Bovine tuberculosis, caused by Mycobacterium bovis and other closely related mycobacteria of the Mycobacterium tuberculosis complex, is endemic in many countries. These mycobacteria can infect a wide range of domestic and wild animals [1–3]. Wild animals become increasingly important in the spread and maintenance of M. bovis infection, especially when the efforts to eradicate the disease in livestock have reduced its incidence in domestic cattle [2, 4]. The existence of wildlife tuberculosis (TB) reservoirs and the difficulty of controlling the disease in these species is the most important complication in eradication programs [3]. Well known examples of wildlife TB reservoirs include the badger (Meles meles) in the United Kingdom and Ireland [5, 6], the brushtail possum (Trichosurus vulpecula) in New Zealand [3], the white-tailed deer (Odocoileus virginianus) in the north of the United States of America [7], the buffalo (Syncerus caffer) in South Africa [8, 9], or the bison (Bison bison) in Canada [10].

In Spain, TB prevalence is relatively low in cattle (0.42 in 2006), but the infection persists in other livestock including goats and free-ranging swine, and there is a wide range of wild animal species susceptible to this disease [11]. Previous research suggested inter-specific transmission of the M. tuberculosis complex among wild ungulates and livestock [11–14]. The European wild boar (Sus scrofa) is one of the ungulates involved in the epidemiology of tuberculosis in Spain. Recent epidemiological, pathological and microbiological evidence strongly suggests that, at least in Spanish Mediterranean ecosystems, wild boar are able to maintain TB infection in the wild and most likely can transmit the disease to other species, acting as a true wildlife reservoir [15]. Depending on risk factors such as host age and management including feeding and fencing, wild boar TB prevalence ranges based on gross pathology from 18 to 100% [16, 17]. The diagnosis of M. bovis infection in live animals generally depends on the cellular immune response to M. bovis antigens in the first stages of the infection [18]. The most usual technique is the hypersensitivity test, based on the intradermal injection of raw antigens [19–21]. This skin testing technique, described by Robert Koch, is still the most widely used tuberculosis diagnostic method in livestock. It is also used in wild ruminants [22, 23]. However, skin tests have a limited sensitivity, and non specific reactions may occur in animals sensitized by mycobacteria other than those of the M. tuberculosis complex [24, 25].

In wild animals, any diagnostic test has an associated risk during the capture, both for the people who handle the animal and for the animal itself, due to handling stress and injuries. Moreover, preliminary results of skin testing in wild boar of known TB status suggest a low sensitivity (unpublished data). Thus, the possibility of a test based on a single sampling would be highly desirable for assessing the prevalence, studying the mechanisms of transmission and monitoring the effects of control measures.

While the delayed-type hypersensitivity reaction is indicative of infection or exposure, antibody formation appears to be more closely related to the extent of bacterial multiplication and antigenic load in the infected individual. ELISA testing is not routinely used in bovine TB control programs mainly due to a reduced sensitivity [26], although it has been suggested to be used as a complement to the tuberculin test, especially for the detection of anergic tuberculous cattle [27, 28].

The aim of this study was to develop and validate an ELISA test for the detection of Mycobacterium bovis antibodies in wild boar serum. To achieve this goal, the humoral immune response measured by this test was first measured in captive wild boar sensitized with inactivated bacterial antigens and then results were validated with sera obtained from wild boar of known microbiological TB status.

To date, studies are not available on the experimental use of antigens to characterize the immune response of wild boar and pigs against M. bovis. Herein, inactivated mycobacterial antigens were successfully used to stimulate a specific humoral immune response in healthy wild boar. Injection of the antigens caused an increase of specific antibodies, but the administration of a second antigen dose did not raise the antibody levels so much. A strong cross reaction between antibodies specific for M. avium and M. avium paratuberculosis (WB 2 and WB 3) and the bovine PPD ELISA was not observed except to some extent for WB2 after the boosting. Thus, the immunization assay provided negative and positive M. bovis control sera to use in the development of the ELISA, as well as for reference in the future.

It is generally recognized that humoral immunity is not important for tuberculosis diagnosis [29], but it may nonetheless be useful to detect animals with severe illness since antibody concentration is related to lesion distribution and severity, as well as to the number of bacilli [18, 22, 26, 30–32]. Mycobacterial infections induce antibody production in ruminants, but the profile of immunoglobulin (Ig) expression in M. bovis infected animals is poorly understood [33–36]. In previous studies, Ig heavy and light chains were up-regulated in European wild boar infected with M. bovis [37]. However, serum determinations suggested elevated levels of IgG in uninfected wild boar when compared to M. bovis infected animals [38]. The mechanism of Ig differential expression in M. bovis infected wild boar is unknown but may reflect different stages during mycobacterial infection. Furthermore, differential gene expression analysis in response to mycobacterial infection in wild boar suggest that antibody responses against M. bovis may be important in natural infections of wildlife species and may be used for bTB surveillance and treatment monitoring [35, 39–41].

Previous studies using ELISA tests achieved 74% sensitivity and 90% specificity in cattle [26], and 79% to 98% specificity and 37% sensitivity in ELISA used in badgers [41–43], although the latter was increased for badgers with progressive tuberculosis [44]. In addition, a positive ELISA result in badgers was correlated with an enhanced likelihood of a future positive culture result [45]. Various mycobacterial antigens have also been used for bovine tuberculosis ELISA in other studies. This includes for example ELISA based on MPB70 and MPB83 in cattle, which have reported specificities of 89% and 96.4%, and sensitivities of 18.1% and 37.5% [46, 47]. The results obtained in the current study, thus, indicate that the ELISA performance was substantially better for wild boar, especially in terms of sensitivity (72.60%) and without a significant loss in specificity (96.43%) despite the fact that it is generally recognized that the sensitivities and specificities of ELISA protocols for serodiagnosis of bovine tuberculosis are low as compared to those for other diseases [48].

At the chosen cut-off, there were a few reference negative animals that had medium level antibody reactivity against bovine PPD. It might be possible that some of these animals were infected individuals without visible lesions and bacteria not found, or true non-infected but exposed to M. bovis infection, that is, potentially resistant animals. Actually, ELISA positive results are usually considered only as evidence of exposure [17] and not necessarily of current infection. Infections with other mycobacteria causing cross-reactivity cannot be completely ruled out although, given the origin of the animals in infected areas and the EI gap to the rest of negative controls, we think they are less likely to be involved. On the other hand there were animals where M. bovis was isolated that had very low EI readings. These animals might represent recently infected individuals not having developed yet a humoral immune response or anergic animals with limited immune responses due to poor body condition.

We conclude that the use of the serological ELISA test developed herein may contribute to the diagnosis of TB in wild boar and probably also in pigs, with an acceptable sensitivity and specificity and without the need to handle the animals twice as in the skin test. This ELISA test could be used in the control of TB in wild boar through "test & cull" schemes and in large-scale surveys [49]. These results support the use of the ELISA test to complement other techniques based on cellular response to characterize mycobacterial infection in wild boar.