animals does not clot after their death because of the anticoagulative effect of the anthrax bacilli, and it is thick and black-red in colour (Gk. anthrax coal).
Pathogenesis and diseases in man. Anthrax is a typical zoonosis. Anthrax is primarily a disease of sheep, goats, cattle, and, to a lesser extent, other herbivorous animals. Although in the United States it is found only in a few areas (Louisiana, Texas, California, Nebraska, and South Dakota), it is a world-wide problem, especially in parts of Europe, Asia, and Africa Once the disease is established in an area, bacterial endospores from infected or dead animals are able to contaminate the soil and, because of the resistant endospores, the pasture areas remain infectious for other animals for many years In most animal infections, the spores enter the body by way of abrasions in the oral or intestinal mucosa, and after entering the bloodstream, they germinate and multiply to tremendous numbers, causing death in 2 to 3 days.
Humans acquire the disease from sick animals or articles and clothes manufactured from contaminated raw materials: sheepskin coats, fur mittens, collars, hats, shaving-brushes, etc. In summer the infection may be transmitted by blood-sucking insects. Anthrax occurs in three main clinical forms: cutaneous, respiratory, and intestinal.
In the cutaneous form the causative agent enters the body most frequently through injured integuments, mainly those parts of the body which are not covered with clothes (face, neck, hands, and forearms). Cutaneous infection is an occupational hazard for persons who handle livestock or work with items derived from contaminated wool or hides. An anthrax carbuncle (malignant pustule) develops at the site of bacilli localization. An initial lesion occuiring at the site of entry soon develops into a black necrotic area (Fig. 3). If the lesion is not treated, the organisms may invade the regional lymph nodes and bloodstream, causing death.
FIGURE. Lesion of cutaneous anthrax (eighth day of illness) on the arm of a woman who had been a carder in a wool factory.
The pulmonary form of the disease occurs less frequently than the cutaneous form but is more serious and carries a higher mortality rate. This form is acquired through the air when working with material contaminated with bacillary spores. The disease assumes the form of a severe bronchopneumonia. The bacilli are discharged in the sputum.
The intestinal form is due to ingestion of meat of sick animals. It is characterized by grave lesions in the intestinal mucosa with haemorrhages and necrotic foci The bacilli are excreted in the faeces. It is considered by a number of authors that the intestinal form of the disease is caused by the bacilli invading the intestine through the blood.
A seemingly rare source of anthrax infection came to the force in 1979 when an epidemic of anthrax occurred in Sverdlovsk in the Soviet Union This epidemic, which is believed to have killed almost 100 people, supposedly originated as gastric anthrax caused by eating anthrax infected meat. In such cases, it is assumed that the organisms enter the bloodstream from the intestine. Although gastric anthrax is rare in the western world, Soviet textbooks have described previous epidemics of gastric anthrax that resulted in 100% mortality.
At present the cutaneous form of anthrax occurs sporadically, while the intestinal form is very rare. Anthrax septicaemia may develop as a complication in any of the clinical forms or in debilitated and emaciated patients.
Until 1955, it was believed that anthrax caused death by its ability to resist phagocytosis, allowing it to grow to tremendous numbers of bacilli that clogged capillaries. This explanation had been termed the “log jam” theory. It is known that this concept is incorrect and that B. anthracis secretes a highly toxic exotoxin.
The toxin consists of a complex of proteins that has been isolated as three components named deem factor (EF), lethal factor (LF), and protective antigen (PA)None of these factors has any biologic activity by itself. An insight into the mechanism of action of this toxin complex was provided by Stephen Leppla when he discovered that EF is an inactive form of adenylate cyclase, which is activated by eucaryotic calmodulin, and that PA is required to facilitate the entry of EF into cells. Thus, it is similar to other A-B toxins in that PA is analogous to what is termed the B subunit, with EF acting as the A subunit of this toxin. It differs in that unlike the A-B toxins already discussed, PA and EF are secreted as separate components. The end result is an increase in cAMP in polymorphonuclear neutrophils, inhibiting their ability to carry out normal phagocytosis. Thus, as described for Bordetella pertussis, the phagocyte impotence seen in anthrax infections results from the secretion of an extracellular adenylate cyclase and its effect on host leukocytes. Table summarizes the effects of these components.
Table
Components of Anthrax Toxin
|
Component |
Function |
|
EE |
Inactive adenylate cyclase activated by calmodulin |
|
LF |
Causes pulmonary edema and death in rats; cytolytic for macrophages |
|
PA |
Required for the binding of both EF and LF to host cell |
EF, edema factor, LF, lethal factor, PA, protective antigen
Interestingly, PA also is required to show a biologic effect of LF, indicating that PA acts as a common binding subunit for both EF and LF. No enzymatic activity is known for LF, but when injected together with PA, LF causes severe pulmonary edema and death in Fisher 344rats. PA and LF together are also rapidly cytolytic for macrophages.
When PA binds to a specific receptor on a host cell, a cell-surface protease cleaves off a peptide of about 20kd to generate a cell-bound 63-kd PA, which possesses a new binding site to which LF and EF bind with high affinity. The resulting complexes of PA63-LF and PA63-EF are then internalized by receptor-mediated endocytosis. Mutants lacking LF produced edema when injected subcutaneously but were not lethal. Mutants missing EF but still producing LF were lethal when injected into susceptible animals.
Notice that the virulence of. B. anthracis is dependent on two separate plasmids, one of which codes for the D-glutamic acid capsule, and the other for the three separate genes of the secreted toxin.
Immunity following anthrax is antimicrobial and depends on the presence of protective antigens. Phagocytosis plays no defensive role in the disease. The protective antigen does not give rise to the production of complete antibodies, but it stimulates the formation of protective (in-complete) antibodies which cause the destruction of the virulent anthrax bacilli.
Serum of individuals who have recovered from anthrax is found to contain substances which are capable of destroying capsular matter and neutralizing aggressins and toxins (lethal factor).
Laboratory diagnosis. In cases of cutaneous anthrax the malignant pustular exudate is examined; it is obtained from the deep layers of the oedematous area where it borders with the healthy tissues. Sputum is examined in cases of the respiratory form, faeces and urine, in intestinal form, and blood is examined in cases of septicaemia.
1. The specimens are examined under the microscope, the smears are Gram-stained, or stained by the Romanowsky-Giemsa method. The presence of morphologically characteristic capsulated bacilli, arranged in chains, allows a preliminary diagnosis.
2. For isolation of the pure culture the specimens are inoculated into meat-peptone agar and meat-peptone broth. The isolated culture is differentiated from other morphologically similar bacteria by its morphological and biochemical properties.
3. Laboratory animals (white mice, guinea pigs and rabbits) are inoculated with the pathological material and with the pure culture de-rived from it. B. anthracis causes the death of white mice in 24-48 hours and of guinea pigs in 2-3 days following inoculation. Microscopic examination of smears made from blood and internal organs reveals anthrax bacilli which are surrounded by a capsule.
A rapid biological test is also employed. The culture obtained which has to be identified is introduced intraperitoneally into white mice. Several hours after inoculation smears are prepared from the peritoneal contents. Detection of typical capsulated bacilli gives a basis for con-firming the final result of the biological test.
The allergic test with anthracin (a purified anthrax allergen) is employed when a retrospective diagnosis is required in cases which have yielded negative results with microscopical and bacteriological examination.
Postmortem material as well as leather and fur used as raw materials are examined serologically by the thermoprecipitin reaction (Ascoli’s test) since isolation of the bacilli is a matter of difficulty in such cases.
As can be seen in Fig. 3, the result in the first test tube (containing the test material) may be either positive or negative, in the second test tube (control) it must be only positive, and in the third, fourth, fifth, and sixth control test tubes the results must always be negative.
Figure. Positive thermoprecipitation reaction (Ascoli’s test)
When employing laboratory diagnosis of anthrax, one must bear in mind the possibility of the presence of bacteria identical with B. anthracis in their biological properties (see Table 1). These sporing aerobes are widely distributed in nature and are normally sporeforming saprophytes. They include B. cereus, B. subtilis, B. megaterium, etc.
The anthrax bacilli may be differentiated from anthracoids (false anthrax organisms) and other similar sporing aerobes by phagodiagnosis. The specific bacteriophage only causes lysis of the B. anthracis culture.
Treatment comprises timely intramuscular injection of antianthraxglobulin, and the use of antibiotics (penicillin, tetracycline, and streptomycin).
Prophylaxis. General measures of anthrax control are carried out in joint action with veterinary workers. These measures are aimed at timely recognition, isolation, and treatment of sick animals. They also include thorough disinfection of premises for live-stock, territory an dall objects found on it, and ploughing over of pastures. Carcasses of animals which have died of anthrax are burnt or buried on specially assigned territory, not less than
The veterinary authorities also enforce regulations banning the use of contaminated meat for food and introduce thorough control of manufactured articles from animal hide and fur which are to be marketed.
Ever since Pasteur’s celebrated field trial, in which animals were successfully immunized with a living attenuated suspension of B. anthracis, efforts have been directed toward the production of effective vaccines that possess little or no toxicity. Because killed vaccines are of little value, two different approaches for the stimulation of artificial immunity have been undertaken: (1) the isolation and use of the protective antigenic component of the anthrax toxin, and (2) the use of attenuated living bacteria to induce antitoxic immunity.
At present, a vaccine, prepared from non-capsulated anthrax bacilli and consisting of a suspension of live spores of vaccine strains, is used. It is employed for immunization of man and domestic animals.
The vaccine is completely harmless, producing immunity quite rapidly (in 48 hours) and for a period of over a year. It is inoculated in a single dose.
Vaccination is carried out among people who work at raw-material processing factories (processing of animal hide and hair), at meat-packing factories, and at farms where anthrax is encountered. Reinoculation is performed after a period of 12 months.
However, all effective living vaccines possess some toxicity, and they have not been used in the United States for humans. The vaccine licensed in the United States for use in humans is an aluminum hydroxide-adsorbed supernatant material from ferment or cultures of a toxigenic, but nonencapsulated strain of B. anthracis. Unfortunately, it induces a short-lived immunity and re-quires annual boosters.
Individuals who have been in contact with material contaminated with anthrax organisms (when dressing infected carcasses or using such meat for food) are given intramuscular injections of 20-25 ml of antianthrax globulin together with penicillin.
PA seems to be the only effective component of the vaccine because neither EF nor LF induced protection. It has been cloned in Bacillus subtilis, and guinea pigs immunized by the intramuscular injection of the B subtilis cell suspension were protected against challenge by B. anthracis. PA also has been cloned in vaccinia virus, where it induced at least partial immunity to challenge in guinea pigs and mice. It is, therefore, possible that a cloned source of PA will be used in future anthrax vaccines for humans. The vaccine used in domestic animals consists of a living spore culture, which is designated the Sterne strain. It still carries the plasmid encoding for PA, EF, and LF, but its avirulence is attributed to the loss of the plasmid encoding the antiphagocytic capsule.
A chemical anthrax vaccine consisting of a protective antigen (a filtrate of non-capsulated non-proteolytic anthrax strains which had been cultivated on synthetic and semisynthetic media) is used in England and the USA, it is as effective as the living vaccine.
Control of anthrax in humans is, therefore, not a simple matter, particularly for those who are occupationally exposed, but sterilization of wool, hair, and other animal materials capable of transmitting the disease does prevent the spread of the bacilli to persons not normally exposed to infected hides. However, not all such products are sterilized, as exemplified by the person who contracted anthrax from contaminated goat hair fringe on a souvenir drum purchased in Haiti. Other Haitian products using goat hair have been found contaminated with anthrax spores, and the Centres for Disease Control in Atlanta has recommended that all such items be considered potentially contaminated and that they be given to local health authorities for disposal.
However, due to the prophylactic measures conducted regularly by the veterinary and public health services, mortality has decreased 130-fold while the disease is a very rare occurrence.
BACILLUS CEREUS. Bacillus cereus is an aerobic, spore-forming, gram-positive rod that is the etiologic agent of a food borne intoxication.
Pathogenesis of B cereus Intoxication. Bacillus cereus produces two distinct clinical forms of food poisoning (1) an illness with an incubation period of 10 to 12 hours, which is characterized by abdominal pain, profuse diarrhea, and nausea, lasting 12 to 24 hours, and (2) an illness with an incubation period of 1 to 6 hours characterized by vomiting, with or without a mild diarrhea.
These two clinical entities result from the production of two different enterotoxins by B cereus. The first, like cholera enterotoxin and LT from Eschenchia coli, stimulates the adenylate cyclase-cyclic AMP system in intestinal epithelial cells. When fed to rhesus monkeys, this heatlabile toxin causes only diarrhea. The second enterotoxin does not stimulate the synthesis of cAMP, and this heatstable toxin causes only vomiting when fed to rhesus monkeys
Epidemiology and Control of B. cereus Intoxication. Bacillus cereus is readily found in soil and on raw and dried foods, including uncooked rice, a major source of B. cereus food poisonings. The spores may not be killed during cooking, and will germinate when the boiled rice is left unrefrigerated (to avoid clumping of grains). Brief warming or flash frying does not always destroy the elaborated enterotoxins, particularly the heat stable toxin. Meat or meat products, as well as cream or pudding preparations, also have been sources of B. cereus food poisoning. A diagnosis usually is based on finding 10sorganisms per gram of the incriminated food.
Prevention is best accomplished by the prompt refrigeration of boiled rice and other dried foods that have been cooked. Because the symptoms are mediated by preformed enterotoxins, antibiotic therapy is of no value.
Laboratory diagnosis in details
The material for examination, depending on the clinical form of the disease (skin, septic), is contents of vesicles, carbuncles, secretion of ulcers, scabs, sputum, faeces, blood, and cerebrospinal fluid. Autopsy material usually studied is blood and pieces of the internal organs.
The material is collected into sterile test tubes or vials. When autopsy material has to be transported to the laboratory, blood, a puncture sample of the spleen, and bone marrow are placed in a thick layer on glass slides (this ensures the formation from vegetative forms of spores with a high rate of survival). After drying, the slides are wrapped with paper, put into a box, sealed, and sent to the laboratory with a courier. In case of epidemiological indications various objects of the environment, as well as the fur and bristle of animals, are examined.
All examinations related to the identification of the culture of anthrax bacilli are carried out in special laboratories.
Bacterioscopic examination. Clinical and autopsy material is examined microscopically. The presence in smears of large Gram-positive bacilli, which form chains surrounded by a capsule), makes it possible to make a preliminary diagnosis of anthrax. Staining of capsules of anthrax bacilli is made by Gins’ method or some other technique. The capsular form of the causative agent is usually detected in blood in a septic form of the disease.
Luminescent microscopy is used as a supplementary method of the diagnosis of anthrax. In this kind of examination anthrax bacilli treated with specific fluorescent serum appear as rods with a rim, which give off green light.
Bacteriological and biological examination. To confirm the diagnosis, inoculation into nutrient media and animals is carried out. The material is introduced into plates with a meat-peptone agar and test tubes with a meat-peptone broth, which are placed into a
In 20-24 hrs the inoculated cultures are examined and dead mice are autopsied (death occurs 24-48 hrs after inoculation). Characteristic rough colonies (“medusa head”) with curly edges are noticed on plates with a meat-peptone agar. A growth resembling a piece of cotton wool forms on the bottom of a test tube with broth; the medium remains transparent. A smear and a hanging-drop preparation are made from the sediment. Acapsular Gram-positive bacilli appearing as long chains are found in the smear. The causative agent of anthrax, unlike mobile soil bacilli, is immobile. To isolate a pure culture, typical colonies are subcultured onto an agar slant.
Of great diagnostic significance is detection of capsule formation outrient media. For this purpose a medium containing Hanks’ solution (or Hottinger’s broth) and 40 per cent of sterile cattle serum, Bouze’s medium (3 per cent hunger agar, pH 7.4, to which 15 per cent of defibrinated sheep blood is added), and other media are employed. The inoculated cultures are incubated for 18-24 hrs at
Dissect the corpses of the mice in 24-48 hrs and look for signs characteristic of anthrax, namely: oedema at the site of material introduction, darkened uncoagulated blood, haemorrhages into fat tissue, loose consistence of the spleen, and a compact red liver. Typical bacilli surrounded with a capsule are found in smears from the internal organs and blood. On the basis of morphological, tinctorial, cultural, and biological data a conclusion is drawn on the detection of B. anthracis.
The isolated culture is identified by inoculating it into gelatin and blood-containing- agar and into animals. B. anthracis induce a characteristic growth in the gelatin column which resembles a rod whose processes go downward in the form of an inverted fir-tree. Later on the gelatin liquefies. There is no haemolysis on a blood-agar.
Study of biological properties of the isolated culture makes it possible to differentiate between B. anthracis and soil bacilli (Table 1).
The penicillin test also allows for the differentiation between the causative agent of anthrax and soil bacilli. It is conducted in the following manner. A three-hour-broth culture of the isolated microorganism is streaked onto plates with a meat-peptone agar containing 0-5 and 0.05 U/ml of penicillin. After 3 hrs of inoculation smears are made and examined microscopically for the appearance of “a pearl necklace”, i.e., disintegration of anthrax bacilli with the formation of balls. Soil bacilli retain the form of chain-arranged rods.
Bacilli of anthrax, in contrast to similar non-pathogenic bacilli are lysed by a specific anthrax phage.
The presence of the anthrax antigen in the decayed carcass of the animal, in the skin (fresh, dry, and tanned) and products from it. as well as in pelts and fur is determined using Ascoli’s thermoprecipitation reaction. The material studied is comminuted, immersed with 10-20-fold volume of isotonic saline and boiled for 10-45 min or kept for 16-20 hrs at 6-
The reaction is considered non-specific if a precipitate forms in 10 min. Simultaneously, control reactions with an extract and precipitating serum are run.
IFT
Serological diagnosis is resorted to in those cases where the causative agent cannot be detected from the material studied. To demonstrate antibodies in the patient’s blood serum, the highly sensitive latex agglutination and passive agglutination reactions with a protective anthrax antigen are employed.
The allergy test is used for detecting immunological changes following a disease and vaccination. The allergen anthraxin is injected intracutaneously in a dose of 0.1 ml. The results are read in 24-48 hrs. The test is considered positive if the patient develops hyperaemia over
SUPPLEMENT
http://en.wikipedia.org/wiki/Yersinia_pestis
http://www.cdc.gov/ncidod/dbmd/diseaseinfo/yersinia_g.htm
http://www.cehs.siu.edu/fix/medmicro/yersi.htm
http://www.cfsan.fda.gov/~mow/chap5.html
http://www.bacteriamuseum.org/species/ypestis.shtml
http://cmr.asm.org/cgi/reprint/10/1/35.pdf
http://en.wikipedia.org/wiki/Francisella_tularensis
http://en.wikipedia.org/wiki/Francisella
http://microbes.historique.net/tularensis.html
http://iai.asm.org/cgi/content/abstract/74/12/6895
http://www.lcusd.net/lchs/mewoldsen/aleach.html
http://en.wikipedia.org/wiki/Brucella
http://www.gsbs.utmb.edu/microbook/ch028.htm
http://medic.med.uth.tmc.edu/path/00001493.htm
http://www.dshs.state.tx.us/idcu/disease/brucella/
http://microbewiki.kenyon.edu/index.php/Brucella
http://en.wikipedia.org/wiki/Bacillus_anthracis
http://textbookofbacteriology.net/Anthrax.html
http://www.bacteriamuseum.org/species/anthrax.shtml
http://www.gsbs.utmb.edu/microbook/ch015.htm
http://en.wikipedia.org/wiki/Clostridia
http://www.gsbs.utmb.edu/microbook/ch018.htm
http://textbookofbacteriology.net/clostridia.html
http://medic.uth.tmc.edu/path/00001496.htm
http://www.bact.wisc.edu/themicrobialworld/clostridia.html
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http://microbewiki.kenyon.edu/index.php/Clostridium
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http://www.lcusd.net/lchs/mewoldsen/tetanus.htm
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