Masking of two in vitro immunological assays for Mycobacterium bovis (BCG) in calves acutely infected with non-cytopathic bovine viral diarrhoea virus.

Acute infection of calves, previously vaccinated with bacille Calmette-Guerin (BCG), with non-cytopathic viral diarrhoea virus (BVDV) resulted in the temporary suppression of two in vitro assays used to monitor Mycobacterium bovis infection. Lymphocyte proliferation and interferon-gamma production by whole blood cultures containing purified protein derivatives prepared from Mycobacterium avium (PPD-A) and M bovis (PPD-B) were markedly suppressed. The implication is that acute infections of cattle with non-cytopathic BVDV may temporarily compromise diagnostic tests for M. bovis infections and result in a failure to identify cattle with tuberculosis.     

Isolation and identification of mycobacteria from cattle slaughtered in Pakistan.

Specimens of lung, liver and mesenteric lymph node from cows and buffaloes slaughtered in the Lahore area were cultured to investigate the type of mycobacteria involved in bovine tuberculosis. Employing the concentration method, 56 out of 530 cattle were found to be culture positive for acid-fast bacteria, 48 being Mycobacterium bovis and eight atypical mycobacteria. No M tuberculosis or M avium was isolated. Most of the isolated M bovis strains were found to be highly virulent for rabbits.     

Method for evaluation of Mycobacterium bovis purified protein derivative tuberculin in experimentally infected cattle.

A method for the evaluation of Mycobacterium bovis purified protein derivative (PPD) tuberculin in experimentally infected cattle is presented. The development of skin test responses in M bovis-infected cattle was determined for International Standard PPD-S, M bovis PPD-2, and M bovis PPD-5 at 24, 48, and 72 hours. Significantly larger reactions (dermal thickness) were observed at 48 and 72 hours than at 24 hours (P = 0.001). Statistically significant differences were not detected in the responses obtained with M bovis PPD-2, M bovis PPD-5, and International Standard PPD-S if comparisons were made at approximately the same concentrations in M bovis-infected cattle (P greater than 0.25). In Mycobacterium avium-infected cattle, M bovis PPD-2 produced skin test responses that were significantly smaller than responses obtained using M avium PPD-2 (P = 0.001). Significant variation was not observed in the PPD-S responses in 2 groups of M bovis-infected cattle (P greater than 0.1).     

Tuberculosis in domesticated red deer: Comparison of purified protein derivative and the specific protein MPB70 for in vitro diagnosis

The use of a Mycobacterium bovis-specific protein, mycobacterial protein bovis 70 (MPB70), was compared with complex, M bovis-derived purified protein derivative (bovine PPD), for its ability to improve the diagnostic precision of in vitro assays for tuberculosis in farmed deer. A combination of lymphocyte transformation and enzyme-linked immunosorbent assay (ELISA) was used to differentiate between specific M bovis reactivity and crossreactivity due to sensitisation with saprophytic mycobacteria such as Mycobacterium avium. In the lymphocyte transformation assay the response of mononuclear cells, from red deer, to MPB70 was found to be more specific, but less sensitive, as an indicator of infection by M bovis when compared with the complex antigen bovine PPD. When used in conjunction with bovine PPD alone, MPB70 was found to increase the specificity of the ELISA in diagnosing animals with disease.     

Diagnosis of Mycobacterium bovis infection in calves sensitized by mycobacteria of the avium/intracellulare group

Because of the frequent exposure of cattle to mycobacteria of the avium/intracellulare group, an investigation was carried out into the possible repercussions thereof on the diagnosis of bovine tuberculosis. Three calves from a bovine tuberculosis-free herd, scored avian reactors in the gamma-interferon assay for bovine tuberculosis, were sedated and inoculated endotracheally with a virulent Mycobacterium bovis strain. Then, three other avian reactors were housed with the above donor calves. Mycobacterium bovis was isolated from the nasal swabs of the three endotracheally infected, donor calves. On these samples, TB complex-specific polymerase chain reaction (PCR) tests for IS6110 were also positive, albeit with a different time kinetics. The three contact-infected calves showed clear immunological signs of infection; however, their nasal swabs were always PCR-negative and only Mycobacterium avium was isolated. In the endotracheally infected donor calves there was a rise of the gamma-interferon responses to avian and bovine purified protein derivative (PPD) tuberculins, which reached the same stable plateau levels over the whole experiment. The above effect was also observed in the contact-infected calves, even though the response to avian PPD tuberculin always remained at a higher level. By using conventional bovine and avian PPD tuberculins, the comparative intradermal test was generally positive in endotracheally infected, as opposed to contact-infected calves; a positive intradermal test for M. bovis was obtained in two contact-infected calves by different bovine PPD tuberculins based on M. bovis bacillus Calmette-Guerin (BCG) secreted or somatic antigens. It was concluded that M. bovis infection may be concealed for some time in cattle sensitized by mycobacteria of the avium/intracellulare group and that different diagnostic procedures should be adopted for such animals.     

Apa antigen of Mycobacterium avium subsp. paratuberculosis as a target for species-specific immunodetection of the bacteria in infected tissues of cattle with paratuberculosis

Comparative genomics of Mycobacterium spp. have revealed conservative genes and respective proteins differently expressed in mycobacteria that could be used as targets for the species-specific immunodiagnostics. The alanine and proline-rich antigen Apa is a mycobacterial protein that present significant variability in primary sequence length and composition between members of M. avium and M. tuberculosis complexes. In this study, the recombinant Apa protein encoded by the MAP1569/ModD gene of M. avium subsp. paratuberculosis (Map) was used to generate a panel of monoclonal antibodies which were shown to recognize the most important veterinary pathogens of the M. avium complex, specifically Map and M. avium subsp. hominissuis, and which did not cross-react with M. bovis or M. tuberculosis. The produced antibodies were demonstrated to be a useful tool for the species-specific immunofluorescence or immunohistochemical detection of Map in experimentally infected cell cultures or intestinal tissues from cattle with bovine paratuberculosis and, additionally, they may be employed for the discrimination of pathogenic M. avium subspecies via Western blotting.     

Mycobacterial disease in cats in Great Britain: I. Culture results, geographical distribution and clinical presentation of 339 cases

This study investigated 339 cases of feline mycobacterial disease from cats with cutaneous lesions or masses found at exploratory laparotomy. Tissue samples were submitted to the Veterinary Laboratories Agency for mycobacterial culture over a 4-year period to December 2008. The study assessed which species of culturable mycobacteria were involved, where the cats lived, and their clinical presentation (physical findings, serum biochemistry, radiography, feline leukaemia virus and feline immunodeficiency virus status). Mycobacterium microti was cultured from 19%, Mycobacterium bovis 15%, Mycobacterium avium 7%, non-M avium non-tuberculous mycobacteria 6%, with no growth in 53% of samples. M microti, M bovis and M avium were found in almost mutually exclusive clusters within Great Britain (GB) (ie, M bovis in South-West England/Wales/Welsh Border, M avium in eastern England and M microti south of London and in South-West Scotland). While differences were seen in the clinical presentation and distribution of lesions caused by the different infections, these were not sufficiently different to be diagnostic. Cats commonly presented with single or multiple cutaneous lesions (74%), which were sometimes ulcerated or discharging, located most frequently on the head (54%). Lymph nodes were usually involved (47%); typically the submandibular nodes. Systemic or pulmonary signs were rarely seen (10-16%). When a cat is suspected of having mycobacteriosis, accurate identification of the species involved helps to determine appropriate action. Our findings show that knowing the cat's geographic location can be helpful, while the nature of the clinical presentation is less useful. Most cases of feline mycobacterial disease in GB are cutaneous.     

Zoonotic aspects of Mycobacterium bovis and Mycobacterium avium-intracellulare complex (MAC)

Pathogens that are transmitted between the environment, wildlife, livestock and humans represent major challenges for the protection of human and domestic animal health, the economic sustainability of agriculture, and the conservation of wildlife. Among such pathogens, the genus Mycobacterium is well represented by M. bovis, the etiological agent of bovine tuberculosis, M. avium ssp. paratuberculosis (Map) the etiological agent of Johne disease, M. avium ssp. avium (Maa) and in a few common cases by other emergent environmental mycobacteria. Epidemiologic surveys performed in Europe, North America and New Zealand have demonstrated the existence and importance of environmental and wildlife reservoirs of mycobacterial infections that limit the attempts of disease control programmes. The aim of this review is to examine the zoonotic aspects of mycobacteria transmitted from the environment and wildlife. This work is focused on the species of two main groups of mycobacteria classified as important pathogens for humans and animals: first, M. bovis, the causative agent of bovine tuberculosis, which belongs to the M. tuberculosis complex and has a broad host range including wildlife, captive wildlife, domestic livestock, non-human primates and humans; the second group examined, is the M. avium-intracellulare complex (MAC) which includes M. avium ssp. avium causing major health problems in AIDS patients and M. avium ssp. paratuberculosis the etiological agent of Johne disease in cattle and identified in patients with Crohn disease. MAC agents, in addition to a broad host range, are environmental mycobacteria found in numerous biotopes including the soil, water, aerosols, protozoa, deep litter and fresh tropical vegetation. This review examines the possible reservoirs of these pathogens in the environment and in wildlife, their role as sources of infection in humans and animals and their health impact on humans. The possibilities of control and management programmes for these mycobacterial infections are examined with regards to the importance of their natural reservoirs.     

Use of DNA amplification for the rapid identification of Mycobacterium bovis

DNA amplification using the polymerase chain reaction technique was evaluated for rapid identification of Mycobacterium bovis. Two oligonucleotide primers of 20 bases in length were constructed to target a region of the gene encoding the M. bovis secretory protein, MPB70. The amplification reaction produced a single product 372 bp in size which was readily detected by agarose gel electrophoresis. All 84 strains of M. bovis tested produced a positive signal in the amplification reaction. In addition all isolates fro the M. tuberculosis complex tested, with the exception of M. microti, gave a single band at 372 bp. No amplified product was detected when 24 other species of mycobacteria and species from four other genera were tested. The sensitivity of the test was such that a single viable cell could be detected in the reaction. This technique provides a simple and extremely sensitive method of identifying isolates of M. bovis and other pathogenic M. tuberculosis complex organisms.     

An outbreak of tuberculosis in pigs and cattle caused by Mycobacterium africanum.

An outbreak of tuberculosis in pigs and cattle caused by Mycobacterium africanum produced lesions in the pigs similar to those caused by M tuberculosis, M bovis and M avium, with caseation in the lymph nodes of the head and in the jejunal lymph nodes. Bacteriological examination of the dysgonic mycobacteria isolated showed that they were M africanum I. The intradermal tuberculin test was very reliable in pigs, differentiating between mammalian and avian reactions, and the results of the test were in accordance with the lesions found at meat inspection. No clinical signs were observed during the outbreak, and the infection was neither serious nor progressive. There were no lesions in the tuberculin-positive cattle. The source of the infection remains unknown.     

Production and partial characterization of monoclonal antibodies to the neotype strain of Mycobacterium bovis

Six monoclonal antibodies (MAB) to virulent Mycobacterium bovis ATCC 19210 were produced, using a suspension of heat-inactivated whole cells. Immunoglobulin isotype for MAB VMB6, VMB73, and VMB93 was IgG1, and for VMB31, VMB99, and VMB119, it was IgG2a. Monoclonal antibodies were examined for cross-reactivity to M tuberculosis, M kansasii, M fortuitum, M paratuberculosis, M avium serovars 1, 2, 4, 8, and 10, M chelonei, M phlei, M scrofulaceum, M smegmatis, Nocardia asteroides, and Rhodococcus equi. Monoclonal antibodies could be grouped on the basis of binding activity by ELISA and immunoblot analysis, in which MAB VMB6, VMB31, and VMB119 had binding activity to M bovis; MAB VMB93 and VMB99 detected M bovis and M tuberculosis antigens, and MAB VMB73 reacted with other mycobacterial species, as well as with N asteroides and R equi. Apparent molecular mass of antigens was 30 to 25 kilodaltons (kD) for VMB6, VMB31, and VMB119 and 63 kD for VMB93 and VMB99, and ranged from greater than 200 to 31 kD for VMB73, as estimated by immunoblot analysis. Monoclonal antibody binding activity to 18 field isolates of M bovis was evaluated, using ELISA. Each of 18 field isolates was detected, using MAB VMB6, VMB31, or VMB119; 10 isolates were detected, using MAB VMB93/VMB99, and 14 were detected by use of MAB VMB73. Use of MAB in ELISA failed to detect antigens from M bovis strain AN-5.     

利用聚合酶链反应技术检测牛结核杆菌病的研究

应用聚合酶链反应（ Ｐ Ｃ Ｒ） 技术检测牛结核分枝杆菌纯化 Ｄ Ｎ Ａ, 其敏感性为２５０ｆｇ 。所用引物序列对９种抗酸分枝杆菌 Ｄ Ｎ Ａ 进行扩增, 经琼脂糖凝胶电泳证实, 只有人型结核分枝杆菌、牛型结核分枝杆菌产生了３１７ｂｐ 的特异性扩增带。将 Ｐ Ｃ Ｒ 法与皮内变态反应试验（ Ｐ Ｐ Ｄ） 检测方法比较; ５４ 份血样标本中 Ｐ Ｃ Ｒ 的阳性率为１ ％ , Ｐ Ｐ Ｄ 试验的阳性率为０ 。同时对奶样标本的检测与血样结果一致。结果表明, Ｐ Ｃ Ｒ 在直接检测患牛血样、奶样标本中显示出快速、敏感、特异的优点。为今后牛结核病的检测工作提供了一条新的途径。     

Systemic Mycobacterium avium infection in a dog diagnosed by polymerase chain reaction analysis of buffy coat

Dogs may be infected by Mycobacterium (M.) tuberculosis, M. bovis, and M. avium complex, and the clinical signs associated with each of these infections may be indistinguishable. Rapid speciation of the infecting organism is desirable because of the public health concerns associated with M. bovis and M. tuberculosis infections. A mycobacterial infection was suspected in the dog of this report based on acid-fast staining of organisms in macrophages obtained from liver aspirates and buffy-coat preparations. Polymerase chain reaction (PCR) analysis of a buffy-coat preparation identified M. avium.     

Real-time polymerase chain reaction testing for the detection of Mycobacterium genavense and Mycobacterium avium complex species in avian samples

Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR. In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products. The TaqMan PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan PCR products. The TaqMan PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation.     

Mycobacterial diseases of deer

The most significant mycobacterial diseases of free-living, captive and farmed deer are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp paratuberculosis (basonym M. paratuberculosis), and avian tuberculosis, caused principally by M. avium subsp avium. The first case of M. bovis infection in farmed deer was identified in New Zealand in 1978. In 1983, a voluntary scheme was introduced in New Zealand to control tuberculosis in farmed deer, followed by a compulsory tuberculosis control scheme in 1990. The primary control measure is the slaughter of infected animals, detected by skin testing and blood testing, together with movement control and vector control. The number of infected deer herds peaked in the mid 1990s at over 160 herds, but by 30 June 2002 this had been reduced to 79 (1.45%), and to 67 (1.23%) by June 2003. Deer-to-deer transmission occurs, but the majority of herd breakdowns are believed to be from infected vectors. Factors likely to affect the susceptibility of deer include age, environment, population density, exposure and genetics. Avian tuberculosis occasionally causes clinical disease in wild, captive and farmed deer in New Zealand and overseas. Mycobacterium intracellulare, and subspecies of M. avium other than M. paratuberculosis, are widespread throughout New Zealand and are thought to be largely responsible for the high level of sensitisation to avian purified protein derivative (PPD), which is used for comparison purposes in tuberculosis skin testing of deer in this country. Infections with these organisms are usually subclinical in farmed deer, although M. avium subsp avium commonly causes lesions in retropharyngeal, mesenteric and ileocaecal lymph nodes. These lesions cause problems because of their gross and microscopic similarity to those due to M. bovis infection. Birds and domestic animals are most likely to become infected via environmental contamination of food, water, bedding litter or soil, while carnivores or scavengers may also become infected by ingesting infected carcasses. Johne's disease has been reported in deer in the wild and in zoos, especially in North America, the United Kingdom (UK) and Europe. Since first being confirmed in farmed deer in New Zealand in 1979, the incidence of Johne's disease has increased steadily. To date, M. paratuberculosis has been identified in >600 farmed deer on 300 properties. The majority of cases have been identified from suspected tuberculous lesions submitted from deer slaughter plants. Clinically, Johne's disease in deer is similar to the disease in sheep and cattle, with typical signs of loss of weight and condition, and diarrhoea. However, outbreaks of Johne's disease frequently occur in young red deer, 8-15 months of age, whereas the clinical disease in sheep and cattle is sporadic and usually affects adults 3-5 years of age. The disease is characterised by a chronic granulomatous enteritis and lymphadenitis, especially affecting the jejunum and ileum and the mesenteric lymph nodes. Deer affected subclinically may have lesions in these lymph nodes at slaughter, which are grossly indistinguishable from those due to bovine tuberculosis. Because of the antigenic similarity between M. intracellulare and all the subspecies of M. avium, including M. paratuberculosis, the diagnostic tests for Johne's disease lack sensitivity and specificity, making control difficult.     

Antigenic characterization of mycobacteria from South American wild seals.

Tuberculosis-producing mycobacteria have been previously described in marine mammals (Cousins et al., 1990, 1993; Romano et al., 1995; Bernardelli et al., 1996). The strains belonged to the M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti and M. africanum), but showed genetic and biochemical differences. The antigenic composition of mycobacteria isolated from wild seals was analyzed by Western blots, using antibodies against some selected antigens. The antigenic content was compared with that of M. bovis, M. tuberculosis and M. microti isolates. The lack of Hsp65 protein in supernatants suggested a low degree of cell lysis in the three-week cultures used. SOD, P27 lipoprotein, MPB64 and antigen 85 were observed in all the strains studied. The wild seal strains, as well as M. tuberculosis, did not produce MPB70 and MPB83. Only very weak bands of P36 antigen were observed in culture supernatants from wild seal mycobacteria. Summarizing, the antigenic composition of mycobacterial strains from wild seals is different from M. bovis strains.     

Isolation of Mycobacterium bovis and other mycobacterial species from ferrets and stoats

As part of wildlife surveillance for bovine tuberculosis, pooled lymph nodes from 21,481 ferrets, 1056 stoats and 83 weasels were cultured for mycobacteria. A total of 268 isolates of Mycobacterium bovis were obtained from ferrets, 2 from stoats and none from weasels, demonstrating the presence of a wildlife reservoir of infection in ferrets. DNA typing by restriction endonuclease analysis (REA) of 48 selected isolates of M. bovis revealed 23 REA types. Twenty-one of these types had previously been isolated from cattle and farmed deer, demonstrating a complex cycle of infection involving wildlife and domestic animals. Apart from M. bovis, a further 208 mycobacterial isolates were obtained, the majority of which (178) were members of the M. avium complex. Speciation of the remaining 30 mycobacterial isolates by DNA sequencing of the 16s rRNA gene, identified half the isolates as M. triplex. Other species identified included M. fortuitum, M. florentinum, M. interjectum, M. intracellulare, M. holsaticum, and M. septicum/M. peregrinum.     

An outbreak of tuberculosis by Mycobacterium bovis in coatis (Nasua nasua)

Mycobacterium tuberculosis complex, which includes Mycobacterium bovis, infrequently causes severe or lethal disease in captive wildlife populations. A dead coati from a wildlife triage center showing pulmonary lesions compatible with tuberculosis had raised suspicion of a potential disease caused by mycobacteria species and was further investigated. Four native coatis (Nasua nasua) with suspected mycobacterial infection were sedated, and bronchoalveolar lavages and tuberculin skin tests (TSTs) were performed. All animals tested positive upon TST. Mycobacterial culturing, Ziehl-Neelsen staining, and genetic testing were performed on postmortem samples and the etiologic agent was identified as M. bovis. Molecular genetic identification using a polymerase chain reaction panel was crucial to achieving a definitive diagnosis.     

牛分枝杆菌与鸟型分枝杆菌2型重组蛋白Ag85b的血清学交叉反应研究

牛分枝杆菌(M.bovis)是引起牛结核病的常见病原,而鸟分枝杆菌(M.avium)2型则通常交叉感染牛,但不会导致严重的病变.为鉴定M.bovis和M.avium的免疫交叉反应情况,本研究分别以M.bovis和M.avium 2型的基因组为模板,扩增ag85b基因,分别构建了M.bovis和M.avium的原核和真核表达重组质粒进行表达.以原核表达纯化的两种重组蛋白Ag85b (rAg85b)分别作为包被抗原,交叉检测两种真核重组质粒免疫豚鼠制备的抗血清.结果表明,原核表达的M.bovis和M.avium rAg85b与真核重组质粒免疫豚鼠制备的两种抗血清之间存在较强的免疫交叉反应.研究结果揭示M.bovis和M.avium的Ag85b蛋白存在很强的血清学交叉反应,这将严重干扰M.bovis Ag85b作为候选疫苗的免疫监测.     

Estimation of the spread of pathogenic mycobacteria in organic broiler farms by the polymerase chain reaction

Organic poultry breeding allows for increased exposure of birds to soil, faeces, and wildlife, which have been associated with the transmission of mycobacterial infections. Therefore the aim of this study was to investigate the spread of the major pathogenic mycobacteria in organically reared broilers in Greece using a diagnostic algorithm that relied on a combination of the polymerase chain reaction (PCR) and the restriction fragment length polymorphism analysis (RFLP). Liver, spleen and gonads from 81 to 150 days old broilers were aseptically collected post-mortem. 500 broilers from a population of 35,370, reared in the 25 registered as organic farms in Greece for the 2005 were used. DNA was isolated and incorporated to PCR targeted to 16S-rRNA gene (for Mycobacterium spp.), IS6110 (for Mycobacterium tuberculosis complex-MTBc), IS1245 (for Mycobacterium avium complex-MAC), IS901 (for M. avium subsp. avium-MAA) and hsp65 (for Mycobacterium genavense, by PCR-RFLP). The mean prevalence of mycobacteria detected by PCR with a 95% confidence interval was estimated to 4.4-8.8%. The relevant percentage with regard to the mycobacterial species that were included in this study was 0.17-2.03% for MAC, 2.11-3.39% for MTBc and 0.66-3.08% for mycobacteria not belonging to any of the above groups. None of the mycobacteria detected were identified as MAA or M. genavense. Considering that avian tuberculosis has been eradicated from conventional farms, the level and the pattern of positivity recorded here, indicates that our results may be associated with the specific conditions that apply to organic breeding.