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.     

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.     

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.     

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.     

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.     

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.     

Serological investigation of horse sera for antibodies against mycoplasmas and acholeplasmas

Sera from horses with respiratory disease (RD) have been investigated using the complement fixation test, indirect hemagglutination test, enzyme immune assay, and the metabolic inhibition test, and sera from mares after abortion, using the complement fixation test, indirect hemagglutination test and enzyme immune assay, for antibodies against Mycoplasma equirhinis, M. subdolum, M. equigenitalium, M. pulmonis, M. felis, Acholeplasma laidlawii, A. hippikon and A. equifetale. Antibodies were found against all mycoplasma and acholeplasma species tested, more often against acholeplasmas. The antibody pattern was quite similar for horses with RD and for mares after abortion. The results of the four serological tests performed showed only a limited correlation and the percentage of sera with antibodies detected by the four tests used differed widely.     

Ovine brucellosis in alberta

Two parallel surveys of rams from Alberta sheep flocks were conducted to determine the presence of infection with Brucella ovis. In a retrospective study over a period of 24 months, using complement fixation test, 12 flocks out of 142 tested were considered infected. In another 17-month survey of slaughter rams by serology and culture methods 11 flocks out of 124 were found to be infected. The overall prevalence of ovine brucellosis was 8.6% of the flocks tested which represented 12.5% of the estimated sheep flocks in Alberta. Up to 67% of rams in infected flocks reacted to complement fixation test.

The complement fixation test was evaluated for its efficiency in the diagnosis of ovine brucellosis and compared with a limited number of an enzyme-linked immunosorbent assay (ELISA) results and clinical criteria. The complement fixation test as well as ELISA identified all culture positive rams. Both serological tests appeared satisfactory for the diagnosis of B. ovis epididymitis when the results could be interpreted in the light of flock history and clinical findings.

     

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.     

Observations on the eradication of Brucella ovis infection from a ram flock

The measures taken to eradicate Brucella ovis infection from a naturally infected flock of 64 rams are described. Lesions of epididymitis were detected in 18 rams, all of which gave either positive or suspicious reactions in the complement fixation test. A further 20 rams gave serological reactions in the complement fixation test. Subsequently, semen was collected from 14 of these 20 rams and B. ovis was cultured from the semen of all 14 rams. Serum samples from two rams failed to react in the complement fixation test. However, they were identified as infected with the aid of an enzyme-linked immunosorbent assay and the subsequent culture of semen samples. It is suggested that, when eradicating B. ovis infection from ram flocks, the enzyme-linked immunosorbent assay be used in addition to both the complement fixation test and the physical examination. Using a combination of tests as described can increase the likehood of an earlier eradication of B. ovis infection.     

A radial growth precipitation test and its comparison with certain other serological tests for the detection of antibody in bovine sera to Mycoplasma agalactiae subsp. bovis

A radial growth precipitation test is described for measuring antibody to Mycoplasma agalactiae subsp. bovis. The test is quantitative and appears to depend on the production of soluble antigen by growing organisms. When compared with indirect haemagglutination, complement fixation and inhibition of film production, for measuring antibody to M. agalactiae subsp. bovis in bovine sera, it was found to have a sensitivity comparable to that of the complement fixation test.     

Evaluation of the cold complement fixation test for diagnosis of ovine brucellosis

SUMMARY A cold complement fixation test for ovine brucellosis in which fixation of complement occurs for 16 h at 4°C was compared with the conventional warm complement fixation test in which fixation takes place for 40 minutes at 37°C. The cold complement fixation test detected experimentally infected animals up to one week sooner than the warm complement fixation test and the titres were approximately one twofold dilution higher. Similar results were obtained when the 2 tests were compared using 11,922 serum samples collected from 700 ram flocks over a 2-year period. False positive reactions were observed in less than 0.1% of cases.     

Detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis

A highly sensitive indirect sandwich enzyme-linked immunosorbent assay suitable for adoption as the routine diagnostic and typing test for foot-and-mouth disease virus of all seven serotypes is described. The assay uses rabbit and guinea pig antisera raised against inactivated 146S virus antigens. Strong homotypic and minimal heterotypic reactions with both whole virion 146S and derived virion subunit 12S antigens achieved a detection sensitivity approximately 125 times that of the complement fixation test. When applied to diagnostic material with positive-negative threshold criteria generated from testing a large number of negative samples, a positive result was obtained on 83.3 per cent of original virus-positive epithelia, three times the rate for the complement fixation test, and all were typed after one passage in culture.     

Avian influenza: eradication from commercial poultry is still not in sight

Avian influenza viruses are highly infectious micro-organisms that primarily affect birds. Nevertheless, they have also been isolated from a number of mammals, including humans. Avian influenza virus can cause large economic losses to the poultry industry because of its high mortality. Although there are pathogenic variants with a low virulence and which generally cause only mild, if any, clinical symptoms, the subtypes H5 and H7 can mutate from a low to a highly virulent (pathogenic) virus and should be taken into consideration in eradication strategies. The primary source of infection for commercial poultry is direct and indirect contact with wild birds, with waterfowl forming a natural reservoir of the virus. Live-poultry markets, exotic birds, and ostriches also play a significant role in the epidemiology of avian influenza. The secondary transmission (i.e., between poultry farms) of avian influenza virus is attributed primarily to fomites and people. Airborne transmission is also important, and the virus can be spread by aerosol in humans. Diagnostic tests detect viral proteins and genes. Virus-specific antibodies can be traced by serological tests, with virus isolation and identification being complementary procedures. The number of outbreaks of avian influenza seems to be increasing - over the last 5 years outbreaks have been reported in Italy, Hong Kong, Chile, the Netherlands, South Korea, Vietnam, Japan, Thailand, Cambodia, Indonesia, Laos, China, Pakistan, United States of America, Canada, South Africa, and Malaysia. Moreover, a growing number of human cases of avian influenza, in some cases fatal, have paralleled the outbreaks in commercial poultry. There is great concern about the possibility that a new virus subtype with pandemic potential could emerge from these outbreaks. From the perspective of human health, it is essential to eradicate the virus from poultry; however, the large number of small-holdings with poultry, the lack of control experience and resources, and the international scale of transmission and infection make rapid control and long-term prevention of recurrence extremely difficult. In the Western world, the renewed interest in free-range housing carries a threat for future outbreaks. The growing ethical objections to the largescale culling of birds require a different approach to the eradication of avian influenza.     

Outbreaks of foot-and-mouth disease in Peninsular Malaysia from 2001 to 2007

This is a retrospective study of the outbreaks of foot-and-mouth disease (FMD) in Peninsular Malaysia between 2001 and May 2007. In total, 270 outbreaks of FMD were recorded. Serotype O virus (89.95 %) and serotype A (7.7 %) had caused the outbreaks. Significant differences on the occurrence of FMD were found between the years (t?=?5.73, P?=?0.000, df?=?11), months (t?=?4.7, P?=?0.000, df?=?11), monsoon season (t?=?2.63, P?=?0.025, df?=?10) and states (t?=?4.84, P?=?0.001, df?=?10). A peak of outbreaks observed in 2003 could be due to increased animal movement and the other peak in 2006 could be due to a compromised FMD control activities due to activities on the eradication of highly pathogenic avian influenza. Cattle (86 % of outbreaks) suffered the most. However, no difference in disease occurrence between species was observed. The populations of cattle (r?=?0.672, P?=?0.023) and sheep (r?=?0.678, P?=?0.022) were significantly correlated with occurrence of FMD. Movement of animals (66 % of outbreaks) was the main source for outbreaks. A combination of control measures were implemented during outbreaks. In conclusion, the findings of this study show that FMD is endemic in Peninsular Malaysia, and information gained could be used to improve the existing control strategy.     

A latex agglutination test for field diagnosis of contagious caprine pleuropneumonia

Latex beads were sensitised with a polysaccharide isolated from a F38 culture supernatant and used in a slide agglutination test to detect serum antibodies in goats with contagious caprine pleuropneumonia. The latex agglutination test detected antibodies in the sera of goats by 22 +/- 2 (mean +/- 1 sd) days after contact exposure to contagious caprine pleuropneumonia, whereas the complement-fixation test detected antibodies by 24 +/- 4 days after contact exposure. Both tests were negative with 181 sera from a farm which was free of the disease. When the same tests were done on 763 sera from two different farms with outbreaks of classical contagious caprine pleuropneumonia, 63 per cent were positive by the latex agglutination test and 23 per cent were positive by the complement-fixation test. Besides being more sensitive than complement fixation, the latex agglutination test can be performed in the field using undiluted serum or whole blood and a result obtained within two minutes.     

Haemophilus somnus Infections II. A Canadian Field Trial of a Commercial Bacterin: Clinical and Serological Results

To evaluate the efficacy of a Haemophilus somnus bacterin a total of 1114 beef calves or yearling bulls were used in the province of Saskatchewan. The six herds were included in a vaccination trial in which the vaccine was administered subcutaneously in one or two doses. The prevalence of H. somnus in nasal swabs of these animals at the time of the initial vaccination was 0.35% Postvaccination information on morbidity and mortality for a four month period was requested from the five ranches where there were nonvaccinated control calves. Postvaccination outbreaks of infectious thromboembolic meningoencephalitis occurred in two of these herds and, although the numbers were limited, there was a trend to reduced morbidity and mortality in the vaccinated animals compared to controls. Seroconversion rates, as determined by the complement fixation test, in the six herds were 28.3% for control calves, 57% for animals vaccinated once and 80.3% for animals vaccinated twice. On the basis of these results the bacterin was considered to be sufficiently efficacious to warrant its further evaluation under field conditions.     

Future options for brucellosis surveillance in New Zealand beef herds

It is probable that bovine brucellosis will be eradicated from New Zealand by 1990. However, continued surveillance for the disease will have to be maintained for a number of years. This paper examines alternative methods of surveillance in beef herds with a view to ensuring that the most cost effective method is used. The three surveillance methods examined are the present automated complement fixation test, abattoir surveillance, and a system based on a delayed hypersensitivity skin test using a purified Brucella protein antigen. It is concluded that despite the relatively low sensitivity of the skin test the probability of its identifying herds as infected is likely to be greater in practice than abattoir surveil- lance. The skin test is cheaper than the present complement fixation test.     

Comparison of the enzyme-linked immunosorbent assay and complement fixation test for detecting Brucella ovis antibodies in sheep

A solid-phase, indirect, enzyme-linked immunosorbent assay (ELISA) was compared with the microtitre complement fixation test for detecting Brucella ovis antibodies in 220 ram sera. The ELISA was more sensitive than the complement fixation test; it demonstrated antibodies in 11 sera from known infected or vaccinated rams that were complement fixation test negative. No false positives were recorded with the ELISA and, in 36 sera positive to both tests, the ELISA titres were consistently higher than the corresponding complement fixation test titres.