Christian Gortazar
email: christian.gortazar@uclm.es

Joaquín Vicente

Mariana Boadella

Cristina Ballesteros

Ruth C Galindo

Joseba Garrido

Alicia Aranaz

José De La Fuente

Progress in the Control of Bovine Tuberculosis in Spanish Wildlife

Keywords: Cervus elaphus, Disease control, Meles meles, Sus scrofa, Wildlife epidemiology

Despite the compulsory test and slaughter campaigns in cattle, bovine tuberculosis (bTB) is still present in Spain, and the role of wildlife reservoirs is increasingly recognized. We provide an update on recent progress made in bTB control in Spanish wildlife, including aspects of epidemiology, surveillance, host-pathogen interaction and wildlife vaccination.

At the high densities and in the particular circumstances of Mediterranean environments, wild ungulates, mainly Eurasian wild boar and red deer, are able to maintain M. bovis circulation even in absence of domestic livestock. Infection is widespread among wild ungulates in the south of the country, local infection prevalence being as high as 52% in wild boar and 27% in M a n u s c r i p t 3 bovis BCG from faeces, internal organs at necropsy and the environment, even after oral delivery of very high doses. Finally, preliminary vaccination and challenge experiments suggested that a single oral BCG vaccination may protect wild boar from infection by a virulent M. bovis field strain.

red deer. Risk factors identified include host genetic susceptibility, abundance, spatial aggregation at feeders and waterholes, scavenging, and social behaviour. An increasing trend of bTB compatible lesions was reported among wild boar and red deer inspected between 1992 and 2004 in Southwestern Spain. Sporadic cases of badger TB have been detected, further complicating the picture.

Gene expression profiles were characterized in European wild boar and Iberian red deer naturally infected with M. bovis. The comparative analysis of gene expression profiles in wildlife hosts in response to infection advanced our understanding of the molecular mechanisms of infection and pathogenesis, revealed common and distinctive host responses to infection and identified candidate genes associated with resistance to bTB and for the characterization of host response to infection and vaccination.

Ongoing research is producing valuable knowledge on vaccine delivery, safety and efficacy issues. Baits for the oral delivery of BCG vaccine preparations to wild boar piglets were developed and evaluated. The use of selective feeders during the summer was found to be a potentially reliable bait-deployment strategy. Safety experiments yielded no isolation of M. Bovine tuberculosis (bTB), a chronic infectious disease shared between livestock and wildlife, has a complex epidemiology, often with climate and habitat-mediated peculiarities.

Four potential wildlife bTB reservoirs (as reviewed in EFSA 2009) exist in mainland Spain:

Eurasian wild boar (Sus scrofa), red deer (Cervus elaphus), fallow deer (Dama dama) and Eurasian badger (Meles meles). Eurasian badgers are scarce in Mediterranean habitats and more abundant in Atlantic ones [START_REF] Revilla | Spatial organization, group living and ecological correlates in low-density populations of Eurasian badgers, Meles meles[END_REF], and wild ungulates are continuously expanding in range and in numbers throughout the whole Peninsula (e.g. Gortazar et al. 2000[START_REF] Delibes-Mateos | Longterm changes in game species over a long period of transformation in the Iberian Mediterranean landscape[END_REF]), but are largely absent from the Islands.

Spain is a major livestock producer within the European Union. There are about 6,250,000 cattle in 143,000 herds. Since test and slaughter campaigns became compulsory, bTB in cattle declined significantly from 12% herd prevalence in 1987 to 1.68% in 2008. However, cattle bTB distribution is not uniform in the country. Island regions are almost bTB free, and most of the northern Spanish mainland (Atlantic climate region) is also almost bTB free. In contrast, several regions of central and southern Spain still have high bTB prevalence (Figure 1). In infected regions, bTB is consistently more prevalent among beef cattle and bullfighting cattle, which often share wildlife habitats, than among the generally fenced dairy cattle (http://rasve.mapa.es//Publica/Sanidad/documentos/INFORME%20FINAL%20TÉCNICO%2 0TB%202008.pdf).

Moreover, bTB is also present among goat and pig livestock, although information on prevalence is lacking. About 2,500,000 pigs are raised in open air systems. Most belong to the Iberian pig breed, which is raised in open evergreen oak woodland habitats of the south western Iberian Peninsula. Contact with domestic ruminants and with wildlife occurs in this ecosystem, and cases of M. bovis infection have been recorded (Gómez-Laguna et al. 2010).

Molecular typing suggests that M. bovis strains of pigs may be shared with livestock and A c c e p t e d M a n u s c r i p t 5 wildlife species [START_REF] Parra | Molecular epidemiology of bovine tuberculosis in wild animals in Spain: A first approach to risk factor analysis[END_REF]. Regarding goats, current numbers in Spain are around 3,000,000 (http://www.mapa.es/estadistica/pags/anuario/2008/). Not only M. caprae, but also M. bovis strains are identified in goats [START_REF] Gutiérrez | Brief communications and case reports: Cryptococcus neoformans and Mycobacterium bovis causing granulomatous pneumonia in a goat[END_REF].

The role of wildlife reservoirs in bTB epidemiology is increasingly recognized, worldwide [START_REF] Efsa ; Fernández-De-Mera | Differential expression of inflammatory and immune response genes in mesenteric lymph nodes of Iberian red deer (Cervus elaphus hispanicus) naturally infected with Mycobacterium bovis[END_REF]. However, the definition of a wildlife reservoir is somewhat controversial. In a broad sense, a reservoir is defined as one or more epidemiologically connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population [START_REF] Haydon | Identifying reservoirs of infection: A conceptual and practical challenge[END_REF]. For being a competent bTB reservoir, any host species must be susceptible, able to transmit the disease, and abundant enough [START_REF] Corner | The role of wild animal populations in the epidemiology of tuberculosis in domestic animals: how to assess the risk[END_REF]. In Spain, the wildlife component of this reservoir is composed of one or more sympatric host species, with marked regional differences.

Essentially, wild ungulates are responsible for bTB maintenance in Mediterranean regions of continental Spain, and the badger could have some relevance in the more humid Atlantic regions (see below).

Herein, we provide an update on recent progress made in bTB control in Spanish wildlife, including aspects of epidemiology, surveillance, host-pathogen interaction and wildlife vaccination.

The wildlife factor in bTB epidemiology in Spain

The Iberian Peninsula in the southwestern end of Europe is largely dominated by Mediterranean climate, with mild to cold, dry winters, hot and dry summers, and limited rainfall (usually less than 600 mm per year), which is concentrated in spring and autumn. The northern strip of the Peninsula, from Portugal to the Pyrenees, is characterized by an Atlantic climate, with up to 2000 mm rainfall per year. Islands in the Atlantic) are almost bTB free. This is interesting from a wildlife perspective, since these regions are lacking all four potential wildlife reservoirs. For comparison, bTB was diagnosed among feral black pigs from the Italian Mediterranean island Sicily and the disease still constitutes a problem for livestock in this region (Di [START_REF] Marco | Black wild pigs of Nebrodi Park in Sicily: evidence of their possible role as reservoir of Mycobacterium tuberculosis complex infection[END_REF]).

Atlantic Spain

It is not known if wildlife represents a significant bTB reservoir in northern Spain. Eurasian badgers, a well known bTB reservoir in Ireland and the UK, are more common and abundant in Atlantic than in Mediterranean habitats in Spain [START_REF] Revilla | Spatial organization, group living and ecological correlates in low-density populations of Eurasian badgers, Meles meles[END_REF], and sporadic cases of M. bovis infection have been detected in different Spanish regions, even in the north (Sobrino et al. 2008, A. Balseiro pers. comm.). However, prevalence figures based on large enough sample sizes, which are difficult to obtain in this protected species, are currently lacking. Moreover, recent monitoring data suggests that badger densities are increasing (Sobrino et al. 2009).

Very few cases of M. bovis infection have been reported among wild ungulates from Atlantic habitats in Spain, despite locally intense sampling. However, the bTB outbreak among red deer and wild boar from the Brotonne forest in France shows that stable situations with a high infection prevalence may well occur in Atlantic habitats (Zanella et al. 2008). One spillover case has been reported in a roe deer (Capreolus capreolus, [START_REF] Balseiro | Tuberculosis in roe deer from Spain and Italy[END_REF].

Considering together both the continued reduction of cattle bTB prevalence up to levels close to official eradication, and the nearly absence of wildlife bTB cases detected in Atlantic Spain, the current role of wildlife in bTB epidemiology seems of little relevance in this A c c e p t e d M a n u s c r i p t 7 region, and eradication among cattle will hopefully be achieved soon. However, sporadic cases diagnosed in Eurasian badgers, along with increasing demographic trends in several potential bTB hosts (Gortazar et al. 2000[START_REF] Acevedo | Factors affecting wild boar abundance across an environmental gradient in Spain[END_REF], Sobrino et al. 2009), indicate that maintaining a targeted surveillance is advisable (see below).

Mediterranean Spain

In semiarid Mediterranean habitats of central and southern Spain, several wild ungulates contribute to bTB maintenance in a multi-host system [START_REF] Gortazar | Bovine Tuberculosis in Doñana Biosphere Reserve: The role of wild ungulates as disease reservoirs in the last Iberian lynx strongholds[END_REF]). The epidemiologically most relevant wildlife hosts include the Eurasian wild boar (Naranjo et al. 2008a), and two cervids of the subfamily cervinae: the red deer and the fallow deer. Spillover hosts include the red fox (Vulpes vulpes;Millán et al. 2008) and the endangered Iberian lynx (Lynx pardinus; [START_REF] Peña | Histopathological and immunohistochemical findings in lymphoid tissues of the endangered Iberian lynx (Lynx pardinus)[END_REF]), among others. Habitat constraints determine that badgers exist only at very low densities in Mediterranean Spain [START_REF] Revilla | Spatial organization, group living and ecological correlates in low-density populations of Eurasian badgers, Meles meles[END_REF].

Bovine TB is widespread among wild ungulates in central and southern Spain (Vicente et al. 2006), local prevalence of culture confirmed infection being as high as 52% in wild boar and 27% in red deer [START_REF] Gortazar | Bovine Tuberculosis in Doñana Biosphere Reserve: The role of wild ungulates as disease reservoirs in the last Iberian lynx strongholds[END_REF]. At the high densities and in the particular circumstances of Mediterranean environments, Eurasian wild boar and red deer are able to maintain Mycobacterium tuberculosis complex (MTBC) circulation in the absence of domestic livestock, for instance in fenced estates (Figure 2, panel a) and protected natural areas with no domestic ruminants [START_REF] Acevedo-Whitehouse | Genetic resistance to bovine tuberculosis in the Iberian wild boar[END_REF]. Red deer alone and wild boar alone maintain bTB circulation in the absence of other wildlife hosts (Vicente et al. 2006). The situation is worst among farm-like hunting enclosures called "cercones", where virtually all wild boar become infected [START_REF] Acevedo | Estimation of European wild boar relative abundance and aggregation: a novel method in epidemiological risk assessment[END_REF]). The reservoir role of fallow deer has been less studied, but is possibly relevant at a local scale [START_REF] Aranaz | Bovine tuberculosis (Mycobacterium bovis) in wildlife in Spain[END_REF], Gortazar et al. 2008, A c c e p t e d M a n u s c r i p t 8 Jaroso et al. unpublished data). Table 1 displays relevant facts about these three host species in Mediterranean habitats.

Individual risk factors for M. bovis infection in wild ungulates include the host species, wild boar consistently showing higher M. bovis infection prevalence than deer (Vicente et al. 2006, 2007b, Gortazar et al. 2008), and red deer showing higher prevalence than fallow deer [START_REF] Gortazar | Bovine Tuberculosis in Doñana Biosphere Reserve: The role of wild ungulates as disease reservoirs in the last Iberian lynx strongholds[END_REF]; sex in red and fallow deer (males more prevalent, Vicente et al. 2006, Gortazar et al. 2008); age in deer and in wild boar (age-increasing trends; Vicente et al. 2006, Gortazar et al. 2008); and host genetic susceptibility (e.g. [START_REF] Acevedo-Whitehouse | Genetic resistance to bovine tuberculosis in the Iberian wild boar[END_REF]and Naranjo et al. 2008ain wild boar, Fernández-de-Mera et al. 2009b, in red deer).

In multi-host systems such as those occurring in Mediterranean Spain, the infection levels of sympatric host species do also contribute to the risk factors for a given one (e.g. wild boar on red deer and vice versa; Vicente et al. 2007b). Belonging to an M. bovis-infected social group is a significant risk factor for infection in red deer and wild boar, but not for fallow deer (Gortazar et al. submitted).

Environmental risk factors are often, but not always, linked to artificial management of wildlife habitats and wildlife populations in high-wire fenced estates (Vicente et al. 2007a(Vicente et al. , 2007b)). Fencing (Figure 2, panel a) can affect bTB epidemiology through a reduced host genetic variability. Using microsatellite markers, [START_REF] Acevedo-Whitehouse | Genetic resistance to bovine tuberculosis in the Iberian wild boar[END_REF] found that genetic variability, fencing and wild boar abundance had significant effects on bTB infection. The strongest effects were observed for genetic heterozygosity, with relatively less heterozygous wild boar being more likely to be infected. Fencing may increase the chances of mating amongst close relatives, and contribute to a reduced genetic variability and reduced disease resistance. This was also suggested in red deer, where Fernández- de-Mera et al. (2009ade-Mera et al. ( , 2009b) ) showed significant loss in variability of the drb2 MHC-II locus over only 16 years in a fenced red deer population.

A c c e p t e d M a n u s c r i p t 9 Moreover, fencing is commonly associated with other well known bTB risk factors such as feeding and translocating (Figure 2, panel c), and in general with wildlife overabundance (Gortazar et al. 2006). Artificial feeding and watering causes spatial aggregation and allows maintaining ungulate densities above the carrying capacity of a given habitat. Disease prevalence has been linked to spatial aggregation and high densities [START_REF] Acevedo | Estimation of European wild boar relative abundance and aggregation: a novel method in epidemiological risk assessment[END_REF], Vicente et al. 2007a, 2007b). However, high M. bovis prevalences are found even in protected areas where no feeding and no translocations of wild ungulates take place, such as the Doñana National Park (DNP, [START_REF] Gortazar | Bovine Tuberculosis in Doñana Biosphere Reserve: The role of wild ungulates as disease reservoirs in the last Iberian lynx strongholds[END_REF], possibly because of the high densities and habitat mediated spatial aggregation occurring in DNP. Aggregation and M. bovis transmission may occur more often in certain habitats. For instance hardwood Quercus spp.

forest availability was associated with increased bTB risk in red deer and wild boar in central Spain (Vicente et al. 2007b). [START_REF] Miller | Evaluation of the influence of supplemental feeding of white-tailed deer (Odocoileus virginianus) on the prevalence of bovine tuberculosis in the Michigan wild deer population[END_REF] suggested that woodland areas provide shady, moist conditions under which M. bovis might survive longer in the environment.

Finally scavenging, including hunting gut-pile consumption (Figure 2, panel d), is most probably a significant risk factor for wild boar and for carnivores. Infection through consumption of contaminated materials may increase the probability of contacting M. bovis [START_REF] Gortazar | Bovine Tuberculosis in Doñana Biosphere Reserve: The role of wild ungulates as disease reservoirs in the last Iberian lynx strongholds[END_REF]). Ongoing research is evidencing that the amount of carrion scavenged by wild boar in Mediterranean Spain compares to that by specialized scavengers (vultures), and even locally this suid becomes the top consumer (unpublished data). Figure 2 suggests that gut-piles may occasionally attract herbivores such as red deer.

The main purpose of controlling bTB in Spanish wildlife is to prevent the infection of the domestic stock, and also humans. In this sense, ongoing research is addressing wildlifedomestic livestock interaction to elucidate shared epidemiology and to identify control measures such as those related to safe husbandry practices. Also, large scale studies on spatiotemporal incidence patterns of bTB in livestock herds are including wildlife related

Wildlife disease surveillance

Defining the concepts

Two important concepts in wildlife disease epidemiology need to be defined: surveillance and management. Surveillance is the ongoing recording of diseases in wildlife populations with a view to disease management. Surveillance data are used to identify the areas to be targeted for control, and to anticipate spatial and temporal trends so that pre-emptive management interventions can be used to reduce disease risks [START_REF] Artois | Wildlife disease surveillance and monitoring[END_REF]. Wildlife disease management begins with surveillance; knowing if diseases are present, their past and current distribution, and the trends in their prevalence. Possibilities for disease management, always in addition to surveillance, include disease control through different means, disease eradication, which is usually not realistic, or taking no action, if the cost-benefit analysis suggests that this is the most convenient option (Wobeser, 1994).

Methods in bTB surveillance

The development of effective schemes for the surveillance of disease in wildlife populations is becoming increasingly important [START_REF] Artois | Wildlife disease surveillance and monitoring[END_REF]. Tools available for bTB surveillance include visual inspection for macroscopic bTB-compatible lesions or combinations of visual inspection with culture (e.g. Vicente et al. 2006); systematic culture of selected tissues (e.g. [START_REF] Gortazar | Bovine Tuberculosis in Doñana Biosphere Reserve: The role of wild ungulates as disease reservoirs in the last Iberian lynx strongholds[END_REF]; and even serology or combinations of serology and culture (e.g. [START_REF] Aurtenetxe | Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in European Wild Boar[END_REF], Vicente et al. 2007b). In Spain, surveillance is based on large post- Otherwise, the inspected organs should be clearly stated to allow comparisons. In the wild boar visible lesions are most often (i.e. in 92-100% of cases) located in the mandibular lymph nodes (Gortazar et al. 2003[START_REF] Parra | An epidemiological evaluation of Mycobacterium bovis infections in wild game animals of the Spanish Mediterranean ecosystem[END_REF][START_REF] Naranjo | Proteomic and transcriptomic analyses of differential stress/inflammatory responses in mandibular lymph nodes and oropharyngeal tonsils of European wild boars naturally infected with Mycobacterium bovis[END_REF]). This makes targeted surveillance in this host easy.

However, for epidemiological purposes it is interesting to sample at least also the left tracheobronchial LN of wild boar, to estimate the number of individuals with thoracic extended lesions [START_REF] Naranjo | Proteomic and transcriptomic analyses of differential stress/inflammatory responses in mandibular lymph nodes and oropharyngeal tonsils of European wild boars naturally infected with Mycobacterium bovis[END_REF]. Ideally, lesion identification should be carried out by trained staff in a systematic manner, and the presence of infection at the local level should later be confirmed by culture. Such information is considered to be valuable for exploring the magnitude and general distribution of bTB in wildlife, provided a large enough sample size is obtained from an extensive area (Vicente et al. 2006). Further, molecular characterization of the isolates from different species (including livestock) would provide additional information to clarify both the local epidemiology of bTB and large spatiotemporal patterns [START_REF] Acevedo-Whitehouse | Genetic resistance to bovine tuberculosis in the Iberian wild boar[END_REF]. bovis has been observed in wild boar [START_REF] Aurtenetxe | Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in European Wild Boar[END_REF][START_REF] Lyashchenko | Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife[END_REF]. More data on the utility of these techniques for other hosts such as red and fallow deer are needed.

Prevalence trends among Spanish wildlife

An increasing trend of bTB compatible lesions was reported among wild boar and red deer inspected between 1992 and 2004 in Extremadura, south-western Spain [START_REF] Parra | An epidemiological evaluation of Mycobacterium bovis infections in wild game animals of the Spanish Mediterranean ecosystem[END_REF]. In Doñana NP (Southern Spain), M. bovis infection prevalence increased from 1998-2003 to 2006-2007 by 100% in wild boar and by 50% in red deer [START_REF] Romero | Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation[END_REF], Gortazar et al. 2008). Among 14 wildlife populations included in a 10 year survey in south central Spain, 11 presented increasing levels of bTB compatible lesions and of incidence among juvenile wild boar, in contrast to only 3 sites with decreasing bTB (unpublished data; Figure 3). However, no nationwide figures on bTB trends are available for wildlife.

Field methods for estimating wild ungulate abundance and spatial aggregation (e.g. [START_REF] Acevedo | Estimation of European wild boar relative abundance and aggregation: a novel method in epidemiological risk assessment[END_REF][START_REF] Acevedo | Estimating red deer abundance in a wide range of management situations in Mediterranean habitats[END_REF] have been implemented, allowing matching population monitoring with disease surveillance. At a national scale, a Wildlife Disease Surveillance Program is being set up. This scheme is expected to provide quality information on disease trends in Spanish wildlife.

Host-pathogen interactions

A c c e p t e d M a n u s c r i p t 13 Substantial evidence suggests that genetic and environmental factors contribute to the pathogenesis and differences in susceptibility of humans and mice to M. tuberculosis (Fernando and Britton, 2006). In Eurasian wild boar, genetic factors have been associated with resistance to bTB at the population level [START_REF] Acevedo-Whitehouse | Genetic resistance to bovine tuberculosis in the Iberian wild boar[END_REF]. In this study, wild boar microsatellite marker variability was correlated with bTB providing evidence of both general and single-locus associative effects on bTB, and several loci revealed high homology to regions of the genome with known immune function [START_REF] Acevedo-Whitehouse | Genetic resistance to bovine tuberculosis in the Iberian wild boar[END_REF].

Recent studies demonstrated by suppression-subtractive hybridization and proteome analysis differential gene expression in tonsils and mandibular lymph nodes of tuberculous and nontuberculous wild boar exposed to natural M. bovis infection (Naranjo et al. 2006a;2007a).

Microarray gene expression profiling also showed differential gene expression in Iberian red deer lymph nodes and wild boar peripheral blood mononuclear cells in response to natural M. bovis infection (Fernández de [START_REF] Efsa ; Fernández-De-Mera | Differential expression of inflammatory and immune response genes in mesenteric lymph nodes of Iberian red deer (Cervus elaphus hispanicus) naturally infected with Mycobacterium bovis[END_REF][START_REF] De La Lastra | Expression of immunoregulatory genes in peripheral blood mononuclear cells of European wild boar immunized with BCG[END_REF]). These studies showed tissue-specific expression profiles that suggested differences in the role that tonsils and mandibular lymph nodes play in response to M. bovis infection in wild boar (Naranjo et al. 2007b). Furthermore, these studies suggested candidate gene markers associated with bTB resistance in wild boar and characterized genes that could be used to monitor host response to pathogen infection and vaccination (Naranjo et al. 2006b;2008b;[START_REF] De La Lastra | Expression of immunoregulatory genes in peripheral blood mononuclear cells of European wild boar immunized with BCG[END_REF]). One of these genes, methylmalonyl-CoA mutase (MUT) was upregulated in M. bovisexposed uninfected animals and specific alleles were associated with resistance to bTB in wild boar (Naranjo et al. 2006b;2008b;[START_REF] De La Lastra | Expression of immunoregulatory genes in peripheral blood mononuclear cells of European wild boar immunized with BCG[END_REF]. Other genes such as complement component 3 (C3), interferon gamma (IFN-gamma), interleukin 4 (IL-4) and Regulated on Activation, Normal T Expressed and Secreted cytokine, also known as CCL5 (RANTES) were downregulated in infected wild boar and upregulated in parentally and A c c e p t e d M a n u s c r i p t 14 orally BCG-immunized animals when compared to non-immunized controls (Pérez de la Lastra et al. 2009;Ballesteros et al. 2009c). These results also provided additional evidence that expression of selected genes correlates with protection to M. bovis infection after oral BCG vaccination in wild boar (Ballesteros et al. 2009c).

These studies identified new mechanisms by which wildlife hosts respond to M. bovis infection and how the pathogen circumvents host immune responses to establish infection. Furthermore, gene expression profile in vaccinated animals showed BCG-specific responses that are different from those observed in naturally M. bovis-infected wild boar which may be used to monitor BCG vaccination during experimental vaccine studies in this species. Gene expression studies in naturally-infected wildlife bTB reservoirs are important for functional genomics and vaccine studies to aid in disease control.

Wild boar oral vaccination with BCG

Whether wildlife vaccination will become a realistic option in the frame of bTB control in Spain will depend on factors such as the long term success of cattle bTB control in wildlife habitats, and the results of ongoing vaccination trials in other countries, such as Ireland and UK in badgers and New Zealand in possums (Trichosurus vulpecula). Current vaccination research in Spain is focused on wild boar. Delivery, safety and efficacy issues need to be addressed in order to be prepared for eventually licensing a vaccine. Recently, baits for the oral delivery of vaccine preparations to 2-4 month-old wild boar piglets, the preferred age for vaccination, were developed and evaluated. The use of selective feeders during the summer was found to be a potentially reliable bait-deployment strategy (Ballesteros et al. 2009a;2009b). Safety experiments yielded no isolation of M. bovis BCG from faeces, internal organs at necropsy and the environment, even after oral delivery of very high doses. Finally, A c c e p t e d M a n u s c r i p t 15 preliminary vaccination and challenge experiments suggest that a single oral BCG vaccination may protect wild boar from infection by a virulent M. bovis field strain (Ballesteros et al. 2009c).

Discussion

The ecology of M. bovis in Spain represents a multi-host system, with a relevant role for wildlife, but also for domestic reservoirs and for the environment. There are huge differences between regions, with almost no bTB and no wildlife reservoirs in the islands; almost no bTB and few wildlife and habitat risk factors in the Atlantic mainland; and still high bTB prevalence in the Mediterranean habitats of the mainland, partly explained by wildlife and habitat risk factors. As bTB prevalence has dropped in livestock, the relative importance of a potential wildlife reservoir may increase. Thus, wildlife aspects need to be considered in the strategy to control bTB in Spain (http://ec.europa.eu/food/animal/diseases/eradication/reportsanco-10584-2007btbsubgroupsevillarev110-1-08.pdf ). Moreover, wildlife numbers and wildlife distribution are continuously changing, with potential impacts on epidemiology.

Regarding wildlife, steps have been taken towards disease control. Firstly, new regulations of wild animal translocations (Royal Decree 1082/2009; http://www.boe.es/boe/dias/2009/07/23/pdfs/BOE-A-2009-12206.pdf) and on disposal of hunting carcass remains (both at the EU and the regional level), if properly enforced, will contribute to limit bTB spread. Secondly, new diagnostic tools and setting up a Wildlife Disease Surveillance scheme will allow documenting the spatial and temporal trends of bTB in wildlife. In Spain, the variety of surveillance systems is broad, and a need exists to find effective ways to share and exchange data among regions and coordinate on a global scale.

A c c e p t e d M a n u s c r i p t 16 This will hopefully improve our ability to identify new health risks in wildlife populations and enhance our capability to manage them when necessary [START_REF] Artois | Wildlife disease surveillance and monitoring[END_REF].

Wildlife fencing, feeding, and keeping overabundant populations for hunting or other recreational purposes, is still a problem of paramount importance in disease epidemiology, not only regarding bTB (Gortazar et al. 2006). To contribute to solve this problem, census methods have been developed [START_REF] Acevedo | Estimation of European wild boar relative abundance and aggregation: a novel method in epidemiological risk assessment[END_REF] or adapted to Mediterranean habitats [START_REF] Acevedo | Estimating red deer abundance in a wide range of management situations in Mediterranean habitats[END_REF]. This allows getting accurate estimations of the wildlife densities before and after a given management. Now it is the responsibility of the environment authorities to achieve a reduction of the current wildlife densities to more sustainable levels, and to contribute also to reduce the spatial aggregation of wildlife at feeders or waterholes (Figure 2, panel b). However, the success of any such management action must be assessed critically, including an analysis of the costs, of the ecological consequences and of the animal and human health, welfare, and conservation benefits. This, in turn, urgently requires applied research regarding waterhole ecology, wild boar and deer space use and movements, carrion and gutpile consumption by birds and mammals, and particularly on the effect of any attempt to control bTB in wildlife, for instance through population control or testing the hypothesis that changes in food or water distribution may affect transmission rates. Ongoing research is also addressing wildlife-domestic livestock interaction in order to provide safer husbandry practices.

Despite the initial characterization of gene expression profiles in wildlife in response to M. bovis infection and BCG vaccination, further experiments are required to identify specific genes associated with protective response in these animals to monitor immune response after vaccination and to establish gene markers for genetic studies and assisted breeding in farmed game.

  regions (the Balearic Islands in the Mediterranean and the Canary

  will provide a first approximation to the relative contribution of wild animals, and a fine biogeographical picture of the problem.

  from hunter-harvested wildlife. However, alternative techniques need to be used in protected wildlife such as badgers, or in protected areas where hunting is banned. Bacteriological culture is the gold standard test for determining bTB infection prevalence. In DNP red and fallow deer, taking culture samples from both the tonsil and retropharyngeal lymph node increased the rate of isolation of M. bovis by 22% over culture of the retropharyngeal lymph node alone (Martín-Hernando et al. 2010). Similarly, 19% of DNP wild boar yielded M. bovis isolates from the tonsil but not from the mandibular LN samples (unpublished data). Hence, pools of both tissues are now used for surveillance purposes. Investigation of wild deer for bTB compatible lesions should ideally include examination of the medial retropharyngeal, left tracheobronchial, mediastinal, mesenteric and ileocaecal lymph nodes (LNs) (Martín-Hernando et al. 2010).

  of ELISA tests for the detection of antibodies against M. bovis will facilitate studies on large areas and long time series. Two recent studies in wild boar coincide in reporting sensitivity of 73-77% and specificity of 96-97%. A close association between strong antibody response and the presence of gross lesions in individuals infected with M.

  challenge trials are currently being conducted with larger numbers of experimental subjects. In parallel, safety issues are being addressed and, in the field, bait delivery experiments with biomarkers are being analyzed to evaluate the coverage of our vaccination strategy and model the possible impact on bTB control in wildlife under the conditions found in Spain. If these experiments progress as expected, work towards licensing the use of oral BCG in free living wild boar will be the goal. However, vaccination will not be a golden bullet but just one of several tools available for bTB control in wildlife in Spain.

  Figure legends

Figure 2 .

 2 Figure 2.-Risk factors for bovine tuberculosis in Spanish wildlife. (a) High wire fencing

Figure 3 .-

 3 Figure 3.-Ten year trends in the prevalence of bovine tuberculosis compatible lesions in

Acknowledgements

We thank many colleagues at IREC for making this research possible. Studies on TB at IREC are supported by Grupo Santander -Fundacion Marcelino Botin, and by TB-STEP EU FP7, INIA-MICINN FAU 06-017, and PAI07-0062-6611 Castilla -La Mancha.

Conflict of Interest Statement

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Inma Aznar
email: inma.aznar@ucd.ie

Guy Mcgrath

Denise Murphy

Leigh A L Corner

Eamonn Gormley

Klaas Frankena

Simon J More

Wayne Martin

James O'keeffe

Mart C M De Jong

Trial Design to Estimate the Effect of Vaccination on Tuberculosis Incidence in Badgers

The principal wildlife reservoir of Mycobacterium bovis in Ireland is the European badger.

Studies in the Republic of Ireland (RoI) have shown that badgers culled in association with cattle herd tuberculosis (TB) breakdowns (focal culling) have a higher prevalence of infection than the badger population at large. This observation is one rationale for the medium term national strategy of focal badger culling. A vaccination strategy for the control of TB in badgers is a preferred long-term option. The Bacillus Calmette-Guérin (BCG) vaccine has been shown to decrease disease severity in captive badgers under controlled conditions. As the vaccine has been tested in a controlled environment with precise information on infection pressure, it cannot be assumed a priori that the effects of vaccination are similar in the wild, where other environmental

A c c e p t e d M a n u s c r i p t 2 and/or ecological factors prevail. For this reason we have designed a vaccine field trial to assess the impact of vaccination on the incidence of TB infection in a wild badger population.

The selected study area for the vaccine trial (approximately 755 square kilometers) is divided into three zones each of which has similar characteristics in terms of size, number of main badger setts, cattle herds, cattle and land classification type. Three vaccination levels (100%, 50% and 0%) will be allocated to the three zones in a way that a gradient of vaccination coverage North to South is achieved. The middle zone (Zone B) will be vaccinated at a 50% coverage but Zone A and C will be randomly allocated with 100% or 0% vaccination coverage. Vaccination within Zone B will be done randomly at individual badger level.

The objective of this paper is to describe the design of a field TB vaccination trial for badgers, the epidemiological methods that were used to design the trial and the subsequent data analysis. The analysis will enable us to quantify the magnitude of the observed vaccination effect on M. bovis transmission in badgers under field conditions and to improve our knowledge on the biological effects of vaccination on susceptibility and infectiousness.

Keywords: Vaccine trial, badgers, BCG vaccine, Ireland, tuberculosis, M. bovis

Introduction

Ireland initiated an eradication program for bovine tuberculosis (bTB) as early as 1950 [START_REF] More | The tuberculosis eradication programme in Ireland: a review of scientific and policy advances since 1988[END_REF]. The adopted test-and-slaughter policy achieved a 97% reduction in cattle tuberculosis prevalence, from 17% to 0.5% prevalence in the initial ten years [START_REF] Watchorn | Bovine tuberculosis eradication scheme 1954-1965[END_REF].

A c c e p t e d M a n u s c r i p t 3 Since then, bTB prevalence has remained relatively unchanged despite the introduction of a range of measures aimed at reducing cattle to cattle transmission [START_REF] Griffin | The role of cattle-to-cattle transmission of Mycobacterium bovis in the epidemiology of tuberculosis in cattle in the Republic of Ireland: a review[END_REF].

In the 1970s in England, badgers were first suspected as a reservoir for Mycobacterium bovis [START_REF] Krebs | Bovine Tuberculosis in Cattle and Badgers[END_REF]. The first infected badger was discovered in Ireland in 1974 [START_REF] Noonan | Wildlife as a possible reservoir of bovine TB[END_REF].

Since then numerous papers have been published that confirm badgers as the main wildlife M. bovis reservoir in Ireland and England [START_REF] Barrow | Aspects of the epidemiology of bovine tuberculosis in badgers and cattle. I. The prevalence of infection in two wild animal populations in south-west England[END_REF][START_REF] Cheeseman | The population structure, density and prevalence of tuberculosis (Mycobacterium bovis) in badgers (Meles meles) from four areas in south-west England[END_REF][START_REF] Fagan | Tuberculosis in badgers in Ireland: Pathology. In: The badger[END_REF][START_REF] Gallagher | Role of infected, non-diseased badgers in the pathogenesis of tuberculosis in the badger[END_REF]. A recent study carried out in Ireland detected a prevalence of 36.3% in badgers trapped as part of Department of Agriculture, Fisheries and Food (DAFF) culling operations; the prevalence reported here was much higher than in previous studies where less comprehensive bacteriological culture methods had been used [START_REF] Murphy | The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture[END_REF].

The precise role of badgers in the dynamics of bTB is not clear. Several studies in Ireland have linked badger removal with a subsequent reduction in bTB incidence [START_REF] Eves | Impact of badger removal on bovine tuberculosis in east County Offaly[END_REF][START_REF] Griffin | The impact of badger removal on the control of tuberculosis in cattle herds in Ireland[END_REF][START_REF] More | The tuberculosis eradication programme in Ireland: a review of scientific and policy advances since 1988[END_REF][START_REF] Olea-Popelka | Targeted badger removal and the subsequent risk of bovine tuberculosis in cattle herds in county Laois, Ireland[END_REF]. However, in a field trial carried out in Britain, the reduction in cattle TB incidence in culled areas was only modest and an increase in TB incidence, albeit transient [START_REF] Jenkins | The effects of annual widespread badger culls on cattle tuberculosis following the cessation of culling[END_REF], was observed in non culled neighboring areas [START_REF] Woodroffe | Effects of A c c e p t e d M a n u s c r i p t 22 culling on badger abundance: implications for tuberculosis control[END_REF]. [START_REF] Pope | Genetic evidence that culling increases badger movement: implications for the spread of bovine tuberculosis[END_REF] concluded that the increased prevalence observed in neighboring areas was associated with medium and long-distance badger dispersal and emphasized the importance of taking into account the potential negative effects associated with badger dispersal when using culling as a disease control strategy. Although there are discrepancies between different studies about the efficiency of badger culling in the control of bTB, they all provide compelling evidence that badgers play an important role in the maintenance of it. Therefore, addressing infection in badgers is considered vital when trying to control bovine tuberculosis in the aforementioned countries. In the short-to medium-term in Ireland, focused badger culling is being conducted as part of a broader national TB control programme, to limit In 2001, a 10 year work program was designed in Ireland to study the possibility of using Bacillus Calmette-Guérin (BCG) vaccine in badgers to assist in the control and eradication of TB in cattle.

The program consisted of a sequence of experimental studies carried out initially with captive badgers [START_REF] Corner | Experimental tuberculosis in the European badger after endobronchial inoculation of Mycobacterium bovis: I. Pathology and bacteriology[END_REF][START_REF] Lesellier | Immunological responses and A c c e p t e d M a n u s c r i p t 21 protective immunity in BCG vaccinated badgers following endobronchial infection with Mycobacterium bovis[END_REF]. Although vaccines can be tested in a controlled environment for evidence of both protection and decreased transmission, it cannot be assumed a priori that the effects of vaccination are similar in the wild where other factors may play a role. For this reason, a vaccine field trial to assess the impact of vaccination on the incidence of tuberculosis in a wild badger population has been designed as part of the ten year project.

The objective of this paper is to describe a field trial design, the epidemiological methods that were used to design the trial and the subsequent data analysis. The analysis will enable us to quantify the magnitude of the observed vaccination effect on transmission under field conditions and to improve our knowledge on the biological effects of the vaccination on susceptibility and infectiousness of badgers.

3 Theoretical basis: a review what reduction in infectiousness can be achieved. The effect of interest will determine the study unit, parameters of effect, as well as the level of information required [START_REF] Halloran | Study Designs for Evaluating Different Efficacy and Effectiveness Aspects of Vaccines[END_REF]. The main question of interest in our study is to determine the efficiency of badger vaccination in reducing M. bovis transmission. Specifically we are interested in the value of the reproduction ratio (R) at different levels of vaccination (p); ie R(p). We will clarify how to estimate R(p) subsequently.

Initial considerations in designing a vaccine trial

Vaccines that reduce transmission in a population can have a beneficial effect in vaccinated as well as in unvaccinated individuals. Most studies of vaccine efficacy emphasize the direct benefit of vaccination (often called 'protective ability') to vaccinated individuals. Following [START_REF] Halloran | Design and Interpretation of Vaccine Field Studies[END_REF], we denote this as VE S or Vaccine Efficacy for Susceptibility. In addition, there often are indirect benefits for both vaccinated and unvaccinated individuals which Halloran et al. denoted as Vaccine Efficacy for Infectiousness (VE I ). These measures will be estimated in this trial; however, for subsequent modelling purposes we will emphasize R(p)

Parameters of effect

In order to estimate R(p), we define four transmission parameters:

 transmission from a vaccinated to a vaccinated badger (β VV );

 transmission from a vaccinated to an unvaccinated badger (β VU );

 transmission from an unvaccinated to a vaccinated (β UV ), and  transmission from an unvaccinated to an unvaccinated badger (β UU ). represented by R to distinguish them from R 0 . The reproduction ratio as a function of the proportion of vaccinated badgers R(p) can be determined from the transmission rates described above and the time that infected badgers remain infectious (T). Transmission rates are a combination of the infectiousness of the donor and the susceptibility of recipient individuals, and since vaccination with BCG has the potential to affect both of these, R(p) will be an important parameter for understanding the impact of badger vaccination in disease transmission and population dynamics of M. bovis. Ultimately, the R(p) obtained from the badger vaccine trial will be used in further modelling aimed at assessing badger vaccination as a strategy to control/eradicate M. bovis infection in cattle.

As noted above, vaccine efficacy/effectiveness has been traditionally defined as 1 minus some measure of relative risk (RR) of the incidence of disease in the vaccinated group compared to the incidence in the non-vaccinated group [START_REF] Halloran | Design and Interpretation of Vaccine Field Studies[END_REF]. In the past, the main objective of human vaccine studies was to measure individual protection against infection or disease (VE S ).

Perhaps less appreciated, but not less important, is the ability of a vaccine to reduce the duration or severity of the infectiousness of those vaccinates that become infected (VE I ) [START_REF] Longini | Optimal vaccine trial design when estimating vaccine efficacy for susceptibility and infectiousness from multiple populations[END_REF]. The latter effect has been reported in vaccine studies using BCG vaccine by the subcutaneous or mucosal routes in badgers [START_REF] Corner | Vaccination of European badgers (Meles meles) with BCG by the subcutaneous and mucosal routes induces protective immunity against endobronchial challenge with Mycobacterium bovis[END_REF]. In these experiments, M. bovis was recovered from both vaccinated and non-vaccinated badgers after being challenged with the mycobacterium; however, a reduction in the size, number and distribution of gross and histological lesions in vaccinated badgers compared to non-vaccinated badgers was demonstrated.

Vaccination did not confer individual protection against infection in the mentioned study, but this A c c e p t e d M a n u s c r i p t 7 has to be cautiously interpreted as vaccine protection could be dependent on the infection dose. It is not known what the infectious dose is in natural infections. In field vaccine trials in possum in New Zealand, using conjunctival and intranasal vaccination [START_REF] Corner | The efficacy of bacille Calmette-Guérin vaccine in wild brushtail possums (Trichosurus vulpecula)[END_REF] and more recently vaccination by oral delivery [START_REF] Tompkins | Oral vaccination reduces the incidence of tuberculosis in free-living brushtail possums[END_REF], significant protection against natural exposure was seen in the vaccinated group compared to the control groups. Protection was much higher than predicted from previous studies where possums had been experimentally challenged [START_REF] Corner | Aerosol vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin: the duration of protection[END_REF][START_REF] Buddle | Oral vaccination of brushtail possums with BCG: investigation into factors that may influence vaccine efficacy and determination of duration of protection[END_REF].

Estimation of R(p), VE S and VE I will give us a more detailed understanding of the ways BCG vaccine works in a wild badger population.

Epidemiological contribution to the design of the vaccine trial

Study site

Prior to deciding on our study site for the badger vaccine trial, several epidemiological and logistic factors were considered:  Culling history. Because of the badger culling pollicy in Ireland, it was important to have confidence that the area selected for the trial had been protected from culling for some time before the commencement of the trial. Badger culling in that area could have had a negative effect not just in the total number of individuals captured but also on the initial prevalence of TB among badgers.

 Knowledge of sett location. Knowledge of the area in terms of sett location was considered an advantage and helped in dividing the study area into three similar zones (see below). Based on these criteria, the area selected for the vaccine trial is located in County Kilkenny (Figure 1). The size of the area is approximately 755 square kilometers. This area had been part of one of the reference areas in the Four Area Project (FAP) [START_REF] Griffin | The impact of badger removal on the control of tuberculosis in cattle herds in Ireland[END_REF] and will have been protected from culling for at least two years before the start of the vaccine trial. A prevalence of infection of 30% is expected based on historical data from neighbouring areas.

Trial design

A vaccine trial to exclusively determine VE S can be designed by vaccinating one or several populations with the same vaccination coverage, with coverage being strictly inferior to 100% (40%, 50%, 60%, etc) so that a number of infections within the vaccinated group can occur.

Because we aimed to estimate both VE S and VE I , then two populations vaccinated with different vaccination coverage are required [START_REF] Longini | Optimal vaccine trial design when estimating vaccine efficacy for susceptibility and infectiousness from multiple populations[END_REF]. In this paper it is explained how the vaccination fractions for these two populations have to be selected to maximise the differences on indirect effects between the two populations. This can be achieved with one population vaccinated at 0% and the other vaccinated at the highest percentage that allows a minimum number of infections to happen (critical vaccination fraction). Likewise to estimate the four betas (transmission rates), different levels of coverage are needed (DeJong et al., 1996).

Our objectives could have therefore been attained with two populations, one vaccinated at 0% and the other at 100% vaccination coverage (badger trapping rates will never be 100% effective; also the dynamics of the badger population ensures a certain number of susceptible badgers every A c c e p t e d M a n u s c r i p t 9 year and therefore a certain number of infections). We chose though to include a third population vaccinated at 50% to optimise the design in two ways: firstly by making sure that there will be enough new infections even if the "100%" vaccination would lead to R<1 and secondly by allowing us to estimate the area effect (in the case of only two populations, the parameters would be estimated from different populations and it would not be possible to disentangle the area effect).

The trial area is divided into three zones with similar characteristics in terms of size, number of main badger setts, cattle herds, cattle and land classification (Figure 2). In these zones, vaccination coverage will be of allocated 100, 50 and 0%, with the gradient of coverage (either 100% to 0% from north to south, or vice-versa) being allocated randomly at the start of the trial.

Badger data were acquired through rigorous surveying of the entire study area. Previous surveying had been performed in sections of the study area as part of the FAP reference area and through the activities of the DAFF's licensed badger culling policy on setts adjacent to herd bTB breakdowns. Setts previously recorded were revisited during the recent survey and assessed for signs of activity. All bovine data were derived from the Animal Health Computer System (AHCS) and farm outlines were taken from the Land Parcel Identification System (LPIS). Land use classifications were defined using the CORINE dataset (Coordination of Information on the Environment, 2000). Using ArcMap 9.2 (ESRI, Redlands, CA, USA) with geo-rectified colour orthophotography and vectorized 1:50,000 data (Ordnance Survey Ireland, Dublin, Ireland), natural boundaries, where possible, were selected to define the perimeter and internal boundaries of the study area. The aim was to achieve similar infection pressure from cattle and badgers in the three zones.

Badgers will be captured, treated with a vaccine or a placebo depending on the randomly allocated treatment and then released. In zone B (with 50% vaccine coverage), each badger at A c c e p t e d M a n u s c r i p t 10 first capture will be randomly allocated to either vaccine or placebo. In each zone throughout the study period, the treatment will be administered every year to avoid possible waning of vaccine effects. Live M. bovis BCG strain Danish will be used. It will be prepared in a lipid formulation for oral delivery, containing 10 8 colony forming units/ml. A lipid-only placebo with identical visual characteristics, texture and viscosity to the vaccine, and in identical syringes, will also be used (Aldwell et al., 2003a: Aldwell et al., 2003b). Vaccine and placebo control samples will be coded at the laboratory where they are prepared, and neither field staff nor data analysts will be aware of the vaccine status of individual animals.

The trial will employ a capture-tag-release regime with both cages and stopped wire restraints being used. Badgers in the trial will be 'hand vaccinated'; that is, each animal will be individually orally dosed with the vaccine. Each badger will be permanently identified with a tattoo and passive transponder (microchip) when first captured. At each capture, badgers will be examined and a blood sample collected. Humoral immune responses (serologic titres) will be used to determine the badger's infection status and to detect a change in infection status; that is, to detect both pre-existing infection in badgers as they are recruited to the study and the occurrence of new infections on recapture. Key data, including sex, estimated age (cub, juvenile, and yearling, adult and old adult), body weight, presence of injuries and the GPS location of the cage trap or restraint, will be recorded at each badger capture. All data collected in the field will be recorded onto handheld computers. The trial will last four years and there will be two 'catching' sweeps of the entire area each year. At the end of the trial, badgers in the three zones will be depopulated and a detailed post mortem examination will be conducted on all badgers, involving an examination for gross pathology and the collection of samples for histopathology and bacteriology. The severity of infection will be assessed from the number, distribution and the severity of gross lesions, the number and distribution of histological lesions, and the number and distribution of culture positive tissues and the bacterial load in those tissues.

A c c e p t e d M a n u s c r i p t 11 Badger removal will be undertaken within the trial area when three or more standard reactors are disclosed in a herd only if active badger setts are found in the farm and all other sources of infection (residual, purchase and farm to farm spread) have been ruled out by an epidemiological investigation. If culling of badgers is deemed necessary for control of tuberculosis in cattle herds, the culling will be carried out by field staff of the project when they next trap in the designated area.

Analysis of the vaccine trial data

General description

The outcome from the vaccine trial will be in the form of a Bernoulli experiment: as badgers are trapped and a blood sample taken, we will gather information on whether these badgers have become or "are" infected (assigned value 1) or not (assigned value 0) during the time at risk (time between two subsequent trappings). Infection in this case will be defined by serologic results i.e. by sero-positivity. Records on individual badgers will be taken at each successful capture (not necessarily at each trapping exercise) such as location of the badger at the time of the trapping (zone A, B or C) and its vaccination status. Other demographic data will be also recorded.

From the observations at each subsequent capture of each individual badger, the following variables will be extracted: The rate at which vaccinated/non-vaccinated badgers acquire infection from vaccinated and nonvaccinated badgers (β VV , β VU , β UV , β UU ) can be estimated based on the observed probability of becoming infected for each of these individuals. The estimated βs will be used to calculate VE S , VE I and the reproduction ratio as a function of the vaccination coverage (R(p)).

 Time interval (Δt) between the two captures

Estimation of the transmission parameters

The rate at which vaccinated/non-vaccinated badgers acquire infection from vaccinated and nonvaccinated badgers (β VV , β VU , β UV , β UU ) can be estimated based on the observed probability of becoming infected for each of these individuals. For the purposes of explanation, we shall at first ignore the vaccination state of the badgers. A stochastic susceptible-infectious (SI) model can be used then to describe the transmission of M. bovis in the trial where transmission can occur if an infectious and a susceptible individual make contact. The number of infectious contacts (NIC, contacts with an infectious individual) encountered by each randomly chosen susceptible individual in a period of time Δt can be expressed as: From the above it can be derived that the number of 'successful' infectious contacts (infectious contacts that result in transmission events) encountered by this randomly chosen individual in a period of time Δt follows a Bernoulli distribution with probability:

N t I e p / 1       
The observed number of new infections (C) among all susceptible badgers at the end of the time interval between two captures can then be modelled using a binomial distribution where S is the number of susceptible badgers at the beginning of the time interval and the probability that each of these badgers will become infected during that time interval is defined by Let's assume that a badger that was originally trapped in zone A at time 't' is trapped again in zone A at time 't+1' and that this badger had been vaccinated. Vaccination coverage in zone A is targeted to be 100%, but since not all badgers will be trapped in each trapping exercise it will take 2-3 trapping rounds to approach this goal. Our hypothetical badger will then have infection pressure coming from both vaccinated and non-vaccinated infected badgers. The number of infectious contacts encountered by this randomly selected badger in that period of time is defined by:

N t I e / 1       ) 1 ( ) ( / N t I e S C E         A c
t N I N I NIC U UV V VV      ) / * / (  
If we assume that there is a multiplicative effect on NIC as the fraction of infected vaccinated badgers increases, after doing some algebraic manipulations [START_REF] De | Quantitative investigation of population persistence and recurrent outbreaks of bovine respiratory syncytial virus on dairy farms[END_REF]:

F F        ) 1 ( *   or: t evA Pr e NIC VA UV VV UV F ) (          
where F VA is the fraction of infected badgers that are vaccinated in zone A and PrevA is the average prevalence of infection in zone A during Δt. If we set β UV = K 0 and (β VV -β UV ) =K 1 then we can write:

) 1 ( ) ( Pr ) 1 0 ( t evA e V VA F K K e S C E         
Since we will know the observed infection status of the specific badger (C V ) at the end of the time interval (Δt), we can fit a GLM with Log (PrevA Δt) as an offset and calculate K 0 and K 1 . We can subsequently calculate β UV and β VV as:

β UV =Exp[K 0 ] β VV =Exp[K 0 +K 1 ]
If we apply the same logic to model the observed infectious status of an unvaccinated badger (C U ) that was trapped in zone A at time t and at time t+1, we could estimate β UU and β VU as: This way we can estimate the four betas based on incidence data from all three zones. In zone C there will be no (or very few) vaccinated badgers so F VC will be zero and that zone does not contribute to the estimation of k 1 and thus that area does not give information for β VU .

) 1 ( ) ( Pr ) 1 0 ( t evA e U VA F k k e S C E         
Vaccine Efficacy for Susceptibility can be calculated then as:

          )] * * ) 1 ( * * 4 ) * * ) 1 (( * ) 1 [( * 2 1 ) ( 2      
where T is the duration of the "infectious" period of a typical infected individual (for relative comparisons of R(p) knowledge of T is not essential).

A c c e p t e d M a n u s c r i p t 17 For decision making, if p=0 and β UU T <1, then no vaccination is needed; if p=1 and β VV T>1, vaccination will not stop spread of the disease. Otherwise, the vaccination fraction necessary in order to achieve R(p)<1 can be calculated.

Concluding remarks

The aim of this paper was to present the theory applied to the design of a badger vaccine trial, as well as the epidemiological methodology and statistical analysis that will help to interpret the results obtained.

The vaccine trial area was divided into three zones A, B and C, such that differences in infection pressure in the three zones at the beginning of the trial will be minimized. However, there is an inherent assumption that the contact patterns between badgers will be similar in the three zones.

We believe that this is a reasonable assumption as the size of the total area is sufficiently small, and the landscape and distribution of setts is very similar in the three zones. Further, changes with respect to infection pressure in the three zones will occur as the vaccine starts working. Our model is designed to adjust for these changes. For simplicity, we have only presented the analysis relevant to badgers repeated trapped in the same zone. However, the final model can be modified to accommodate other scenarios where badgers move from one zone to another.

Where possible, natural boundaries are used to define the perimeter of the study area. The internal boundaries between the three zones include streams, rivers and roads. The external boundaries, while not bio-secure, should be substantial enough to delineate badger territories. This would reduce the extent of typical badger movement into and out of the study area.

  areas where TB incidence in cattle is high. However, badgers are a protective species in the Republic of Ireland (ROI) under the 1976 Wildlife Act. Consequently, alternative strategies to badger culling are being sought in the long term for the control of bovine tuberculosis.

  (s) of interest is essential when designing a trial to evaluate the effects of vaccination. Different vaccination programs have different aims; the question of interest could vary, for example from how good vaccination is in protecting the individual against infection to

  of secondary cases caused by one typically infected individual in a fully susceptible population (Diekmann and Heesterbeek 2000) is called the Basic Reproduction Ratio (R 0 ). Reproduction ratios of host populations in which interventions are taking place are often

  A c c e p t e d M a n u s c r i p t 8  Community and technical support. Good support from the local farming communityas well as from from both the District Veterinary Offices (DVOs) and Regional Veterinary Laboratories (RVLs) was vital.



  Disease status of the badger at the beginning and at the end of the time interval  Vaccination status of the badger  Zone where the animal was observed at the beginning and end of the time interval (A, B or C)  Average prevalence of infection in the zone where the badger was caught during Δt (PrevA, PrevB, PrevC)  Average fraction of infected badgers that are vaccinated during Δt in the zone where the badger was caught (Fv A , Fv B , Fv C )

  Prevalence of infected badgers* * For our purpose infected badgers are deemed to be infectious

  results we will be able to determine the number of new cases among the susceptible badgers (C). By using a Generalized Linear Model (GLM) with a complementary-loglog link function, a binomial error function, with binomial total S β can be estimated.Following the introduction of the vaccination protocol, there will be heterogeneity in the vaccination status of badgers; we will have four different β values with two sub indexes, of the type β vu . The first sub index will refer to the vaccination state of the infectious badger and the second sub index to the vaccination state of the susceptible animal (to which the first has made contact). Clearly, we know the vaccination status of the receiving susceptible badger and thus we can calculate β .u and β .v separately. However to account for donor (infectious) badger, we use the differences in the fraction of infected badgers that are vaccinated in the different zones.

  =Exp[k 0 ] and β VU =Exp[k 0 + k 1 ]

Figure 2 .

 2 Figure 2. Map showing study area divided into three zones A, B and C (grey, pink and yellow

Although the vaccine trial will not determine whether bTB can be eradicated in cattle and badgers through a strategy of badger vaccination, the parameters obtained in the vaccine trial will be used in a mathematical model of bTB transmission (currently under development) in order to assess different control and eradication options for bTB in cattle in the Republic of Ireland.

Conflict of Interest Statement

The authors have not declared any conflict of interest.