Bovine tuberculosis in a badger (Meles meles) in Spain

C012724 Bovine tuberculosis – 1 Set by: Sue Date: 19.05.08 Proofed:

Short Communications

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Bovine tuberculosis in a badger (Meles meles) from Spain R. Sobrino, M. P. Martín-Hernando, J. Vincente, O. Aurtenetxe, J. M. Garrido, C. Gortázar BOVINE tuberculosis (TB) in badgers (Meles meles) was first diagnosed in Switzerland (Bouvier 1963), a country where no further reports on wildlife bovine TB exist in the scientific literature (Wyss and others 2000). Later, Mycobacterium bovis was isolated from badgers in Gloucestershire (southwest England) in 1971 and in Cork (Ireland) in 1973, both areas with high bovine TB incidence in cattle. Since then, the infection in badgers has been found throughout dense badger populations of south-west England, parts of Wales (Krebs 1997) and Ireland (Dolan 1993). By contrast, there have been no published bovine TB cases in the continent since the first description in Switzerland in the 1960s. This is surprising since many countries in continental Europe have both bovine TB and badgers, although lower badger densities compared with Britain and Ireland may partly explain this absence. In Spanish Mediterranean ecosystems, wild board (Sus scrofa), and probably deer, are able to maintain bovine TB infection in the wild in the absence of domestic livestock and are probably able to transmit the disease to other species, acting as true wildlife reservoirs (Gortazar and others 2005, Vicente and others 2006, Naranjo and others 2008). Recently, a serological survey evidenced antibodies against M bovis MPB70 in badgers (23 per cent), foxes (three per cent) and lynxes (four per cent) from Doñana National Park (Southern Spain), strongly suggesting contact with M bovis (Martín-Atance and others 2006). In this context, the authors describe the first case of clinical bovine TB in a free-living Spanish badger and discuss the implications of this observation for bovine TB control in Spanish wildlife and livestock. An adult female badger (total body length, 64·2 cm) was found moribund in Cabañeros National Park (central Spain) on December 14, 2003 and taken to the nearby IREC laboratory immediately. The badger died during transport. The mandibular and medial retropharyngeal lymph nodes (LNs); tonsils; mediastinal and tracheobronchial LNs; kidneys, adrenals, liver, spleen and their associated LNs; mesenteric LNs and the ileocecal valve; along with the iliac, precapular and poplitean LNs were dissected and sectioned serially into 2-mm-thick slices and examined carefully for gross lesions. Tissue samples from the aforementioned collected LNs, diaphragmatic lung lobes, kidneys, adrenals, liver, spleen and ileocecal valve were fixed in 10 per cent neutral buffered formalin and processed routinely in order to obtain haematoxylin and eosin- and Ziehl-Neelsen-stained slides. Gram and PAS stains were performed to discard non-mycobacterial granulomas. A pool of the LN samples described above was submitted to culture as described by Aduriz and others The Veterinary Record, DATE

Veterinary Record (2008) 163, XXX-XXX R. Sobrino, xxx, xxx, M. P. Martín-Hernando, xxx, xxx, J. Vicente, xxx, xxx, C. Gortázar, xxx, xxx, Instituto de Investigacíon en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13071 Ciudad Real, Spain O. Aurtenetxe, xxx, xxx, J. M. Garrido, xxx, xxx, NEIKER Instituto Vasco de I+D Agraria, Berreaga 1, 48160 Derio, Spain Correspondence to C. Gortázar 1


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FOR PROOFING PURPOSES ONLY. PLEASE PRINT OUT THIS PDF AND RETURN YOUR CORRECTED PROOF TO THE ADDRESS SHOWN ON PAGE 1 (1995). The identification of M bovis was confirmed by PCR as described in Rodriguez and others (1999). Gross pathological evaluation revealed a markedly cachectic carcass with a body weight of only 3·5 kg and no kidney fat (Fig 1). Among the subcutaneous lymph nodes, only one prescapular and one popliteal LN had caseous nodules of 0·2 cm. Multiple caseous and calcified nodules of up to 2 cm diameter were observed in the lung, with numerous pleural adherences (Fig 2). Bronchial LNs also showed nodules of similar appearance. In the abdominal cavity, the omentum and the spleen showed abundant miliary lesions. In the kidneys, whitish areas were evident on the surface and in the parenchyma. The liver was increased in size. No other significant gross lesions were noticed at the time of postmortem examination. Histopathology evidenced bovine TB-compatible lesions in the lung and trachea, liver, spleen, kidney, and five LNs (mandibular, left bronchial, iliac, precapular and poplitean). Severe lesions were observed in the lung, with granulomas of different evolution composed of a mix of epitheloid cells, macrophages and scarce lymphocytes. Most of the lung presented large, sometimes calcified necrotic areas surrounded by epithelioid cells, macrophages and lymphocytes. The affected bronchioli were filed with necrotic debris and epitheloid cells (Fig 3). The alveoli that were around these necrotic areas were oedematous and had numerous active macrophages in their light. In the trachea, a large granuloma was observed with a necrotic calcified centre, surrounded by macrophages and lymphocytes. Four of the examined LNs presented granulomas of medium size, with a small necrotic centre surrounded by epithelioid cells and macrophages (Fig 3a). The kidney medulla and pelvis had non-encapsulated granulomas of medium size, with a necrotic centre also surrounded by epithelioid cells, macrophages and lymphocytes. The tubuli close to the lesion were distended and filled with necrotic debris. Numerous small granulomas were observed in the spleen and the liver. These were composed of a mixture of epithelioid cells, macrophages and lymphocytes, with no necrotic core. Several acid-fast bacilli were observed in Ziehl-Neelsen stains of the trachea, principally in the necrotic calcified centre. Some bacilli were also found in the necrotic area of the lung. Mycobacterium bovis infection was confirmed after isolation and PCR testing of the cultured colonies. This is the first report of clinical bovine TB in a badger from Spain, and also the first report of badger TB in continental Europe in the last 40 years. However, this is not an isolated observation, since a second bovine TB case was detected recently in the León province, north-western Spain. In that case, a badger captured in a dairy farm with a recent history of bovine TB was analysed in the Veterinary Faculty, and a positive M bovis culture was obtained from a pooled LN sample. Molecular typing revealed the same strain as in cattle (F. García-Marín, personal communication). In the present study, the authors found that the badger, considered a reservoir host in other latitudes, may become infected in an area where bovine TB is highly prevalent in ungulates (Vicente and others 2006). The pattern of lesions was similar to that found in Britain (Gavier-Widen and others 2001), which is consistent with this badger being a mycobacteria excretor and potential disseminator of the disease. However, a limited availability of trophic resources determines low

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The Veterinary Record, DATE


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FOR PROOFING PURPOSES ONLY. PLEASE PRINT OUT THIS PDF AND RETURN YOUR CORRECTED PROOF TO THE ADDRESS SHOWN ON PAGE 1 badger densities in Mediterranean habitats (Virgos and others 2004). Densities have been reported to be as low as 0·23 to 0·67 individuals/km2 (Revilla and others 1999). At the current densities found in the present study area, badgers would be more a spill-over host that a true reservoir. Nevertheless, the increase of badger abundance observed in some areas of Spain; for example, in the Aragon region (north-east Spain) from 1992 to 2006 (Sobrino and others, unpublished data) could mean an increased disease risk. Thus, more epidemiological research is needed, and active and passive surveillance of badgers and other wildlife TB reservoirs, mainly wild ungulates, is advisable. Acknowledgements This is a contribution to the agreement between CSIC, OAPN and MAPA on wildlife disease surveillance in Spain, and to the Castilla – La Mancha network on wildlife diseases (JCCM). This is also a contribution to a grant from Grupo Santander – Fundación Marcelino Botín (Spain) and to projects INIA FAU06-016 and AGL2005-07401 , Ministerio de Educación y Ciencia, Plan Nacional and FEDER. RS acknowledges a grant from JCCM and MPMH, and a postdoctoral contract from ISCIII. The research group acknowledges support from Consejería de Sanidad JCCM (GC05-06; SAN06-036). References ADÚRIZ, J. J., JUSTE, R. A. & CORTABARRIA, N. (1995) Lack of micobactin dependence of Mycobacteria isolated on Middlebrook 7H11 from clinical cases of ovine paratuberculosis. Veterinary Microbiology 45, 211-217 BOUVIER G. (1963) Transmission possible de la Tuberculose et de la Brucellose du gibier a l,home et aux animaux domestiques et sauvages. Bulletin de L´Office International des Epizooties 59, 433-436 DOLAN, L. A. (1993) Badgers and Bovine Tuberculosis in Ireland: A Review. Ed T. H. Hayden. Dublin, Royal Irish academy. pp 108-116 GAVIER-WIDEN, D., CHAMBERS, M. A., PALMER, N., NEWELL, D. G. & HEWINSON, R. G. (2001) Pathology of natural Mycobacterium bovis infection in European badgers (Meles meles) and its relationship with bacterial excretion. Veterinary Record 148, 299-304 GORTAZAR, C., VICENTE, J., SAMPER, S., GARRIDO, J. M., FERNÁNDEZDE-MERA, I. G., GAVÍN, P., JUSTE, R. A., MARTÍN, C., ACEVEDO, P., DE LA PUENTE, M. & HÖFLE, U. (2005) Molecular characterization of Mycobacterium tuberculosis complex isolates from wild ungulates in southcentral Spain. Veterinary Research 36, 43-52 KREBS, J. R. (1997) Bovine Tuberculosis in Cattle and Badgers. London. Ministry of Agriculture, Fisheries and Food. p 191 LEÓN-VIZCANO, L. (1991) Informe científico al Patronato del Parque Nacional de Doñana. Sevilla, Spain. Estación Biológica de Doñana (CSIC) MARTÍN-ATANCE, P., LEÓN-VIZCAINO, L., PALOMARES, F., REVILLA, E., GONZÁLEZ-CANDELA, M., CALZADA, J., CUBERO-PABLO, M. J. & DELIBES, M. (2006) Antibodies to Mycobacterium Bovis in wild carnivores from Doñana Nacional Park (Spain). Journal of Wildlife Diseases 42, 704-708 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. Veterinary Microbiology 127, 1-9 REVILLA, E., DELIBES, D., TRAVAINI, A. & PALOMARES, F. (1999) Physical and population parameters of Eurasian badgers, Meles meles, from Mediterranean Spain Zeitschrift für Säugetierkunde 64, 269-276 RODRÍGUEZ, J. G., FISSANOTI, J. C., DEL PORTILLO, P., PATARROYO, M. E., ROMANO, M. I. & CATALDI, A. (1999) Amplification of a 500-base-pair fragment from cultured isolates of Mycobacterium bovis. Journal of Clinical Microbiology 37, 2330-2332


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FOR PROOFING PURPOSES ONLY. PLEASE PRINT OUT THIS PDF AND RETURN YOUR CORRECTED PROOF TO THE ADDRESS SHOWN ON PAGE 1 VICENTE, J., HÖFLE, U., GARRIDO, 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. Veterinary Research 37, 107-119 VIRGÓS, E., MANGAS, J. G., BLANCO-AGUIAR, J. A., GARROTE, G., ALMAGRO, N. & VISO, R. P. (2004) Food habits of European badgers (Meles meles) along an altitudinal gradient of Mediterranean environment: a field test of the earthworm specialization hypothesis. Canadian Journal of Zoology 82, 41-51 WYSS, D., GIACOMETTI, M., NICOLET, J., BURNENS, A., PFYFFER, G. E. & AUDIGÉ, L. (2000) Farm and slaughter survey of bovine tuberculosis in captive deer in Switzerland. Veterinary Record 147, 713-717

(a)

FIG 1: A skinned badger (Meles meles) carcase evidencing emaciation

FIG 2: A lung section from a badger (Meles meles) showing multiple caseous and calcified tuberculosis granulomas

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(b)

FIG 3: Bovine tuberculosis in a badger (Meles meles) from Spain. (a) lymph node section shows granulomas composed of a mixture of epithelioid cells, macrophages and scarce lymphocytes (bar=100 µm). (b) A tuberculosis granuloma in the lung compromises the bronchiolar wall and fills the lumen with cellular detritus and inflammatory cells (bar=xxxx). Haematoxylin and eosin
