Virulence of two strains of mycobacterium bovis in cattle following aerosol infection.

Over the past two decades, highly virulent strains of Mycobacterium tuberculosis have emerged and spread rapidly in man, suggesting a selective advantage based on virulence. A similar scenario has not been described for Mycobacterium bovis infection in cattle (i.e. bovine tuberculosis). An epidemiological investigation of a recent outbreak of bovine tuberculosis in a USA dairy indicated that the causative strain of M. bovis (strain 10-7428) was particularly virulent, with rapid spread within the herd. In the present study, the virulence of this strain (10-7428) was directly compared in the target host with a well-characterized strain (95-1315) of relevance to the USA bovine tuberculosis eradication programme. Aerosol inoculation of 10(4) colony forming units of M. bovis 95-1315 (n = 8) or 10-7428 (n = 8) resulted in a similar distribution and severity of gross and microscopical lesions of tuberculosis as well as mycobacterial colonization, primarily affecting the lungs and lung-associated lymph nodes. Specific cell-mediated and antibody responses, including kinetics of the response, as well as antigen recognition profiles, were also comparable between the two treatment groups. Present findings demonstrate that M. bovis strains 95-1315 and 10-7428 have similar virulence when administered to cattle via aerosol inoculation. Other factors such as livestock management practices likely affected the severity of the outbreak in the dairy.

Performance of immunochromatographic and ELISA tests for detecting fallow deer infected with Mycobacterium bovis.

Fallow deer (Dama dama) are widely distributed as natural or naturalised populations, as well as in game parks and deer farms. We used 157 fallow deer sampled in populations considered to be Mycobacterium tuberculosis complex (MTC) free and 73 Mycobacterium bovis-infected fallow deer confirmed postmortem by culture to evaluate the diagnostic performance of two tests for the detection of anti-mycobacterial antibodies: the dual path platform (DPP) VetTB assay and the bovine purified protein derivative (bPPD) ELISA. We also compared their sensitivity with that of the skin test, analyzed the effect of haemolysis degree on the antibody detection and described the relationship between the test readings and presence/absence of gross tuberculosis (TB) compatible lesions. Sensitivity of bPPD ELISA was 51% at a specificity of 96%. Depending on the cut-off value selected, the sensitivity of DPP VetTB ranged from 62 to 71%, while its specificity was 88-95%. In the subgroup of M. bovis-infected deer for which the skin test data were available (33 of 73); this method detected 76% of culture-positive animals, although the specificity of the intradermal test was not determined in this study. When the DPP VetTB and skin test data were combined, the resulting sensitivity obtained in this sub-group of M. bovis-infected deer increased to 97%. Gross pathology identified TB compatible lesions (TBL) in 89% culture-confirmed fallow deer. The infected animals with visible lesions had significantly higher readings in the DPP VetTB, but not in the bPPD ELISA. Only high levels of haemolysis decreased antibody test sensitivity and this effect was more evident for the bPPD ELISA. The results allowed inferring a number of management recommendations for rapid detection of MTC infection in live fallow deer and in surveys on hunter-harvested cervids.

Ante-mortem testing wild fallow deer for bovine tuberculosis.

This study aimed to maximize the sensitivity of bovine tuberculosis detection in living wild fallow deer (Dama dama) under field conditions. We evaluated the rapid test (RT; CervidTB STAT-PAK Assay, Chembio Diagnostic Systems, Inc., USA) in comparison with the comparative cervical skin test (CCT). A total of 134 fallow deer were captured between January and March 2008. At time 0, 0.1 ml of avian purified protein derivative (avian PPD; Cooper-Zeltia, Spain), 0.1 ml bovine PPD (Cooper-Zeltia, Spain), 0.1 ml negative control PBS and 0.1 ml of a positive control (the mitogen phytohaemagglutinin, PHA; containing 250 mg PHA, diluted in PBS) were injected intradermally at four shaved sites in the neck. The skin fold thickness at each injection site was measured at time 0 and 72 h (3 repeats each time). Animals with a skin test response of 2mm or more at the bovine PPD injection site and animals with any visible reactivity in the RT were necropsied and tissues submitted for culture and for histopathology. A total of 36 fallow deer were considered reactors to bovine PPD or to the RT (apparent prevalence 27%). Regarding both bovine PPD reactivity and the skin fold increase at the PHA injection site, we found significant effects of age and sex by age interaction. Adult males had the largest responses. Mycobacterium bovis was isolated from lymphoid tissues of 21 fallow deer. Skin test sensitivity, as compared to M. bovis culture confirmed deer, was 80.1% (17/21). But, the CCT alone would have missed 4 of 21 culture confirmed animals. RT sensitivity, based on culture confirmed deer, was also 80.1% (17/21). Similarly, the RT alone would have missed another 4 of 21 culture confirmed deer. However, combining the CCT and the RT allowed for detecting all 21 culture positive fallow deer. We conclude that the combined application of the RT and the skin testing can maximize the sensitivity of bTB detection in living fallow deer, thus facilitating control programs for wildlife disease surveillance.

Tuberculosis caused by Mycobacterium microti in South American camelids.

BACKGROUND: Infection with Mycobacterium microti can cause chronic disease in animals and threaten human health through its zoonotic potential. OBJECTIVE: To describe clinical findings, diagnostic investigations, necropsy, and epidemiology results in South American camelids (SAC) infected with M. microti, member of the Mycobacterium tuberculosis complex. ANIMALS: Eleven SAC with tuberculous lesions. METHODS: Description of 10 llamas and 1 alpaca, aged 4-18 years, from 6 herds with a history of wasting and weakness admitted to the Vetsuisse-Faculty of Berne over 8 years. RESULTS: Clinical signs included weight loss, recumbency, and anorexia in late stages of the disease. Respiratory problems were seen in 6 animals of 11. No consistent hematologic abnormalities were identified. Suspect animals were examined in detail by abdominal ultrasonography and thoracic radiology. Abnormal findings such as enlarged mediastinal, mesenteric, or hepatic lymph nodes were seen only in animals with advanced disease. Single comparative intradermal tuberculin test with bovine protein purified derivate (PPD) and avian PPD was negative in all animals. At necropsy, typical tuberculous lesions were found, and confirmed by bacteriological smear and culture, molecular methods, or both. CONCLUSIONS AND CLINICAL IMPORTANCE: Infection caused by M. microti should be considered a differential diagnosis in chronic debilitating disease with or without respiratory signs in SAC. Antemortem confirmation of the diagnosis remains challenging at any stage of infection. Because cases of M. microti infection have been reported in immunocompromized human patients, the zoonotic potential of the organism should be kept in mind when dealing with this disease in SAC.

Humoral immune responses of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis BCG vaccination and experimental challenge with M. bovis.

Monitoring of the kinetics of production of serum antibodies to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and the efficacy of intervention strategies in several species. The humoral immune responses to multiple M. bovis antigens by white-tailed deer vaccinated with BCG orally via a lipid-formulated bait (n=5), orally in liquid form (n=5), and subcutaneously (n=6) were evaluated over time after vaccination and after experimental challenge with virulent M. bovis and were compared to the responses by unvaccinated deer (n=6). Antibody responses were evaluated by using a rapid test (RT), a multiantigen print immunoassay (MAPIA), a lipoarabinomannan enzyme-linked immunosorbent assay (LAM-ELISA), and immunoblotting to whole-cell sonicate and recombinant antigen MPB83. MAPIA and RT detected minimal to no antibody responses over those at the baseline to multiple M. bovis antigens in vaccinated white-tailed deer after challenge. This was in contrast to the presence of more readily detectable antibody responses in nonvaccinated deer with more advanced disease. The LAM-ELISA results indicated an overall decrease in the level of production of detectable antibodies against lipoarabinomannan-enriched mycobacterial antigen in vaccinated animals compared to that in nonvaccinated animals after challenge. Immunoblot data were inconsistent but did suggest the occurrence of unique antibody responses by certain vaccinated groups to Ag85 and HSP70. These findings support further research toward the improvement and potential use of antibody-based assays, such as MAPIA, RT, and LAM-ELISA, as tools for the antemortem assessment of disease progression in white-tailed deer in both experimental and field vaccine trials.

Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife.

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n=1532), white-tailed deer (n=463), brushtail possums (n=129), and wild boar (n=177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.

Antibody responses in New World camelids with tuberculosis caused by Mycobacterium microti.

Antibody responses in New World camelids (NWC) infected with Mycobacterium microti were studied by two serological methods, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT). Serum samples were collected during 2004-2006 from 87 animals including 1 alpaca and 7 llamas with confirmed or suspected M. microti infection, 33 potentially exposed but clinically healthy animals from known infected herds, and 46 control NWC from herds where infection had not been previously diagnosed. The serological assays correctly identified infection status in 97% (MAPIA) or 87% (RT) cases. In three llamas with confirmed M. microti infection and one llama with gross pathology suggestive of disease, for which multiple serum samples collected over time were available, the antibody-based tests showed positive results 1-2 years prior to the onset of clinical signs or being found dead. In MAPIA, MPB83 protein was identified to be an immunodominant serological target antigen recognized in NWC infected with M. microti. With the limited number of animals tested in this study, the serological assays demonstrated the potential for convenient, rapid, and accurate diagnosis of M. microti infection in live llamas and alpacas.

Tuberculosis outbreak in a dromedary racing herd and rapid serological detection of infected camels.

A recent outbreak of tuberculosis (TB) in a dromedary racing herd of 58 animals involved 3 infected animals. Disease was confirmed at necropsy by finding gross lesions from which Mycobacterium bovis (antelope type) was isolated. Sera collected from the camels in this herd were used to evaluate two new serological methods, Multiantigen Print Immunoassay (MAPIA) and rapid test (RT) developed using the lateral-flow technology, in comparison with the intradermal tuberculin tests. Antibodies were found in all three infected dromedaries by both RT and MAPIA, but not in the remaining 55 animals in the herd. With the limited number of animals tested in this study, the serological assays showed the potential for convenient, rapid, and accurate diagnosis of TB in live camels.

Immune responses to defined antigens of Mycobacterium bovis in cattle experimentally infected with Mycobacterium kansasii.

Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.

Early antibody responses to experimental Mycobacterium bovis infection of cattle.

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.

Effects of different tuberculin skin-testing regimens on gamma interferon and antibody responses in cattle experimentally infected with Mycobacterium bovis.

Although tuberculin skin testing has been a hallmark of bovine tuberculosis eradication campaigns, it lacks sensitivity, can be confounded by exposure to nontuberculous mycobacteria, and cannot be repeated for 60 days due to desensitization. To overcome these difficulties, an effective whole-blood cellular immunoassay for bovine gamma interferon (IFN-gamma) has been developed. The IFN-gamma test is commonly used in conjunction with tuberculin skin testing as a confirmatory test following a positive response to the caudal fold test (CFT). The present study was conducted to determine the effect of different tuberculin skin-testing regimens on IFN-gamma and antibody production by using calves that were experimentally infected with Mycobacterium bovis. Holstein calves were CFT tested 60 days after inoculation and the comparative cervical test (CCT) was conducted 7 (7-day CCT) or 55 (55-day CCT) days after the CFT. In both the 7-day CCT and 55-day CCT groups, IFN-gamma responses increased 3 days after the CFT; this was immediately followed by a decrease to pre-skin test levels 7 days after the CFT. In both groups, the application of the CCT at 7 or 55 days after the CFT resulted in no significant increase in IFN-gamma production. The administration of the CFT and the CCT to M. bovis-inoculated cattle boosted antibody responses to M. bovis PPD, rMPB83, ESAT-6, and the fusion protein Acr1-MPB83. The boosting effect was more pronounced in the 55-day CCT group. Increases in either IFN-gamma or antibody production were not seen in noninoculated cattle. Measurement of both IFN-gamma and antibody responses after skin testing may be useful in identifying M. bovis-infected cattle; however, the timing of collection of such samples may influence interpretation.

Antibody responses in reindeer (Rangifer tarandus) infected with Mycobacterium bovis.

Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer.

Antigen recognition by serum antibodies in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis.

White-tailed deer (Odocoileus virginianus) have emerged as reservoirs of bovine tuberculosis in northern America. For tuberculosis surveillance of deer, antibody-based assays are particularly attractive because deer are handled only once and immediate processing of the sample is not required. Sera collected sequentially from 25 Mycobacterium bovis-infected and 7 noninfected deer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for immunoglobulin specific to M. bovis antigens. Various routes of experimental M. bovis infection, such as intratonsillar inoculation (n = 11), aerosol (n = 6), and exposure to infected deer (in contact, n = 8), were studied. Upon infection, specific bands of reactivity at approximately 24 to 26 kDa, approximately 33 kDa, approximately 42 kDa, and approximately 75 kDa to M. bovis whole-cell sonicate were detected by immunoblot. Lipoarabinomannan-specific immunoglobulin was detected as early as 36 days postchallenge, and responses were detected for 94% of intratonsillarly and "in-contact"-infected deer. In MAPIA, sera were tested with 12 native and recombinant antigens coated on nitrocellulose. All in-contact-infected (8 of 8) and 10 of 11 intratonsillarly infected deer produced antibody reactive with one or more of the recombinant/native antigens. Responses were boosted by injection of tuberculin for intradermal tuberculin skin testing. Additionally, three of six deer receiving a very low dose of M. bovis via aerosol exposure produced antibody specific to one or more recombinant proteins. M. bovis was isolated from one of three nonresponding aerosol-challenged deer. Of the 12 antigens tested, the most immunodominant protein was MPB83; however, a highly sensitive serodiagnostic test will likely require use of multiple antigens.

A comparative study of a new rapid and one-step test for the detection of parvovirus in faeces from dogs, cats and mink.

A one-step immunochromatographic test, based on the use of monoclonal antibodies, was developed for the detection of canine parvovirus (CPV) in dog faeces. In addition to canine parvovirus the test can also be used for the diagnosis of infections with viruses causing parvovirus enteritis in cats (feline panleukopenia virus) and mink (mink enteritis virus). Four hundred and forty-three faecal samples were evaluated by comparative testing between this one-step test and three different enzyme-linked immunosorbent assays (ELISA) in Sweden, Denmark and The Netherlands. The result of the evaluation showed an overall relative sensitivity and specificity of 95.8 and 99.7%, respectively. Furthermore, the comparative testing of 83 dog samples in Germany between the one-step test and an immune electron microscopy (IEM) agreed to 85.5%. The sensitivity and specificity were 83.9 and 88.9%, respectively. These results show that the one-step test is a rapid, simple, reproducible and sensitive diagnostic test for the detection of parvovirus in faecal samples of dogs, cats and mink.

Evaluation of a one-step test for rapid, in practice detection of rotavirus in farm animals.

An immunochromatographic test for the detection of group A rotavirus was evaluated against a reference group A rotavirus ELISA, by using a panel of 161 bovine, porcine and equine faecal samples submitted for routine examination. The sensitivity of the test was 89 per cent and the specificity 99 per cent compared with the ELISA. Its reproducibility was 100 per cent. The simplicity and rapidity of the test procedure make it suitable for use in practice.