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.     

Characterization by restriction endonuclease analysis and seroagglutination of strains of Mycobacterium avium and Mycobacterium intracellulare obtained from farmed deer.

Restriction endonuclease analysis and seroagglutination were used to characterize strains of Mycobacterium avium and Mycobacterium intracellulare (collectively, MAI) recovered from 1 local herd and 2 imported shipments of red deer (Cervus elaphus) that developed sensitization to bovine tuberculin during skin testing. A total of 31 MAI strains were isolated from lymph node pools (head, thorax, abdomen, and peripheral regions) of 21 of 29 local deer. Similarly, 15 MAI strains were isolated from the lymph node pools of 12 deer from the 2 imported shipments. Mycobacterial strains were isolated from more than 1 of the lymph node pools of 9 local and 2 imported deer. Most of the strains (59% from local deer, 46% from imported deer) were recovered from the lymph nodes of the head region. After restriction endonuclease analysis of these isolates using the enzymes Bcl I, BstEII, and Pvu II, 26 of the strains from the local herd were separated into 3 groups, each consisting of strains with indistinguishable or closely related patterns. Seroagglutination results indicated that the first of these groups contained strains belonging to serotype 1, the second group contained strains belonging to serotype 8; and the third group of strains belonged to serotype 3 and 9. The 5 remaining strains from the local herd had unrelated restriction patterns. One of these belonged to serotype 3, whereas the remaining 4 could not be serotyped. Restriction analysis of the 15 strains from the imported deer identified 2 groups. Seroagglutination results indicated that 1 group contained strains belonging to serotype 2 and the other group contained strains belonging to serotype 8.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paratuberculosis and avian tuberculosis infections in one red deer farm studied by IS900 and IS901 RFLP analysis

As the attempt to eradicate paratuberculosis in one red deer (Cervus elaphus) farm failed, all 167 red deer of different age groups were slaughtered and examined by culture for mycobacteria, and the farm was closed down. Spleen and hepatic lymph nodes, mediastinal lymph node, ileocecal lymph node, and ileum were collected from each animal and examined (a total of 835 organs). Neither tuberculosis lesions nor pathognomic signs of paratuberculosis were detected. Among all microscopically negative for mycobacteria organs, Mycobacterium avium subsp. paratuberculosis alone was isolated from 165 organs, M. a. avium alone from 41 organs, and both pathogens from four organs. M. a. paratuberculosis alone was detected in 71 red deer, M. a. avium alone in 13 red deer and both pathogens in 18 red deer. Using standardised RFLP methods, three IS900 RFLP types B-C1, B-C16, and B-C32 were identified among 40 M. a. paratuberculosis isolates and four IS901 RFLP types N-B1, N-B3, N-B4, and P-B3 among 17 M. a. avium isolates.     

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.     

Mycobacterium avium abortion in a sow.

A 2-year-old sow aborted her entire near-term litter of 11. Gross and histologic examination of a fetus suggested a tuberculous infection, and a yellow-pigmented Mycobacterium avium serotype 1 was subsequently isolated from the fetal tissue. Efforts to rebreed the sow were unsuccessful. She was anergic to skin tests with purified protein derivative of M. avium on two occasions but had M. avium specific in vitro lymphocyte immunostimulation. Gross granulomatous lesions were found in the liver, kidneys, and endometrium when the sow was necropsied 5 months after the abortion. Histologic examination showed diffuse and focal non-encapsulated granulomas in lymph nodes, tonsils, kidney, liver, spleen, lung, and uterine and vaginal walls. There were a few encapsulated calcified foci in the endometrium. The centers of some granulomas in the tonsils, liver, kidneys, and some lymph nodes were caseated. The yellow-pigmented M. avium was isolated from the reproductive organs and from 11 of 12 other tissues cultured.     

Patterns of lesions of bovine tuberculosis in wild red deer and wild boar

The data obtained from a survey of Mycobacterium bovis infection in wild red deer (Cervus elaphus) and wild boar (Sus scrofa) conducted in France in the 2005/06 hunting season were used to describe and quantify the pathological findings in the two species. The red deer had caseous abscessed lesions in their organs and lymph nodes, whereas in the wild boar the lesions were predominantly caseocalcareous and occurred mainly in the lymph nodes. The severity of the gross tuberculosis-like lesions was estimated on the basis of a numerical score. The significant difference between the distribution of the scores in the two species indicated that the disease was more serious in the red deer than in the wild boar. Unlike the red deer, the wild boar did not show a generalised pattern of disease. Among the lymph nodes examined systematically, gross lesions were most frequently observed in the mesenteric lymph nodes in the red deer and in the retropharyngeal lymph nodes in the wild boar. In both species, the presence of gross lesions showed the closest agreement with the isolation of M bovis from the same lymph nodes. The different patterns of the lesions of tuberculosis in the two species suggest that red deer might play an important role in the intraspecies and interspecies dissemination of the infection, whereas in wild boar the spread of the infection would be more likely to be restricted to other wild boar.     

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.     

Farm and slaughter survey of bovine tuberculosis in captive deer in Switzerland

In 1998, a survey was conducted by postal questionnaire to gather basic knowledge about the management, health and productivity of captive deer in Switzerland. In addition, lymph nodes were collected from slaughtered deer from 124 of the 262 holdings surveyed, and tested for Mycobacterium bovis and Mycobacterium tuberculosis. The total farmed deer population was 8389 animals kept on 485 holdings; 87 per cent were fallow deer, 8 per cent red deer, 4 per cent sika deer, and there were small numbers of other species. The median herd sizes were 12 for fallow deer and eight for red deer. Few owners had handling facilities or crushes. In none of the lymph nodes examined were lesions typical of bovine tuberculosis observed, and neither M bovis nor M tuberculosis was cultivated from any of the samples.     

Experimentally induced Mycobacterium avium serotype 8 infection in swine.

Five of 6 swine experimentally inoculated with Mycobacterium avium serotype 8 had microgranulomas in the cervical or mesenteric lymph nodes at necropsy 92 days later. In vivo tuberculin skin reactivity and in vitro lymphocyte immunostimulation responses were evaluated at 10 and 12 weeks after the pigs were inoculated. Positive responses were obtained on both tests in inoculated pigs, whereas test results in noninoculated pigs and pigs given killed bacterial cells were negative. Mycobacterium avium serotype 8 was isolated at necropsy from the cervical or mesenteric lymph nodes of each of the pigs inoculated with viable microorganisms and from the 2 pigs kept in the pen with inoculated swine. Mycobacteria were not isolated from tissues of the noninoculated swine or those given killed cells.     

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.     

Paratuberculosis in red deer (Cervus elaphus): an immunohistochemical study

In the present study, we compared the utility of immunohistochemistry with serological and histological results for the characterization of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) in tissues of affected red deer. Bacterial isolation was considered the standard reference. Samples were taken from seven clinically affected animals with typical macroscopic lesions. The enzyme linked immunosorbent assay (ELISA) and the gel diffusion tests (GD) were used for serological determinations. Samples from intestine and mesenteric lymph nodes were processed for bacterial isolation and histology. M. paratuberculosis was isolated from all the animals. Histologically, lymph nodes displayed necrosis and mineralization at the cortical and medullar areas. Ziehl-Neelsen stained bacteria were numerous inside macrophages and Langhans-type giant cells. Giant and epithelioid cells and lymphocytes were prominent at the ileal mucous membrane. The immunostaining of M. paratuberculosis was very clear inside epithelioid and giant cells. Image analysis was carried out to determine the immunostained area. There was total agreement among the methods employed. Immunohistochemistry can be very useful when the microorganism cannot be recovered from tissues or faeces.     

Experimental infections in young red deer (Cervus elaphus) with a bovine and an ovine strain of Mycobacterium avium subsp paratuberculosis

AIMS: To compare the virulence of a 'bovine' and an 'ovine' strain of Mycobacterium avium subsp paratuberculosis (M. ptb) in red deer (Cervus elaphus) after experimental inoculation orally, and to examine the relationship between the dose of the bovine strain given and immunological, clinical and histopathological outcomes in young red deer. METHODS: Newly-weaned 4-month-old male red deer (n=81) were randomly assigned to one of five groups. Three groups (n=16) received high (10(9) colony forming units (cfu); HB), medium (10(7) cfu; MB) or low (10(3) cfu; LB) oral doses of a bovine strain of M. ptb, one group (n=16) received medium (10(7) cfu; MO) doses of an ovine strain of M. ptb, and a Control group (n=17) was not dosed. The HB and Control groups were grazed together, the MB and LB groups were grazed together, and the MO group was grazed alone, in separate small paddocks on a quarantined area of the farm for 45 weeks. Liveweight, clinical signs and immunoglobulin G1 (IgG1) antibody levels were monitored for up to 45 weeks. Deer affected with Johne's disease were euthanised when they showed obvious clinical signs. Unaffected deer were slaughtered at the end of the trial (Week 45), and all deer were necropsied. Faeces and tissue samples were cultured for M. ptb, and fixed tissues were examined for histopathology. RESULTS: Between 21 and 38 weeks post-challenge (pc), 5/16 animals in the HB group developed early signs of Johne's disease and were euthanised. The remaining deer in the five groups were all apparently healthy and reached good liveweights (approximately 100 kg average), and were euthanised and examined 45 weeks pc. Three deer (two HB and one MB) had small caseous lesions in their jejunal lymph nodes (JJLNs) and one HB animal had a small caseous lesion in a retropharyngeal lymph node. The remaining animals had no grossly-visible lesions. Mycobacterium avium subsp paratuberculosis was cultured from samples from 100% of the HB and MB animals, 50% of the LB group, 69% of the MO group and all Control animals. Thus all Control deer were infected by natural transmission from the HB group but none developed signs of clinical disease. Examination of histological sections of jejunum, ileocaecal valve (ICV) and associated lymph nodes showed a gradation of severity of lesions that was positively correlated (p<0.001) with dose of the bovine strain administered; mean lesion severity scores were 4.8, 2.9 and 0.9 for HB, MB and LB groups, and 2.2 and 0.9 for the Control and MO groups, respectively. IgG1 antibody levels at the time of euthanasia were also correlated with lesion severity scores at slaughter (p<0.001). CONCLUSIONS: The ovine strain of M. ptb used in this study was less virulent for red deer than the bovine strain. The correlation between dose of the bovine strain and the severity of lesions suggests that clinical Johne's disease in yearling red deer likely results from high oral challenge with a bovine strain whilst they are young. The minimum oral infective dose may be close to 10(3) cfu for this bovine strain.     

Pathogenesis of systemic Mycobacterium avium infection in pigs through histological analysis of hepatic lesions

Mycobacterium avium causes systemic infections through primary intestinal lesions in pigs. However, its pathogenesis is not well understood. The aim of this study was to confirm the effects on swine after enteral infection. One hundred and twelve pigs with hepatic lesions infected with M. avium were used in this study. We investigated the involvement of other organs and the distribution of hepatic lesions in the lobular structure. Most lesions involved the mesenteric lymph nodes. Hepatic lymph nodes were the secondary nodes involved. In 74 cases (66.1%), the hepatic lesions were predominantly distributed in the portal tract of the affected livers. The other 38 cases (33.9%) showed granulomatous lesions in the hepatic lobule. Many cases showed interface hepatitis. There was a significant relationship between focal lesions within hepatic lobule and splenic lesions. These findings suggest that granulomatous lesions formed in hepatic lobules upon establishment of bacteremia in pigs systemically infected with M. avium.     

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.     

Paratuberculosis in Red Deer (Cervus elaphus hippelaphus) in the Western Alps

During the hunting seasons 1995–96 to 1997–98, 19 red deer from the Upper Susa Valley (Cottian Alps) were examined for paratuberculosis (Johne's disease). Specific DNA amplification on mesenteric lymph nodes detected Mycobacterium avium paratuberculosis in 17 animals. Ten of these red deer were tested for serum antibodies by the AGID and ELISA tests, nine being negative. Three isolates from infected deer were genetically characterized by an arbitrarily primed polymerase chain reaction, and showed similar genetic polymorphism to that of bovine strains isolated in different Italian areas. The study showed that paratuberculosis is present in red deer of the Upper Susa Valley and that serological tests are not an efficient means for monitoring this infection.     

Isolation and characterisation of Mycobacterium avium and Rhodococcus equi from granulomatous lesions of swine lymph nodes in Slovenia

Granulomatous lesions in bovine and especially swine lymph nodes are still frequently observed during routine veterinary meat inspections even though Mycobacterium bovis infections are no longer detected in domestic animals in Slovenia. Different lymph nodes of pigs (n = 260) were investigated using classical bacteriological and molecular methods. Mycobacterium avium alone was isolated in 47.3% of pigs and in mixed infection with Rhodococcus equi in 3.9% of pigs. R. equi alone was isolated in 27.3% and in mixed infection with mycobacteria other than M. avium in 1.5% of pigs. A total of 133 M. avium isolates were typed using the IS1245, IS901 and FR300 PCR. Almost two thirds (60.9%) of isolates belonged to M. avium hominissuis (IS901-, IS1245+ genotype), 33.8% of isolates belonged to M. avium avium (IS901+, IS1245+ genotype) and 5.3% of isolates remained non-typed. Fifty out of 85 R. equi isolates were tested for the virulence-associated antigens (VapA and VapB). Nearly two thirds (60.0%) were positive for VapB while all the other isolates were VapA- and VapB-negative.     

Swine tuberculosis in South Dakota.

Mycobacteria were isolated from 195 of 200 lesions in lymph nodes identified as granulomatous by meat inspectors at 4 abattoirs in South Dakota. Mycobacterium avium serotypes 1 and 2 accounted for 89% of the isolants. Mycobacteria were isolated more frequently from lesions than acid-fast bacilli were observed on microscopic examination (P less than 0.001). The frequency with which mycobacteria was isolated was similar to the occurrence of granulomatous lesions. The numbers of the various kinds of mycobacteria isolated at each of the 4 abattoirs and for the 3 meat inspection disposition classes were not significantly different.     

Fatal mycobacteriosis with hepatosplenomegaly in a young dog due to Mycobacterium avium.

Cases of disseminated Mycobacterium avium infections in dogs are rare because it appears that the species is innately resistant to infection. A 2-year-old, castrated, 5 kg Shih Tzu-Poodle-cross developed anemia, abdominal pain, lethargy, and splenomegaly. Histological examination of surgically removed spleen indicated marked granulomatous splenitis with myriad intracytoplasmic acid-fast bacterial rods. Ultrastructural examination revealed the presence of 3-4-microm-long mycobacteria in phagolysosomes of epithelioid macrophages. Tissue extract of lightly fixed spleen was positive for M. avium 16S ribosomal RNA and negative for M. tuberculosis complex IS6110 DNA by polymerase chain reaction testing. Anemia was associated with the presence of mycobacteria-infected macrophages in bone marrow. The animal's condition deteriorated, and euthanasia was performed after a clinical course of 2 months. The principal morphological findings at necropsy were severe diffuse granulomatous hepatitis, enteric lymphadenomegaly, and segmental granulomatous enteritis with intralesional mycobacteria present. Mycobacterium avium was cultured from enteric lymph nodes sampled at necropsy. The source of infection was not established but was presumed to be environmental with an enteric portal of entry.     

Experimental deer-to-deer transmission of Mycobacterium bovis

OBJECTIVE: To determine whether Mycobacterium bovis can be transmitted from experimentally infected deer to uninfected in-contact deer. ANIMALS: Twenty-three 6-month-old white-tailed deer. PROCEDURE: On day 0, M bovis (2 X 10(8) colony-forming units) was administered by intratonsillar instillation to 8 deer; 3 control deer received saline (0.9% NaCl) solution. Eight in-contact deer were comingled with inoculated deer from day 21. On day 120, inoculated deer were euthanatized and necropsied. On day 180, 4 in-contact deer were euthanatized, and 4 new in-contact deer were introduced. On day 360, all in-contact deer were euthanatized. Rectal, oral, and nasal swab specimens and samples of hay, pelleted feed, water, and feces were collected for bacteriologic culture. Tissue specimens were also collected at necropsy for bacteriologic culture and histologic analysis. RESULTS: On day 90, inoculated and in-contact deer developed delayed-type hypersensitivity (DTH) reactions to purified protein derivative of M bovis. Similarly, new in-contact deer developed DTH reactions by 100 days of contact with original in-contact deer. Tuberculous lesions in in-contact deer were most commonly detected in lungs and tracheobronchial and medial retropharyngeal lymph nodes. Mycobacterium bovis was isolated from nasal secretions and saliva from inoculated and in-contact deer, urine and feces from in-contact deer, and hay and pelleted feed. CONCLUSIONS AND CLINICAL RELEVANCE: Mycobacterium bovis is efficiently transmitted from experimentally infected deer to uninfected in-contact deer through nasal secretions, saliva, or contaminated feed. Wildlife management practices that result in unnatural gatherings of deer may enhance both direct and indirect transmission of M bovis.