LABORATORY DIAGNOSIS OF BOVINE TUBERCULOSIS

Lesions of suspected bovine tuberculosis were examined by culture, histopathology and auramine-O (AO) stained smears and the findings correlated with field aspects of the disease. Of 642 lesions considered to be tuberculous, 62.0% yielded M. bovis and 4.5% other mycobacteria (OM). M. bovis and OM were recovered also from 0.6% and 3.6% respectively of 165 cattle which gave tuberculin reactions but had no visible lesions at slaughter. Of 262 lesions in which a histopathological diagnosis other than tuberculosis was made, 1.5% and 3.0% yielded M. bovis and OM respectively. All OM isolates tested belonged to the Mycobacterium-avium-intracellulare-scrofulaceum (MAIS) complex with a predominance of serotype 2. A good relationship was found between the recovery of mycobacteria and histopathology but examination of smears revealed 22.0% apparent false negatives. Apparent false negative culture results were also reported for 35.8% of lesions positive on histopathology and smear examination. The majority of herds yielding M. bovis contained reactors to the tuberculin test and many of these had lesions of tuberculosis. In contrast, herds yielding OM seldom contained reactors to the tuberculin test and rarely reactors with tuberculous lesions. The thoracic cavity was the main site of lesions from infections by M. bovis and OM.     

Diagnosis of tuberculosis due to Mycobacterium bovis in New Zealand red deer (Cervus elaphus) using a composite blood test and antibody assays

A blood test for tuberculosis in deer was developed as an ancillary test to clarify the status of skin test-positive deer, with non-specific sensitisation following exposure to saprophytic mycobacteria. The blood test incorporates the measurement of the relative humoral and cellular immunological responses to Mycobacterium bovis and M. avium antigens to provia composite test with high levels of sensitivity (>95%) and specificity (>98%). The specificity of the test has allowed it to be used in parallel with the skin test to salvage thousands of tuberculosis-free deer with non-specific skin test-positive reactions, while its high sensitivity has consistently identified M. bovis-specific reactivity in tuberculous skin test-positive animals. The rules for establishing the diagnostic parameters for the cellular and antibody assays were developed by retrospective analysis of the laboratory results using blood samples from many thousand tuberculous or disease-free deer. The sensitivity of the blood test was tested in this study using 150 animals with tuberculosis diagnosed by the isolation of M. bovis. It had sensitivity values of 95.7–95.9% in herds with a low (<2.0% ) or a high (>30.0%) incidence of tuberculosis. The test had a specificity of 98.0% when tested on 218 disease-free animals, 118 of which were skin test-positive.

An antibody test was developed to diagnose M. bovis in skin test-negative “anergic” deer from tuberculosis infected herds. When this test was used with deer blood taken 10 days after reading the skin test, it had a sensitivity of 85.3% for 102 M. bovis-positive deer. When used in combination with skin test, the antibody test complemented the skin test to raise the sensitivity of the combined tests to 95.0%) when antibody-positive or skin test-positive tests were used to diagnose tuberculosis. The specificity of the antibody test was 100% when used to evaluate 218 disease-free deer from non-infected herds.     

A SURVEY OF MYCOBACTERIOSIS OF FERAL PIGS IN THE NORTHERN TERRITORY

Seven hundred and fifty-one feral pigs from the subcoastal plains of the Northern Territory were examined. The sample population consisted of 52.4% females and 47.6% males. They ranged in age from newborn piglets to mature animals of over 72 months. Of the pigs examined 47.7% had macroscopic abscesses and of these 80.2% were probably caused by mycobacteria. Tissues from 193 pigs were examined bacteriologically and 93 strains of mycobacteria were isolated. These were typed as M. bovis (37 strains); M. avium serotype 2 (1); M. intracellulare serotypes 6 (2), 7 (3), 9 (1) and 18 (1); M. intracellulare double serotypes 6 + 12 (1), 8 + 12 (1) and 11 + 25 (1); M. intracellulare unclassified serotype (4); M. scrofulaceum serotype 41 (1); M. scrofulaceum unclassified serotype (7); M. gordonae (2); M. kansasii (1); M. simiae (2); M. szulgai (2); M. vaccae (1); and M. xenopi (2). Additionally, 3 strains were unidentifiable members of the M. avium-M. intracellulare-M. scrofulaceum (MAIS) complex, one strain was a Runyon's group IV and 4 strains were typed as members of the genus Rhodococcus. Five strains were non-viable on subculture and 10 did not conform to any currently recognised species of mycobacteria. Of the 93 strains, 3 were isolated from tissue that did not contain macroscopic lesions, viz. M. simiae, Runyon's group IV and an unidentifiable member of the MAIS complex. It was concluded that the feral pig is probably an end host for both M. bovis and atypical mycobacteria and not a significant source of infection for cattle. M. bovis is not a significant cause of mortality in feral pigs but mycobacterioses are a significant cause of morbidity. With increasing age, the proportion of pigs having lesions increased whereas the proportion of lesions from which mycobacteria could be isolated decreased.     

Prevalence of Mycobacterium bovis skin positivity and associated risk factors in cattle from Western Uganda

Purpose

A cross-sectional study was undertaken to determine the prevalence of Mycobacterium bovis skin positivity and associated risk factors in cattle in western Uganda.

Methods

Herds were selected using multi-stage cluster sampling. The comparative cervical intradermal tuberculin test (CCT) was used to determine cattle tuberculosis status using US Department of Agriculture protocols. Risk factor data were collected from cattle owners through questionnaires collected by in-person interviews. Multivariable logistic regression models were used to measure the association between risk factors and herd CCT reactor prevalence.

Results

A total of 525 cattle from 63 herds were screened for M. bovis infection. Of the 525 cattle tested, 2.1 % were CCT reactors and 15.43 % were CCT suspects. Of herds tested, 14.28 % had at least 1 CCT reactor. Using a private water source for cattle and not introducing new cattle into the farm were associated with lower prevalence of M. bovis skin positivity. The herd-level prevalence of M. bovis reactors in Kashaari County of Mbarara District was 14.5 %, and the individual cattle prevalence was low (2.1 %).

Conclusions

Using communal sources of drinking water for cattle and introducing new cattle on the farm were farm management practices associated with increased risk of M. bovis exposure in cattle. Despite the low prevalence of bovine tuberculosis (TB), there is a need to educate the populace on the possibility of human infection with zoonotic TB and for educating farmers on practices to reduce the risk of acquiring M. bovis in the Mbarara District.     

The prevalence of <Emphasis Type="Italic">Mycobacterium bovis</Emphasis>-infection and atypical mycobacterioses in cattle in and around Morogoro,Tanzania

A study was conducted to determine the prevalence of Mycobacterium bovis-infection and atypical mycobacterioses in different cattle herd management systems in and around Morogoro, Tanzania. Between April and June 2005, a total of 728 bovines from 49 herds were tested for M. bovis-infection and atypical mycobacterioses. Milk samples were taken from tuberculin positive animals and analysed for the presence of mycobacteria. Total prevalences of 2.5% and 10.1% were found for M. bovis-infection and atypical mycobacterioses respectively, with more M. bovis-infection in cattle in the extensive management system and more atypical mycobacterioses in cattle in the intensive management system. From 8 out of 42 milk samples (19%) atypical mycobacteria were cultured. A higher prevalence of M. bovis-infection in the extensive sector could be due to several factors. In addition, such high prevalence puts herd owners and their families at risk for BTB. Therefore control of BTB, as well as education of cattle owners is important, especially in the extensive sector.     

Prevalence of Mycobacterium bovis infection in traditionally managed cattle at the wildlife-livestock interface in South Africa in the absence of control measures

Cattle are the domestic animal reservoir for Mycobacterium bovis (M. bovis) which also affects other domestic animals, several wildlife species and humans leading to tuberculosis. The study area is in a resource-poor community that is surrounded by several game parks, where M. bovis infection has been previously diagnosed in wildlife. A cross-sectional study was carried out to determine the prevalence of M. bovis infection in 659 cattle from a total of 192 traditionally managed herds using the BOVIGAM® interferon gamma assay (IFN-γ). Infection was confirmed by post mortem examination and M. bovis isolation from three test-positive cattle. Genotyping of the M. bovis isolates was done using spoligotyping and VNTR (variable number of tandem repeats typing). The apparent M. bovis prevalence rate in cattle at animal level was 12% with a true population prevalence of 6% (95% Confidence interval (C.I) 3.8 to 8.1) and a herd prevalence of 28%. Spoligotyping analysis revealed that the M. bovis isolates belonged to spoligotype SB0130 and were shared with wildlife. Three VNTR profiles were identified among the SB0130 isolates from cattle, two of which had previously been detected in buffalo in a game reserve adjacent to the study area. The apparent widespread presence of M. bovis in the cattle population raises a serious public health concern and justifies further investigation into the risk factors for M. bovis transmission to cattle and humans. Moreover, there is an urgent need for effective bTB control measures to reduce infection in the communal cattle and prevent its spread to uninfected herds.

     

Increased prevalence of Mycoplasma bovis in the Netherlands

Summary

The epidemiology, therapy, and prevention of M. bovis infections are briefly reviewed In a survey begun in 1982 M. bovis was found frequently in the respiratory of veal calves and beef cattle with respiratory problems. In replacement calves infected with respiratory disease in dairy herds, however, the organism has only been detected since 1986. Respiratory tract specimens collected from calves with respiratory disease were submitted for examination for M. bovis from 1986 to 1991 and originated from 83 herds. Mycoplasma bovis was detected in specimens from 59 of the herds, 20% of which were dairy herds and 80% fattening herds. Arthritis caused by M. bovis was observed in 12 herds until July 1991. Since 1976 when the first mastitis outbreak caused by M. bovis was diagnosed M. bovis has caused 14 more outbreaks. The number of diseased cattle varied from 1 tot 16 per farm, and clinical signs of mastitis varied from mild to severe. In all instances the infection has been eradicated from the herds. Because M. bovis can cause great losses in intensively reared cattle herds, it is advisable to separate purchased veal calves and beef cattle from dairy cattle to prevent further spread of M. bovis.     

Sensitive diagnosis of bovine tuberculosis in a farmed cervid herd with use of an MPB70 protein fluorescence polarization assay

After histopathological examination of a lesion found in a herd member returned a diagnosis of mycobacteriosis, a farmed herd (n = 47) of elk (Cervus elaphus nelsoni) and red deer (C. elaphus elaphus) was investigated for bovine tuberculosis with a battery of antemortem and postmortem diagnostic tests. Every animal was tested with the mid-cervical tuberculin skin test; all 47 had negative results. All of the 16 adult animals and 15 of the 31 calves (approximately 2-years-old) were blood-tested with a lymphocyte stimulation test (LST) and a fluorescence polarization assay (FPA), which detects antibody to the MPB70 protein antigen. At necropsy of the 31 blood-tested animals, tissues were harvested for histopathological examination and culture of mycobacteria. Mycobacterium bovis was isolated from 16 of the 31 animals, and a scotochromogen was also isolated from 1 of the 16 whose tissues yielded M. bovis. Each of these 16 animals, 15 of which were calves, also received a histopathological diagnosis of mycobacteriosis. Other species of mycobacteria, including those belonging to the M. avium and M. terrae complexes, were isolated from an additional 7 animals. The FPA was scored “positive” or “suspect” for 16 animals, 13 (81%) of which were culture-positive for M. bovis. The other 3 animals that were culture-positive for M. bovis had negative FPA results. Of the 3 FPA-positive or FPA-suspect animals that were culture-negative, 2 were suspected to have mycobacteriosis on the basis of the histopathological examination. The 7 animals from which Mycobacterium species other than M. bovis were cultured were all FPA-negative. The only animal with positive LST results was also FPA-positive and culture-positive for M. bovis. The M. bovis isolates had an identical spoligotype pattern, with an octal code of 664073777777600. This is the first report of the isolation and identification of this strain type in Canada.     

Tuberculosis Infection in Animal and Human Populations in Three Districts of Western Gojam,Ethiopia

Tuberculosis concurrent infection in cattle and their respective owners in North‐western Ethiopia had been investigated. Two hundred and ten cattle owners and 1220 heads of their cattle were included in the study to determine degree of tuberculosis infection in cattle owned by tuberculosis patients and tuberculosis patients. Comparative intradermal tuberculin test, bacteria culturing, acid fast staining and biochemical tests were used to conduct the study. The prevalence of tuberculosis was significantly (P < 0.001) higher in cattle owned by tuberculosis patients than in cattle owned by non‐tuberculosis owners, and infection with tuberculosis was threefold greater in cattle owned by tuberculosis‐positive owners. Further more, cattle owners who consumed raw milk were at higher risk (P < 0.001, OR = 3.23) for tuberculosis infection than those who consumed boiled milk. Mycobacterium tuberculosis (15.4%), Mycobacterium bovis (44.1%) and atypical mycobacteria (38.5%) were identified from milk collected from tuberculin‐positive cows using biochemical tests. Similarly M. tuberculosis (74.5%), M. bovis (14.9%) and atypical mycobacteria (8.5%) were identified from sputum and fine needle aspiration specimens of tuberculosis patient cattle owners. Mutual transmission of mycobacterium from animals to humans and vice versa has been signified.     

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White‐tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programmes. One approach in dealing with wildlife reservoirs of disease is to interrupt inter‐species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white‐tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 10⁷ CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 10⁷ CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid‐fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white‐tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.     

Extent of Mycobacterium bovis infection in dairy cattle herds subject to partial culling as determined by an interferon-gamma assay

The interferon-gamma (IFN-γ) assay is employed as a complementary diagnostic test for bovine tuberculosis (BTB) in many countries. To simplify this assay, we established a 96-well plate format using the ESAT-6 and CFP-10 antigens and then employed it to determine the extent of Mycobacterium (M.) bovis infection in dairy herds with a history of BTB outbreaks in a country where only selective culling is practiced. The sensitivity and specificity of this IFN-γ assay were 85.9% and 100%, respectively, based on comparison with the conventional single intradermal tuberculin test (SIDT). The IFN-γ assay was also positive in 30.4% and 36.8% of SIDT-negative animals from herds with recent and remote BTB outbreaks, respectively. Of 14 SIDT-negative, IFN-γ positive cattle, five (35.7%) were culture positive and an additional six were positive based on a polymerase chain reaction-based test for M. bovis. Therefore, the IFN-γ assay has the potential to serve as a specific and sensitive test for M. bovis infection in dairy cattle. Further, the results indicated that a substantial portion of SIDT-negative animals in herds with previous BTB outbreaks were actually infected with M. bovis. Accordingly, the present selective-culling strategy may require modifications to include this more sensitive assay.     

Prevalence and risk factors of mycobacterial infections in farm and trade cattle in southwestern Nigeria

We evaluated the prevalence of mycobacterial infections (i.e., Mycobacterium bovis and non-tuberculous mycobacteria [NTM]) and their associated risk factors among cattle herds and trade cattle in southwestern Nigeria. Through cross-sectional study design, cattle herds from three locations were screened using the single intradermal comparative cervical tuberculin test based on two diagnostic standards; more than 4 mm (? 4 mm) and more than 2 mm (? 2 mm) cut-off points. Abattoir study involved screening trade cattle for tuberculous lesions. Overall, 515 cattle from 45 herds were screened. Using >?4 mm, animal level and herd prevalence of 11.7 and 46.7% were recorded, respectively. Applying the ? 2 mm cut-off, animal level and herd prevalence increased to 31.1 and 60.0%, respectively. Significantly, using the ? 2 mm cut-off, cattle in medium size herds/extensive management system (OR?=?1.6; 95% CI 1.1–2.5) and Sokoto Gudali (OR?=?2.3; 95% CI 1.4–3.8) were more at risk of being positive reactors, while Rahaji (OR?=?0.3; 95% CI 0.1–0.7) breeds of cattle and cows in the peri-urban area (OR?=?0.4; 95% CI 0.2–0.9) were less at risk of being positive reactors. Again, M. avium reactor of 21.7% was observed. In the abattoir, 1797 cattle were examined with 126 lesions suggestive of tuberculosis (TB). Culture/molecular analyses confirmed 2.2% M. bovis and 0.9% NTM infections. Risk factors associated with bovine TB among trade cattle were sex (OR?=?4.0; 95% CI 1.2–13.5) and age (OR?=?0.3; 95% CI 0.1–0.9). We confirm 11.7% prevalence of mycobacterial infections among populations of cattle screened with breed and herd size being major risk factors.     

Bovine tuberculosis in South Darfur State, Sudan: an abattoir study based on microscopy and molecular detection methods

Bovine tuberculosis (BTB) is a widespread zoonosis in developing countries but has received little attention in many sub-Saharan African countries including Sudan and particularly in some parts such as Darfur states. This study aimed to detect bovine tuberculosis among caseous materials of cattle slaughtered in abattoirs in South Darfur State, Sudan by using microscopic and PCR-based methods. The study was a cross-sectional abattoir-based study which examined a total of 6,680 bovine carcasses for caseous lesions in South Darfur State between 2007 and 2009. Collected specimens were examined for the presence of acid-fast bacilli (AFB) by using microscopic and culture techniques. Isolated mycobacteria were identified by selected conventional cultural and biochemical tests in comparison to a single tube multiplex PCR (m-PCR) assay which detect Mycobacterium bovis-specific 168-bp amplicons. Of the total 6,680 slaughtered cattle examined in South Darfur, 400 (6 %) showed caseations restricted to lymph nodes (86.8 %) or generalized (13.2 %). Bovine tuberculosis was diagnosed in 12 (0.18 %), bovine farcy in 59 (0.88 %), unidentified mycobacteria in 6 (0.09 %), and missed or contaminated cultures in 7 (0.1 %). Out of 18 cultures with nonbranching acid-fast rods, 12 amplified unique 168-bp sequence specific for M. bovis and subsequently confirmed as M. bovis. With the exception of the reference M. tuberculosis strains, none of the remaining AFB amplified the 337-bp amplicon specific for M. tuberculosis. It could be concluded that bovine tuberculosis is prevalent among cattle in South Darfur representing 4.5 % from all slaughtered cattle with caseous lesions. The study sustains microscopy as a useful and accessible technique for detecting AFB. m-PCR assay proved to be valuable for confirmation of BTB and its differentiation from other related mycobacteriosis, notably bovine farcy.     

AN ABATTOIR SURVEY OF TUBERCULOSIS IN FERAL BUFFALOES

Tuberculosis lesions were found in 193 (1.7%) of 11,322 buffaloes examined during routine post-mortem inspection at 2 export abattoirs. The prevalence of tuberculosis in buffaloes supplied from 17 separate farms ranged from 0.3% to 8.22%, with the highest levels occurring on the coastal plains. Lesions were confined to one major body region in 50 of 72 randomly chosen cases of tuberculosis and to 2 or more regions in 22 cases. Thoracic lesions occurred in 65 of the 72 cases, abdominal lesions in 19, head lesions in 18 and carcase lesions in 9. In the thoracic cavity, lesions occurred most frequently in mediastinal and bronchial lymph nodes. In the head region the retropharyngeal lymph node was most frequently involved, in the abdominal cavity, the liver, and in the carcase, the deep inguinal lymph node. Tuberculosis lesions in buffaloes had a lardaceous consistency and were paler in colour and less calcified than those normally exhibited by cattle. Mycobacteria were isolated from 30 of 31 lesion samples submitted for bacteriological examination. Of the isolates, 25 were identified as Mycobacterium bovis, 3 as M. avium-intracellulare-scrofulaceum complex, one as M. fortuitum and one as M. flavescens. The M. bovis isolates from buffaloes showed minor cultural differences to those normally characteristic of bovine isolates.     

Isolation of Mycobacterium bovis from brushtail possums with non-visible lesions

AIM: To determine the prevalence of Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) that did not have macroscopic lesions of bovine tuberculosis, and to evaluate culture of pooled tissues from multiple possums as a method for determining the M. bovis-infection status of wildlife populations in New Zealand.

METHODS: Pools of selected tissues were collected from possums from four different populations known to be infected with M. bovis. Tissue pools from individual animals, and combined pools from multiple animals, were cultured for M. bovis.

RESULTS: In the four populations investigated, the prevalence of possums with macroscopic lesions confirmed by culture to be infected with M. bovis ranged from 1 to 19 (mean 31/283; 10.9)%. The prevalence of possums with non-visible lesions that were culture positive for M. bovis in the same populations ranged from 4 to 10 (mean 24/283; 8.5)%. The mean of the log10 cfu of M. bovis of the macroscopic lesions and of the culture-positive samples that did not have visible lesions was 3.85 (SE 0.26) and 1.46 (SE 0.26) log₁₀ cfu, respectively (p<0.01). Mycobacterium bovis was cultured from pools of 30–50 animals in the four populations studied.

CONCLUSIONS: The finding of M. bovis infection in possums with non-visible lesions identified a potential deficiency of declaring possum populations free of M. bovis on the basis of absence of macroscopic lesions. The culturing of pools of selected tissues from multiple animals without visible lesions can be used to reduce laboratory costs of possum surveys without a major reduction in the ability to detect M. bovis infection.     

Serological reactivity to Mycobacterium bovis protein antigens in cattle

The serological response to 12 purified Mycobacterium bovis antigens were examined in an ELISA assay. These antigens included the majority of M. bovis protein antigens described to date and in most cases they were very similar to the M. tuberculosis antigens of the same molecular mass.The purified antigens were tested against sera from M. bovis infected cattle, M. bovis culture-negative cattle from infected herds and animals infected with related microorganisms, mainly other mycobacterial species. All the antigens gave strong reactions with at least some sera from the M. bovis infected group and showed cross-reactivity with some of the sera from the other two groups. The antigen with the highest specificity reacted strongly with only 60% of the M. bovis infected sera. Antigens that reacted with most or all of the M. bovis infected sera also gave the highest cross-reactivity with sera from the other two groups. These results indicate that a serological test based on any one or a combination of these antigens, without removal of the cross-reacting epitopes, would be unsatisfactory.     

Evaluation of the Abbott LCx Mycobacterium Tuberculosis Assay for Direct Detection of Mycobacterium Bovis in Bovine Tissue Samples

The commercial LCx amplification assay, usually employed to detect the Myocobacterium tuberculosis complex in respiratory specimens, was evaluated by comparing the results it gave with those obtained using Löwenstein-Jensen solid medium and pathological findings on 55 lymph nodes from cattle with positive and 10 lymph nodes from cattle with negative skin tests for tuberculosis. Fifty-three cultures (51 and 2, respectively) were positive for M. bovis, while the results for the LCx assay and the histological method were positive in 48 (45, 3) and 24 (20, 4) samples, respectively. None of the samples from cattle from certified tuberculosis-free herds were positive by any of the procedures. The results obtained with the LCx assay, compared with the culture procedure, regarded as the gold standard among the diagnostic techniques, gave a specificity of 91.6% and sensitivity of 90.5%. Although the sensitivity of LCx was suboptimal, DNA of M. bovis was detected in 81.8% of the skin test-positive animals. Amplification techniques could provide a rapid and reasonably reliable tool for detecting bovine tuberculosis.     

Molecular Typing of Mycobacterium bovis Isolates in Argentina: First Description of a Person‐to‐Person Transmission Case

Bovine tuberculosis is caused by Mycobacterium bovis, a mycobacterium highly similar to M. tuberculosis that belongs to the M. tuberculosis complex. The main host of M. bovis is cattle but it also affects many other mammalians including humans. Tuberculosis in humans caused by either M. bovis or M. tuberculosis is clinically hard to distinguish. During 2004–2005, samples from 448 patients with diagnosis of TB were collected from different regions of Argentina. The PRA technique identified 400 isolates with representative patterns of mycobacterium. The predominant ones were the M. tuberculosis complex, the M. avium–M. intracellulare complex and M. gordonae. Samples with M. tuberculosis complex PRA restriction profiles were analyzed with a multiplex PCR to differentiate between M. tuberculosis and M. bovis. Multiplex PCR identified nine M. bovis. The results allowed the possibility to establish that 2% of pulmonary tuberculosis was due to M. bovis. Isolates of M. bovis from humans were examined using spoligotyping. These isolates presented five different spoligotypes. The main spoligotype was also the most frequently one found in cattle. The remaining human spoligotypes (grouped in clusters) are occasionally found in cattle. Variable number tandem repeat (VNTR) analysis identified five different patterns. By combining the results of spoligotyping and VNTR analysis, we were able to differentiate seven M. bovis isolates. The remaining two M. bovis samples showed the same spoligotype and VNTR profile and belonged to household contacts. An MDR‐M. bovis was isolated from the samples of these household contacts. The identification of two epidemiologically linked cases of human M. bovis infection suggests person‐to‐person transmission of an MDR‐M. bovis.     

The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p < 0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.