Waldo L. García-Jiménez

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.

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.

Severity of bovine tuberculosis is associated with co-infection with common pathogens in wild boar

Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa), a recognized reservoir of bovine tuberculosis (bTB) in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location (cervical lymph nodes), or more severe patterns affecting more than one location (mainly cervical lymph nodes and lungs), was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2), Aujeszkys disease virus (ADV), swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures under controlled conditions. Furthermore, more research including other important pathogens, such as gastro-intestinal nematodes, will be necessary to complete this picture.

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.

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.

Genotypic characterization by spoligotyping and VNTR typing of Mycobacterium bovis and Mycobacterium caprae isolates from cattle of Tunisia

This work is an approach to the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) bovine infections in Tunisia. A total of 35 MTBC isolates from both lateral retropharyngeal lymph node samples of cattle slaughtered in different Tunisian regions were genotyped by spoligotyping and variable number tandem repeat typing (VNTR)-typing. Spoligotyping allowed to identify two profiles not previously registered, namely SB2024, a Mycobacterium caprae isolate from Nabeul Region (North East Tunisia), the first description of this species in the country, and SB2025 (Mycobacterium bovis) from Sfax Region (Southern Tunisia). A second M. caprae isolate with a spoligotyping profile previously described in Europe mainland, SB0418, was also isolated from a bovine of Sfax region. Both isolates suggest the possibility of a widespread distribution of this species in the country. The predominant spoligotype was SB0120, present in all Tunisian regions selected for the study but Nabeul. Molecular typing also allowed to describe a mixed infection caused by two different M. bovis isolates (SB0120 and SB0848) in the same animal. VNTR typing was highly discriminant by testing a panel of six loci. Loci QUB3232 and QUB11b were the most discriminant, whereas ETR-D and QUB11a had the lower diversity index. The value of allelic diversity can significantly vary among countries; thus, it is important to standardize a panel of loci for future inter-laboratory comparisons. Although VNTR typing proved to be useful for an efficient discrimination among MTBC isolates, especially in combination with spoligotyping, further studies are needed in order to assess the genetic diversity of the MTBC in Tunisia.

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.

Susceptibility of Mycobacterium tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNFα

ABSTRACT An important feature of Mycobacterium tuberculosis pathogenesis is the ability to control cell death in infected host cells, including inhibition of apoptosis and stimulation of necrosis. Recently an alternative form of programmed cell death, necroptosis, has been described where necrotic cell death is induced by apoptotic stimuli under conditions where apoptotic execution is inhibited. We show for the first time that M. tuberculosis and TNFα synergise to induce necroptosis in murine fibroblasts via RIPK1-dependent mechanisms and characterized by phosphorylation of Ser345 of the MLKL necroptosis death effector. However, in murine macrophages M. tuberculosis and TNFα induce non-necroptotic cell death that is RIPK1-dependent but independent of MLKL phosphorylation. Instead, M. tuberculosis-infected macrophages undergo RIPK3-dependent cell death which occurs both in the presence and absence of TNFα and involves the production of mitochondrial ROS. Immunocytochemical staining for MLKL phosphorylation further demonstrated the occurrence of necroptosis in vivo in murine M. tuberculosis granulomas. Phosphorylated-MLKL immunoreactivity was observed associated with the cytoplasm and nucleus of fusiform cells in M. tuberculosis lesions but not in proximal macrophages. Thus whereas pMLKL-driven necroptosis does not appear to be a feature of M. tuberculosis-infected macrophage cell death, it may contribute to TNFα-induced cytotoxicity of the lung stroma and therefore contribute to necrotic cavitation and bacterial dissemination.

Fatal outbreak of systemic pasteurellosis in a wild boar (Sus scrofa) population from southwest Spain.

Pasteurella multocida is a common pathogen of swine that causes specific diseases with great economic impact. However, the importance of this pathogen in wild boar is still unknown. In the current work, an outbreak of systemic pasteurellosis in wild boar with a high mortality rate is described. A total of 23 wild boar of all ages were found dead over a 5-day period on a game estate in southwest Spain (11.11% mortality). Three animals were necropsied and showed subcutaneous edema, a generalized congestion, and fibrin deposits in the peritoneal cavity. Hemorrhages, general congestion, and intravascular thrombosis were microscopically observed. Pasteurella multocida type B was isolated from all of the studied organs. Outbreaks of systemic pasteurellosis have been described in domestic pigs from Asia and Australia, but not to date in Europe. This outbreak suggests that systemic pasteurellosis affecting wild boar populations may be an important cause of mortality.