Isolation of Mycobacterium bovis from the respiratory tracts of skin test-negative cattle.

Mycobacterium bovis was isolated from the respiratory tracts of three cattle which registered negative to tuberculin testing; no tuberculous lesions were found and the culture of lymph nodes and other tissues proved negative. One animal was from a group of five calves which had been inoculated intranasally with M bovis, and the organism was recovered once only from nasal mucus sampled 100 days after inoculation. The second animal had had contact with experimentally infected cattle which were excreting M bovis and the third was from a commercial farm. The results of ELISAS for antimycobacterial antibodies and interferon-gamma, and of lymphocyte transformation assays are presented. The animals' immune responses provided evidence that each of them had been challenged.     

Pulmonary lesions and Mycobacterium bovis excretion from the respiratory tract of tuberculin reacting cattle

Although it is generally recognised that tuberculous lesions are present in lymph nodes associated with the respiratory tract in approximately 90 per cent of reactors with confirmed infection, lung lesions are found in only 1 to 2 per cent of such cases during abattoir examination. When lung lesions are not detected, it has been claimed that such cattle are non-excretors and thus unimportant in the epidemiology of the disease. In this study the lungs of 55 reactor cattle were sliced into sections approximately 0.5 cm thick. Tuberculous lesions were evident in over 70 per cent of lungs from reactors with concurrent lesions in lymph nodes of the respiratory system. Further, M bovis was isolated from single samples of nasal and, or, tracheal mucus taken at slaughter in 19 per cent of confirmed cases. Several of these reactors had a clear tuberculin test less than six months previously indicating recent infection. This study confirms the continued importance of the infected bovine in the epidemiology and current eradication of bovine tuberculosis. It is suggested that all tuberculous cattle with lesions in respiratory lymph nodes, rather than being regarded as non-excretors, should be considered as possible excretors and thus important sources of infection for other cattle both within and between herds.     

Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 gene in cattle from herds with naturally occurring tuberculosis

OBJECTIVE: To determine necropsy and Mycobacterium bovis culture results in cattle from herds with tuberculosis, the role of the bovine NRAMP1 gene in resistance and susceptibility to infection with M bovis, and the association between magnitude of the tuberculous lesions and various types of M bovis isolates. ANIMALS: 61 cattle from herds with tuberculosis in Texas and Mexico. PROCEDURE: 61 cattle were evaluated by necropsy; 59 had positive and 2 had negative caudal fold tuberculin intradermal test (CFT) results. Thirty-three cattle with positive CFT results were genotyped to evaluate polymorphism of the 3' untranslated region of the bovine NRAMP1 gene, using single-stranded conformational analysis, 9 were resistant to M bovis with no tuberculous lesions and negative M bovis culture results, and 24 were susceptible with tuberculous lesions and positive M bovis culture results. Isolates of M bovis were analyzed by restriction fragment length polymorphism (RFLP) on the basis of IS6110 sequences and direct-repeat fingerprinting patterns. RESULTS: 21 (35.6%; 21/59) cattle with positive CFT results had tuberculous lesions or positive culture results; in addition, 1 of 2 cattle with negative CFT results had tuberculous lesions and positive culture results. Tuberculous lesions were most common in the thorax (35/63; 55.5%) and lymphoid tissues of the head (10/63; 15.9%). Tuberculous lesions varied from 1 to 11/animal; 8 of 21 (38.1%) had solitary lesions. Associations were not found between resistance or susceptibility to infection with M bovis and polymorphism in the NRAMP1 gene or between the magnitude of the lesions and various RFLP types of M bovis isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The NRAMP1 gene does not determine resistance and susceptibility to infection with M bovis in cattle.     

Further evaluation of an indirect enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis

The sensitivity and specificity of an ELISA for the detection of bovine IgG anti-Mycobacterium bovis antibodies were 73.6% and 94.1%, respectively, as determined in 53 bacteriologically confirmed tuberculous cattle and 101 healthy cattle from a tuberculosis-free area. In addition, the results of ELISA and tuberculin tests in 149 cattle were compared with those of subsequent necropsy studies. Both tests failed to detect 2 animals with tuberculous lesions and positive culture; 3/12 cattle with M. bovis isolation and no lesions, and 2/7 with atypical mycobacterial infection reacted to tuberculin, but none had antibodies; in 128 cattle with neither lesions nor mycobacterial isolation, 6 were tuberculin reactors and 7 others had antibodies. Negative results were obtained by ELISA in 21/22 paratuberculous cattle. Antibodies were not detected in 88.9% to 96.4% of 697 cattle from two tuberculin negative herds of an endemic area. In a herd with proved M. bovis infection, distribution of seropositive animals in tuberculin and non-tuberculin reactors was similar. Antibody responses to cutaneous tuberculin stimuli were observed in 4 experimentally infected cattle, but only in 2/10 healthy controls after repeated PPD stimuli. Nine controls which had either received a single tuberculin dose or none showed no increase in antibody levels. The low sensitivity of this ELISA limits its usefulness as a diagnostic tool for bovine tuberculosis eradication campaigns. However, it could be helpful in epidemiological surveillance if its efficiency to identify infected herds is demonstrated.     

An ELISA for the detection of anergic tuberculous cattle

An enzyme-linked immunosorbent assay (ELISA) for bovine antibody to antigens in unheated Mycobacterium bovis culture filtrate was standardised against a reference serum from an experimentally infected cow. Two Northern Territory herds with a total of 561 cattle were tested. All cattle reacting in the caudal fold tuberculin test, those giving strong reactions in the ELISA and those with visible lesions of tuberculosis were subjected to a detailed bacteriological examination. Of the 19 cattle which yielded isolates of M. bovis, only 4 were positive to the tuberculin test. Serum samples from 5 cattle gave ELISA values greater than 7.0 units. None of these 5 reacted in the tuberculin-test and 2 had no visible lesions. Of the 10 remaining cattle from which M. bovis was isolated, 3 had ELISA values between 6.5 and 7.0 units and were also without visible lesions. The ELISA values for the remaining 7 infected cattle ranged down to 4.6 units. Forty cattle yielded no M. bovis on culture of their tissues. They included 7 which were reactors in the tuberculin test and 23 with ELISA values of 7.0 units or more. The evident low specificity and sensitivity of the ELISA make it of little value as an alternative to the tuberculin test, but it can detect some anergic cattle at the cost of increasing the number of false positive reactors. This may be acceptable in some circumstances and would justify the use of the ELISA as a complement to the tuberculin test or to an in vitro assay of T-cell immunity. In the 2 Northern Territory herds described, the removal of 5 of the anergic cattle would have required a cull of 28 animals of 5% of the total. A cut off value of 6.5 units would have eliminated 3 more, but at the cost of culling 80 animals or nearly 15% of the cattle. Even so, 7 cattle from which M. bovis was isolated would have remained undetected by either test.     

Investigations into an outbreak of tuberculosis in a flock of sheep in contact with tuberculous cattle

Tuberculosis, associated with Mycobacterium bovis infection, occurs infrequently in sheep. A sheep flock, which was potentially exposed to a high level of infection from in-contact tuberculous cattle, was examined for evidence of infection. Six sheep that had given a positive reaction to the comparative intradermal tuberculin test were examined post mortem. Tuberculous lesions were present in four of these sheep. Lesion morphology and distribution in the sheep was similar to that in cattle. M. bovis was cultured from the lesions and the isolates were strain typed by spoligotyping and variable number of tandem repeats (VNTR) typing. Tuberculin-reacting sheep also reacted positively to an assay for in vitro release of interferon-gamma. This paper describes the first report of an outbreak of tuberculosis in sheep in either Britain or Ireland. The report describes immunology and pathology findings and, using molecular typing techniques, suggests that the sheep had been infected from in-contact cattle.     

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.     

Tonsillar lesions in cattle naturally infected with Mycobacterium bovis

The upper respiratory tract surfaces, the palatine and pharyngeal tonsils and associated lymph nodes of 32 tuberculin reactor cattle were examined pathologically and bacteriologically. Tuberculous lesions were observed histologically in the palatine tonsils of five animals and in both the palatine and pharyngeal tonsils of a sixth. Mycobacterium bovis was cultured from the tonsils of four of these animals and from the palatine or pharyngeal tonsils of a further eight cattle in which no lesions were observed. The upper respiratory tract surfaces of 10 animals were M bovis-positive.     

A review of M. bovis BCG protection against TB in cattle and other animals species

Bovine tuberculosis (TB) causes severe economic losses in livestock due to low production, animal deaths and condemnation of carcasses. It is also an important constraint in international trade of animals and animal products. A scientific committee in Great Britain in 1997 concluded that the development of a cattle vaccine would be the best option for long-term control of TB. However, vaccination of cattle currently is not accepted because the vaccine interferes with the skin reaction to the tuberculin test in the field. Efficacy of M. bovis BCG in protecting bovine and other animal species against tuberculous infection has received much study. Vaccination of cattle prevents the spread of the disease in populations by reducing the number and size of the lesions, and the load of bacteria (rather than by preventing infection). We review the literature about the efficacy of BCG in protecting cattle and other animal species against infection with field strains of M. bovis and discusses its potential use in programs of TB control in high-prevalence populations.     

Cattle-to-cattle transmission of bovine tuberculosis

In developed countries, Mycobacterium bovis infection in cattle is now mostly confined to the respiratory system, which reflects transmission and establishment of infection mainly by this route. A single bacillus transported within a droplet nucleus is probably sufficient to establish infection within the bovine lung. Infected cattle should always be considered as potential sources of infection, since studies have demonstrated that a significant proportion of tuberculous cattle excrete M. bovis.In general, the dynamics of M. bovis transmission are poorly understood and the conditions under which a tuberculous animal becomes an effective disseminator of infection are currently not defined although environmental contamination appears to be a less effective method of disease transmission. Field studies indicate a wide spectrum of transmission rates but generally the spread of M. bovis infection is still considered to be a relatively slow process. Slaughter of diseased cattle detected by tuberculin testing and at meat plant inspection has been shown to be an effective policy for tuberculosis eradication, provided there are no other reservoirs of infection and all involved in the cattle industry are committed to a policy of eradication. Epidemiological approaches, particularly case-control studies, seem to provide the best method for quantifying the relative importance of the various sources of M. bovis transmission to cattle and modelling techniques can be used to assist in the design of cost-effective control measures that may lead to tuberculosis eradication.     

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.     

Specific antibody responses to Mycobacterium bovis in infected cattle analysed with six mycobacterial antigens in enzyme-linked immunosorbent assays

A total of 23 (15.3 per cent) of 150 cattle infected with Mycobacterium bovis and which had never been tuberculin tested showed specific antibody responses to M bovis. Their sera may be important keys to the identification of unique M bovis antigens for use in specific serodiagnostic tests. Assessment of specific and non-specific responses was done by screening sera in six indirect anti-IgG enzyme-linked immunosorbent assays using whole cell sonicates of M bovis and five members of the Mycobacterium avium-intracellulare-scrofulaceum complex as respective antigens. Sera from 16 infected cattle that had been tuberculin tested positive and nine uninfected cattle (never tuberculin tested) were also assayed for specific and non-specific responses. Three other findings emerged. First, 43 of the 150 infected animals (28.7 per cent) showed no antibody responses to any of the mycobacterial antigens used. Secondly, the cattle showing the highest antibody levels were associated with the greatest cross reactivity. Lastly, the results indicated that tuberculin injections may increase antibody responses to shared, rather than specific, M bovis antigens in infected cattle.     

PATHOLOGY AND TUBERCULIN SENSITIVITY IN CATTLE INOCULATED WITH MYCOBACTERIUM AVIUM COMPLEX SEROTYPES 6, 14 AND 18

SUMMARY Three strains of Mycobacterium avium complex organisms, serotypes 6, 14 and 18 isolated from typical tuberculous lesions in cattle were examined for pathogenicity and ability to sensitise cattle to avian and bovine tuberculin. Each strain caused tuberculoid granulomas at the site of subcutaneous inoculation but no lesions elsewhere. Sensitisation to bovine tuberculin was detected in the caudal fold test in 11 of 18 inoculated animals 8 weeks after injection. In a simultaneous comparative cervical test, reactions to avian tuberculin were much larger than reactions to bovine tuberculin in all inoculated animals.     

The epizootiological significance of positive bacteriological findings on Mycobacterium tuberculosis and Mycobacterium bovis in humans

The relation between the active form of tuberculosis in persons working in agriculture and incidence of tuberculosis in cattle was analyzed in 1974 to 1978, i.e. in the period after the elimination of bovine tuberculosis in Czechoslovakia (in 1968). M. tuberculosis was isolated in 15 cases and M. bovis in four cases of persons employed by the farms on which the Regional Hygienic Station, Brno, was responsible for the microbiological diagnostics of tuberculosis. Direct contact with animals was demonstrated in eight patients; M. tuberculosis was isolated from seven of these patients and M. bovis from one. Seven cattle herds were exposed to spontaneous infection by M. tuberculosis and in one of them tuberculosis was not demonstrated during complex examination. In three herds the examination revealed only a sensitivity of cattle to mammalian tuberculin. In other three herds tuberculosis was detected by allergic tests, patho-anatomic examination and bacteriological examination. M. tuberculosis in cattle was detected in two herds. The occurrence of bovine tuberculosis caused by a cattle tender with a positive finding of M. bovis in sputum was demonstrated in one herd. Virulence for the tested cattle was found in one strain (isolated from a mesenterial lymph node of cattle) of the four strains of M. tuberculosis used for the experimental infection of 17 animals. On the other hand, in three strains of M. tuberculosis, trials with experimental infection demonstrated only allergy to mammalian tuberculin and changes at the sites of subcutaneous inoculation of mycobacteria of regressive nature; these mostly disappeared within 90 days from infection.     

Experimental Mycobacterium bovis infection of cattle: effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of 18-month-old cattle were inoculated intratracheally with 5 x 10(5) colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21-28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23-24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-gamma responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-gamma responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.     

Examination of the role of Mycoplasma bovis in bovine pneumonia and a mathematical model for its evaluation

The authors screened 34 large cattle herds for the presence of Mycoplasma bovis infection by examining slaughtered cattle for macroscopic lung lesions, by culturing M. bovis from lung lesions and at the same time by testing sera for the presence of antibodies against M. bovis. Among the 595 cattle examined, 33.9% had pneumonic lesions, mycoplasmas were isolated from 59.9% of pneumonic lung samples, and 10.9% of sera from those animals contained antibodies to M. bovis. In 25.2% of the cases M. bovis was isolated from lungs with no macroscopic lesions. The proportion of seropositive herds was 64.7%. The average seropositivity rate of individuals was 11.3% but in certain herds it exceeded 50%. A probability model was developed for examining the relationship among the occurrence of pneumonia, the isolation of M. bovis from the lungs and the presence of M. bovis specific antibodies in sera.     

Prevalence of bovine tuberculosis in slaughter cattle in Kenya: a postmortem, microbiological and DNA molecular study

A study to determine the presence and prevalence of bovine tuberculosis in slaughter cattle in Kenya was carried out in two abattoirs from July to November 2009. Routine postmortem meat inspection was performed on a subpopulation of 929 cattle selected randomly from among 4,984. Carcases were inspected for gross tuberculous lesions which were then examined for acid-fast bacilli, (AFB), cultured for isolation of mycobacteria and the isolates characterised by DNA molecular analysis. Of the carcases examined, 176 (18.95?%, 95?% CI) had lesions suggestive of tuberculosis. AFB were observed in 63/176 of the lesioned cattle and mycobacteria were isolated from 64 of them. The isolates were identified as Mycobacterium bovis (19/64), Mycobacterium tuberculosis, (2/64) and mycobacteria other than tuberculosis (43/64). The prevalence of M. bovis by molecular analysis was 2.05?% (95?% CI). This study documents for the first time the presence of bovine tuberculosis among slaughter cattle in Kenya. There is therefore a need to formulate and implement control programmes in order to minimise transmission among animals and to humans. Isolation of M. tuberculosis from cattle underscores the risk tuberculous humans pose to animals.     

Controlling tuberculosis in a llama (Lama glama) herd using clinical signs, tuberculin skin testing and serology

An outbreak of tuberculosis (TB), caused by Mycobacterium bovis, was investigated in a small herd of llamas (Lama glama). Based on three ante-mortem diagnostic methods (clinical signs, tuberculin skin test reactions, and 'Rapid Test' serology), 12 llamas were selected for examination post-mortem. Grossly visible lesions suspicious of TB were observed in eight animals, four of which had exhibited clinical signs, one was a skin test 'reactor', and three had been seropositive. M. bovis was isolated from seven of these eight animals. Clinical signs combined with serology were found to be useful in identifying infected animals, but tuberculin skin testing had limited negative predictive value as four llamas that were subsequently confirmed as infected were not detected using this assay.     

Pathogenesis of Mycobacterium bovis infection in American bison

Eighteen 12-month-old bison were randomly placed in each of 3 groups (6 animals/group): group-1 bison were exposed to live Mycobacterium bovis, group-2 bison were inoculated with killed M bovis in oil, and group-3 bison were noninfected controls. Six, 6-month-old, tuberculin test-negative calves were placed (pen contact) with group-1 bison 30 days after exposure to M bovis. Tuberculin skin test responses (caudal fold and/or comparative cervical) were detected in all bison in groups 1 and 2 at 2, 4, 6, 10, and 12 months. Tuberculin skin test responses were observed in 2 of 6 calves at 9 and 11 months after pen contact with M bovis-exposed bison (group 1). Statistically significant lymphocyte blastogenic responses to M bovis purified protein derivative were detected in group-1 bison exposed to live M bovis at 2 months after exposure (P less than 0.025). Significant ELISA reactions were detected in sera of bison at 2 months after exposure to killed M bovis in oil (P less than 0.005) and in bison 2 months after exposure to live M bovis (P less than 0.01). Significant tuberculin skin responses, ELISA reactions, or lymphocyte blastogenic responses to M bovis purified protein derivative were not observed in the 6 control bison. Grossly visible tuberculous lesions were observed in lymph nodes and/or lung collected at necropsy in 4 of 6 bison at 12 months after exposure to live M bovis. Microscopic granulomas compatible with tuberculosis were detected in 5 of 6 bison; M bovis was isolated from tissues of each of the 6 bison.(ABSTRACT TRUNCATED AT 250 WORDS)     

ANERGY TO TUBERCULIN IN BEEF CATTLE

SUMMARY Various aspects associated with anergy to tuberculin were investigated in a total of 78 tuberculous cattle. Forty-six (59%) had single lesions and 32 (41%) had multiple lesions. Forty-three (55.1%) had lesions in the lungs or associated lymph nodes and 23 (29.5%) showed multiple involvement of these tissues. Nine anergic cattle had generalised tuberculosis. Histological examination of lesions from 38 animals showed that the majority were of the progressive type.