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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.
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.
A polymerase chain reaction (PCR) assay targeting the flagellin P (fliP)-I S407A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP-IS407A sequence of B. mallei American Type Culture Collection (ATCC) 23344(T) allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of B. mallei ATCC 23344(T). B. mallei DNA was also amplified from various tissues of horses with a generalized B. mallei infection. The developed PCR assay can be used as a simple and rapid tool for the specific and sensitive detection of B. mallei in clinical samples.
MANY COMMON FACTORS: Glanders and melioidosis are infectious diseases that are caused by the bacteria of the Burkholderia species. These infections are endemic in tropical regions and can lead to la broad spectrum of common clinical manifestations. TWO PRINCIPLE CLINICAL FORMS: The most frequent clinical presentation is the pulmonary form, which can mimic pulmonary tuberculosis. The septicemic form is the most severe form, and lethal in nearly 50% of cases. WEAPONS FOR BIOTERRORISM AND WAR: Very few organisms are required to cause disease by aerosolisation, which could be the main route of contamination for humans after a deliberate release. This property has permitted yet the use of these bacteria as biological warfare weapon during the past century. We have to consider these agents as possible biological warfare agents. Europeans guidelines for treatment and post-exposure prophylaxis are detailed.
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.
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.
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.
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.
From the many existing documents on the history of glanders, it is possible to detail the practical measures adopted for disease surveillance and control from antiquity until the 19th century, principally in European countries. Surveillance is based on clinical diagnosis, post-mortem examination, animal inoculation and knowledge of the conditions under which infection occurred: aetiology, pathogenesis, susceptible species, virulent material, mode of infection, incubation period, etc. The historical data are assembled and compared, with comments on each of these points. Control is based on the application of general disease control measures and attempts at vaccination and treatment. A study of these procedures enables a comparison of their efficacy and a description of the major steps in their implementation.
The authors present the results of in vitro determination of the sensitivity of the causative agent of glanders and melioidosis to 8 preparations-5-nitrofuran derivatives, and also to negram and PASK. The most active against M. mallei and Ps. pseudomallei were furazonal and furacrillin; negram was less active. No naturally resistanct strains to furacrylin and furazonal were revealed among the M. mallei and Ps. pseudomallei strains studied.
Pathogenic Burkholderia--Burkholderia mallei and Burkholderia pseudomallei--are causative agents of glanders and melioidosis, severe infectious diseases of man and animals. They are regarded as potential agents of bioterrorism. The existing bacteriological and immunological methods of identification of B. mallei and B. pseudomallei are not efficient enough for the rapid diagnosis and typing of strains. Described in the paper are molecular methods of detection of the agents by PCR, hybridization and strain typing made on the basis of bacterial total cell protein profiles, RAPD, ribotyping as well as of plasmid and DNA microrestriction analyses.
Criteria for the evaluation of the plasmocoagulase activity of natural isolates and mutant strains of the causative agents of glanders and melioidosis were worked out, which made it possible to subdivide them by this sign into pathogens with high, moderate and low activity. Plasmocoagulase produced by pathogenic Burkholderia was shown to be a thermolabile enzyme, comparatively stable with respect to the action of such chemico-biological agents as hydrogen peroxide and chloramine.
The interaction of the aqueous saline extracts of 55 plant species with the antigens of the causative agents of plague, glanders and melioidosis in the reaction of immunodiffusion (RID) in gel has been studied. The aqueous saline extracts obtained from the seeds of 12 plant species have been revealed to possess precipitating capacity. At the same time these extract have been found to ensure, as a rule, RID with several antigens under test.
The authors have demonstrated the possibility of using solid-phase enzyme immunoassay to detect specific circulating immune complexes without their preliminary separation in guinea pigs infected with Pseudomonas mallei and Pseudomonas pseudomallei. They have examined the prospects of using neutrophil injury test and solid-phase enzyme immunoassay for the detection of specific immune complexes as additional tests used to predict the outcome of an infectious process in glanders and melioidosis.
Combinations of sulfazine or sulfamonomethoxine with trimethoprim were found to be efficient in the prophylaxis and treatment of experimental glanders simulated by various strains and doses of the causative agent, the treatment being started and terminated at various periods. The efficacy indices and treatment schemes were elaborated. By the efficacy indices the sulfazine combination with trimethoprim was shown to be 2-3 times more efficient than the sulfamonomethoxine combination with trimethoprim.
The pathomorphology and cell-mediated response of the body to Burkholderia mallei in laboratory animals, highly sensitive and resistant to these bacteria. In the comparative study of the pathomorphology and pathogenesis of glanders in golden hamsters and white rats quantitative and qualitative differences in the histogenesis of response reaction and the morphology of immunocompetent organs were established. Cell-mediated reactions play a greater role in the limitation of the early spread of B. mallei in the host body than antigen-mediated mechanisms.
Unlike the glanders agent, the superficial structures of the melioidosis agent were demonstrated to be responsible for marked was immunosuppressive activity. Some antigenic fractions suppressing the blast transformation of lymphocytes, reducing the count of T helpers and profoundly potentiating the infection in vivo were isolated from P. pseudomallei cells. The immunogenic and immunosuppressive activities of both agents' superficial structures were studied by high performance chromatography. Antigenic complexes that were able to protect immunized laboratory animals against fatal infections and to prevent bacterial carriage due to the activation of T cells and to the bacterial activity of macrophages were identified. A composition comprising several immunogens was found to provide an additive protective action against both causative agents. Therefore, the composition may be considered to be a prototype of a molecular antipseudomonadic vaccine.
In multiple experiments on golden hamsters, highly sensitive to glanders, the method of the transfer of antibacterial resistance was worked out. The most positive effect was obtained after the subcutaneous injection of saline-suspended marrow cells, obtained from white rats, infected with Burkholderia mallei C5 in a single inoculation and passaged through noninfected white rats 5 times. Out of 60 golden hamsters, treated with such material and subsequently (on day 21) injected with 1.0 x 10(2) B. mallei cells, 42 animals survived (70%).