Histological and immunohistochemical characterisation of Mycobacterium bovis induced granulomas in naturally infected Fallow deer (Dama dama)

Veterinary Immunology and Immunopathology 149 (2012) 66–75

Contents lists available at SciVerse ScienceDirect

Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm

Research paper

Histological and immunohistochemical characterisation of Mycobacterium bovis induced granulomas in naturally infected Fallow deer (Dama dama) W.L. García-Jiménez a,f,∗ , P. Fernández-Llario a , L. Gómez b , J.M. Benítez-Medina a , A. García-Sánchez c , R. Martínez a , D. Risco a , J. Gough f , A. Ortiz-Peláez d , N.H. Smith e , J. Hermoso de Mendoza a , F.J. Salguero f a

Red de Grupos de Investigación Recursos Faunísticos, Facultad de Veterinaria, Universidad de Extremadura, 10003 Cáceres, Spain Unidad de Anatomía Patológica, Departamento de Medicina Animal, Facultad de Veterinaria, Universidad de Extremadura, 10003 Cáceres, Spain c Producción Animal. Centro de Investigación “Finca La Orden Valdesequera”, 06187 Badajoz, Spain d Centre for Epidemiology and Risk Analysis, Animal Health and Veterinary Laboratories Agency, AHVLA-Weybridge, KT15 3NB, Addlestone, Surrey, United Kingdom e Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, AHVLA-Weybridge, KT15 3NB, Addlestone, Surrey, United Kingdom f Pathology and Host Susceptibility Department Animal Health and Veterinary Laboratories Agency, AHVLA-Weybridge, KT15 3NB, Addlestone, Surrey, United Kingdom b

a r t i c l e

i n f o

Article history: Received 4 November 2011 Received in revised form 21 May 2012 Accepted 6 June 2012 Keywords: Fallow deer (Dama dama) Tuberculosis Mycobacterium bovis immunohistochemistry Granuloma

a b s t r a c t Mycobacterium bovis infections in fallow deer have been reported in different countries and play an important role in the epidemiology of bovine tuberculosis (bTB), together with other deer species. There is little knowledge of the pathogenesis of bTB in fallow deer. The aim of this study was to perform a histopathological characterisation of the granulomas induced by M. bovis in this species and the immunohistochemical distribution of different cell subsets (CD3+, CD79+, macrophages) and chemical mediators (iNOS, TNF-␣, IFN-␥) in the different developmental stages of granulomas. Stage I/II granulomas showed a marked presence of macrophages (MAC387+) expressing high iNOS levels while stage III/IV granulomas showed a decrease in the number of these cells forming a rim surrounding the necrotic foci. This was correlated with the presence of IFN-␥ expressing cell counts, much higher in stage I/II than in stage III/IV. The number of B cells increased alongside the developmental stage of the granuloma, and interestingly the expression of TNF-␣ was very low in all the stages. This characterisation of the lesions and the local immune response may be helpful as basic knowledge in the attempts to increase the vaccine efficacy as well as for disease severity evaluation and for the development of improved diagnostic tools. Immunohistochemical methods using several commercial antibodies in fallow deer tissues are described. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author at: Red de Grupos de Investigación Recursos Faunísticos, Facultad de Veterinaria, Universidad de Extremadura, 10003 Cáceres, Spain. Tel.: +34 927 257114; fax: +34 927 257110. E-mail address: [email protected] (W.L. García-Jiménez). 0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2012.06.010

Tuberculosis remains a significant disease of animals and humans worldwide. Bovine tuberculosis is caused by Mycobacterium bovis with an extremely wide host range and has a serious, although to date probably underdiagnosed, zoonotic potential (Humblet et al., 2009; Good and Duignan, 2011).

W.L. García-Jiménez et al. / Veterinary Immunology and Immunopathology 149 (2012) 66–75

The potential role of wildlife in the maintenance and spread of M. bovis infection in domestic livestock has been widely reported in various countries, e.g. possums (Trichosurus vulpecula) in New Zealand (Porphyre et al., 2008), Eurasian badgers (Meles meles) in British Isles (Corner, 2006), European wild boar (Sus scrofa) in Spain (Hermoso de Mendoza et al., 2006; Martín-Hernando et al., 2007; Naranjo et al., 2008) and different species of cervids, like red deer (Cervus elaphus) in New Zealand and in Britain (Delahay et al., 2001; Mackintosh et al., 2004; Delahay et al., 2007) or white-tailed deer (Odocoileus virginianus) in Michigan, USA (O’Brien et al., 2001). M. bovis has also been reported to infect fallow deer in the wild (Wilson and Harrington, 1976; Robinson et al., 1989; Rhyan and Saari, 1995; Wahlström et al., 1998; Delahay et al., 2002, 2007; Johnson et al., 2008). Cervids seem to be highly susceptible to bTB (Jaroso et al., 2010) and its role as a wild reservoir might be very important due to the lesion patterns that can facilitate the spread of infection within the population (Zanella et al., 2008). Results from a survey of 4715 wild mammals from South-West England concluded that deer species should be considered as potential, although probably localized, sources of M. bovis infection for cattle (Delahay et al., 2007). There are some circumstances in areas of Central, Southern, and Western Spain that may favour infection transmission between species (Aranaz et al., 2004; Hermoso de Mendoza et al., 2006). There is a wide variety of wild animal species, and many of them are susceptible to infection by M. bovis. On the other hand, game is essential for the sustainability of agriculture in these regions of Spain, which in some areas has led to overgrown populations, due to the lack of natural predators and specific farming practices (extensive management) that allows grazing cattle to overlap wildlife habitats (Aranaz et al., 2004). In these areas the prevalence of cattle herds with positive intradermal tuberculin (IDTB) test in 2009 was between 3.78% and 10.27%, much higher than in other areas where the potential wild reservoir is absent (0.00–0.91%), like the islands and the north of Spain (MARM, 2011). In 2009, the herd prevalence (IDTB) in the study region (Central Spain) was 5.54% (MARM, 2011). There is little knowledge about the pathogenesis of M. bovis infection in fallow deer. Most of the available literature reports only focus on the histological characterisation of the induced lesions (Rhyan and Saari, 1995; Johnson et al., 2008; Martín-Hernando et al., 2010). However, other authors have carried out more extensive analyses of the development of lesions in different livestock species (Liébana et al., 1999; Wangoo et al., 2005; Johnson et al., 2006; Liébana et al., 2007; Palmer et al., 2007). The indepth knowledge of the granuloma in cattle has proven to be very helpful to get further insight into the pathogenesis of the disease, to improve the diagnostic tools and to augment the efficacy of vaccines (Johnson et al., 2006). In line with this, vaccines for wildlife are being developed to control bTB in specific areas (Lesellier et al., 2011) and deer species might be in a similar situation in the near future. The main aim of this study was to characterise the granulomas of naturally infected fallow deer in Central

67

Spain by means of histological and immunohistochemical analyses. 2. Materials and methods 2.1. Animals A total of 112 wild fallow deer (32 male and 80 female, mixed ages) from a large estate close to Madrid, Spain, were investigated for bTB lesions between October and November 2010 during two different hunting game seasons. A high prevalence of M. bovis had been previously described in the wildlife of the study area (Aranaz et al., 2004; Jaroso et al., 2010). Post-mortem examination was performed in the field, with detailed macroscopic inspection of lymph nodes and thoracic and abdominal organs. This examination routinely included retropharyngeal and submandibular lymph nodes in the head and neck, tracheobronchial and mediastinal lymph nodes and lungs in the thorax, and hepatic and mesenteric lymph nodes, liver and spleen in the abdomen. Gross lesions in other locations were also recorded and taken to the laboratory for detailed examination. 2.2. Bacterial culture and spoligotyping Tissue samples were sectioned and dissected by trimming fat and connective tissue, using sterile scissors and forceps for each individual sample. Two grams of each sample were homogenized in 10 ml of sterile water with 0.2% bovine serum albumin (Sigma–Aldrich, USA) for 4 min in a mechanic homogenizer (Smasher, AES Laboratories). The homogenized material was decontaminated with hexadecyl piridinium chloride as previously described (Corner and Trajstman, 1988). Finally, two Lowenstein–Jensen slants (Pronidasa, Madrid, Spain), with piruvate and without glycerol were inoculated in parallel and incubated for six to eight weeks. For each isolate a loopful of growth from the surface of a media slope was suspended in 150 ␮l of distilled water and heated at 99 ◦ C for 10 min. The suspension was centrifuged at 10,000 rpm for 5 min and cooled down to room temperature. For further examination the supernatant was used. Identification of isolates belonging to the M. tuberculosis complex was carried out by PCR following standard methods, amplification of Mycobacterium genus-specific 16S rRNA fragment and MPB20 sequences (Cousins et al., 1991; Liébana et al., 1996). The spoligotyping technique was applied according to previous studies (Kamerbeek et al., 1997). DNA from M. bovis BCG Pasteur II and Mycobacterium tuberculosis H37Rv were used as reference isolates and positive controls in this study; and water as a negative control. Spoligotype patterns were assigned international names by www.Mbovis.org (Smith and Upton, 2012). 2.3. Histopathology Tissue samples from thirteen fallow deer lungs with TB-like lesions were fixed in two different fixatives:

68

W.L. García-Jiménez et al. / Veterinary Immunology and Immunopathology 149 (2012) 66–75

neutral-buffered formalin (4% formaldehyde) for 7 days and Zinc salt based fixatives (ZSF) prepared according to Hicks et al. (2006). For ZSF fixation, samples were cut into 5–6 mm blocks and fixed in freshly prepared ZSF for 8 h at room temperature. Tissues then were trimmed to a 2–3 mm thickness, transferred to fresh ZSF and fixed for an additional 72 h before embedded in paraffin wax. Sections (4 ␮m) were cut and stained with hematoxylin and eosin (H&E) for histopathological examination and by the Ziehl–Neelsen (ZN) method to detect acid-fast bacilli (AFB). Similar sections were placed on Vectabondtreated slides (Vector Laboratories, Peterborough, UK) for immunohistochemistry. In H&E sections, four stages of granulomas were identified and termed as previously described in cattle samples (Wangoo et al., 2005; Johnson et al., 2006): Stage I (initial): Irregular unencapsulated clusters of epithelioid macrophages, with interspersed lymphocytes and few admixed neutrophils. Langhan’s multinucleated giant cells may be present, but necrosis is not present. Stage II (solid): Granulomas composed primarily of epithelioid macrophages and enclosed partly or completely by a thin capsule. Infiltration of lymphocytes, neutrophils and often Langhan’s multinucleated giant cells with minimal necrotic areas are sometimes present, generally composed of necrotic inflammatory cells. Stage III (minimal Necrosis): The granuloma is fully encapsulated, with central necrotic areas, which is caseous and minimally mineralized. Epithelioid macrophages admixed with Langhan’s multinucleated giant cells surrounded the necrotic areas. A peripheral zone of macrophages mixed with clusters of lymphocytes and scattered neutrophils extended to the fibrous capsule. Stage IV (necrosis and mineralization): Thickly encapsulated, large, irregular, multicentric granulomas with prominent caseous necrosis and extensive islands of mineralization comprising the greatest area of the lesion. Epithelioid macrophages and multinucleated giant cells surrounded the necrosis, with particularly dense clusters of lymphocytes near the peripheral fibrotic capsule. Acid-fast bacilli were quantified in the ZN stained slides by counting the total number of bacteria present in each granuloma at high power filed (HPF). Numbers of bacilli present were recorded on a scale of 0–3 (0 = no bacilli; 1 = 1–10; 2 = 11–50 and 3 ≥ 50 bacilli). Multinucleated giant cells identified in H&E stained sections were counted in a similar fashion and represent a total number for the entire granuloma. They were scored on a scale of 0–2 (0 = no multinucleated giant cells, 1 = 1–10 and 2 ≥ 10 cells).

sections in citric acid buffer (2.1 g citric acid (Fisher Scientific, Loughborough, Leicestershire, UK) in 100 ml distilled water), pH 6.0 for 18 min at 100 ◦ C, 90% effect (780 W). The sections were then mounted in a Sequenza Immunostaining Centre (Shandon Scientific, Runcorn, UK) and rinsed with Tris buffered saline (TBS) pH 7.6, 0.005 M (Sigma–Aldrich, USA). Primary antibody cross-reactivity with tissue constituents was prevented using 1.5% normal serum block which matched the host species in which the link antibody was applied to the sections for 20 min. Details of primary antibodies used, specificity, concentration and incubation time are summarized in Table 1. All primary antibodies had been previously screened to determine the optimum dilution and incubation temperature. The sections were washed in TBS and then incubated for 30 min with the appropriate biotinylated secondary link antibody (Vector Laboratories) before being washed twice in TBS again. The sections were incubated for 30 min at room temperature with Avidin Biotin complex (Vector Elite Kit, Vector Laboratories) and the signal was detected using 3,30-diaminobenzidine tetrahydrochloride (DAB) finally the sections were lightly counterstained with Mayer’s hematoxylin (Surgipath, Peterborough, UK) for 5 min, dehydrated in absolute alcohol and cleared in xylene before being coverslipped. Appropriate controls were included in each immunohistochemical run. These included sequential sections with an isotype control for each primary antibody, and the omission of the primary antibody. All the techniques were performed in a GLP, ISO 9001:2008 and ISO 17025 compliant laboratory facility Most of the immunohistochemical techniques applied in this study, have been successfully used in bovine tissue samples (CD3, CD79a, IFN␥) or in multi-species samples (iNOS, MAC387) previously by our group (GómezVillamandos et al., 2001, 2003; Wangoo et al., 2005; Johnson et al., 2006; Gómez-Laguna et al., 2010; Barranco et al., 2011). The use of a commercial anti-bovine antibody to detect TNF-␣ in formalin fixed-paraffin embedded fallow deer tissues is also described in this article (Table 1). This antibody has also been found to be valuable for bovine tissues using the same protocol than in fallow deer (data not shown). Other anti-bovine antibodies (CD68, CD4, and CD8) that has been used previously with bovine tissues by our group (Johnson et al., 2006; Liébana et al., 2007) did not display any positive reaction in fallow deer. This can be due to differences in the epitope that the antibodies are targeting.

2.4. Immunohistochemistry 2.5. Image analysis The avidin biotin complex (ABC Vector Elite; Vector laboratories) method was used for immunolabelling. The sections were dewaxed, rehydrated and then treated in hydrogen peroxide 3% in methanol for 15 min to eliminate endogenous peroxidase activity. The tissue sections were then pretreated for antigen retrieval by either enzymatic digestion with trypsin/alpha-chymotrypsin (0.5% trypsin and 0.5% alpha-chymotrypsin; (Sigma–Aldrich, Gillingham, Dorset, UK) at 37 ◦ C for 10 min or microwaving the

The immunolabelled sections were examined by light microscopy and by digital image analysis (Lucia, Laboratory Imaging Ltd., Prague, CZ). In each slide multiple granulomas were analysed (40×) to determine the area covered by total cells and immunolabelled cells. The percentage of the granuloma area covered by positive cells was calculated (immunolabelled cells/total cells × 100).

W.L. García-Jiménez et al. / Veterinary Immunology and Immunopathology 149 (2012) 66–75

69

Table 1 Antisera used for immunohistochemistry. Target

Specifity

Source

Epitope demasking

Primary antibody concentration (incubation time)

Human CD3 Human CD79 Human MAC387 Bovine IFN-␥ iNOS Bovine TNF-␣

Pan T cell marker (Rabbit pc) Pan B cell marker (clone HM57) Macrophages/monocytes (clone MAC387) Interferon ␥ (clone 7B6) Inducible nitric oxide synthase (Rabbit pc) Tumour necrosis factor ␣ (clone CC327)

DAKO DAKO Serotec Serotec Chemicon Serotec

Trypsin/␣-chymotrypsin Microwave-mediated AR Trypsin/␣-chymotrypsin Microwave-mediated AR Dako high pH microwave buffer Trypsin/␣-chymotrypsin

1 ␮g/ml (O/N at 4 ◦ C) 2.1 ␮g/ml (1 h at RT) 2 ␮g/ml (1 h at RT) 4 ␮g/ml (1 h at RT) 1 ␮g/ml (1 h at RT) 4 ␮g/ml (1 h at RT)

O/N: over night; RT: room temperature.

2.6. Statistical analysis For the histopathological study a total number of 78 TB granulomas from lungs of 13 fallow deer have been analysed testing the association between the granuloma stage (four categories: stage I, II, III and IV) and the number of acid fast bacterial count measured by two variables: categorical and numeric as described in Section 2.3; and the number of multinucleated giant cells, which was aggregated in three categories as described in Section 2.3. For the immunohistochemical study normality of the outcome variable (cell marker or chemical mediator detected) was assessed using a test for normality based on skewness and transformation of the variable according to the result of the test was conducted. In order to assess the effect of the different immunohistochemical markers, individual linear regression models with robust estimation of the standard errors were fitted for each immunohistochemical marker to test the association between the percentage of positivity and stage of the granuloma accounting for the effect of individual animal. All analyses were conducted using Stata© 10 (StataCorp. 2007. Stata Statistical Software: Release 10. College Station, Texas, USA). 3. Results

histopathological features of the tuberculous lesions in the lungs were similar to those observed for bovine tissues (Wangoo et al., 2005). Four developmental stages were easily identified and often, more than one category was observed in the same slide. Large stage III and IV granulomas were often surrounded by satellite small stage I and II ones (Fig. 1). Table 2 shows descriptive statistics of the acid fast bacterial counts for each category of granuloma stage. Given the similar mean number of bacteria in granulomas of stage I and II, these have been grouped into a single category (I–II). Applying the t test of independent samples assuming unequal variance (Welch correction), the results of the pairwise comparisons of the mean bacterial counts by granuloma stage revealed that the mean bacterial count is not significantly different in granulomas of stages I, II and III (P = 0.15). The mean bacterial count of granulomas of stage IV is higher than those of stage III (P = 0.04) and those of stage I and II (P = 0.03). Looking at the categories of bacterial count (1–2, 3 and 4), the stage of the granuloma is positively associated with the category of bacterial count (Fisher’s exact test: P < 0.05). The number of Langhans’ multinucleated giant cells is not associated with the stage of the granuloma as in the categorical variable defined above (Fisher’s exact test: P = 0.229).

3.1. Gross lesions and histopathology 3.2. Bacterial culture and spoligotyping Lesions compatible with bTB were found in 12/32 (37.5%) males and 14/80 (17.5%) female. The total prevalence of compatible bTB lesions was 26/112 (23.21%). Lesions observed were similar to lesions described by Zanella et al. (2008) in naturally infected red deer in France. However we observed that, the respiratory system was the most frequently affected in fallow deer, with parenchymatous caseous lung lesions varying from 1 to 10 cm in diameter (Fig. 1A). Generalized disease was frequent, with 9 animals (34.6%) with large and fibrously encapsulated granulomas similar to abscesses nodes that varied in size (up to 20 cm) and contained creamy yellowish caseum. Two animals presented caseous lesions from 1 to 5 cm in diameter in the liver. From these 26 fallow deer with lesions compatible with bTB, we selected samples from 13 lungs for the histopathological study and a total of 78 individual granulomas were used to identify the developmental stage, the number of AFBs and Langhan’s multinucleated giant cells. The

All animals with gross pathology (n = 26) were positive for M. bovis culture in this study and strains with two

Table 2 Descriptive statistics of AFBs counts for each category of granuloma stage. Granuloma stage

Number of observations

Mean

STD

Min

Max

Stage I Stage II Stage I–II Stage III Stage IV

38 13 51 19 8

0.07 0.07 0.07 1.9 23

0.27 0.27 0.27 5.4 23.8

0 0 0 0 0

1 1 1 23 51

The mean bacterial count is not significantly different in granulomas of stages I, II and III (P = 0.15). The mean bacterial count of granulomas of stage IV is higher than those of stage III (P = 0.04) and those of stage I and II (P = 0.03) (t test with Welch’s correction). The stage of the granuloma is positively associated with the category of bacterial count (Fisher’s exact test: P < 0.05).

70

W.L. García-Jiménez et al. / Veterinary Immunology and Immunopathology 149 (2012) 66–75

Fig. 1. (A) Caseous granulomas in the lung of a fallow deer. (B) Stage II granuloma containing multiple macrophages, neutrophils and some lymphoctes. H&E 200×. (C) Stage IV granuloma with a caseous necrotic centre with mineralization, surrounded by a rim of epitheliod cells and multinucleated cells and an outer layer of collagen and fibroblasts (from right to left). H&E 100×. (D) Detail of the outer layers of a stage IV granuloma with epithelioid macrophages and giant cells surrounding necrosis and a thick rim collagen encapsulating the granuloma (from left to right). H&E 400×.

different spoligotype patterns were isolated, SB0339: 12/26 (46.2%) and SB1142: 14/26 (53.8%).

3.3. Cell subsets and iNOS positive cell distribution MAC387-positive macrophages were very abundant in stage I and II granulomas while stage III and IV showed less positive cells. The positive cells were diffusely distributed within the granulomas of early stages while they were present in the outer layers in more advanced stage in the development of the granulomas (Fig. 2A). CD3+ cells were abundantly labelled in stage I and II granulomas distributed throughout and surrounding epithelioid macrophages and giant cells (Fig. 2C). In advanced granuloma stages, central caseous necrosis was rimmed predominantly by epithelial macrophages and giant cells, with most of the CD3+ cells interspersed among macrophages but also at the peripheral margin of the granuloma (Fig. 2D) CD79 antibody showed positive B lymphocytes and plasma cells. CD79-positive cells were scattered throughout the clusters of epitheliod macrophages in early stage granulomas. However, in granulomas with advanced necrosis, CD79-positve cells were scattered within peripheral subcapsular margins, but often forming clusters or

bigger nests of cells at the periphery of the granuloma (Fig. 2B). iNOS-positive cells consisted of macrophages and multinucleated giant cells (Fig. 2E and F). Immunoreactvitiy was high in stage I and II granulomas, mainly within epitheloid macrophages in the centre of the granuloma (Fig. 2E). Stage III and IV granulomas showed a rim of iNOS-positive macrophages surrounding the necrotic centres. The presence of MAC387-positive cells and the iNOS expressing cells were following a similar trend.

3.4. Distribution of IFN- and TNF-˛ expressing cells The majority of IFN-␥ expressing cells were lymphocytes but also a small proportion of macrophages, including epithelioid cells and multinucleated giant cells were found positive. The positive reaction was higher in stage I/II granulomas, within scattered lymphocytes and macrophages, occasionally forming clusters of positive cells (Fig. 2G). Stage III/IV granulomas showed positive lymphocytes in the outer internal layers of the granulomas and to a lesser extent in epithelioid macrophages and Langhans’ multinucleated giant cells surrounding the necrotic foci. TNF-␣ was expressed by few macrophages in all the developmental stages of the granuloma (Fig. 2H)

W.L. García-Jiménez et al. / Veterinary Immunology and Immunopathology 149 (2012) 66–75

71

Fig. 2. (A) MAC387+ cells in a stage IV granuloma. Multiple macrophages, epithelioid cells and multinucleated giant stained positive cells. ABC. 200×. (B) CD79+ cells in the outer layers of a stage IV granuloma forming a big nest of B lymphocytes. ABC. 200×. (C) stage II granuloma showing a large amount of CD3+ cells interspersed with macrophages. ABC. 200×. (D) Multiple CD3+ cells in the outer layers of a stage IV granuloma. ABC. 100×. (E) Numerous iNOS+ macrophages in a stage I granuloma. ABC. 200×. (F) iNOS+ macrophages and giant cells surrounding the necrotic centre of a stage III granuloma. ABC. 400×. (G) Abundant IFN-␥ positive cells in a stage II granuloma. ABC. 200×. (H) TNF-␣ expressing cells in the outer layers of a stage IV granuloma. ABC. 100×.

72

W.L. García-Jiménez et al. / Veterinary Immunology and Immunopathology 149 (2012) 66–75

3.5. Association of percentage of positivity of immunohistochemical marker and the stage of the granuloma The percentage of positivity was not normally distributed (test for normality based on skewness: P < 0.05) and a new predictor was created using the square root of the percentage of positivity. The six linear regression models, one per immunohistochemical marker, showed the significant association between the percentage of positivity (normalized) and stage of the granuloma adjusted by the animal effect. CD79 is the only marker in which higher percentage of positivity was significantly associated to granulomas stages III and IV compared to those of the baseline category: stage I, adjusted by animal. There were no significant differences of percentage of positivity between stages II and I in CD79. Stage IV of granulomas of the TNF␣ marker were significantly associated, albeit marginally, with higher percentage of positivity, compared to granuloma stage I. In granulomas of the markers IFN-␥, iNOS and MAC387, the effect was the opposite. Higher granuloma stages (III and IV) were significantly associated with lower percentage of positivity, compared to stage I. For the CD3 marker, the different granuloma stages were not significantly associated with the percentage of positivity. Fig. 3 shows the mean percentage of positivity plus the standard deviation against stage of the granulomas by immunohistochemical marker. 4. Discussion The role of wild cervids as reservoirs of bTB in some regions of Central and South of Spain has been previously described (Aranaz et al., 1996; Gortazar et al., 2003; Hermoso de Mendoza et al., 2006). Management practices aimed to increase the game population and thus enhance the economic returns from hunting are common in these regions and include the building of perimeter fencing around estates, shrub removal and the provision of supplementary feed during periods of food shortage. The effect has been an increase in deer population densities to extremely high levels, up to 25–40 deer/100 ha (Carranza et al., 1990; Martínez et al., 2002). Fallow deer can adapt to a broad range of habitats and maintain high densities even in spatially limited environments (Morse et al., 2009). In Spain, over 10,000 wild deer are captured and translocated yearly (Soriguer et al., 1998). This fact added an additional constraint for the bTB eradication programs in cattle in Spain and as a result, the regulations with regards to the requirements for transporting live animals has been recently updated (MARM, 2009). In this study we have isolated two different strains with spoligotype patterns, SB0339 and SB1142. Strains with similar spoligotype patterns are present in high prevalence in Monte El Pardo Nature Reserve, Madrid (Aranaz et al., 2004). We observed a prevalence of tuberculous visible lesions of 23.21%. Our result is slightly higher than the 18.5% reported in previous studies in other areas of Southern

˜ National Park (Gortazar et al., 2008). Other Spain as Donana authors found a slightly higher prevalence in the same area as our study (27.3%) by using skin test reaction against PPD (Jaroso et al., 2010). Several studies have described non-visible lesion tuberculosis (NVL Tb) in deer species. For example in captive elk, 7% of culture-positive animals showed no macroscopic lesions at inspection in an abattoir (Rohonczy et al., 1996). Higher proportions of NVL Tb in red deer were found in Spain, like the studies conducted in 2006–2007 ˜ in Donana National Park (Spain), an endemic bTB area with a high prevalence of the disease in wild ungulates, where 30% of the infected red deer (Cervus elaphus) and fallow deer (Dama dama) had NVL Tb (C. Gortázar, personal communication). In a closed population with a lower prevalence of bTB, sampled from 2000 to 2007, 50% of all M. bovis-infected red deer had NVL Tb (C. Gortázar, personal communication). A potential explanation for this finding is that visible tuberculous lesions in deer may be missed if the inspection does not include the head, thoracic cavity and abdominal cavity (Vicente et al., 2006; Gavier-Widén et al., 2009). In cases of NVL Tb in red deer, histology may reveal early granulomas or aggregations of macrophages in lymph nodes, mainly in the medial retropharyngeal (M.P. MartínHernando, personal communication). In white-tailed deer, histological lesions in cases with NVL Tb were present only in oropharyngeal tonsils, and ranged from simple necrosis to caseation, suppuration, and formation of granulomas. AFBs were found rarely (O’Brien et al., 2004). Likewise, early microscopic lesions were present in the tonsils of red deer with NVL Tb, and consisted of aggregations of macrophages, (Gavier-Widén et al., 2009), resembling stage I granulomas described by our group. Pathology studies can indicate the likelihood of mycobacterial shedding. For example, disseminated disease with extensive macroscopic lesions, with poor fibrotic containment of the granulomas, abundant AFBs, and ulceration into the lumina of airways are conducive to aerogenous shedding (Gavier-Widén et al., 2001). Hosts of M. bovis that contaminate the environment or transmit infection directly to other animals of the same or other species generally show severe disease (Gavier-Widén et al., 2009). Another study showed that more than 70% of the histologically studied granulomas in deer species contained far greater number of AFBs than the typically reported in cattle (Martín-Hernando et al., 2010). Red and fallow deer also had the largest number of poorly encapsulated granulomas often containing many hundreds of bacilli (De Lisle et al., 2002; Johnson et al., 2008; Martín-Hernando et al., 2010). The presence of poorly encapsulated granulomas has been associated with British deer being a potential source of environmental contamination and onward transmission to other species (Johnson et al., 2008). The use of immunohistochemical markers in M. bovis infection models has proven to be a very useful tool to augment the evaluation of vaccine efficacy and disease severity in cattle (Johnson et al., 2006). In this sense, there are very few examples in the literature of the use of commercial antibodies for immunohistochemistry in fallow deer tissues. As this species is closely related to bovine, many of the

W.L. García-Jiménez et al. / Veterinary Immunology and Immunopathology 149 (2012) 66–75

73

Fig. 3. Histogram representing the percentage of positivity in each granuloma stage for all the immunohistochemical markers studied. Bars represent mean + standard deviation. * P < 0.05.

antibodies used previously for cattle tissues cross-reacted successfully with the fallow deer tissues used in this study. These markers, together with other ones used for multiple species by our group have been useful to develop a panel of antibodies that cross-react with fallow deer tissues (Gómez-Villamandos et al., 2001, 2003; Wangoo et al., 2005; Johnson et al., 2006; Gómez-Laguna et al., 2010; Barranco et al., 2011). The characteristic cellular composition of initial stage I and II granulomas is associated with an initial immune response from the host. These granulomas are primarily composed by macrophages and a large proportion of CD3+ cells are also observed, suggesting an early involvement of T cells in the development of the granuloma. Interestingly, these stage I and II granulomas were expressing more IFN-␥ than stage III and IV (Fig. 3). The early production of IFN-␥ is associated with the early Th1 response by the host and has been correlated with increased pathogenicity in M. bovis infection in cattle (Villarreal-Ramos et al., 2003). Even though the majority of IFN-␥ expressing cells were lymphocytes, some macrophages, epitheliod and giant cells were also found to express this cytokine. The expression of IFN-␥ by macrophages has been described in several bacterial and viral infections (Gessani and Belardelli, 1998; Munder et al., 1998; Acosta-Iborra et al., 2009). The biological relevance of macrophage IFN-␥ is still unclear but it has been related to an autocrine macrophage activation (Munder et al., 1998). The presence of abundant CD79+ cells in the outer layers of the stage III and IV granulomas forming nests of cells suggests that this structure is similar to that of active follicles found in secondary lymphoid organs, with B cells at many different stages of maturation present, that might contribute to the coordination of the host immune response with the CD3+ cells (Ulrichs et al., 2004). Infection with Mycobacterium spp. can stimulate the production of nitric oxide by macrophages, which play an important role in intracellular killing of mycobacteria as it is cytotoxic at high concentrations (Hernández-Pando et al., 2001). Stage I and II showed a large proportion of macrophages expressing iNOS and to a lesser extent in a rim surrounding the necrotic centre in stage III and IV granulomas, as an attempt from the host to control the mycobacterial infection and spread.

The results of this study revealed little differences in TNF-␣ expression among the stages of granuloma development, but it was present in all of them as it is an important cytokine produced mainly by macrophages with a role in the granuloma formation and maintenance (Algood et al., 2003; Boddu-Jasmine et al., 2008). In conclusion, we have found many similarities between granuloma development and immunohistochemical characterisation of the lesions in both cattle (Wangoo et al., 2005; Johnson et al., 2006; Liébana et al., 2008) and deer. The immunohistochemical techniques described here can be used in further studies involving this species. Conflict of interest The authors have not declared any conflict of interest. Acknowledgments This study was supported by Ministerio de Ciencia e ˜ Innovación (Gobierno de Espana) PS0900513, Junta de Extremadura (PDT09A046 and GRU10142), and by the European Community‘s Seventh Framework Programme (FP7/2007–2013) under grant agreement n◦ 228394 (NADIR). NHS is funded by the Department of Environment, Food and Rural Affairs, UK (project SB4020). Mr. García-Jiménez is a recipient of a PhD studentship grant from Junta de Extremadura (PRE07024). Mr. BenítezMedina acknowledges the Junta de Extremadura and FSE for his research fellowship (PRE08042). References Acosta-Iborra, B., Elorza, A., Olazabal, I.M., Martín-Cofreces, N.B., MartinPuig, S., Miró, M., Calzada, M.J., Aragonés, J., Sánchez-Madrid, F., Landázuri, M.O., 2009. Macrophage oxygen sensing modulates antigen presentation and phagocytic functions involving IFN-gamma production through the HIF-1 alpha transcription factor. J. Immunol. 182, 3155–3164. Algood, H.M.S., Chan, J., Flynn, J.L., 2003. Chemokines and tuberculosis. Cytokine Growth Factor Rev. 14, 467–477. Aranaz, A., Liébana, E., Mateos, A., Domínguez, L., Vidal, D., Domingo, M., González, O., Rodriguez-Ferri, E.F., Bunschoten, A.E., Van Embden, J.D.A., Cousins, D., 1996. Spacer oligonucleotide typing of Mycobacterium bovis strains from cattle and other animals: A tool for studying epidemiology of tuberculosis. J. Clin. Microbiol. 34, 2734–2740.

74

W.L. García-Jiménez et al. / Veterinary Immunology and Immunopathology 149 (2012) 66–75

Aranaz, A., De Juan, L., Montero, N., Sánchez, C., Galka, M., Delso, C., Alvarez, J., Romero, B., Bezos, J., Vela, A.I., Briones, V., Mateos, A., Domínguez, L., 2004. Bovine tuberculosis (Mycobacterium bovis) in wildlife in Spain. J. Clin. Microbiol. 42, 2602–2608. Barranco, I., Gómez-Laguna, J., Rodríguez-Gómez, I.M., Salguero, F.J., Pallarés, F.J., Bernabé, A., Carrasco, L., 2011. Immunohistochemical detection of extrinsic and intrinsic mediators of apoptosis in porcine paraffin-embedded tissues. Vet. Immunol. Immunopathol. 139, 210–216. Boddu-Jasmine, H.C., Witchell, J., Vordermeier, M., Wangoo, A., Goyal, M., 2008. Cytokine mRNA expression in cattle infected with different dosages of Mycobacterium bovis. Tuberculosis 88, 610–615. Carranza, J., Alvarez, F., Redondo, T., 1990. Territoriality as a mating strategy in red deer. Anim. Behav. 40, 79–88. Corner, L.A.L., 2006. The role of wild animal populations in the epidemiology of tuberculosis in domestic animals: how to assess the risk. Vet. Microbiol. 112, 303–312. Corner, L.A., Trajstman, A.C., 1988. An evaluation of 1hexadecylpyridinium chloride as a decontaminant in the primary isolation of Mycobacterium bovis from bovine lesions. Vet. Microbiol. 18, 127–134. Cousins, D.V., Wilton, S.D., Francis, B.R., 1991. Use of DNA amplification for the rapid identification of Mycobacterium bovis. Vet. Microbiol. 27, 187–195. De Lisle, G.W., Bengis, R.G., Schmitt, S.M., O’Brien, D.J., 2002. Tuberculosis in free-ranging wildlife: detection, diagnosis and management. OIE Revue Scientifique et Technique 21, 317–334. Delahay, R.J., Cheeseman, C.L., Clifton-Hadley, R.S., 2001. Wildlife disease reservoirs: the epidemiology of Mycobacterium bovis infection in the European badger (Meles meles) and other British mammals. Tuberculosis 81, 43–49. Delahay, R.J., De Leeuw, A.N.S., Barlow, A.M., Clifton-Hadley, R.S., Cheeseman, C.L., 2002. The status of Mycobacterium bovis infection in UK wild mammals: a review. Vet. J. 164, 90–105. Delahay, R.J., Smith, G.C., Barlow, A.M., Walker, N., Harris, A., CliftonHadley, R.S., Cheeseman, C.L., 2007. Bovine tuberculosis infection in wild mammals in the South-West region of England: a survey of prevalence and a semi-quantitative assessment of the relative risks to cattle. Vet. J. 173, 287–301. Gavier-Widén, D., Chambers, M.A., Palmer, N., Newell, D., Hewinson, R.G., 2001. Pathology of natural Mycobacterium bovis infection in Eurasian badgers (Meles meles) and its relationship with bacterial excretion. Vet. Rec. 148, 299–304. Gavier-Widén, D., Cooke, M.M., Gallagher, J., Chambers, M.A., Gortazar, C., 2009. A review of infection of wildlife hosts with Mycobacterium bovis and the diagnostic difficulties of the ‘no visible lesion’ presentation. N. Z. Vet. J. 57, 122–131. Gessani, S., Belardelli, F., 1998. IFN-gamma expression in macrophages and its possible biological significance. Cytokine Growth Factor Rev. 9, 117–123. Gómez-Laguna, J., Salguero, F.J., Barranco, I., Pallarés, F.J., RodríguezGómez, I.M., Bernabé, A., Carrasco, L., 2010. Cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus. J. Comp. Pathol. 142, 51–60. Gómez-Villamandos, J.C., Ruiz-Villamor, E., Bautista, M.J., Sánchez, C.P., Sánchez-Cordón, P.J., Salguero, F.J., Jover, A., 2001. Morphological and immunohistochemical changes in splenic macrophages of pigs infected with classical swine fever. J. Comp. Pathol. 125, 98–109. Gómez-Villamandos, J.C., Salguero, F.J., Ruiz-Villamor, E., Sánchez-Cordón, P.J., Bautista, M.J., Sierra, M.A., 2003. Classical swine fever: pathology of bone marrow. Vet. Pathol. 40, 157–163. Good, M., Duignan, G., 2011. Perspectives on the history of bovine TB and the role of tuberculin in bovine TB eradication. Veterinary Medicine International, http://dx.doi.org/10.4061/2011/410470, Article ID 410470. Gortazar, C., Vicente, J., Gavier-Widén, D., 2003. Pathology of bovine tuberculosis in the European wild boar (Sus scrofa). Vet. Rec. 152, 779–780. Gortazar, C., Torres, M.J., Vicente, J., Acevedo, P., Reglero, M., 2008. Bovine ˜ tuberculosis in Donana Biosphere Reserve: the role of wild ungulates as disease reservoirs in the last Iberian Lynx strongholds. PLoS ONE, 3. ˜ Hermoso de Mendoza, J., Parra, A., Tato, A., Alonso, J.M., Rey, J.M., Pena, J., García-Sánchez, A., Larrasa, J., Teixidó, J., Manzano, G., Cerrato, R., Pereira, G., Fernández-Llario, P., Hermoso de Mendoza, M., 2006. Bovine tuberculosis in wild boar (Sus scrofa), red deer (Cervus elaphus) and cattle (Bos taurus) in a Mediterranean ecosystem (1992–2004). Prev. Vet. Med. 74, 239–247. Hernández-Pando, R., Schön, T., Orozco, E.H., Serafin, J., Estrada-García, I., 2001. Expression of inducible nitric oxide synthase and nitrotyrosine

during the evolution of experimental pulmonary tuberculosis. Exp. Toxicol. Pathol. 53, 257–265. Hicks, D.J., Johnson, L., Mitchell, S.M., Gough, J., Cooley, W.A., La Ragione, R.M., Spencer, Y.I., Wangoo, A., 2006. Evaluation of zinc salt based fixatives for preserving antigenic determinants for immunohistochemical demonstration of murine immune system cell markers. Biotech. Histochem. 81, 23–30. Humblet, M.F., Boschiroli, M.L., Saegerman, C., 2009. Classification of worldwide bovine tuberculosis risk factors in cattle: a stratified approach. Vet. Res. 40, 50, http://dx.doi.org/10.1051/vetres/2009033. Jaroso, R., Vicente, J., Martín-Hernando, M.P., Aranaz, A., Lyashchenko, K., Greenwald, R., Esfandiari, J., Gortázar, C., 2010. Ante-mortem testing wild fallow deer for bovine tuberculosis. Vet. Microbiol. 146, 285–289. Johnson, L., Gough, J., Spencer, Y., Hewinson, G., Vordermeier, M., Wangoo, A., 2006. Immunohistochemical markers augment evaluation of vaccine efficacy and disease severity in bacillus Calmette-Guerin (BCG) vaccinated cattle challenged with Mycobacterium bovis. Vet. Immunol. Immunopathol. 111, 219–229. Johnson, L.K., Liebana, E., Nunez, A., Spencer, Y., Clifton-Hadley, R., Jahans, K., Ward, A., Barlow, A., Delahay, R., 2008. Histological observations of bovine tuberculosis in lung and lymph node tissues from British deer. Vet. J. 175, 409–412. Kamerbeek, J., Schouls, L., Kolk, A., van Agterveld, M., van Soolingen, D., Kuijper, S., Bunschoten, A., Molhuizen, H., Shaw, R., Goyal, M., van Embden, J., 1997. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35, 907–914. Lesellier, S., Palmer, S., Gowtage-Sequiera, S., Ashford, R., Dalley, D.J., Davé, D., Weyer, U., Salguero, F.J., Nunez, A., Crawshaw, T., Corner, L.A.L., Hewinson, R.G., Chambers, M.A., 2011. Protection of Eurasian badgers (Meles meles) from tuberculosis after intra-muscular vaccination with different doses of BCG. Vaccine 29, 3782–3790. Liébana, E., Aranaz, A., Francis, B., Cousins, D., 1996. Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex. J. Clin. Microbiol. 34, 933–938. Liébana, E., Girvin, R.M., Welsh, M.D., Neill, S.D., Pollock, J.M., 1999. Generation of CD8+ T-cell responses to Mycobacterium bovis and mycobacterial antigen in experimental bovine tuberculosis. Infect. Immun. 67, 1034–1044. Liébana, E., Marsh, S., Gough, J., Nunez, A., Vordermeier, H.M., Whelan, A., Spencer, Y., Clifton-Hadley, R., Hewinson, G., Johnson, L., 2007. Distribution and Activation of t-lymphocyte subsets in tuberculous bovine lymph-node granulomas. Vet. Pathol. 44, 366–372. Liébana, E., Johnson, L., Gough, J., Durr, P., Jahans, K., Clifton-Hadley, R., Spencer, Y., Hewinson, R.G., Downs, S.H., 2008. Pathology of naturally occurring bovine tuberculosis in England and Wales. Vet. J. 176, 354–360. Mackintosh, C.G., de Lisle, G.W., Collins, D.M., Griffin, J.F.T., 2004. Mycobacterial diseases of deer. N. Z. Vet. J. 52, 163–174. MARM, 2009. Real Decreto 1082/2009, de 3 de julio, por el que se establecen los requisitos de sanidad animal para el movimiento de animales de explotaciones cinegéticas, de acuicultura continental y de núcleos zoológicos, así como de animales de fauna silvestre. In: Ministerio de Medio Ambiente. MARM, 2011. Programa Nacional de Erradicacion de Tuberculosis Bovina ˜ para el ano, ˜ 2011. presentado por Espana Martínez, J.G., Carranza, J., Fernández-García, J.L., Sánchez-Prieto, C.B., 2002. Genetic variation of red deer populations under hunting exploitation in southwestern Spain. J. Wildl. Manage. 66, 1273–1282. Martín-Hernando, M.P., Höfle, U., Vicente, J., Ruiz-Fons, F., Vidal, D., Barral, M., Garrison, J., de la Fuente, J., Gortazar, C., 2007. Lesions associated with Mycobacterium tuberculosis complex infection in the European wild boar. Tuberculosis 87, 360–367. Martín-Hernando, M.P., Torres, M.J., Aznar, J., Negro, J.J., Gandía, A., Gortázar, C., 2010. Distribution of lesions in Red and Fallow Deer naturally infected with Mycobacterium bovis. J. Comp. Pathol. 142, 43–50. Morse, B.W., Nibbelink, N.P., Osborn, D.A., Miller, K.V., 2009. Home range and habitat selection of an insular fallow deer (Dama dama L.) population on Little St. Simons Island, Georgia, USA. Eur. J. Wildl. Res., 55. Munder, M., Mallo, M., Eichmann, K., Modolell, M., 1998. Murine macrophages secrete interferon gamma upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation. J. Exp. Med. 187, 2103–2108. Naranjo, V., Gortazar, C., Vicente, J., de la Fuente, J., 2008. Evidence of the role of European wild boar as a reservoir of Mycobacterium tuberculosis complex. Vet. Microbiol. 127, 1–9.

W.L. García-Jiménez et al. / Veterinary Immunology and Immunopathology 149 (2012) 66–75 O’Brien, D.J., Fitzgerald, S.D., Lyon, T.J., Butler, K.L., Fierke, J.S., Clarke, K.R., Schmitt, S.M., Cooley, T.M., Berry, D.M., 2001. Tuberculous lesions in free-ranging white-tailed deer in Michigan. J. Wildl. Dis. 37, 608–613. O’Brien, D.J., Schmitt, S.M., Berry, D.E., Fitzgerald, S.D., Vanneste, J.R., Lyon, T.J., Magsig, D., Fierke, J.S., Cooley, T.M., Zwick, L.S., Thomsen, B.V., 2004. Estimating the true prevalence of Mycobacterium bovis in hunter-harvested white-tailed deer in Michigan. J. Wildl. Dis. 40, 42–52. Palmer, M.V., Waters, W.R., Thacker, T.C., 2007. Lesion development and immunohistochemical changes in granulomas from cattle experimentally infected with Mycobacterium bovis. Vet. Pathol. 44, 863–874. Porphyre, T., Stevenson, M.A., McKenzie, J., 2008. Risk factors for bovine tuberculosis in New Zealand cattle farms and their relationship with possum control strategies. Prev. Vet. Med. 86, 93–106. Rhyan, J.C., Saari, D.A., 1995. A comparative study of the histopathologic features of bovine tuberculosis in cattle, fallow deer (Dama dama), sika deer (Cervus nippon), and red deer and elk (Cervus elaphus). Vet. Pathol. 32, 215–220. Robinson, R.C., Phillips, P.H., Stevens, G., Storm, P.A., 1989. An outbreak of Mycobacterium bovis infection in fallow deer (Dama dama). Aust. Vet. J. 66, 195–197. Rohonczy, E.B., Balachandran, A.V., Dukes, T.W., Payeur, J.B., Rhyan, J.C., Saari, D.A., Whiting, T.L., Wilson, S.H., Jarnagin, J.L., 1996. A comparison of gross pathology, histopathology, and mycobacterial culture for the diagnosis of tuberculosis in elk (Cervus elaphus). Can. J. Vet. Res. 60, 108–114. Smith, N.H., Upton, P., 2012. Naming spoligotype patterns for the RD9-deleted lineage of the Mycobacterium tuberculosis complex; www.Mbovis.org. Infect Genet. Evol. 12, 873–876.

75

Soriguer, R.C., Marquez, F.J., Perez, J.M., 1998. Las translocaciones (introducciones y reintroducciones) de especies cinegéticas y sus efectos medioambientales. Galemys 10 (2). Ulrichs, T., Kosmiadi, G.A., Trusov, V., Jörg, S., Pradl, L., Titukhina, M., Mishenko, V., Gushina, N., Kaufmann, S.H.E., 2004. Human tuberculous granulomas induce peripheral lymphoid follicle-like structures to orchestrate local host defence in the lung. J. Pathol. 204, 217–228. Vicente, J., Höfle, U., Garrison, J.M., Fernández-De-Mera, I.G., Juste, R., Barral, M., Gortazar, C., 2006. Wild boar and red deer display high prevalences of tuberculosis-like lesions in Spain. Vet. Res. Commun. 37, 107–119. Villarreal-Ramos, B., McAulay, M., Chance, V., Martin, M., Morgan, J., Howard, C.J., 2003. Investigation of the role of CD8+ T cells in bovine tuberculosis in vivo. Infect. Immun. 71, 4297–4303. Wahlström, H., Englund, L., Carpenter, T., Emanuelson, U., Engvall, A., Vågsholm, I., 1998. A Reed–Frost model of the spread of tuberculosis within seven Swedish extensive farmed fallow deer herds. Prev. Vet. Med. 35, 181–193. Wangoo, A., Johnson, L., Gough, J., Ackbar, R., Inglut, S., Hicks, D., Spencer, Y., Hewinson, G., Vordermeier, M., 2005. Advanced granulomatous lesions in Mycobacterium bovis-infected cattle are associated with increased expression of type I procollagen. (WC1+) T cells and CD 68+ cells. J. Comp. Pathol. 133, 223–234. Wilson, P., Harrington, R., 1976. A case of bovine tuberculosis in fallow deer. Vet. Rec. 98, 74. Zanella, G., Duvauchelle, A., Hars, J., Moutou, F., Boschiroli, M.L., Durand, B., 2008. Patterns of lesions of bovine tuberculosis in wild red deer and wild boar. Vet. Rec. 163, 43–47.