Differential Cell Composition and Cytokine Expression Within Lymph Node Granulomas from BCG-Vaccinated and Non-vaccinated Cattle Experimentally Infected with Mycobacterium bovis.

Cattle vaccination against bovine tuberculosis (bTB) has been proposed as a supplementary method to help control the incidences of this disease. Bacillus Calmette-Guérin (BCG) is currently the only viable candidate vaccine for immunization of cattle against bTB, caused by Mycobacterium bovis (M. bovis). In an attempt to characterize the differences in the immune response following M. bovis infection between BCG-vaccinated and non-vaccinated animals, a combination of gross pathology, histopathology and immunohistochemical (IHC) analyses was used. BCG vaccination was found to significantly reduce the number of gross and microscopic lesions present within the lungs and lymph nodes. Additionally, the microscopically visible bacterial load of stages III and IV granulomas was reduced. IHC using cell surface markers revealed the number of CD68+ (macrophages), CD3+ (T lymphocytes) and WC1+ cells (γδ T cells) to be significantly reduced in lymph node granulomas of BCG-vaccinated animals, when compared to non-vaccinated animals. B lymphocytes (CD79a+) were significantly increased in BCG-vaccinated cattle for granulomas at stages II, III and IV. IHC staining for iNOS showed a higher expression in granulomas from BCG-vaccinated animals compared to non-vaccinated animals for all stages, being statistically significant in stages I and IV. TGFß expression decreased alongside the granuloma development in non-vaccinated animals, whereas BCG-vaccinated animals showed a slight increase alongside lesion progression. IHC analysis of the cytokines IFN-γ and TNF-α demonstrated significantly increased expression within the lymph node granulomas of BCG-vaccinated cattle. This is suggestive of a protective role for IFN-γ and TNF-α in response to M. bovis infection. Findings shown in this study suggest that the use of BCG vaccine can reduce the number and severity of lesions, induce a different phenotypic response and increase the local expression of key cytokines related to protection.

Non-tuberculous mycobacteria in wild boar (Sus scrofa) from Southern Spain: epidemiological, clinical and diagnostic concerns.

Non-tuberculous mycobacteria (NTM) are widely distributed in the environment, particularly in wet soil, marshland, rivers or streams, but also are causative agents of a wide variety of infections in animals and humans. Little information is available regarding the NTM prevalence in wildlife and their effects or significance in the bovine tuberculosis (bTB) epidemiology and diagnosis. This research shows the most frequently NTM isolated in lymph nodes of wild boar (Sus scrofa) from southern Spain, relating the NTM presence with the individual characteristics, the management of animals and the possible misdiagnosis of Mycobacterium bovis in concurrent infections. A total of 219 NTM isolates were obtained from 1249 wild boar mandibular lymph nodes sampled between 2007 and 2011. All but 75 isolates were identified by the PCR-restriction analysis-hsp65, and a partial sequencing of the 16S rDNA was carried out to identify the rest of the isolates. Results showed that Mycobacterium chelonae was the most frequently isolated NTM specie (133 isolates, 60.7%), followed by Mycobacterium avium (24 isolates, 11%). No relation was found regarding sex, body condition and management, but M. chelonae was more frequently detected in adults, whereas M. avium was more prevalent in subadults. The high NTM prevalence observed in the studied wild boar populations could make difficult the bTB diagnostic.

Influence of porcine circovirus type 2 infections on bovine tuberculosis in wild boar populations.

The wild boar is an important reservoir of bovine tuberculosis (bTB) in south-western Spain. Some risk factors such as wild boar density or age have been associated with the presence of high prevalences of bTB in wild boar. However, the influence of other risk factors such as co-infections with other pathogens has not yet been studied. This work aims to assess the influence of porcine circovirus type 2 (PCV-2) infection on bTB prevalence and bTB lesional patterns observed in wild boar. The presence of bTB-like lesions was evaluated in 551 hunted wild boar from 11 different game estates in south-western Spain, with a known history of bTB. Tuberculosis prevalences in each estate were calculated based on the percentage of animals found with bTB-like lesions. The percentage of animals with generalized bTB lesional patterns (bTB lesions in more than one organ) was also assessed. The prevalence of PCV-2 was studied in each estate using a specific PCR assay. The relationship between PCV-2 and bTB prevalences and between PCV-2 infections and the presence of generalized lesional patterns in wild boar were analysed. A statistical relationship between the prevalences of bTB and PCV-2 was found, with bTB prevalences being higher in estates where prevalences of PCV-2 were high. On the other hand, animals infected with PCV-2 were more likely to develop a generalized lesional pattern. Porcine circovirus type 2 prevalences seem to be associated with prevalences of bTB in wild boar. PCV-2 infection may aggravate the development and severity of bTB, favouring the presence of generalized lesional patterns and raising the risk of contagion in these estates. The implementation of sanitary measures that focus on the control of PCV-2 infection may be necessary as a preliminary measure in bTB control programmes for wild boar.

Immunopathology of granulomas produced by Mycobacterium bovis in naturally infected wild boar.

Limited information has been published on the wild boar immune response against bovine tuberculosis (bTB) and the immunopathogenesis of the pathological hallmark (granuloma) in this species. The main objectives of this study were, on the one hand, to characterize the histopathological features (number of acid-fast bacilli (AFB) and multinucleated giant cells (MNGCs) and the immunohistochemical distribution of different cell subsets (CD3+, CD79a+ and MAC387+) and chemical mediators (iNOS and IFN-γ) in the different developmental stages of granulomas produced by the natural infection of Mycobacterium bovis (M. bovis) in wild boar. On the other hand, the study also aimed to analyze the mechanisms underlying the marked differences in the typical lesional patterns observed in M. bovis infections of wild boar (contained, not generalized) and those previously described in fallow deer (poorly contained, generalized). The majority of granulomas analyzed (95.3%) did not show any AFB with the ZN stain and a low number of MNGCs were identified in the different granuloma stages. The immunohistochemical analysis showed that MAC387 was the only immune marker that produced decreasing positivity by granuloma stage, being statistically significantly lower in stages III and IV when compared to stage I and II. Immune markers for lymphocyte cells (CD3 and CD79a) showed a slight rise in the positivity (which was not statistically significant) in the advanced granuloma stages. In keeping with the presence of large numbers of T cells and macrophages, there was a consistently high level of expression of IFN-γ at all stages of granuloma development without a statistical significant decrease in advanced stages. Also related with the higher presence of macrophages in stage I and II, the expression of iNOS was higher in early stages and sustained until stage III, showing a non statistical significant decrease in stage IV. The macrophage and iNOS activity are more intense and sustained along the granuloma development than those described in fallow deer. Immunohistochemical protocols with a panel of markers for wild boar different cells subsets (CD3+, CD79a+ and MAC387+) and chemical mediators (iNOS and IFN-γ), and their use to further investigate the immune response in this species are provided.

Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain.

Research on management of bovine tuberculosis (bTB) in wildlife reservoir hosts is crucial for the implementation of effective disease control measures and the generation of practical bTB management recommendations. Among the management methods carried out on wild species to reduce bTB prevalence, the control of population density has been frequently used, with hunting pressure a practical strategy to reduce bTB prevalence. However, despite the number of articles about population density control in different bTB wildlife reservoirs, there is little information regarding the application of such measures on the Eurasian wild boar (Sus scrofa), which is considered the main bTB wildlife reservoir within Mediterranean ecosystems. This study shows the effects of a management measure leading to a radical decrease in wild boar population density at a large hunting estate in Central Spain, in order to assess the evolution of bTB prevalence in both the wild boar population and the sympatric fallow deer population. The evolution of bTB prevalence was monitored in populations of the two wild ungulate species over a 5-year study period (2007-2012). The results showed that bTB prevalence decreased in fallow deer, corresponding to an important reduction in the wild boar population. However, this decrease was not homogeneous: in the last season of study there was an increase in bTB-infected male animals. Moreover, bTB prevalence remained high in the remnant wild boar population.

Comparative pathology of the natural infections by Mycobacterium bovis and by Mycobacterium caprae in wild boar (Sus scrofa).

The potential role of wild animals in the maintenance and spread of tuberculosis (TB) infection in domestic livestock is of particular importance in countries where eradication programs have substantially reduced the incidence of bovine tuberculosis but sporadic outbreaks still occur. Mycobacterium bovis is the agent mainly isolated in wildlife in Spain, but recently, infections by Mycobacterium caprae have increased substantially. In this study, we have analysed 43 mandibular lymph nodes samples containing TB-like lesions from 43 hunted wild boar from Madrid and Extremadura (central and south-western regions of Spain). After isolation, identification and typing of Mycobacterium tuberculosis complex isolates, we found that 23 mandibular lymph nodes involved M. caprae infections and 20 M. bovis. The lesions were compared for histopathology (different granuloma stage and number of multinucleated giant cells (MNGCs)), and acid-fast bacilli (AFBs) were quantified in the Ziehl-Neelsen-stained slides. Granulomas produced by M. caprae showed more stage IV granulomas, more MNGCs and higher AFBs counts than those induced by M. bovis. In conclusion, lesions caused by M. caprae would be more prone to the excretion of bacilli, and infected animals result as a high-risk source of infection for other animals.

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

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

Management practices and their association with Mycobacterium tuberculosis complex prevalence in red deer populations in Southwestern Spain.

Intensification of game management may increase the prevalence of tuberculosis (TB) in wildlife despite eradication programs implemented in cattle herds in the same areas. In this cross-sectional study, we investigated the association between wild game management practices and the presence of tuberculosis in red deer populations in Southwestern Spain. Five hundred and fifty-one animals were examined by necropsy to detect tuberculosis-like lesions in the main lymph nodes. Prevalence, as determined by TB-like lesions, was estimated to be 5.1% of animals, with 77% of TB-like lesions confirmed by PCR. Our results suggest that population density, in addition to factors which promote the local aggregation of animals, is factors associated with increased prevalence of TB in red deer populations. We suggest that management practices including supplementary feeding, fencing, water ponds and interaction with domestic livestock should be revised in order to prevent TB in wild deer both.