Equine Glanders Research Articles

Use of a Recombinant Burkholderia Intracellular Motility A Protein for Immunodiagnosis of Glanders

Glanders, caused by the Gram-negative, nonmotile bacterium Burkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance and B. mallei is listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) have been linked to the use of crude test antigens. We expressed a novel recombinant Burkholderia intracellular motility A (rBimA) protein in Escherichia coli for the diagnosis of equine glanders. Purified rBimA was used in an indirect ELISA format. All of the 21 true-positive serum samples used in the study tested positive, whereas only 17 of the 1,524 potentially negative sera tested positive by indirect ELISA, thus exhibiting 100% sensitivity and 98.88% specificity. Also, rBimA protein did not react with melioidosis patient and normal healthy human serum samples, showing its high specificity. The developed assay can be used as a simple and rapid tool for diagnosis of glanders in equine serum samples. An Indian patent (1328/DEL/2010) has been filed for the reagent.

Biological terrorism and the allergist's office practice

During the anthrax outbreak and threat in Trenton (2001), our allergy practice experienced increased visits from approximately 50 of our regular patients with symptoms they believed resulted from anthrax exposure. In all cases, their symptoms were caused by a combination of an exacerbation of their underlying allergic disease and anxiety because of possible exposure to anthrax. Our objective is to present an orderly approach to the allergist's outpatients presenting with possible exposure to a bioterrorist's agent. The 10 precepts of approach to the management of a biological casualty (index of suspicion, protect yourself, patient assessment, decontaminate, diagnose, treat, infection control, alert authorities, assist in investigation, and maintain proficiency) and the epidemiological characteristics of a biological attack are discussed. In table form, we compared the signs and symptoms of the most common outpatient consultations to an allergist's office practice (chronic rhinitis, asthma, food allergy, venom allergy, atopic dermatitis, drug allergy, chronic urticaria, acute urticaria, immunodeficiency, and anaphylaxis) with those of likely bioterrorism threats. Descriptions of smallpox, plague, tularemia, anthrax, viral hemorrhagic fevers, Q fever, brucellosis, Venezuelan equine encephalitis, glanders, and melioidosis are presented. Patients may readily mistake their allergic symptoms with those of infection with a bioterrorist's agent. At the same time, the allergist may be faced with one of his own chronic patients presenting with symptoms resembling their allergic disease but actually caused by one of the aforementioned pathogens.

Antimicrobial Susceptibility of 41 Burkholderia mallei Isolates From Spontaneous Outbreaks of Equine Glanders in Punjab, Pakistan

Minimum inhibitory concentrations (MICs) of 20 antimicrobial agents for 41 isolates of Burkholderia mallei from natural outbreaks of equine glanders were determined by agar dilution. All isolates were intrinsically resistant to ampicillin (MIC 90 ≥128). Resistance to other antimicrobials was as follows: 95.1% to amoxicillin and cephradine, 85.4% to cefuroxime and norfloxacin, 68.3% to ceftizoxime and ceftriaxone, 61.1% to ceftiofur, 58.5% to oxytetracycline, 41.5% to ciprofloxacin, 58.5% to roxithromycin, 17.1% cefotaxime, and 12.2% clarithromycin. Overall resistance patterns revealed that 17% of isolates were simultaneously resistant to amoxicillin, cephradine, cefuroxime, ceftizoxime, ceftriaxone and norfloxacin. None of the isolates were resistant to amoxicillin-clavulanic acid, doxycycline, chloramphenicol, gentamicin or trimethoprim-sulphadiazine. Mode MICs for these antimicrobials were 2, 1, 8, 4 and 1 μg/ml, respectively. A majority of the isolates (∼ 94%) were susceptible to both enrofloxacin and ofloxacin. These data provide an updated perspective on susceptibility profiles of current strains of B. mallei in an endemic area.

Comparative Evaluation of Rose Bengal Plate Agglutination Test, Mallein Test, and Some Conventional Serological Tests for Diagnosis of Equine Glanders

The Rose Bengal plate agglutination test (RBT) was evaluated for the diagnosis of equine glanders, and its diagnostic efficiency was compared with that of mallein and other serological tests, including indirect hemagglutination test (IHAT), complement fixation test (CFT), and modified counter immunoelectrophoresis test (mCIET). Sera from 70 naturally infected culture-positive, 96 potentially exposed cohorts, and 110 healthy equines were tested. All tests but mCIET showed 100% specificity when testing the sera from glanders-negative equines. The calculated sensitivities of RBT, IHAT, CFT, mCIET, and mallein test when testing culture-positive equines were 90.0, 97.1, 91.4, 81.4, and 75.7%, respectively. The RBT was significantly (P < 0.05) more sensitive than the mallein test and mCIET. The positive and negative predictive values of each test (RBT, IHAT, CFT, mallein test, and mCIET) were as follows: 100 and 94, 100 and 98.2, 100 and 96.7, 100 and 86.6, and 90.5 and 88.6, respectively. On comparing glandered and nonglandered animals, the highest agreement (0.987) was found between RBT and CFT followed by RBT and IHAT (0.940), RBT and mallein test (0.871), and RBT and mCIET (0.852). Because the RBT is simpler and rapid to perform, the inclusion of the test as a supplementary test for the diagnosis of glanders in field conditions is recommended.

Burkholderia Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis.

BackgroundThe bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.ResultsUsing bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis.ConclusionMembers of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study.

Detection of the reemerging agent Burkholderia mallei in a recent outbreak of glanders in the United Arab Emirates by a newly developed fliP-based polymerase chain reaction assay

A polymerase chain reaction (PCR) assay targeting the flagellin P ( fliP)-I S 407A 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-IS 407A 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.

Characterization of experimental equine glanders

Considerable advances in understanding of the disease caused by Burkholderia mallei have been made employing a combination of tools including genetic techniques and animal infection models. The development of small animal models has allowed us to assess the role of a number of putative virulence determinants in the pathogenesis of disease due to B. mallei. Due to the difficulties in performing active immunization studies in small animals, and due to the fact that the horse is the target mammalian species for glanders, we have initiated experimental studies on glanders in horses. Intratracheal deposition of B. mallei produced clinical glanders with organisms being recovered from tissues of infected horses. The model should prove to be of considerable value in our ongoing studies on the pathogenesis and vaccine development for glanders.

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....

Clinico-Microbiological and Therapeutic Aspects of Glanders in Equines.

The role of glanders therapy as an alternative to test and slaughter policy was investigated in 13 draught equines (1 horse, 2 mares and 10 mules). The disease was diagnosed clinically and confirmed by isolation of Burkholderia mallei on blood agar and sterile potato slices. Mallein test was positive in 6 (46.15 %) of the 13 animals. All isolates from the 13 equines were sensitive to chloramphenicol, danofloxacin, norfloxacin and co-trimoxazole whereas penicillin and cephalexin were the least effective in vitro antibiotics. These animals were treated for 4 days with Ringer's-lactate-dextrose 500 ml + dimetyl-sulfoxide 60-80 ml intravenously and Inj. Norfloxacin 5% 35-50 ml intramuscularly (1st round of treatment). Five animals were treated again similarly (2nd round) after two weeks of the first round. All treated animals showed a dramatic improvement in the severity of the disease condition which lasted for 2-3 weeks after which the signs rebounded. Even the second round of treatment was met with the same fate. The treatment was not associated with any side-effect except in two cases which suffered injection site swelling. The possibility of a cure in clinical glanders using a longer course of treatment is proposed.

Potency of partially purified malleo-proteins for mallein test in the diagnosis of glanders in equines

Malleo-proteins from synthetic broth mallein of six strains of Pseudomonas mallei were separated by trichloroacetic acid precipitation, amonium sulfate precipitation and Ultrogel AcA 34 gel filtration chromatography. When tested comparatively with Dutch PPD mallein as standard on P. mallei-sensitized and normal horses all the strains were found to be malleinogenic, trichloroactive acid precipitated proteins were comparable to Dutch PPD mallein in potency and innocuity whereas ammonium sulfate-precipitated proteins elicited non-specific reactions. Ultrogel AcA 34 chromatographed high molecular weight proteins (MW range > 350 000) were having equal or higher potency and without adverse reactions and low molecular weight proteins (MW range 120 000–170 000) nearly inactive. Ultrogel AcA 34 column fractionation studeis revealed that mallein activity was associated with high molecular weight proteins and represented a purer sensitin than PPD mallein for the purpose of mallein test.

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 avidinbiotin 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-completement activity, and it was able to detect antibodies at an early stage. Testing of serum at 1:200 dilution is proposed for epidemiological screening.