Lesion development in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis

The recent discovery of tuberculosis in free-living white-tailed deer in northeastern Michigan underscores the need for increased understanding of the pathogenesis of tuberculosis in wildlife species. To investigate lesion development in white-tailed deer, 32 deer were experimentally infected by intratonsilar instillation of 300 colony-forming units of Mycobacterium bovis. Three deer each were euthanatized and examined at days 15, 28, 42, and 56 after inoculation, and five deer each were euthanatized and examined at days 89, 180, 262, and 328 after inoculation. Microscopic lesions first were seen in the medial retropharyngeal lymph node and lung 28 and 42 days after inoculation, respectively. Lung lesions were present in 12 (38%) of 32 deer, involving 23 lung lobes. Left caudal and right middle and caudal lobes were involved in 17 (74%) of the 23 affected lung lobes. Lesions in the medial retropharyngeal lymph node first appeared as granulomas composed of aggregates of macrophages and Langhans-type giant cells. Some early granulomas contained centrally located neutrophils. As granulomas developed, neutrophils were replaced with a central zone of caseous necrosis that first showed signs of mineralization 42 days after inoculation. Granulomas increased in size as the zone of caseous necrosis expanded. Peripheral fibrosis, first seen at 56 days after inoculation, progressed to only a thin fibrous capsule by 328 days after inoculation. By the termination of the study, the central necrotic core of the granuloma contained abundant liquefied necrotic material and grossly resembled an abscess. Although tuberculous lesions in white-tailed deer follow a developmental pattern similar to that in cattle, fibrosis is less pronounced and the advanced lesions may liquefy, a change seldom reported in cattle. An understanding of lesion development will aid in the identification of the spectrum of disease that may be seen in this important wildlife reservoir of tuberculosis.     

Lesion development and immunohistochemical changes in granulomas from cattle experimentally infected with Mycobacterium bovis

Mycobacterium bovis, the causative agent of bovine tuberculosis, persists within granulomas. Formation of granulomas involves a complex array of immune activation and cellular migration. To examine temporal changes in granuloma development, we inoculated 32 cattle with M. bovis of deer origin. Tissues from 4 calves each were examined at 15, 28, 42, 60, 90, 180, 270, and 370 days after inoculation. Granulomas in the medial retropharyngeal lymph node were staged (I-IV) on the basis of cellular composition and the presence or absence of necrosis and peripheral fibrosis. Immunohistochemistry for inducible nitric oxide synthase (iNOS), CD68, CD4, CD8, and gamma/delta T cells was performed. Fifteen days after inoculation only stage I granulomas were seen, while between 28 and 60 days, there was a steady progression through granuloma stages such that by day 60, granulomas of all 4 stages were seen. Acid-fast bacilli were present in moderate-to-large numbers in stage I granulomas 15-60 days after inoculation. Stage IV granulomas contained large numbers of acid-fast bacteria. Abundant iNOS immunoreactivity was associated with granulomas from day 15 through day 60 but was minimal from day 90 to the termination of the experiment. The relative number of CD4+ and CD68+ cells remained constant throughout the study. In contrast, at time points >60 days, numbers of CD8+ and gamma/delta T cells diminished. Tuberculous granulomas are dynamic lesions that follow an orderly progression through disease stages. Diminished expression of iNOS and reduced numbers of CD8+ and gamma/delta T cells late in the progression of tuberculous granulomas may represent a failure of the host response to control 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.     

Immunohistochemical markers augment evaluation of vaccine efficacy and disease severity in bacillus Calmette-Guerin (BCG) vaccinated cattle challenged with Mycobacterium bovis

Development of necrotic granulomas in response to Mycobacterium bovis infection in cattle is pathognomonic for bovine tuberculosis. Previously our laboratory reported on M. bovis granuloma classification by stage of lesion advancement within bovine lymph nodes and developed immunohistochemical markers to further characterize these granulomas. In this study of bovine lymph node granulomas we applied this classification system to assess the dynamics of vaccination challenge. Lymph nodes collected from cattle vaccinated with M. bovis bacillus Calmette-Guerin (BCG) and subsequently challenged with virulent M. bovis were compared to lymph nodes from unvaccinated, challenged cattle. Expression of interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), type I procollagen and cell marker identification of T cells, B cells, macrophages and WC1(+)gammadelta TCR+ cells were assessed. Granulomas formed in vaccinated cattle were greatly reduced in number, area, degree of necrosis and peripheral fibrosis and contained fewer Langhans' giant cells, acid fast bacilli, WC1(+)gammadelta TCR+ cells and less TGF-beta expression in comparison to controls. B cells clustered intensely along the outer granuloma margins within vaccinated calves, with significantly more IFN-gamma producing cells identified in the medullary regions of lymph nodes from BCG-vaccinated animals compared to unvaccinated controls. This may be indicative of immune activation and surveillance in regions not directly associated with ongoing disease. Lymph node evaluation using light microscopy and immunohistochemical markers is useful to assess the immune response and discriminate granulomas to determine vaccine efficacy and disease severity.     

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.     

Epidemiology of Mycobacterium bovis infections of pigs and wild boars using a molecular approach

A molecular epidemiological approach was applied to establishing a possible role for the wild boar as a natural reservoir of Mycobacterium bovis in Sierra de Villuercas, Western Spain; an area free of farmed cattle and wild deer populations. Spoligo and VNTR typing were used over a three year period to study the epidemiological relationship between the occurrence of bovine tuberculosis (TB) in extensively bred Iberian pigs and indigenous wild boar. The 37 sampled wild boar showed different degree of calcified granulomatous lesions in retropharyngeal, mediastinal and pulmonary lymph nodes. The 25 sampled Iberian pigs showed calcified lesions, mainly in the respiratory tract. Lesions located in the mesenteric lymph nodes appeared secondarily. M. bovis was isolated from all affected animals. Twenty-five and 37 isolates of M. bovis were obtained from domestic pigs and wild boar, respectively. Our findings provide evidence that supports the possibility of cross infection between wild boar and domestic pig populations. This is contrary to the generally held belief that swine represent an epidemiological dead end host and play no role in the epidemiology of M. bovis.     

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.     

The pathogenesis of experimental endo-bronchial Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula)

AIM: To study the nature and development of experimentally induced respiratory tuberculosis in possums and compare the lesions observed with those seen in the natural disease. METHODS: Thirty-three adult possums were inoculated via the endo-bronchial route with 20-100 colony forming units of Mycobacterium bovis. The possums were killed at 1,2,3 and 4 weeks after inoculation and the nature and distribution of the lesions studied in detail histopathologically. Alveolar macrophages recovered from the infected possums were also studied ultrastructurally. RESULTS: Macroscopic lesions were largely confined to the respiratory tract, increasing in size and number with time. Histology greatly increased the detection of the total number of lesions. The most common sites affected outside the respiratory tract were the liver and hepatic lymph nodes, but lesions were less common in peripheral lymph nodes than is observed in the natural disease. Intra-pulmonary lesions were centred on blood vessels and their associated lymphatics. Peripheral blood lymphocyte blastogenic responses to M. bovis antigens were first detected at 3 weeks after inoculation, which was 1 week after lymphocyte infiltrations were detected in the lungs, but 1 week before the majority of infections became generalised. CONCLUSIONS: Differences in the nature of pulmonary lesions and the distribution of lesions were observed between experimentally induced and the natural disease. Rapid haematogenous and lymphatic spread occurs early in the experimentally induced disease.     

Pathology of naturally occurring bovine tuberculosis in England and Wales

The aim of this study was to obtain a contemporary data set of pathology in tuberculin reactor and in-contact cattle in England and Wales. Four hundred animals (200 reactors and 200 in-contacts) from 242 farms located in 14 counties in Western England and Wales were examined. The mean number of lymph nodes (LNs) with tuberculosis (TB)-like lesions per TB-confirmed animal was 1.7 in reactors and 1.5 in in-contact animals. Tuberculous lesions in both reactor and in-contact animals were most commonly observed in the LNs of the thorax, followed by the head and abdomen, particularly the mediastinal, retropharyngeal and tracheobronchial LNs. Twenty-five reactors had macroscopic lesions in the palatine tonsils. Among TB-confirmed cattle, 27% of reactors and 9% of in-contact animals had gross TB-like lesions in the lungs, particularly in the caudal lobes. Gross lesions that were not TB-confirmed were parasitic granulomas (45%), bacterial or mycotic club-forming pyogranulomas (27%) and bacterial abscesses (23%). Diagnostic sensitivity was maximised when bacteriology and histopathology were used concurrently. Stage IV granulomas, alone or in combination with other stages, constituted 63% of lesions, while 16% of lesions were stage I/II granulomas. Caseous necrosis and calcification were common features of the granulomas encountered in natural Mycobacterium bovis infections, even with pathology limited to a small number of sites. Granulomas often covered large areas of histological sections and typically contained only small numbers of acid fast bacilli.     

Experimentally induced infection of reindeer (Rangifer tarandus) with Mycobacterium bovis.

In the USA, all species of Cervidae are included in the USDA's uniform methods and rules for the eradication of bovine tuberculosis and, therefore, are subject to regulations regarding intradermal tuberculin testing. In reindeer (Rangifer tarandus), infection with Mycobacterium bovis is exceedingly rare and the response of reindeer to infection with M. bovis in pathologic and immunologic terms is unknown. The objectives of the study reported here were to describe the pathologic changes associated with M. bovis infection in reindeer and evaluate the effectiveness of intradermal tuberculin testing as a means of diagnosis of tuberculosis in reindeer. Thirteen reindeer were inoculated intratonsilarly with 10(5) colony-forming units (CFU) of M. bovis, and 4 noninoculated reindeer served as negative controls. The comparative cervical test (CCT) was done on all reindeer 90 and 240 days after inoculation. Thirteen months after inoculation, all reindeer were euthanized and examined. All experimentally inoculated reindeer developed lesions in the medial retropharyngeal lymph nodes. The CCT accurately identified all M. bovis-inoculated reindeer, but false-positive results were common among negative-control reindeer. Modifications of the method for interpretation of the CCT decreased false-positive results without increasing false-negative results. Reindeer are susceptible to infection with M. bovis; however, lesions are fewer in number, less severe in nature, and less widely disseminated than are those seen in white-tailed deer (Odocoileus virginianus). Comparative cervical skin testing of reindeer can be highly sensitive, but has low specificity. Specificity can be improved by modification of criteria for interpretation of the CCT.     

Virulence-associated protein characterisation of Rhodococcus equi isolated from bovine lymph nodes

Rhodococcus equi has a low pathogenicity in cattle, but it occasionally causes lymph node granulomas, which are detected at abattoir post mortem inspection, and must be distinguished from tuberculous granulomas. Lymph node lesions were detected in 6719 cattle, from a total of 3,263,622 cattle examined post mortem in abattoirs, in the Republic of Ireland, during 1997 and 1998. Histological examination was performed on all lesions, principally for the purpose of identifying animals with tuberculosis. A total of 1122 of the lesions were cultured on blood agar and on Stonebrinks and Lowenstein-Jensen medium containing pyruvate, because the histological findings were difficult to interpret or were suggestive of R. equi infection. R. equi was isolated from 264 lesions. Almost all of the R. equi granulomas were confined to a single lymph node, and were present predominantly in the retropharyngeal, bronchial and mediastinal lymph nodes. R. equi granulomas were present in a significantly higher proportion of the lesions detected in steers and heifers compared to cows. The prevalence in the total population of 3.3 million cattle examined post mortem was 0.008%. The 15-17kDa antigens, associated with virulence in this organism, and the 20kDa antigen, associated with intermediate virulence, were not detected in isolates from 146 cattle, analysed by immunoblot assays. A PCR assay to detect the plasmid gene encoding the 15-17kDa antigens was also negative for isolates from these 146 animals. Plasmids were not detected in 30 isolates which were examined.     

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.     

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)     

Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection. METHODS: Possums were inoculated with 5 x 106 colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7),orally (n=10), via the endobronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 106 cfu and half of which received 5 x 107 cfu of BCG). The possums were humanely killed between 1-4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically. RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endobronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i. CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intradermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.     

Cytopathic effects of Moraxella bovis on cultured bovine neutrophils and corneal epithelial cells

The effects of Moraxella bovis on the morphologic features of purified bovine neutrophils and bovine corneal epithelial cells were examined, using transmission and scanning electron microscopy and light microscopy. Within 2 minutes after incubation of bovine neutrophils with living M bovis, electron microscopic cellular changes included vacuolation, swelling, and loss of microplicae. Most of the neutrophils were lysed by 10 minutes of incubation. Human neutrophils phagocytosed the M bovis and remained intact, even after 30 minutes of incubation with the bacteria. Living M bovis killed bovine corneal epithelial cells in vitro. Sterile filtrates prepared from 6-hour shaker cultures of M bovis also killed bovine corneal epithelial cells, but the cytotoxic activity was less than that produced by the living bacteria. Cellular changes were first observed in specimens collected 1 hour after corneal cell monolayers were inoculated with sterile culture filtrates. The changes in these cells included pit-like lesions on the cellular surface, cellular separation, and vacuolation.     

Mycobacterium avium infection in a farmed deer herd

Tuberculous lesions were identified over a 2-year period in 36 clinically normal red deer from a single herd. The lesions were only present in the retropharyngeal lymph nodes and lymph nodes draining the intestinal tract, indicating infection by the oral route. Mycobacterium avium was isolated from 27 of 29 lesions examined by bacterial culture. Grossly and histologically, the lesions were indistinguishable from those caused by Mycobacterium bovis. DNA restriction endonuclease analysis revealed that all the 26 M. avium isolates available for examination had identical cleavage patterns. These patterns were identical to a New Zealand M. avium serotype 2 isolate from a pig and were very similar to a reference strain of M. avium serotype 2. The DNA examinations indicated that the deer were infected from a common source that was not identified.     

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.     

Mycobacterium bovis-infected white-tailed deer (Odocoileus virginianus): detection of immunoglobulin specific to crude mycobacterial antigens by ELISA.

White-tailed deer (Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis infection for cattle within North America. The objective of this study was to evaluate the antibody response of M. bovis-infected deer to crude mycobacterial antigens. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis-infected (i.e., in contact) deer. To determine the time course of the response, including the effects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. The reactivity to M. bovis purified protein derivative (PPDb) exceeded (P < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of the infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis strain 1315 exceeded (P < 0.05) the mean of the prechallenge responses to this antigen at approximately 1 month after inoculation and throughout the remainder of the study (i.e., approximately 11 months). This response also exceeded (P < 0.05) that of the uninfected deer. Although this is encouraging, further studies are necessary to validate the use of the proteinase K-digested M. bovis antigens in the antibody-based assays of tuberculosis.     

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.     

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.