Shedding of Mycobacterium bovis in the nasal mucus of cattle infected experimentally with tuberculosis by the intranasal and intratracheal routes

Four groups of six calves were infected experimentally with either a low dose of approximately 10(4) colony-forming units (cfu) or a high dose of approximately 10(6) cfu of Mycobacterium bovis. Each dose was delivered by the intranasal and intratracheal routes. More severe disease was observed in the groups inoculated with the high dose. Visible lesions were identified in 21 of the 24 animals, all of which also gave positive skin tests and interferon-gamma (IFN-gamma) responses. Nasal shedding was detected in 15 of the 24 animals and the frequency of shedding was influenced by both the route and the dose of infection; no shedding was observed in the group infected intratracheally with the low dose. Two of the 15 confirmed shedders had no visible lesions at postmortem examination; both of these calves gave IFN-gamma responses but only one was skin test positive.     

Within-herd contact structure and transmission of Mycobacterium avium subspecies paratuberculosis in a persistently infected dairy cattle herd

Within-herd transmission of pathogens occurs either by direct or by indirect contact between susceptible and infected animals. In dairy herds that are structured into groups, the way in which animals encounter each other and share an environment can affect pathogen transmission. Dairy cattle are heterogeneous in terms of susceptibility and infectivity with respect to Mycobacterium avium subspecies paratuberculosis (Map) transmission. It is mainly young animals that are susceptible and adults that are infectious. Both vertical and horizontal transmission through the ingestion of Map shed into the environment by adults and transiently infected calves can occur. Our objective was to assess the effect of contact structure on Map transmission in persistently infected dairy herds and to examine the effect of isolating calves from other calves or from adults before weaning. We developed a stochastic compartmental model of Map transmission in a closed dairy herd. The model reflects the Map infection process and herd management characteristics. Indirect transmission via the environment was modelled explicitly. Six infection states (susceptible, resistant, transiently infectious, latently infected, subclinically infected, and clinically affected) and two contaminated farm area environments (whole farm and calf area) were modelled. Calves were housed in hutches, individual indoor pens, or group indoor pens. Two different levels of exposure of calves to a farm environment contaminated by adults were possible: no exposure and indirect exposure through fomites. Three herd sizes were studied. We found that contacts between calves before weaning did not influence Map transmission in a herd, whereas the level of exposure of calves to an environment contaminated by adults and the starting age of exposure of calves to adults were pivotal. Early culling of clinically affected adults led to a lower prevalence of infectious adults over time. The results were independent of herd size. Despite the many transmission routes that are known, the best control approach is to limit the exposure of calves to adult faeces through the systematic separation of adults and calves in combination with hygiene measures. Reducing contact between calves does not appear effective.     

Description of the infection status in a Norwegian cattle herd naturally infected by Mycobacterium avium subsp. paratuberculosis

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-gamma (IFN-gamma) immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-gamma immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test, 10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-gamma and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-gamma immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.     

Application of the direct leukocyte-migration agarose test in cattle from a Mycobacterium paratuberculosis-infected herd

Using johnin as the specific antigen, the direct leukocyte-migration agarose test was applied to 10 cows from a Mycobacterium paratuberculosis-infected herd. Nine of the cows did not have signs of paratuberculosis; 1 cow was clinically affected. The cows were slaughtered and, on the basis of histopathologic and cultural examinations of the intestines, were classified as infected or noninfected. Six of the healthy cows from which the bacterium was not isolated and the clinically affected cow reacted with migration indices lower than those of 3 healthy animals that were found to harbor the bacterium. It is suggested that the long incubation period of paratuberculosis could to some extent be due to immunologic tolerance.     

Effect of paratuberculosis on the diagnosis of bovine tuberculosis in a cattle herd with a mixed infection using interferon-gamma detection assay

Interferon-gamma (IFN-gamma) detection assay is being applied as an ancillary test to tuberculin tests in the diagnosis of bovine tuberculosis to detect the maximum number of infected animals. Among possible factors influencing the performance of tuberculosis-diagnostic tests, paratuberculosis, a widespread disease in Spain and other European countries, has been pointed out as a cause of false positive reactions. Still, its effect on the sensitivity of these tests in cattle has yet to be fully characterized. The impact of paratuberculosis in the apparent sensitivity of IFN-gamma assay was studied in a bullfighting cattle herd with a mixed tuberculosis-paratuberculosis infection, using culture of Mycobacterium bovis and Mycobacterium avium paratuberculosis as the gold standard to determine the infection status of every animal. A total of 218 animals were slaughtered and sampled for bacteriology after blood sampling. IFN-gamma assay showed a lower apparent sensitivity in animals with a mixed infection (50%) compared to all animals suffering tuberculosis (78.3%). This finding indicates that the presence of paratuberculosis in tuberculosis-infected herds could imply a serious impairment in the sensitivity of IFN-gamma detection test.     

Investigations of cattle herd breakdowns with bovine tuberculosis in four counties of England and Wales using VETNET data

Cattle herd breakdown (HBR) with bovine tuberculosis (BTB) was investigated for farms in four counties of England and Wales outside southwest England from 1986 to early 2000. Data from the national database of TB testing history (VETNET) were used. Factors that influenced HBR included calendar time, herd size, number of cattle tested, the test type, the inter-test interval and spatial grouping of farms. Herd tests other than routine herd tests had an increased risk of HBR in all four counties. In all counties, the risk of HBR increased with calendar time and in Shropshire a test interval of 3 years was associated with an increased risk of HBR compared with a 1-year test interval. In Staffordshire and Sussex, a 4-year test interval was associated with a lower risk of HBR compared with a 1-year test interval. There was no evidence of spatial clustering of HBR in West Glamorgan (equal spatial risk in a 15–30 km radius) and weak evidence of spatial clustering in Shropshire (7–15 km) and Sussex (5–10 km). In Staffordshire, there was evidence of spatial (2–4 km) and time (3–4 years) clustering of HBR. The locally increased rate of testing following a confirmed HBR increased the detection of infected herds but did not prevent local spread in two of the four counties (Shropshire and Staffordshire) since the rate of HBR increased linearly from 1988 to 2000. The main conclusion is that there were both local and distant components of spread.     

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.     

Eradication of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) from a herd of beef cattle

Infectious bovine rhinotracheitis virus was eradicated from a 150 cow beef herd at the Animal Diseases Research Institute, Lethbridge, Alberta. Tests used to accomplish this included standard and modified serum-virus neutralization tests and an enzymelinked immunosorbent assay. These results and those of preliminary pilot studies in the herd and in a nonvaccinated, infectious bovine rhinotracheitis-infected 450 cow beef herd suggest that eradication of infectious bovine rhinotracheitis infection can be considered as a practical control alternative to vaccination, and that young animals in purebred herds could be monitored serologically and isolated, to enhance their eligibility for entry into artificial insemination studs or for export.     

Seroprevalence of bovine immunodeficiency-like virus and bovine leukemia virus in a dairy cattle herd.

To determine the prevalence of single vs. dual infection with bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV), sera (n = 95) from a dairy cattle herd were analyzed for anti-BIV and anti-BLV antibodies by an enzyme linked immunosorbent assay. Twenty-one percent (20/95) of samples were BIV-seropositive, while 52% (49/95) of the same samples were BLV-seropositive. A significantly greater percentage of BIV-seronegative samples were BLV-seropositive, 57% (43/75), than were BIV-seropositive samples, 30% (6/20). There was no significant correlation between data ranked from least to greatest amount of anti-viral antibody. Five cattle had persistent lymphocytosis (PL); all five were BLV-seropositive and two were BIV-positive. The mean anti-BLV titer was significantly greater in PL cattle, as compared at non-PL cattle, whereas there was no significant difference between the mean anti-BIV titer in PL cattle, as compared with non-PL cattle. These results provide additional information on the seroprevalence of naturally occurring BIV infection, and indicate that BIV can exist independent of other common infectious agents, such as BLV. Further, the results suggest that infection with BIV is not associated with an increased rate of infection with other infectious agents such as BLV.     

Molecular studies of T-lymphocytes from cattle infected with bovine leukemia virus

Although bovine leukemia virus (BLV) is mainly associated with infections of B-lymphocytes, we have previously reported the statistically significant increase in the T-lymphocytes obtained from BLV-infected asymptomatic aleukemic (AL) cattle. In this report the presence of BLV provirus in the DNA of immunoaffinity purified T-lymphocytes from AL animals was assessed using a highly specific radiolabelled (32P) BLV-DNA provirus probe and solid phase DNA hybridization. The BLV provirus was found in the DNA of the peripheral blood mononuclear cells of all AL animals tested and three of the four purified T-lymphocyte preparations from these animals. The purified T-lymphocyte preparations used in this study contained less than 4% detectable B-lymphocytes. One animal had no detectable B-lymphocytes in the purified T-lymphocyte preparation and the DNA from these cells also gave positive hybridization results. The lymphocyte blastogenesis assay was then used as an indicator of the functional ability of lymphocytes from these BLV-infected AL cattle to respond to mitogenic stimuli. The responsiveness of lymphocytes from these animals to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweek mitogen (PWM) was comparable to that of lymphocytes from BLV-negative animals when changes in 3H-thymidine uptake (c.p.m.) were used as measurement of mitogenic-induced blastogenesis. This indicated that infection of the T-lymphocytes by BLV does not appear to alter the overall response of the lymphocyte populations to mitogenic stimuli. High levels of spontaneous blastogenesis in the absence of mitogenic stimulation were observed for lymphocyte preparations of AL animals. The reason for this proliferation of lymphocytes is unclear; however, sera from these AL animals were found to contain a blastogenesis-augmenting factor(s) when added to lymphocytes from BLV-negative control animals in the presence of Con A, PHA and PWM.     

Prevalence of the plasma bovine leukaemia virus blocking factor in cattle from a commercial dairy herd

The prevalence of the plasma bovine leukaemia virus blocking factor was examined in a commercial herd. The detection of the blocking factor activity depended on the nature of the infected target cells used in the assay. Using the sensitive bovine leukaemia virus-infected target lymphocytes, the factor was found to be significantly more prevalent in infected cattle than in virus-free cattle. Furthermore, in infected cattle, no correlation was observed between the level of bovine leukaemia virus antibody and the presence of the blocking factor.     

Temporal patterns in bovine tuberculosis in a brushtail possum population contiguous with infected cattle in the Ahaura Valley, Westland

AIMS: To determine temporal and spatial patterns of bovine tuberculosis (Tb) in a population of brushtail possums (Trichosurus vulpecula) free from commercial and recreational hunting and living contiguously with livestock, and to relate these patterns to measures of possum health and density. METHODS: Possums were trapped on 11 lines located on a forest-rough grazing margin in the Ahaura Valley in Westland in 1979-80 and each August from 1992 to 1996, and were examined post mortem for gross lesions indicative of Tb. Infection levels in possums were compared with Th test data from cattle grazing in the same area. RESULTS: Tuberculosis was identified from the trapped sample of possums in 1980. Trapping of further possums on the same trap lines confirmed the presence of the disease each year from 1992 to 1996, and suggested a clustering of infection in time and space. The prevalence of Tb in possums declined with increasing possum population size and well being, and decreasing age. The prevalence of Tb in possums was highest in 1980 and 1992, and appeared to roughly coincide with similar upsurges in Tb in nearby cattle. CONCLUSIONS: Our results suggest that the exceptional levels of Tb recorded in possums in 1992 had a direct effect on possum numbers, and gave rise to very low trap catches in 1993-94. They also suggest that Tb can persist for several years in possum populations existing well below the 5% trap catch targeted by regional councils for Tb possum control.     

Antibodies to mycobacteria in cattle not infected with Mycobacterium bovis

An indirect anti-IgG enzyme-linked immunosorbent assay (ELISA) using a whole cell sonicate of Mycobacterium bovis as the coating antigen, was used to detect anti-mycobacterial antibodies in cattle not infected with M. bovis. False positive M. bovis ELISA scores were produced in 6 cattle experimentally inoculated with Mycobacterium avium-intracellulare-scrofulaceum (MAIS) serovars 2, 8, 9, 14 and 18 and Mycobacterium flavescens, respectively. False positive ELISA results were also found in 39.5% of cattle from which other mycobacteria were cultured and in 56.4% of necropsied cattle with other pathological conditions. No M. bovis was cultured from these animals. Other groups of animals, with no pathological conditions, which had been tuberculin-tested negative, tuberculin-tested positive and never tuberculin tested showed positive ELISA results in 15.4%, 73.6% and 42.4% of the respective groups. The variation of these non-specific responses in uninfected cattle highlights the need for careful selection of negative controls in evaluating ELISAs for the diagnosis of bovine tuberculosis.     

Mycobacterium bovis infection and tuberculosis in cattle

This review considers the possible events that can occur when cattle are exposed to Mycobacterium bovis and, where appropriate, draws on principles accepted for tuberculosis infection in humans and laboratory animal models. Consideration is given to the many complex factors which influence the outcome of challenge with tubercle bacilli. These include features inherent to the mycobacterium, the host and the environment. It is apparent that clinical disease probably occurs only in a relatively small, but undetermined, proportion of cattle that are exposed to Al. bovis. The majority of animals may clear infection or control the bacilli, possibly in a condition of latency. It is concluded that a better understanding of the dynamics of the events following M. bovis exposure and subsequent infection in cattle would be of significant benefit in developing new tools appropriate for disease control and to designing optimal approaches for their application.     

Investigation of bovine viral diarrhea virus infections in a range beef cattle herd

Bovine viral diarrhea virus (BVDV) infections resulting in clinical disease developed in calves, despite vaccination of dams and high maternal BVDV antibody titers in calves. Eight persistently infected (PI) calves born to immunocompetent dams were identified in the herd. Neutralizing BVDV antibody titers of PI calves had decreased greatly by the time the calves were 1 to 2 months old. Antibody titers of PI calves decreased more rapidly than antibody titers of calves that were not PI. Reduced antibody titers in PI calves allowed detection of BVDV in serum specimens of all PI calves by the time they were 8 weeks old. Persistent infection in suspect calves was detectable serologically and was confirmed by virologic examination of serum specimens 4 months after weaning, when the calves were 9 months old. Growth rates were reduced in viremic calves.     

Longitudinal study of ELISA seroreactivity to Mycobacterium avium subsp. paratuberculosis in infected cattle and culture-negative herd mates.

Two thousand nine hundred fifty-two serum samples, collected once or twice annually from 545 cows of known fecal culture status were tested for antibodies to Mycobacterium avium subsp. paratuberculosis using a commercially available enzyme-linked immunosorbent assay (ELISA) test. Overall, 13.5% of the samples from 282 infected cows had positive ELISA results, but when tested multiple times, 38.3% of the cows had at least 1 serum sample with positive results. Among 263 fecal culture-negative cows, 98.1% of the serum samples had negative ELISA results, but when tested multiple times, 7.8% of the cows had at least 1 positive ELISA sample. Fecal culture was positive on a test before the first positive ELISA in 50 cows, ELISA was positive before fecal culture in 12 cows, and in 38 cows, both tests became positive at the same testing time. An additional 174 cows were positive on fecal culture and always negative on ELISA until culled. For cows that had ELISA sample:positive (S/P) ratios below the cutoff point, the change in S/P between sequential tests was evaluated to determine whether a rise in S/P could predict infection status. In this study, change in S/P was not a useful predictor of infection status in seronegative cows.     

Individual animals of a cattle herd infected with the same Mycobacterium bovis genotype shows important variations in bacteriological, histopathological and immune response parameters

Cattle are the host and main reservoir of the etiologic agent of bovine tuberculosis, Mycobacterium bovis; although other mammalian species, including humans, are susceptible. The tuberculin test and/or slaughterhouse surveillance is the diagnostic method used by control programs all around the world to control and eradicate the disease. In order to compare different tuberculosis diagnostic tests and to reach disease confirmation, a study was performed in a group of 14 steers of Friesian breed, reacting positively to tuberculin test. Three ante-mortem assays were performed according to the type of sample: the gamma interferon (IFN-gamma) test (which quantifies the release of this cytokine by sensitized lymphocytes in whole blood in response to purified protein derivative (PPD) and recombinant ESAT-6 and CFP10 proteins); PCR and bacteriologic culture from nasal swab and intradermal tuberculin test. These assays were taken at different times to assess the evolution of clinical parameters. Post-mortem examination showed macroscopic and microscopic tuberculosis lesions with acid-fast bacillus and positive cultures. By spoligotyping, we observed that all the isolates showed the same pattern. The positive results based on comparison to lesions observed ranged from 58% to 75% for the IFN-gamma assays, to 72% for cultures, and ranged from 50% to 90% for PCR in nasal swabs. In conclusion, in a herd infected by the same strain, ante-mortem direct and immune-diagnostic parameters change, suggesting that several tests are needed for a faster control of infection at herd level.     

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.