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[Glanders--a potential disease for biological warfare in humans and animals].

Infection with Burkholderia mallei (formerly Pseudomonas mallei) can cause a subcutaneous infection known as "farcy" or can disseminate to condition known as Glanders. It is primarily a disease affecting horses, donkeys and mules. In humans, Glanders can produce four types of disease: localized form, pulmonary form, septicemia, and chronic form. Necrosis of the tracheobronchial tree and pustular skin lesions characterize acute infection with B. mallei. Other symptoms include febrile pneumonia, if the organism was inhaled, or signs of sepsis and multiple abscesses, if the skin was the port of entry. Glanders is endemic in Africa, Asia, the Middle East, and Central and South America. Glanders has low contiguous potential, but because of the efficacy of aerosolized dissemination and the lethal nature of the disease, B. mallei was considered a candidate for biological warfare. During World War I, Glanders was believed to have been spread to infect large numbers of Russian horses and mules on the Eastern front. The Japanese infected horses, civilians and prisoners of war during World War II. The USA and the Soviet Union have shown interest in B. mallei in their biological warfare program. The treatment is empiric and includes mono or poly-therapy with Ceftazidime, Sulfadiazine, Trimethoprim + Sulfamethoxazol, Gentamicin, Imipenem etc. Aggressive control measures essentially eliminated Glanders from the west. However, with the resurgent concern about biological warfare, B. mallei is now being studied in a few laboratories worldwide. This review provides an overview of the disease and presents the only case reported in the western world since 1949.

Animals↗

[Characteristics of the infectious process in animals susceptible and resistant to glanders].

Combined bacteriological, pathomorphological, luminescent-serological, and electron microscopic studies were used to elucidate some aspects of pathogenesis of glanders in animals with different susceptibility to this infection. The animals highly susceptible to glanders (golden hamsters) were shown to develop the infection of the sepsis type with granulomas of the exudative nature. In the animals highly susceptible to glanders the bacteria were proved experimentally to parasitize in a rod-shaped form within macrophages of the spleen, liver, and lungs as well as extracellularly. In white mice resistant to glanders the infection developed as chronic septicemia with granulomas of the exudative-proliferative nature. Glanders bacteria in this animal species undergo L-transformation.

Animals↗

[Obtaining spheroplasts from the agents of glanders and melioidosis and separation of membrane structures from them].

Spheroplasts were obtained from the causative agents of glanders and melioidosis under the effect of lysozyme and antibiotics. In the capacity of an inducing agent lysozyme was effective in high concentration only (0.4%); preliminary washing and incubation in sucrose were necessary to obtain glanders spheroplasts. Of the antibiotics studied penicillin was more useful for obtaining melioidosis spheroplasts and ampicillin--for glanders spheroplasts. Membrane preparations were derived from the spheroplasts of glanders and melioidosis causative agents.

Ampicillin↗

Comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders.

Melioidosis and glanders are caused by the closely related species Burkholderia pseudomallei and Burkholderia mallei, respectively. Whereas melioidosis is a significant cause of morbidity in south-east Asia, glanders is extremely rare. The efficacies of ciprofloxacin and doxycycline were assessed against a strain of B. pseudomallei and a strain of B. mallei which were susceptible to both antimicrobials in vitro. Porton outbred mice and Syrian hamsters were given 40 mg/kg of either doxycycline or ciprofloxacin twice daily by sc injection according to one of three regimens: dosing starting 48 h before challenge and continuing for 5 days postchallenge; 5 days' therapy starting immediately after challenge; 5 days' therapy starting 24 h after challenge. Mice were challenged ip with B. pseudomallei 4845 and hamsters were challenged ip with B. mallei 23344. Antimicrobial efficacy was determined by the shift in the median lethal dose (MLD). Ciprofloxacin prophylaxis and immediate therapy both raised the MLD of B. pseudomallei to 4 x 10(6) cfu from 19 cfu in untreated animals, but therapeutic ciprofloxacin only raised the MLD to 180 cfu. The results for doxycycline were similar. Ciprofloxacin prophylaxis raised the MLD of B. mallei 23344 to 4.6 x 10(5) cfu compared with 4 cfu in untreated controls. Immediate therapy raised the MLD to 7.0 x 10(4) cfu and therapy raised the MLD to 1.6 x 10(3) cfu. All regimens of doxycycline protected hamsters against challenges of up to 2 x 10(7) cfu. Despite using a susceptible strain of B. pseudomallei, neither antimicrobial was effective when used therapeutically. The timely administration of either antimicrobial, however, was effective in preventing symptomatic infection. Doxycycline was the superior of the two antimicrobials against experimental glanders although relapse did occur in treated animals approximately 4-5 weeks after challenge.

Animals↗

Equine glanders in Turkey.

In the course of an epidemiological study of glanders on a number of Turkish islands in the Sea of Marmara, 1128 horses were examined by using the intracutaneous mallein test. Thirty-five (3-1 per cent) developed an increase in rectal temperature and a swelling at the point of injection. Ten of these horses were killed and glanders was confirmed in five cases by the presence of lesions and by the immunohistological demonstration of the causative agent, Burkholderia mallei. Clinical and pathological findings indicated that in all cases the infection was restricted to the mucous membrane of the nasal cavity with its parasinus, the nostrils and the upper lips. It was confirmed that equine glanders is endemic in Turkey.

Animals↗

Laboratory-acquired human glanders--Maryland, May 2000.

On May 5, 2000, the Baltimore City Health Department was notified by hospital infection-control staff of a serious systemic febrile illness in a microbiologist whose research at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) involved several pathogenic bacteria, including Burkholderia mallei, the causative agent of glanders. This report summarizes the first human case of glanders in the United States since 1945, and emphasizes the importance of considering occupational exposures among laboratory workers with a febrile illness, the difficulty of characterizing unusual agents, including potential agents of biological terrorism such as glanders using routine laboratory techniques, the appropriate isolation practices for patients who may be infected by these agents, and laboratory safety.

Burkholderia↗

[Rayer's studies on the contagion of glanders (1837-1843)].

P. Rayer (1795-1867) had never thoroughly published his experimental studies on the contagion of glanders. His recently un-earthed hand written papers allow us to depict his experimental approach and its results. He was not the first who transmitted glanders from a patient to horses or donkeys. But he did it systematically with glander secretions from acute and chronic cases. Whatever was the disease of the donors the transmitted forms were unpredictably either chronic or acute. His conclusion was that the two forms were two symptomatic aspects of a unique disease. Clinically dormant states were shown to be also contagious. He demonstrated it through deliberately altering healthy and sick horses inside the stable and by using saddles, bridles and brushes of sick horses on healthy ones. Moreover he excluded other causative factors tentatively proposed, peculiarly food products. The systematically logical and rigorous experimental approach used by Rayer for this research is a mile stone, 30 years before Pasteur. This methodology is still nowadays used to study the epidemiology of diseases such as Prion Diseases, Mad Cow for instance.

Animals↗

[Antibiotic sensitivity of the causative agent of glanders].

Sensitivity of the glanders causative agent to 20 antibiotics representing different groups is described. The results of the in vitro studies showed that most of the antibiotics were not active against the glanders causative agent, except for polymyxin M, levomycetin and their combination. Some relative activity was observed with neomycin. The "indicators" method used by the authors for determination of the M. mallei sensitivity to the antibiotics allowed the shortening of the time of the test by 8 to 10 hours.

Actinobacillus↗

[Identification of the causative agents of glanders and melioidosis by polymerase chain reaction].

Burkholderia mallei and B. pseudomallei are causative agents of glanders and melioidosis, respectively, i.e. severe and fatal infection diseases of man and animal. The computer-based analysis of the 23S rRNA gene sites was used for selecting the primers. Two pairs of primers were chosen for the identification of B. mallei and Bpseudomallei. DNAs from 48 B. pseudomallei and 15 strains of B. mallei, unlike from other geterological bacteria, were positively amplified. Therefore, the method of polymerase chain reaction can be used in laboratory diagnosis of glanders and melioidosis.

Animals↗

[Membrane antigens of the agents of glanders and melioidosis].

Double immunodiffusion in gel test was used to determine the antigenic composition of the preparations of membranes isolated from the lysozyme spheroplasts of glanders (strain No. 10230) and melioidosis (strain No. C-141) causative agents. The membranes of these microbes proved to contain antigens of cell walls, lipopolysaccharides and the thermolabile membrane antigen proper. A study of antimembrane sera in the agglutination and immunofluorescence tests demonstrated a heterogeneity of the glanders and melioidosis strains under study by the membrane thermolabile antigen.

Animals↗

[The early laboratory diagnosis of the pulmonary form of glanders and melioidosis by using rapid methods of immunochemical analysis].

The problems of the sanitary guarding of frontiers comprise such aspects as the early recognition of infectious diseases, even those which are considered exotic in the Russian Federation. These diseases include glanders and melioidosis, infections which are endemic for some frontier regions. The study performed on experimental models of the pulmonary form of glanders and melioidosis with the acute course of the diseases demonstrates that the use of such laboratory diagnostic methods as immunosorbent variants of the enzyme-linked, chemiluminescent and immunofluorescent immunoassays permits making reliable diagnosis as early as 3-12 hours after aspiration infection.

Acute Disease↗

[The principles of the therapy of glanders in monkeys].

The effect of pathogenetic therapy in the normalization of homeostasis disturbances in monkeys has been shown under experimental conditions. Data on the possibility of using hemosorption in the treatment of severe forms of glanders are presented. The conclusion on the necessity of using complex treatment for the effective therapy of glanders in humans has been made.

Acute Disease↗

Development of an avidin-biotin dot enzyme-linked immunosorbent assay and its comparison with other serological tests for diagnosis of glanders in equines.

A dot enzyme-linked immunosorbent assay (dot ELISA) was developed for diagnosis of glanders in equines. The test was based on the detection of IgG antibodies to Pseudomonas mallei antigens bound to nitrocellulose coated on plastic strips (dipsticks), the reaction being amplified by an avidin-biotin system with biotinylated anti-horse IgG and horseradish peroxidase-avidin D. Sera from 810 normal, six naturally infected and 48 sensitized equines were tested by this assay, and results were compared with complement fixation, indirect haemagglutination and counter-immunoelectrophoresis tests. Dot ELISA had the highest sensitivity, and was superior to other tests in that it was rapid and easy to perform, the results were easy to interpret, the assay was not influenced by anti-complement activity, and it was able to detect antibodies at an early stage. Testing of serum at 1:200 dilution is proposed for epidemiological screening.

Animals↗

The use of animal infection models to study the pathogenesis of melioidosis and glanders.

The use of animal infection models is central to the study of microbial pathogenesis. In combination with genetic, immunological and antigen purification techniques, much can be learned regarding the pathogenesis of diseases caused by microorganisms. This update focuses on the recent use of animal infection models to study the pathogenesis of melioidosis and glanders.

Animals↗

Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei.

A collection of 147 isolates of Burkholderia pseudomallei, B. mallei, and B. thailandensis was characterized by multilocus sequence typing (MLST). The 128 isolates of B. pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of B. thailandensis and B. pseudomallei was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the B. pseudomallei isolates formed a cluster of closely related lineages that were fully resolved from the cluster of B. thailandensis isolates, confirming their separate species status. However, isolates of B. mallei, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the B. mallei isolates clustered within the B. pseudomallei group of isolates. Alleles at six of the seven loci in B. mallei were also present within B. pseudomallei isolates, and B. mallei is a clone of B. pseudomallei that, on population genetics grounds, should not be given separate species status.

Alleles↗

The hamster model of intraperitoneal Burkholderia mallei (glanders).

Thirty-one female Syrian hamsters (Mesocricetus auratus) were inoculated intraperitoneally with a lethal dose of Burkholderia mallei (Budapest strain). Hamsters were killed postinoculation on days 0 through 6. Lesions were first noted in the spleens on postinoculation day 1, and in mediastinal and mesenteric lymph nodes, mediastinum, liver, and bone marrow on day 2. Lesions were present in the lung and submandibular lymph nodes on day 3, and in the brain on day 5. The characteristic histopathologic change was necrotizing pyogranulomatous inflammation, often with hemorrhage. Lesions indicative of impaired vascular perfusion, such as ischemia and infarction, were evident at the later time points. Pathologic changes generally increased in severity and distribution with time, and almost all tissues were ultimately affected. Our findings suggest that intraperitoneal bacteria were rapidly transported to mediastinal lymph nodes by transdiaphragmatic lymphatics and ultimately seeded other tissues hematogenously. The results of the study indicate that the Syrian hamster is a useful small animal model for glanders.

Animals↗

Mouse model of sublethal and lethal intraperitoneal glanders (Burkholderia mallei).

Sixty male BALB/c mice were inoculated intraperitoneally with either a sublethal or a lethal dose of Burkholderia mallei China 7 strain, then killed at multiple time points postinoculation. Histopathologic changes were qualitatively similar in both groups and consisted of pyogranulomatous inflammation. In sublethal study mice, changes were first seen at 6 hours in mediastinal lymph nodes, then in spleen, liver, peripheral lymph nodes, and bone marrow at day 3. These changes generally reached maximal incidence and severity by day 4 but decreased by comparison in all tissues except the liver. Changes were first seen in lethal study mice also at 6 hours in mediastinal lymph nodes and in spleens. At day 1, changes were present in liver, peripheral lymph nodes, and bone marrow. The incidence and severity of these changes were maximal at day 2. In contrast to sublethal study mice, the incidence and severity of the changes did not decrease through the remainder of the study. The most significant difference between the two groups was the rapid involvement of the spleen in the lethal study mice. Changes indicative of impaired vascular perfusion were more frequently seen in the sublethal study mice. Our findings indicate that mice are susceptible to B. mallei infection and may serve as an appropriate model for glanders infection in a resistant host such as human beings. Additionally, by immunoelectron microscopy, we showed the presence of type I O-antigenic polysaccharide (capsular) antigen surrounding B. mallei.

Animals↗