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), Aujeszky''s 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.     

Analysis of serum biochemical parameters in relation to Mycobacterium bovis infection of European wild boars (Sus scrofa) in Spain

Bovine tuberculosis (bTB) caused by Mycobacterium bovis (Mycobacterium tuberculosis complex) is a zoonotic disease that affects cattle and wildlife worldwide. European wild boar (Sus scrofa) is a major reservoir host of M. bovis in south-central Spain. The identification of biomarkers to predict bTB in wild boars by dependable methods that do not require killing the host would greatly contribute to the implementation of effective control programs for bTB in this region. In this study, we have characterized serum biochemical values in European wild boars in Spain to determine whether biochemical parameters in the serum varied significantly with the presence of bTB in this species. Although apolipoprotein A1 and IgG levels were higher in uninfected boars, the results did not support good predictive value for serum biochemical parameters studied for European wild boars in relation to bTB in Spain.     

Spatio-Temporal Trends of Iberian Wild Boar Contact with Mycobacterium tuberculosis Complex Detected by ELISA

The continuing expansion of Eurasian wild boar (Sus scrofa) populations raises concerns regarding disease transmission. In south-central Spain, overabundant wild boar are reservoirs of Mycobacterium bovis, and related members of the Mycobacterium tuberculosis complex (MTBC), the causative agents of bovine tuberculosis (bTB). An indirect enzyme-linked immunosorbent assay using bovine-purified protein derivative was applied to determine the spatial and temporal distribution of wild boar contact with MTBC in the Iberian Peninsula and to model and identify the associated risk factors. Wild boar apparent seroprevalence was 22%. Seropositives were detected in 71% of 81 sites, including 23 sites where wildlife was thought to be bTB free. The results described a new geographic range of wild boar contact with MTBC and a stable prevalence in this wildlife reservoir that contrasts with the success of bTB control in cattle. Inference of which host (wild boar or cattle) is driving bTB maintenance was not possible with our correlational results. The possibility of a wild boar bTB emergence in non-endemic regions should urgently be taken into account to avoid a future scenario resembling the current situation in south-central Spain.     

Combined evaluation of bovine tuberculosis in wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) and red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis>) from Central-East Portugal

Bovine tuberculosis (bTB), widely considered a disease of livestock, can also affect large game species despite their poorly understood role in the epidemiology of the endemic state of the disease. The Idanha-a-Nova County, situated in the eastern region of central Portugal, is one of the most important regions for large game hunting activity. In order to investigate the role of large game and the presence of bTB in Idanha-a-Nova, 132 wild boar (Sus scrofa) and 339 red deer (Cervus elaphus) were inspected for bTB-compatible lesions (bTBCL) during the 2008–2009 hunting season. Tissue samples with bTBCL were collected and processed for bacteriological confirmation and spoligotyping of Mycobacterium bovis isolates. In wild boar, bTB lesions were single, mostly calcified (21/29, 72.4%) and situated in the mandibular lymph nodes. Lesions were more severe and largely non-calcified (36/40, 90%) in red deer. Wild boar presented a consistently higher bTB infection rate than the red deer; however, the difference was only significant in the southern B area. This area was significantly affected by bTB for both animal species (up to 72.7% in wild boar and 34.8% in red deer) and requires the urgent implementation of intervention measures to contain the disease. Wild boar may assume an important role in the emergence of new disease foci in distant areas that infected red deer cannot reach. Spoligotyping of M. bovis isolates grouped the strains into three clusters, revealing patterns common to wild boar, red deer, and also to local cattle, indicating possible transmission among different animal species.     

Unusual lesion morphology and skin test reaction for Mycobacterium avium complex in macaques

Three rhesus (Macaca mulatta) and one cynomolgus (M. fascicularis) monkey were euthanized because of positive reactions to intradermal tests with mammalian Old Tuberculin (mOT). All the animals had gross or microscopic lesions consisting of unitized macro- or microgranulomas with central necrosis, involving the lung or mesenteric lymph nodes. Small numbers of acid fast organisms were demonstrated in the lesions. Mycobacterium avium complex, serotype 2 was isolated from three of the cases. The cases were unusual because the lesion morphology and mOT hypersensitivity were more typical of mammalian tuberculosis than avian tuberculosis in monkeys.     

Tuberculosis in wild olive baboons, Papio cynocephalus anubis (Lesson), in Kenya

Several wild olive baboons from a single troop in the Masai Mara Game Reserve, Kenya were observed to be lethargic and emaciated. Five were trapped and tuberculin tested by intradermal inoculation of 0.1 cc (100 IU) mammalian old tuberculin in the upper eyelid. Two of the five showed positive reaction at 72 hr and were examined at necropsy. Gross lesions in both animals consisted of multiple nodules with caseation in the lung, spleen and tracheobronchial lymph nodes. There were multiple granulomas throughout the lung, spleen and the lymph nodes. Tissues were cultured on Lowenstein-Jensen media with and without pyruvic acid. Isolates were typed as Mycobacterium bovis.     

Influence of methylmalonyl-CoA mutase alleles on resistance to bovine tuberculosis in the European wild boar (Sus scrofa)

An association study was carried out to examine the influence of methylmalonyl-CoA mutase (MUT) polymorphisms on the susceptibility of a well-studied wild boar population from southern Spain to develop bovine tuberculosis (bTB). To this end, we examined polymorphisms at a closely linked dinucleotide microsatellite flanking exon 2 of the MUT gene in 37 wild boars with bTB and 36 non-infected individuals. The microsatellite showed low polymorphism in the studied population, with only three alleles (MUTm-A, MUTm-B and MUTm-C) found, in contrast to the 11 alleles previously reported for domestic pigs. Our case-control study showed that the MUTm-B allele was associated with disease in a dominant pattern (odds ratio = 3.36; 95% CI = 1.05-10.72; P = 0.04), while the MUTm AA genotype appeared to have a protective effect against bTB infection (odds ratio = 4.33; 95% CI = 1.20-14.96; P = 0.02). Interestingly, infected wild boars heterozygous for MUTm AB are at an advantage (11-fold) to contain the systemic spread of the disease when compared to other genotypes, implying that a balanced polymorphism may be present in the population. These results strengthen previous observations regarding the importance of the MUT gene on bTB resistance in wild boars and indicate that polymorphisms at this locus will influence the risk of acquiring and maintaining bTB in the studied population.     

Genetic resistance to bovine tuberculosis in the Iberian wild boar

Bovine tuberculosis (bTB) is an important re-emerging zoonotic disease, causing major economic losses and constraining international trade of animals and their products. Despite eradication programmes, some countries continue to encounter outbreaks, mainly due to wildlife acting as primary hosts or reservoirs. While the genetic component of tuberculosis in humans and cattle is well documented, the role of genetic factors as modulators of bTB resistance remains unclear for natural populations. To address this issue, we investigated the relative contribution of host genetic variability to susceptibility to bTB infection and disease progression in wild boars from southern Spain. We found that genetic heterozygosity is an important predictor of bTB, not only modulating resistance to infection but also influencing containment of disease progression in infected individuals. Our results provide further evidence that host genetic variability plays a central role in natural populations. Testing each marker separately reveals evidence of both general and single-locus associative effects on bTB and several loci reveal high homology to regions of the genome with known immune function. Our results may prove to be crucial for understanding outbreaks of bTB in wildlife that could potentially affect domestic livestock and humans.     

Seroprevalence and risk factors associated to Mycobacterium bovis in wild artiodactyl species from southern Spain, 2006-2010

The control of bovine tuberculosis (bTB) is at a critical point in the last stage of eradication in livestock. Wildlife species recently have emerged infected with TB in Europe, particularly ungulates in the Iberian Peninsula. Epidemiological information regarding TB in wild ungulates including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in these countries. A cross-sectional study was carried out on wild artiodactyl species, including Eurasian wild boar (Sus scrofa) red deer (Cervus elaphus), roe deer (Capraelus capraelus), fallow deer (Dama dama), Spanish ibex (Capra pyrenaica hispanica) and mouflon (Ovis musimon), in Spain to assess the seroprevalence against Mycobacterium bovis or cross-reacting members of the Mycobcaterium tuberculosis complex (MTBC), and to provide information on associated risk factors. Previously, two in-house indirect enzyme linked immunosorbent assays (bPPD-ELISA and MPB83-ELISA) were developed using known TB status sera. Positive reference sera were selected from infected animals confirmed by culture. The M. bovis isolates belonged to spoligotypes SB0121, SB0120, SB0295, SB0265 and SB0134. Two hundred and two out of 1367 (7.5%; 95% CI: 6.1-8.9) animals presented antibodies against M. bovis by both bPPD-ELISA and MPB83-ELISA. Significantly higher TB seroprevalence was observed in wild boar compared to the other species analyzed. Interestingly, seropositivity against M. bovis was not found in any out of 460 Spanish ibex analyzed. The logistic regression model for wild boar indicated that the seropositivity to M. bovis was associated with age, location and year of sampling, while the only risk factor associated with M. bovis seroprevalence in red deer and fallow deer was the age. The seroprevalence observed indicates a widespread exposure to MTBC in several wild artiodactyl species in southern Spain, which may have important implications not only for conservation but also for animal and public health.     

Bovine tuberculosis (Mycobacterium bovis) in British farmland wildlife: the importance to agriculture

Bovine tuberculosis (bTB) is an important disease of cattle and an emerging infectious disease of humans. Cow- and badger-based control strategies have failed to eradicate bTB from the British cattle herd, and the incidence is rising by about 18%per year. The annual cost to taxpayers in Britain is currently 74 million UK pounds. Research has focused on the badger as a potential bTB reservoir, with little attention being paid to other mammals common on farmland. We have conducted a systematic survey of wild mammals (n=4393 individuals) present on dairy farms to explore the role of species other than badgers in the epidemiology of bTB. Cultures were prepared from 10397 samples (primarily faeces, urine and tracheal aspirates). One of the 1307 bank voles (Clethrionomys glareolus) live-sampled, and three of the 43 badgers (Meles meles), yielded positive isolates of Mycobacterium bovis. This is the first time the bacterium has been isolated from the bank vole. The strain type was the same as that found in cattle and badgers on the same farm. However, our work indicates that the mean prevalence of infectious individuals among common farmland wildlife is extremely low (the upper 95% confidence interval is < or =2.0 for all of the abundant species). Mathematical models illustrate that it is highly unlikely the disease could be maintained at such low levels. Our results suggest that these animals are relatively unimportant as reservoirs of bTB, having insufficient within-species (or within-group) transmission to sustain the infection, though occasional spill-overs from cattle or badgers may occur.     

Bovine tuberculosis in a free ranging red fox (Vulpes vulpes) from Doñana National Park (Spain)

During 1997 and 1998, a survey of Iberian carnivores was conducted to study the epidemiology of bovine tuberculosis in the Do?ana National Park and surrounding areas in southwestern Spain. Post-mortem examinations were done on seven red foxes (Vulpes vulpes), two Egyptian mongoose (Herpestes ichneumon), one weasel (Mustela nivalis), two genets (Genetta genetta), one Iberian lynx (Lynx pardinus), one Eurasian badger (Meles meles), and two polecats (Mustela putorius). Lesions suggestive of bovine tuberculosis were not detected but, in culture, Mycobacterium bovis was isolated from the retropharyngeal lymph nodes of one adult male red fox. This is the first report of M. bovis infection in red fox in Spain.     

Bovine tuberculosis in “Eurasian” badgers (Meles meles) in France

In France, several surveillance programmes have been carried out to monitor the presence of bovine tuberculosis (bTB) in badgers in regions where cattle or wildlife show high bTB prevalence (Seine-Maritime, Côte d’Or, Dordogne/Charente). Diagnostic methods include post-mortem examination, polymerase chain reaction (PCR) and/or microbiological culture. The frequency of visible lesions was significantly higher in Dordogne/Charente (14/283, 4.9 %) than in Côte d’Or (19/1146, 1.7 %) and Seine-Maritime (2/160, 1.25 %) (Fisher’s exact test, p?<?0.001). Lesions were mainly located in cephalic and thoracic lymph nodes with one badger showing generalised tuberculosis. Near infected cattle farms or pastures, Mycobacterium bovis was detected using the culture method in roughly 1 % (1/103) of the badgers sampled in Seine-Maritime and in 5.6 % (49/878) of the badgers sampled in Côte d’Or. In Dordogne/Charente, the prevalence determined by PCR was 13.3 % (29/218). M. bovis was not found in badgers trapped in areas where there are no infected cattle. Spoligotyping and multiple locus variable number tandem repeat analysis showed that all M. bovis strains isolated from badgers were of the same genotype as those isolated from cattle in the vicinity, demonstrating an epidemiological link between both species. These are the first cases of bTB in badgers reported in France. Further surveys and research are needed to investigate the role badgers play in the epidemiology of bTB.     

Susceptibility of raccoons (Procyon lotor) to infection with Mycobacterium bovis

Tuberculosis due to Mycobacterium bovis infection is endemic in white-tailed deer (Odocoileus virginianus) in the northeastern portion of the lower Michigan peninsula (USA). Various wild carnivores and omnivores, including raccoons (Procyon lotor), are infected with M. bovis within the endemic area. To investigate the pathogenesis of tuberculosis in raccoons and the likelihood of M. bovis transmission from infected raccoons to other susceptible hosts, we experimentally inoculated raccoons with single oral doses of M. bovis (ranging from 30 to 1.7 x 10(5) colony forming units [CFU]), five daily oral doses of M. bovis (ranging from 10 to 1 x 10(5) CFU), or a single intravenous (i.v.) dose of 1 x 10(5) CFU of M. bovis, from November 1998 through December 2000. Granulomatous lesions consistent with tuberculosis, or tissue colonization with M. bovis, were seen in one of five raccoons in the single low oral dose group, one of five raccoons in the multiple low oral dose group, two of five raccoons in the multiple medium oral dose group, five of five raccoons in the multiple high oral dose group, and five of five raccoons in the i.v. inoculated group. In oral inoculated raccoons, lesions were most common in the tracheobronchial and mesenteric lymph nodes and lung. Excretion of M. bovis in saliva or nasal secretions was noted in all i.v. inoculated raccoons and two of five multiple low oral dose raccoons. Mycobacterium bovis was not isolated from urine or feces from any experimentally inoculated raccoons. The need for multiple large oral doses to establish infection, and the low number of orally inoculated raccoons that excreted M. bovis in nasal secretions or saliva, suggest that wide-spread tuberculosis among raccoons is unlikely.     

Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda

BackgroundBovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health.Materials and methodsA cross-sectional study employing a systematic random sampling of cattle (n = 300) with the collection of retropharyngeal lymph nodes and tonsils (n = 300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence.FindingsOf the 300 sample pools, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. Mycobacterium bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistant (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle.ConclusionsThis study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is present in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.     

Chemokine/cytokine production by mononuclear cells from human lymphoid tissues and their modulation by Mycobacterium tuberculosis antigens

The majority of knowledge about the role of cytokines and chemokines in controlling Mycobacterium tuberculosis infection mainly derives from animal models. In humans, this knowledge is still mainly limited to the blood compartment or accessible lymphoid organs, such as tonsils. Here, we studied cytokine and chemokine production and their modulation by M. tuberculosis antigens in mononuclear cells from human blood, spleen and hilar lung lymph nodes. Results show that the kinetics and magnitude of cytokine and chemokine production varied according to the tissue of cell origin. Mycobacterium tuberculosis antigens enhanced cytokine and chemokine production in blood, but the enhancement was restricted in spleen and hilar lung lymph node cells. We show, for the first time in humans, differences in cytokine and chemokine microenvironments according to lymphoid tissues, and suggest that these differences may affect the way cells respond to M. tuberculosis infection.     

Host status of wild roe deer in bovine tuberculosis endemic areas

Rarely found infected, roe deer (Capreolus capreolus) are not considered a bovine tuberculosis (bTB) reservoir. However, serial cases discovered between 2010 and 2014 in one of the bTB endemic regions in France threw doubt on the epidemiological role played by this small wild cervid in ecosystems where cattle and other wild animals are both infected. Our objective was to analyse the bTB host status of roe deer as regards infection prevalence within the population and Mycobacterium bovis-induced pathology in this species. From November 2001 to March 2016, 668 roe deer were analysed (culture and/or PCR) through active surveillance implemented in three bTB endemic areas in France (Brotonne forest, Dordogne and Côte d’Or) and 132 through passive surveillance (presence of macroscopic lesions) in the whole country. Only seven roe deer were found infected by M. bovis, exclusively in bTB endemic areas, one through active surveillance (Brotonne forest; n = 203, apparent prevalence 0.49%). On the basis of these results, observed pathological patterns (mainly pulmonary lesions), species social behaviour (less gregarious than other ungulates) and food behaviour (mainly a selective browser), roe deer does not appear to be a true reservoir of bTB. However, once infected, it develops lesions reflecting a clear ability for bacterial excretion and therefore transmission to other species, most likely by indirect contact. It could thus be a spillover host included in a multi-host component reservoir in endemic areas. Consequently, passive surveillance is essential to detect infection and to implement specific management to limit interactions with cattle, where infected roe deer are found.     

Expression levels of 10 candidate genes in lung tissue of vaccinated and TB‐infected cynomolgus macaques

The expression of ten tuberculosis candidate genes in lung and lymph nodes of cynomologus macaques vaccinated and experimentally infected with Mycobacterium tuberculosis (Mtb) was quantified. The expression of TNFα, IL10, IL1β, TLR4, IL17, IL6, IL12, and iNOS in the lungs of vaccinated animals was higher than that of non‐vaccinated animals.     

Paratuberculosis in saiga antelope (Saiga tatarica) and experimental transmission to domestic sheep.

Mycobacterium paratuberculosis was isolated in low numbers from the small intestine and associated mesenteric lymph nodes of a saiga antelope (Saiga tatarica) using routine culture techniques in spite of histologic evidence of high numbers of acid-fast bacteria in these tissues. Two newborn domestic sheep were fed the ground intestinal tissue containing acid-fast bacteria and the progression of the experimental disease was followed by fecal culture, immunodiffusion (AGID) and lymphocyte stimulation (LST) tests. One experimentally infected sheep developed progressive clinical illness 1 yr postinoculation. Few M. paratuberculosis were isolated from feces or tissues although an extensive granulomatous mycobacterial enteritis, lymphadenitis and lymphangitis were observed containing large numbers of typical acid-fast organisms. No clinical illness was observed in the second inoculated sheep after 18 mo of observation, although infection was demonstrated at necropsy. Both sheep developed AGID and LST reactions indicative of paratuberculosis. This study demonstrated that a difficult to culture isolate of M. paratuberculosis was responsible for paratuberculosis in captive wild ruminants and was transmissible to domestic sheep. Diagnosis of paratuberculosis in four of eight of the imported saiga antelope and in eleven of their 18 offspring indicates the importance of this disease in management of captive wild ruminants and the ease with which this organism can be transmitted.