The emergence of Mycobacterium paratuberculosis in farmed deer in New Zealand - a review of 619 cases

AIM: To review cases in which Mycobacterium paratuberculosis was identified in farmed deer in New Zealand. METHODS: Case histories were reviewed where M. paratuberculosis was identified in deer by either culture or a polymerase chain reaction (PCR) test using primers from IS900. RESULTS: Between 1986 and 2000, M. paratuberculosis was identified by bacterial culture and/or PCR in 619 farmed deer from 299 herds, representing approximately 6% of deer herds in New Zealand. Over 85% of cases were identified during the last 6 years. In 60% of the infected herds, only one infected animal was identified. The maximum number of cases identified in a single deer herd was 47, and these were identified over a period of 8 years. Only 36 (5.8%) cases came from clinically affected animals identified on farms by veterinarians. The majority (89.7%) of the 619 cases were identified from lesions in mesenteric lymph nodes, including the ileocaecal lymph nodes, identified at meat inspection as being macroscopically either typical or equivocal of bovine tuberculosis (M. bovis). While the overwhelming majority of lesions were identified in mesenteric lymph nodes, M. paratuberculosis was also identified in 27 lesions in lymph nodes of the head, especially the retropharyngeal lymph node. CONCLUSIONS: The figures presented underestimate the true prevalence of infection with M. paratuberculosis, especially since not all suspect cases were submitted for culture or PCR. However, they do show that M. paratuberculosis appears to be spreading in farmed deer in New Zealand and highlight the possibility that Johne's disease is emerging as a potential major problem affecting this species. Identification of the organism by bacterial culture or PCR is required in many cases to distinguish lesions in mesenteric lymph nodes and lymph nodes of the head caused by M. paratuberculosis from those caused by M. bovis and M. avium.     

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.     

Epidemiology and pathogenesis of Mycobacterium bovis infection of red deer (Cervus elaphus) in New Zealand

AIMS: This study was initiated to investigate aspects of the epidemiology, pathogenesis and transmission of tuberculosis in wild red deer, with the aim of determining whether this species may be considered a reservoir host of Mycobacterium bovis in New Zealand. METHOD: One hundred and six wild red deer (Cervus elaphus) carcasses from the Castlepoint and Hauhungaroa Range areas, which are endemic for bovine tuberculosis, were examined for the presence of M. bovis infection. Samples were also examined from 46 skin test-positive farmed deer killed at two deer slaughter premises. Where possible, a standard set of tissues and excretion site samples was collected for mycobacteriological examination. RESULTS: Fifty-eight infected deer were identified, and of these 28% showed no gross lesions. The prevalence of tuberculosis confirmed by culture in the wild deer was 32%. Only one of 18 deer younger than 1 year was infected. Mature deer (>2 years) were 12 times more likely to be infected than those under 1 year of age. Infected older deer were less likely to show typical gross lesions than younger animals. Mycobacterium bovis was isolated from the oropharyngeal tonsil of 34 of 56 (61%) of the infected deer, and this was the most commonly infected site. Gross lesions were found in 18 of the 34 infected tonsils and only one of these showed a purulent tonsillitis. Mycobacterium bovis was recovered from four of 53 nasopharyngeal tonsils, four of 53 oropharyngeal swabs, one of 53 tracheal and nasal swabs, and one of 46 faecal samples, but not from any urine specimens. CONCLUSION: These findings suggest that significant bacillary excretion from infected deer was uncommon, and is more likely to occur in severely affected animals. This study has confirmed the importance of mucosa-associated lymphoid tissues (MALT), particularly the oropharyngeal tonsil, in the pathogenesis of tuberculosis in deer. The findings justify investigation of the hypotheses that the prevalence of tuberculosis in wild deer in New Zealand is high due to transmission of infection from possums, and that in the absence of an infected possum population, the prevalence of tuberculosis in deer is likely to be low, and spatially patchy. CLINICAL RELEVANCE: The results suggest that about one quarter of infected deer show no detectable gross lesions. This implies that many infected carcasses may enter the food chain unrecognised and that the estimated sensitivity and specificity of diagnostic tests may be erroneous if there is a difference in test performance between those conducted on deer with or without gross lesions. Diagnostic sensitivity following slaughter may be improved by routine culture of oropharyngeal tonsils and careful examination of lungs for adhesions and small subpleural tubercles.     

Mycobacterium bovis infection in a captive herd of Sika deer.

Infection with Mycobacterium bovis was diagnosed in a small privately owned herd of Sika deer. After postmortem examination of a deer with progressive pulmonary disease, diagnosis of infection with M bovis was confirmed by bacteriologic culture. The 2 remaining deer in this herd were euthanatized, necropsied, and confirmed to be infected with M bovis. Three cats in contact with the deer were also euthanatized and necropsied. One of these cats had lesions suggestive of mycobacterial infection in the colon and mesenteric lymph nodes. Infection of this cat with M bovis was not confirmed by bacterial culture. Mycobacteriosis, infrequently encountered in clinical veterinary practice, may be confused with disease caused by other infective agents or neoplasia. The zoonotic potential of these bacteria and a recent increase in human tuberculosis warrants continued surveillance of companion and food animal populations for mycobacterial infection.     

Prevalence of Mycobacterium bovis infection in cervids on privately owned ranches

OBJECTIVE: To determine prevalence of tuberculosis caused by infection with Mycobacterium bovis in cervids on privately owned ranches in northeastern lower Michigan. DESIGN: Epidemiologic survey. ANIMALS: Cervids on 96 privately owned ranches. PROCEDURES: A combination of slaughter and skin tuberculin testing was used to collect data. Infection with M. bovis was confirmed by use of standard necropsy and bacteriologic culture techniques. RESULTS: Cervids with tuberculosis were detected on 1 of the 96 ranches. The apparent prevalence of tuberculosis in cervids from the 96 ranches was 1.1 cases/100 cervids (21 cases/1,867 cervids tested). For the ranch with infected cervids, prevalence of infection with M. bovis was 12.1 cases/100 cervids (21 cases/174 cervids tested). No obvious gross lesions were seen in 8 of 21 white-tailed deer and 1 coyote with culture-confirmed M. bovis infection. CONCLUSIONS AND CLINICAL RELEVANCE: The lack of visible lesions in a substantial proportion of infected animals should be taken into consideration in studies involving detection and prevalence of tuberculosis.     

Naturally occurring tuberculosis in white-tailed deer

OBJECTIVE: To determine the distribution of lesions and extent of tissues infected with Mycobacterium bovis in a captive population of white-tailed deer. DESIGN: Cross-sectional study. ANIMALS: 116 captive white-tailed deer. PROCEDURE: Deer were euthanatized, and postmortem examinations were performed. Tissues with gross lesions suggestive of tuberculosis were collected for microscopic analysis and bacteriologic culture. Tissues from the head, thorax, and abdomen of deer with no gross lesions were pooled for bacteriologic culture. Tonsillar, nasal, oral, and rectal swab specimens, fecal samples, and samples of hay and pelleted feed, soil around feeding sites, and water from 2 natural ponds were collected for bacteriologic culture. RESULTS: Mycobacterium bovis was isolated from 14 of 116 (12%) deer; however, only 9 of 14 had lesions consistent with tuberculosis. Most commonly affected tissues included the medial retropharyngeal lymph node and lung. Five of 14 tuberculous deer had no gross lesions; however, M bovis was isolated from pooled tissue specimens from the heads of each of these deer. Bacteriologic culture of tonsillar swab specimens from 2 of the infected deer yielded M bovis. Mean (+/- SEM) age of tuberculous deer was 2.5 +/- 0.3 years (range, 0.5 to 6 years). Mycobacterium bovis was not isolated from feed, soil, water, or fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Examination of hunter-killed white-tailed deer for tuberculosis commonly includes only the lymph nodes of the head. Results of such examinations may underestimate disease prevalence by as much as 57%. Such discrepancy should be considered when estimating disease prevalence.     

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 provide a 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.     

Failure to identify non-bovine reservoirs of Mycobacterium bovis in a region with a history of infected dairy-cattle herds

The State of Texas had the most (cumulative) tuberculous cattle herds of any state in the United States during the decade ending in 1997. Of the cumulative 18 infected herds in Texas, 12 herds were concentrated in El Paso County (designated the 'El Paso milkshed'). To identify whether non-bovine reservoirs were a source of Mycobacterium bovis infection of cattle in this region, an investigation was conducted on the premises of 14 dairy herds (12 tuberculous and 2 non-affected herds) between May 1995 and June 1997. None of the 670 mammalian, avian and environmental (soil, water and air) samples collected and cultured from the premises of these herds was positive for the presence of M. bovis. None of the 119 human urine samples obtained from employees of these dairies was culture positive for M. bovis. Of 124 dairy-farm workers with tuberculin skin-test results, 48 showed positive test results. There was, however, no difference in percentages of positive skin-test results between farms without, and farms having, bovine tuberculosis within the last two years or longer. The percentage of positive reactions did not increase with length of time employed at a dairy with a history of confirmed tuberculosis. These findings suggest that non-bovine reservoirs appear not to be a factor responsible for tuberculosis of cattle in the El Paso milkshed.     

Surveillance of wildlife for Mycobacterium bovis infection using culture of pooled tissue samples from ferrets (Mustela furo)

AIM: To compare culture results of homogenates of pooled lymph nodes from individual ferrets with and without macroscopic lesions of bovine tuberculosis for the presence of Mycobacterium bovis, and to determine whether homogenates from 10-30 ferrets could be combined and cultured without loss of sensitivity as a possible method for improving cost-effectiveness of surveillance for M. bovis infection in wildlife populations. METHODS: Numbers of colony forming units (cfu) of M. bovis present in cultures of homogenates of pooled lymph nodes from individual ferrets known to be infected and having no visible lesions (NVL) or macroscopic lesions consistent with bovine tuberculosis were determined. Prevalences of M. bovis infection in populations of ferrets in the Marlborough region of the South Island of New Zealand were determined by culturing homogenates of pooled lymph nodes from individual animals. Samples from homogenates from North Canterbury were combined to form pools representing 10, 20 and 30 animals and also cultured for M. bovis. RESULTS: Fewer M. bovis cfu were isolated from ferrets with NVL (mean=0.77 log10) compared with ferrets with macroscopic lesions (mean=3.22 log10; p<0.05). The mean prevalence of infection in eight different surveys involving 427 ferrets from the Marlborough region was 18% (range 8-44%), which included a small number of animals with macroscopic lesions of tuberculosis. Pooling of samples from up to 30 different ferrets with NVL did not reduce the sensitivity of detecting M. bovis infected populations. CONCLUSION: Culturing of pools of lymph node samples detected a significant proportion of M. bovis-infected ferrets that would otherwise have gone unnoticed based on samples that had only macroscopic lesions. Culturing of samples pooled from up to 30 different ferrets could provide significant cost savings in surveys of wildlife for the presence of M. bovis infection without any apparent loss of sensitivity.     

A study of bovine tuberculosis in domestic animals and wildlife in the MacKenzie Basin and surrounding areas using DNA fingerprinting

The MacKenzie Basin, an area of about 5150 km2 in the South Island of New Zealand, was free of bovine tuberculosis prior to 1980. During the next 13 years, the majority of the cattle and deer herds in this area became infected with Mycobacterium bovis. The history of infection in the MacKenzie Basin has all the characteristics of a newly developed region of endemic tuberculosis with a wildlife reservoir of M. bovis. Tuberculous possums and ferrets were found in the MacKenzie Basin and both may have been a source of infection for domestic animals. DNA fingerprinting of 125 isolates of M. bovis from domestic animals and wildlife by restriction endonuclease analysis revealed two major groups of isolates. The same groups were identified using IS6110 as a DNA probe. Restriction endonuclease analysis enabled one group to be subdivided into seven restriction types and the other group into eight types. Mycobacterium bovis isolates with the most common restriction types were present in both domestic animals and wildlife, indicating that infection had spread between these two groups of animals. DNA fingerprinting also revealed that M. bovis was introduced into the MacKenzie Basin from at least two distinct sources. Furthermore, DNA finger-printing was able to identify probable sources of infection.     

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.     

Investigation of the transmission of Mycobacterium bovis from deer to cattle through indirect contact

OBJECTIVE: To investigate the infection of calves with Mycobacterium bovis through oral exposure and transmission of M. bovis from experimentally infected white-tailed deer to uninfected cattle through indirect contact. ANIMALS: 24 11-month-old, white-tailed deer and 28 6-month-old, crossbred calves. PROCEDURE: In the oral exposure experiment, doses of 4.3 x 10(6) CFUs (high dose) or 5 x 10(3) CFUs (low dose) of M. bovis were each administered orally to 4 calves; as positive controls, 2 calves received M. bovis (1.7 x 10(5) CFUs) via tonsillar instillation. Calves were euthanatized and examined 133 days after exposure. Deer-to-cattle transmission was assessed in 2 phases (involving 9 uninfected calves and 12 deer each); deer were inoculated with 4 x 10(5) CFUs (phase I) or 7 x 10(5) CFUs (phase II) of M. Bovis. Calves and deer exchanged pens (phase I; 90 days' duration) or calves received uneaten feed from deer pens (phase II; 140 days' duration) daily. At completion, animals were euthanatized and tissues were collected for bacteriologic culture and histologic examination. RESULTS: In the low- and high-dose groups, 3 of 4 calves and 1 of 4 calves developed tuberculosis, respectively. In phases I and II, 9 of 9 calves and 4 of 9 calves developed tuberculosis, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that experimentally infected deer can transmit M. bovis to cattle through sharing of feed. In areas where tuberculosis is endemic in free-ranging white-tailed deer, management practices to prevent access of wildlife to feed intended for livestock should be implemented.     

Tuberculin skin testing in white-tailed deer (Odocoileus virginianus).

The comparative cervical skin test for antemortem diagnosis of tuberculosis was done 169 times on 116 different white-tailed deer of known Mycobacterium bovis infection status. The sensitivity and specificity were 97 and 81%, respectively. The magnitude of change in skin thickness at test sites was not significantly influenced by dosage of inoculum, dissemination of the disease process, or repeated skin testing. However, the magnitude of change in skin thickness was significantly greater in deer infected for less than 109 days than in deer infected for more than 109 days. As used in the present study, the comparative cervical skin test is a sensitive method of antemortem diagnosis of M. bovis infection in white-tailed deer.     

Dominance hierarchies in cattle and red deer (Cervus elaphus): their possible relationship to the transmission of bovine tuberculosis

A behavioural study was conducted to assess the dominance structure of cattle and deer herds and to assess the possible relationship of dominance to the risk of becoming infected with bovine tuberculosis. Five groups of cattle containing newly identified intradermal tuberculin test reactors were evaluated to determine the dominance hierarchy, and then exposed to sedated possums to assess the response of reactors and non-reactors. Eighty-six percent of the tuberculin test-positive cattle were among the 20% most dominant animals in their herds. In four of the five herds, the dominant animals investigated the sedated possum most actively, and in three of these four the reactors were in the investigating group. Six deer were exposed to a naturally tuberculosis-infected possum population, and the four highest animals in the dominance hierarchy (which also showed strong investigative behaviour when exposed to simulated terminally ill tuberculous possums) all subsequently became infected with tuberculosis. The fifth animal in the hierarchy became test-positive for tuberculosis later than the first four, but was subsequently also shown to be culture-positive for M. bovis. The lowest animal in the hierarchy, which showed no active interest in simulated tuberculous possums, did not become infected. This study strongly suggests a central role for terminally ill tuberculous possums in the transmission of tuberculosis to cattle and farmed deer. Management techniques designed to reduce contact between these few possums and farmed livestock may be expected to reduce the incidence of tuberculosis.     

Evaluation of an in vitro blood-based assay to detect production of interferon-gamma by Mycobacterium bovis-infected white-tailed deer (Odocoileus virginianus).

Tuberculosis due to Mycobacterium bovis in captive Cervidae was identified as an important disease in the United States in 1990 and prompted the addition of captive Cervidae to the USDA Uniform Methods and Rules for eradication of bovine tuberculosis. As well, M. bovis infection was identified in free-ranging white-tailed deer in northeast Michigan in 1995. Tuberculosis in both captive and free-ranging Cervidae represents a serious challenge to the eradication of M. bovis infection from the United States. Currently, the only approved antemortem tests for tuberculosis in Cervidae are the intradermal tuberculin skin test and the blood tuberculosis test (BTB). At present, the BTB is not available in North America. Tuberculin skin testing of Cervidae is time-consuming and involves repeated animal handling and risk of injury to animals and humans. This study evaluated the potential of a new blood-based assay for tuberculosis in Cervidae that would decrease animal handling, stress, and losses due to injury. In addition, a blood-based assay could provide a more rapid diagnosis. Twenty 6-9-month-old white-tailed deer, male and female, were experimentally inoculated by instillation of 300 colony-forming units of M. bovis in the tonsillar crypts. Seven, age-matched uninfected deer served as controls. Blood was collected on days 90, 126, 158, 180, 210, 238, 263, and 307 after inoculation and was analyzed for the production of interferon-gamma (IFN-gamma) in response to incubation with M. bovis purified protein derivative (PPDb), M. avium PPDa, pokeweed mitogen (PWM), or media alone. Production of IFN-gamma in response to PPDb was significantly greater (P < 0.05) at all time points in samples from M. bovis-infected deer as compared with uninfected control deer, whereas IFN-gamma production to PWM did not differ significantly between infected and control deer. Measurement of IFN-gamma production to PPDb may serve as a useful assay for the antemortem diagnosis of tuberculosis in Cervidae.     

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.     

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

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.     

Mycobacteria isolated from deer in New Zealand from 1970-1983

Mycobacterium bovis was isolated from 504 deer from 1970 to 1983. It was first isolated from feral red deer (Cervus elaphus) in New Zealand in 1970, and from farmed deer in 1978. Cervine tuberculosis has emerged as a significant problem in farmed deer and in 1983 M.bovis was found on 40 different farms. Thirty-five isolates of Mycobacterium avium-intracellulare have been cultured from deer but were associated with clinical disease in only four cases. Mycobacterium nonchromogenicum, Mycobacterium diernhoferi, Mycobacterium gastri, Mycobacterium chelonei, Mycobacterium smegmatis and Mycobacterium vaccae were isolated from deer but were not considered to be pathogenic.     

Natural infection of red deer with bovine tuberculosis

Six bovine tuberculosis-free red deer hinds were introduced in October 1993 to a 1.8 ha enclosure, within a larger field study site known to contain tuberculous possums, and kept there for 9 months. A Mycobacterium bovis-infected possum was found in the vicinity of the deer enclosure 3 weeks after the introduction. Subsequently, a further eleven infected possums were found in the area. The deer were monitored by repeated composite antibody detection ELISA and lymphocyte transformation assays for tuberculosis, interpreted in parallel, by skin testing and by routine culturing of samples collected from potential excretion sites. Lymphocyte transformation assay evidence of M. bovis infection in four hinds was first observed 4 months after introduction. One other hind became bovine tuberculin lymphocyte transformation assay positive in the 5th month. Positive or equivocal bovine reactivity remained evident at most test episodes. A comparative cervical skin test performed in July 1994, shortly before slaughter, was positive in these five hinds. Mycobacterium bovis was recovered off swabs from the oropharyngeal tonsils of two hinds during routine sampling. Detailed necropsy of the six deer revealed a single typical tuberculous lesion in only one, but culturing of various tissue specimens ascertained that the five blood test and comparative cervical skin test-positive animals were all infected. Mycobacterium bovis was cultured from the oropharyngeal tonsils of four and medial retropharyngeal lymph nodes of two of the deer with no typical gross lesions. Six additional tuberculosis-free hinds were introduced to the enclosure in April 1994 and kept there for 12 months. Four of these animals showed a positive lymphocyte transformation assay response to M. bovis after 9 weeks, but no significant reactivity thereafter. Concurrent observational studies suggest that five of the first six deer probably became infected through close inspection and investigation of the tuberculous possums, although the possibility of deer-to-deer transmission cannot be totally excluded. The likely deer-possum contact, and thus exposure to M. bovis, was related to the curiosity and social ranking of the hinds. The second group appear to have had transient exposure to M. bovis, possibly caused by direct contact with the infected hinds introduced earlier. This group never showed any curiosity toward, or interaction with, possums during the periods of observation.