Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation

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Abstract

The role of wildlife in tuberculosis epidemiology is being widely studied since it can affect the effectiveness of eradication campaigns in cattle. The health problem is enhanced when it concerns also wildlife welfare and biodiversity conservation. This study was performed to understand the epidemiology of Mycobacterium bovis population affecting livestock and wild animals in the Doñana National Park using bacteriology and molecular characterisation techniques. Tuberculosis research was performed on 1209 cattle and wild animals (artiodactyla and carnivore) collected over 6 years in the Park. One hundred and sixty-three animals were found to be infected with M. bovis, comprising 7.96% of the cattle and 20.53% of the wild animals tested. Spoligotyping revealed nine patterns, being SB1232 and SB1230 the most prevalent (77.30% and 15.34% of infected animals, respectively). MIRU-VNTR analysis of a selected panel of 92 isolates showed eight different profiles, including several spoligotypes within the same MIRU-VNTR profile. The discriminatory capacity of both techniques in this panel was similar. The results obtained by combination of both techniques corroborate that wildlife species are infected with the M. bovis strains which are more prevalent in cattle and reveal their persistence. Genotype variation between isolates strongly suggests micro-evolutionary events in the M. bovis population in the same area. This study in the Doñana National Park exposes the risk of introduction of domestic animals into wildlife areas when there is not a warranty of disease freedom, appropriate diagnostic techniques and control measures.

Introduction

Tuberculosis in wildlife, caused mainly by Mycobacterium bovis, has been reported worldwide (Livingstone, 2000, Bengis et al., 2002, Corner, 2006). The first concern about involvement of feral animals was their role in tuberculosis transmission, limiting the effectiveness of eradication campaigns in cattle. For example, in the UK and the Republic of Ireland, maintenance of bovine tuberculosis has been attributed to the presence of infected badgers (Meles meles) (Gallagher and Clifton-Hadley, 2000, Carter et al., 2007), and in Michigan (USA) the re-emergence of tuberculosis in cattle is thought to have been due to a reservoir in white-tailed deer (O’Brien et al., 2002). On the other hand, this disease has an enormous impact on wildlife welfare and biodiversity conservation which has been disregarded frequently, and even threatens endangered and emblematic species (Briones et al., 2000, Michel et al., 2006). Moreover, tuberculosis is also a zoonotic disease that can be transmitted by aerosols, skin abrasions or consumption of uncooked products (Winters et al., 2005, Evans et al., 2007).

In Spain, recent studies support the role of artiodactyls as wild hosts of tuberculosis. Red deer (Cervus elaphus), fallow deer (Dama dama) and the European wild boar (Sus scrofa) are game species with the highest densities in the Iberian Peninsula that have been found to be infected with M. bovis (Aranaz et al., 2004a, Parra et al., 2006, Vicente et al., 2006, Martín-Hernando et al., 2007, Naranjo et al., 2008).

Molecular epidemiology tools can provide a valuable insight into the importance of different hosts in the maintenance and spread of the infection although the information obtained cannot indicate a route of transmission. Mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) have been proposed as an alternative (Skuce et al., 2005, Allix et al., 2006) to the spoligotyping method (Kamerbeek et al., 1997) because the latter has low discrimination power when M. bovis isolates are closely related to each other (Cousins et al., 1998, Skuce et al., 2002). However MIRU-VNTR analysis is not yet standardised in M. bovis and allelic diversity can vary among countries.

The aim of this report was to study the epidemiology of M. bovis in livestock and wild animals in the Doñana National Park. We used spoligotyping and MIRU-VNTR typing in order to compare their efficiency to discriminate between M. bovis isolates and to gain an insight into its route of transmission between wild and domestic animals within a restricted closed geographical area over a long period of time.

Section snippets

Sample collection

This study was performed in the Doñana National Park, located in western Andalucía (south of Spain) (http://whc.unesco.org/en/list/685). During 1998–2003, samples from 1209 animals were collected in the National Park (Table 1), comprising domestic cattle (Bos taurus) (n = 678) and wild animals (n = 531) [red deer (C. elaphus) (n = 168), fallow deer (D. dama) (n = 134), wild boar (S. scrofa) (n = 214), Iberian lynx (Lynx pardinus) (n = 10), and fox (Vulpes vulpes) (n = 5)]. Cattle in this study were animals

Bacteriology

From the samples collected in the Doñana National Park from 1209 domestic and wild animals, we isolated Mycobacterium spp. from 213 animals: 163 isolates were identified as members of the M. tuberculosis complex and 50 isolates were atypical mycobacteria (Table 1). Fifty-four out of 678 cattle (7.96%) and 109 out of 531 wild animals (20.53%) were found to be infected by M. tuberculosis complex isolates.

Spoligotyping

The 163 isolates belonging to M. tuberculosis complex were characterised by spolygotyping and

Discussion

The presence of tuberculosis in a population of wild animals may interfere with the eradication scheme of bovine tuberculosis and raises problems of wildlife management and public health. In this study we used molecular techniques to analyse the M. bovis population isolated from livestock and wild animals from the Doñana National Park. The combination of spoligotyping and MIRU-VNTR analysis corroborates that wildlife (artiodactyla, carnivores) is infected with the M. bovis strains which are

Conclusion

Spoligotyping and MIRU-VNTR analysis provides further evidence of the transmission of M. bovis population between cattle and wildlife species (artiodactyla and carnivores) sharing an ecosystem. This M. bovis population is persistent and undergoes micro-evolutionary steps over long period of time. The likelihood of inter-species pathogen transmission should be considered before introducing animals in areas that must be preserved from infectious diseases that have detrimental effects in both

Acknowledgments

This research was supported by projects AGL 2006-06206 of the Spanish Ministry of Science and Technology and TB-STEP 212414 of the European Union, and by the Spanish Ministry of Agriculture, Fisheries and Food. B. Romero is recipient of a research contract funded by Comunidad de Madrid (IV regional framework programme of research and technological innovation 2005–2008). We thank the guidelines and suggestions by partners of the EU coordination action “Veterinary European Network on Mycobacteria

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    1

    Present address: Ordway Research Institute, Inc., Albany, NY 12208, USA.

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    Present address: Subdirección General de Sanidad Animal, MAPA, 28071 Madrid, Spain.

    3

    Present address: Clínica Veterinaria Noé Montequinto, Dos Hermanas, 41089 Sevilla, Spain.

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