Performance of complement fixation test and confirmatory immunoblot as two-cascade testing approach for serodiagnosis of glanders in an endemic region of South East Asia

Various serological tests were used for the diagnosis of glanders in the past but still complement fixation test (CFT) is the internationally prescribed test for trading equines. A new immunoblot (IB) technique has recently been introduced to overcome the well known shortcomings of CFT i. e. a considerable number of false positive and negative results and anticomplementary effects of sera. The objective of this study was the comparative evaluation of two glanders CFT antigens commercially available at Central Veterinary Institute ofWageningen UR, Lelystad, NL (CIDC) and at c.c.pro GmbH, Oberdorla, DE (c.c.pro) in a glanders endemic area regarding specificity and sensitivity. A total of 1678 serum samples from the endemic region (Province Punjab, Pakistan) and a non-endemic area (Germany) were analysed. All sera tested positive or suspicious with CFT were analysed by the confirmatory IB to exclude CFT false positive results. Both CFT antigens showed 100% sensitivity. The use of CIDC or c.c.pro antigen resulted in specificities of 77.45% or 75.71% for sera from endemic area and 93.75% or 94.79% for sera from non-endemic areas, respectively. The results demonstrate the different performances of identical tests in different epidemiologically settings. Based on these results, the combined use of CFT and IB is highly suggestive for the serodiagnosis of glanders. Good agreement was calculated between CFT (using either c.c.pro or CIDC antigen) and immunoblot.     

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 mallei infection in a horse imported from Brazil

A horse imported from Brazil developed a respiratory illness 2 weeks after arrival in Germany. After an initial but inefficient treatment glanders was diagnosed based on serological and molecular biological findings. The present case highlights the potential risk of an importation of glanders in free areas. The fact that veterinarians in countries where glanders has been eradicated for decades are not familiar with the clinical symptoms of the disease, can favour the entry of the disease. In order to prevent the spread of glanders, the sanctions of the veterinary authorities in such cases of the infection are of utmost importance.     

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.     

Glanders--a comprehensive review

Since 1990 the number of glanders outbreaks in race, military and pleasure horses in Asia and South America is steadily increasing. Glanders, which is eradicated in Western Europe, Australia and Northern America, is currently considered a re-emerging disease. Consequently, the disease may be introduced into glanders-free regions by subclinical carriers at any time. The causative agent of glanders, Burkholderia (B.) mallei, is highly contagious and leads to chronic disease in horses whereas in donkeys and mules the disease is acute and often fatal. Occurrence of the disease leads to international trading restrictions and infected animals immediately have to be culled and safely disposed off. In humans B. mallei infection results in a severe clinical course, and is fatal without appropriate therapy. Its pathogenicity makes B. mallei a potential biological agent that may be used in bioterroristic attacks. Due to the eradication of glanders in the second half of the last century, veterinarians in western European countries are no longer familiar with its clinical presentation in solipeds. Having these facts in mind, this review describes the epidemiology, clinical signs, pathology and the current eradication strategy of this interesting zoonosis. Pictures of imported endurance horses infected with glanders taken during an eradication campaign in Dubai, United Arab Emirates, in 2004 illustrate most typical clinical findings.     

On the Current Situation of Glanders in Various Districts of the Pakistani Punjab

Glanders is a highly infectious and zoonotic disease of solipeds caused by Burkholderia mallei. Progressive loss of efficiency and fatal outcome resulted in massive economic losses, which forced veterinary authorities throughout the world to implement disease control measures; these measures included mass testing using the complement fixation test and/or malleinization, and the culling of positives. This led to the eradication of glanders from Western Europe and North America in the 1950s. However, in the last decade, the number of outbreaks in Asia and South America increased steadily, and glanders regained the status of a re-emerging transboundary disease. Pakistan has been an endemic country for the past 120 years, but concise data on the presence of disease are not available. A total of 533 serum samples were collected from draught equines, a suspected risk group for glanders, from various districts of Punjab in Pakistan. The complement fixation test and the highly sensitive Western blot technique were used for serodiagnosis. No animal (horse, mule, and donkey) was found to be positive for infection. Glanders seems to be restricted to remote, sporadic pockets of endemicity and may cause outbreaks after being introduced into naive populations by (asymptomatic) shedders.     

马鼻疽弱毒菌苗对马体的免疫试验

应用鼻疽杆菌C67-4060Co(7)兼高温178代和C67-40高温180代两株弱毒菌株,对8匹马进行了免疫试验,其中安全试验组2匹,免疫试验组4匹,对照试验组2匹。安全试验组马经70d左右的观察后,人工迫杀进行病理剖检和组织学检查,均未发现鼻疽性病变,细菌学检查阴性,表明该弱毒菌苗安全性好。免疫试验组马经3个月左右的观察后,以不同剂量强毒菌株攻击,除1匹马出现一侧性鼻漏外,其余马匹均无临床症状,细菌学检查4匹马均为阴性,人工迫杀后经病理学和免疫组织学检查等综合性判定,免疫马有3匹耐过强毒菌的攻击而获得保护。而强毒菌攻击的2匹对照马均发病,经病理剖检和组织学检查均有鼻疽性病变,细菌学检查阳性。     

Identification of a new diagnostic antigen for glanders using immunoproteome analysis

Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n = 49) and negative (n = 79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.     

Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses.

Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses. Apparent test specificities for the B. equi, B. caballi, T. equiperdum, and B. mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively. Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B. equi, B. caballi, T. equiperdum, and B. mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively. The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use. Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera.     

Comparative evaluation of three commercially available complement fixation test antigens for the diagnosis of glanders

The sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true-negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinically positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as the gold standard test. Highest sensitivity was shown for the CIDC antigen (100 per cent) followed by the c.c.pro antigen (99.39 per cent). However, the USDA antigen showed substantially less (p<0.05) sensitivity (62.19 per cent). Highest specificity was found for the USDA antigen (100 per cent) followed by the CIDC (97.5 per cent) and c.c.pro antigen (96.5 per cent). Positive and negative predictive values (assumed glanders prevalence of <0.1 per cent) for each antigen were calculated to be 95.88 and 99.48 (c.c.pro), 97.04 and 100 (CIDC), 100 and 76.33 per cent (USDA), respectively. Almost perfect agreement (0.96) was found between CFT using either c.c.pro or CIDC and IB.     

Development and clinical evaluation of a PCR assay targeting the metalloprotease gene (mprA) of B. pseudomallei

A PCR assay targeting the metalloprotease gene (mprA) of Burkholderia pseudomallei was developed for the specific detection of this organism in pure cultures and clinical samples. All other closely related organisms including B. mallei the causative agent of glanders, and B. thailandensis tested negative. Burkholderia pseudomallei DNA was successfully amplified from paraffin-embedded lung tissue of a camel with a generalized B. pseudomallei infection. The developed PCR assay can be used as a simple tool for the specific and sensitive detection of B. pseudomallei.     

Glanders and the risk for its introduction through the international movement of horses

Glanders is the contagious zoonotic disease caused by infection with Burkholderia mallei. It affects primarily horses, donkeys and mules. The disease was eradicated from large areas of the Western world in the early 20th century, but, over the last 10–20 years, has emerged and re‐emerged in areas in which it was previously unknown or had been eradicated. Although glanders was previously thought to manifest in only acute or chronic presentations, it now appears that B. mallei can produce latent infections similar to those caused by Burkholderia pseudomallei. These latent infections may or may not be detectable by current diagnostic tests. The diagnostic test currently recommended by the World Organisation for Animal Health (Office International des Epizooties [OIE]) for international trade in equids is the complement fixation test (CFT). This test has been shown to have varying sensitivities and specificities depending on the antigen and methodology used. False positives are problematic for the horse‐owner and veterinary authority, whereas false negatives may allow the reintroduction of B. mallei into B. mallei‐free areas. These gaps in knowledge of the epidemiology of glanders, and weaknesses in its diagnosis, coupled with the increased movement of equids, indicate that infection with B. mallei remains a major risk in the context of international movement of equids.     

安徽省马鼻疽防制

安徽省１９５３年由于阜阳地区自外省购入马匹而带入马鼻疽，５０－８０年代该病在淮北及沿淮局部地区流行，疫情发生后，由于各地积极采取检疫、扑杀、隔离、消毒等综合必防治措施，１９９０年后临床再无病例发生,检疫中也再未检出阳性畜。１９９６－１９９８年连续３年在原疫区以鼻疽菌素点眼检疫马属动物７７００５匹，结果均为阴性，１９９９年１１月底农业部专家组验收，达到消灭标准。     

Histofarcin test for the diagnosis of epizootic lymphangitis in Ethiopia: development, optimisation, and validation in the field

Histofarcin, a skin test antigen for the diagnosis of epizootic lymphangitis, was locally produced from the mycelial form of Histoplasma capsulatum var. farciminosum (HCF) in disease-endemic districts of Ethiopia and tested for its application in the field between April 2002 and May 2003. The test was evaluated using 108 mules, 84 in endemic and 24 in disease-free districts. Microscopic and mycological examinations of clinical lesions were used as the "gold standard" for the validation of the test. The concentration of histofarcin that caused an optimum reaction was 0.2-0.4 mg/mL in a 0.1 mL dose and this was attained 24-48 h post-injection. The sensitivity and specificity of the histofarcin test were 90.3% (CI = 73.1, 97.5) and 69% (95%, CI = 48.1, 84.9%) in disease-endemic districts. On the other hand, specificity was 100% (CI=94.8, 100) in disease-free districts. Positive and negative predictive values of the histofarcin test were 77.78% (95% CI = 60.4, 89.3) and 85.71% (95% CI = 62.6, 96.2), respectively. A marginal substantial agreement (kappa = 0.61, P = 0.0000) was observed between the clinical status and the result of the histofarcin test. A large proportion (31%) of 'false positives' was recorded in endemic districts, which could be due to the pre-clinical stage of the disease. The latter ended in lower specificity and positive predictive value of the test since the true infection status of a 'false positive' could not be known on the basis of clinical features. Therefore, standard test validation procedures including slaughtering and isolation of HCF is required. After proper validation, we conclude that the histofarcin test could play a significant role in detecting early infection, and differentiating of EL from glanders, ulcerative lymphangitis, and sporotrichosis.     

Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives

Burkholderia mallei causes glanders or farcy in solipeds, a disease that must be reported to the OIE (Office International des Epizooties, Paris, France). The number of reported outbreaks has increased steadily during the last decade. Serodiagnosis is hampered by the considerable number of false-positives and -negatives of the internationally prescribed tests. The major problem leading to low sensitivity and specificity of complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e. crude preparations of whole cells. Future perspectives for the development and evaluation of serological test kits using well-characterized single antigens are discussed in the light of recent molecular research on B. mallei and the closely related saprozoonotic agent B. pseudomallei.     

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.     

Serodiagnosis of Burkholderia mallei Infections in Horses: State‐of‐the‐art and Perspectives

Burkholderia mallei causes glanders or farcy in solipeds, a disease that must be reported to the OIE (Office International des Epizooties, Paris, France). The number of reported outbreaks has increased steadily during the last decade. Serodiagnosis is hampered by the considerable number of false‐positives and ‐negatives of the internationally prescribed tests. The major problem leading to low sensitivity and specificity of complement fixation test (CFT) and enzyme‐linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e. crude preparations of whole cells. Future perspectives for the development and evaluation of serological test kits using well‐characterized single antigens are discussed in the light of recent molecular research on B. mallei and the closely related saprozoonotic agent B. pseudomallei.     

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.     

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 (MIC₉₀ ≥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.