Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store-operated calcium entry and FAK phosphorylation in colorectal cancer cells.

Store-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in colorectal cancer (CRC) cells. This mechanism, regulated by the filling state of the intracellular Ca2+ stores, is mediated by the endoplasmic reticulum Ca2+ sensors of the stromal interaction molecules (STIM) family [stromal interaction molecule 1 (STIM1) and STIM2] and the Ca2+-release-activated Ca2+ channels constituted by Orai family members, with predominance of calcium release-activated calcium channel protein 1 (Orai1). CRC cells exhibit enhanced SOCE due to remodeling of the expression of the key SOCE molecular components. The enhanced SOCE supports a variety of cancer hallmarks. Here, we show that treatment of the colorectal adenocarcinoma cell lines HT-29 and Caco-2 with inanimate Lacticaseibacillus paracasei (CECT9610) and Lactiplantibacillus plantarum (CECT9608) attenuates SOCE, although no detectable effect is seen on SOCE in normal colon mucosa cells. The effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics was mediated by downregulation of Orai1 and STIM1, while the expression levels of Orai3 and STIM2 remained unaltered. Treatment of HT-29 and Caco-2 cells with inanimate Lacticaseibacillus paracasei and Lactiplantibacillus plantarum impairs in vitro migration by a mechanism likely involving attenuation of focal adhesion kinase (FAK) tyrosine phosphorylation. Cell treatment with the Orai1 inhibitor synta-66 attenuates SOCE and prevents any further effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics. Together, our results indicate for the first time that Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics selectively exert negative effects on Ca2+ influx through SOCE in colorectal adenocarcinoma cell lines, providing evidence for an attractive strategy against CRC.

Wildlife Symbiotic Bacteria Are Indicators of the Health Status of the Host and Its Ecosystem.

Lactic acid bacteria (LAB) are gut symbionts that can be used as a model to understand the host-microbiota cross talk under unpredictable environmental conditions, such as wildlife ecosystems. The aim of this study was to determine whether viable LAB can be informative of the health status of wild boar populations. We monitored the genotype and phenotype of LAB based on markers that included safety and phylogenetic origin, antibacterial activity, and immunomodulatory properties. A LAB profile dominated by lactobacilli appears to stimulate protective immune responses and relates to strains widely used as probiotics, resulting in a potentially healthy wildlife population, whereas microbiota overpopulated by enterococci was observed in a hostile environment. These enterococci were closely related to pathogenic strains that have developed mechanisms to evade innate immune systems, posing a potential risk for host health. Furthermore, our LAB isolates displayed antibacterial properties in a species-dependent manner. Nearly all of them were able to inhibit bacterial pathogens, raising the possibility of using them as an a la carte antibiotic alternative in the unexplored field of wildlife disease mitigation. Our study highlights that microbiological characterization of LAB is a useful indicator of wildlife health status and the ecological origin from which they derive. IMPORTANCE The wildlife symbiotic microbiota is an important component for the greater diversity and functionality of their bacterial populations, influencing host health and adaptability to its ecosystem. Although many microbes are partly responsible for the development of multiple physiological processes, only certain bacterial groups, such as lactic acid bacteria (LAB), have the capacity to overpopulate the gut, promoting health (or disease) when specific genetic and environmental conditions are present. LAB have been exploited in many ways due to their probiotic properties, particularly lactobacilli; however, their relationship with wildlife gut-associated microbiota hosts remains to be elucidated. On the other hand, it is unclear whether LAB such as enterococci, which have been associated with detrimental health effects, could lead to disease. These important questions have not been properly considered in the field of wildlife and, therefore, should be clearly addressed.

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation.

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.

Spread of Antimicrobial Resistance by Salmonella enterica Serovar Choleraesuis between Close Domestic and Wild Environments.

The Salmonellaenterica serovar Choleraesuis affects domestic pig and wild boar (WB), causing clinical salmonellosis. Iberian swine production is based on a free-range production system where WB and Iberian pig (IP) share ecosystems. This study focuses on the negative impact on the pork industry of infections due to this serotype, its role in the spread of antibiotic resistance, and its zoonotic potential. Antibiotic resistance (AR) and genetic relationships were analyzed among 20 strains of S. Choleraesuis isolated from diseased WB and IP sampled in the southwest region of the Iberian Peninsula. AR was studied using the Kirby-Bauer method with the exception of colistin resistance, which was measured using the broth microdilution reference method. Resistance and Class 1 integrase genes were measured using PCR, and the genetic relationship between isolates and plasmid content by pulsed field gel electrophoresis. The results show a higher incidence of AR in isolates from IP. Phylogenetic analysis revealed seven profiles with two groups containing isolates from IP and WB, which indicates circulation of the same clone between species. Most pulsotypes presented with one plasmid of the same size, indicating vertical transmission. AR determinants blaTEM and tetA were routinely found in IP and WB, respectively. One isolate from IP expressed colistin resistance and presented the mcr-1 gene carried by a plasmid. This study suggests that S. Choleraesuis circulates between WB and IP living in proximity, and also that the mobilization of AR genes by plasmids is low. Furthermore, the detection of plasmid-mediated colistin resistance in bacteria from IP is alarming and should be monitored.

Detection of live M. bovis BCG in tissues and IFN-γ responses in European badgers (Meles meles) vaccinated by oropharyngeal instillation or directly in the ileum.

BACKGROUND: Oral vaccination with Mycobacterium bovis Bacille of Calmette and Guerin (BCG) has provided protection against M. bovis to badgers both experimentally and in the field. There is also evidence suggesting that the persistence of live BCG within the host is important for maintaining protection against TB. Here we investigated the capacity of badger inductive mucosal sites to absorb and maintain live BCG. The targeted mucosae were the oropharyngeal cavity (tonsils and sublingual area) and the small intestine (ileum). RESULTS: We showed that significant quantities of live BCG persisted within badger in tissues of vaccinated badgers for at least 8 weeks following oral vaccination with only very mild pathological features and induced the circulation of IFNγ-producing mononuclear cells. The uptake of live BCG by tonsils and drainage to retro-pharyngeal lymph nodes was repeatable in the animal group vaccinated by oropharyngeal instillation whereas those vaccinated directly in the ileum displayed a lower frequency of BCG detection in the enteric wall or draining mesenteric lymph nodes. No faecal excretion of live BCG was observed, including when BCG was delivered directly in the ileum. CONCLUSIONS: The apparent local loss of BCG viability suggests an unfavorable gastro-enteric environment for BCG in badgers, which should be taken in consideration when developing an oral vaccine for use in this species.

Nasal shedding of Mycobacterium tuberculosis in wild boar is related to generalised tuberculosis and concomitant infections.

BACKGROUND: Wild boar is an important reservoir of Mycobacterium tuberculosis variant bovis, the main causative agent of bovine tuberculosis (bTB). A proportion of tuberculosis (TB)-affected wild boars shed M tuberculosis by nasal route, favouring the maintenance of bTB in a multihost scenario. The aim of this work was to assess if M tuberculosis nasal excretion is influenced by factors commonly associated with high TB prevalence in wild boar. METHODS: TB diagnosis and M tuberculosis isolation were carried out in 112 hunted wild boars from mid-western Spain. The association between the presence of M tuberculosis DNA in nasal secretions and explanatory factors was explored using partial least squares regression (PLSR) approaches. RESULTS: DNA from M tuberculosis was detected in 40.8 per cent nasal secretions of the TB-affected animals. Explanatory factors provided a first significant PLSR X's component, explaining 25.70 per cent of the variability observed in M tuberculosis nasal shedding. The presence of M tuberculosis in nasal secretions is more probable in animals suffering from generalised TB and mainly coinfected with Metastrongylus species and porcine circovirus type 2, explaining nearly 90 per cent of the total variance of this model. CONCLUSION: Measures aiming to control these factors could be useful to reduce M tuberculosis shedding in wild boar.

Lactobacilli Isolated From Wild Boar (Sus scrofa) Antagonize Mycobacterium bovis Bacille Calmette-Guerin (BCG) in a Species-Dependent Manner.

Background: Wildlife poses a significant burden for the complete eradication of bovine tuberculosis (bTB). In particular, wild boar (Sus scrofa) is one of the most important reservoirs of Mycobacterium bovis, the causal agent of bTB. Wild boar can display from mild TB lesions, usually found in head lymph nodes, to generalized TB lesions distributed in different anatomical regions; but rarely clinical signs, which complicates the diagnosis of Mycobacterium bovis infection and bTB control. Among the possibilities for this variability in lesion distribution is the influence of the host-beneficial commensal-primed immune barrier. In this respect, beneficial microbes may delay bTB dissemination as a consequence of an antagonistic competition for nutrients and phagocytes. In order to explore this possibility, we have tested whether typical commensals such as lactobacilli have the capacity to reduce the survival rate of the surrogate M. bovis strain Bacillus Calmette-Guerin (BCG); and to modulate its phagocyte intake. Results: Three Lactobacillus species, L. casei, L. plantarum, and L. salivarius, isolated from wild boar feces displayed a pH-dependent inhibitory activity against BCG and influenced its intake by porcine blood phagocytes in a species-dependent manner. All lactobacilli showed a very significant bactericidal effect against BCG at low pH, but only isolates of L. plantarum and L. casei displayed such antimycobacterial activity at neutral pH. The genomes of these isolates revealed the presence of two-peptide bacteriocins whose precursor genes up-regulate in the presence of BCG cells. Furthermore, L. plantarum reduced significantly the BCG phagocytic intake, whereas L. casei had the opposite effect. L. salivarius had no significant influence on the phagocytic response to BCG. Conclusions: Our in vitro results show that lactobacilli isolated from wild boar antagonize BCG as a consequence of their antimycobacterial activity and a competitive phagocytic response. These findings suggest that commensal bacteria could play a beneficial role in influencing the outcome of bTB dissemination. Further work with lactobacilli as a potential competitive pressure to control bTB will need to take into account the complex nature of the commensal microbiome, the specific immunity of the wild boar and the in vivo infection context with pathogenic strains of M. bovis.

Seasonal and dietary effects on Vitamin D deficiencies detected in wild boar from mid-western Spain.

Vitamin D (VitD) is involved in important mammalian physiological mechanisms, such as Ca-P metabolism, bone development and immunological response. VitD deficiencies are frequently detected in domestic animals and related to various health problems (e.g., rickets, bone deformation). However, knowledge about the status of VitD in wildlife species, such as the wild boar, is scarce. The aims of this work were to explore VitD status in wild boar populations from mid-western Spain and to elucidate the influence of daylight exposure and food supplementation in levels of VitD. Serum concentration of VitD (measured as 25-hydroxivitaminD) was assessed in 276 wild boar from 27 game estates located in mid-western Spain using a commercial ELISA kit. In 19 out of 27 estates, the staff supplied a specific VitD-enriched food (2,000 UI/Kg) ad libitum throughout the year, while in the remaining estates (8), no food was supplied. Blood samples were extracted from hunted animals (198) between October and February of hunting seasons 2016/2017 and 2017/2018, and from live wild boar (78) that were captured, sampled and released (March-September of 2017). The percentage of animals with VitD deficiency (<20 ng/ml), VitD insufficiency (20-30 ng/ml) and VitD sufficiency (>30 ng/ml) was estimated, and the relationship of these levels to factors like sex, age and season was assessed using chi-square tests. Furthermore, associations between daylight exposure and supplemental food with VitD levels were explored using linear models. Of the studied wild boar population, 82.2% showed a VitD deficiency or insufficiency. VitD deficiencies were more frequent in animals sampled in winter and spring. Furthermore, levels of VitD positively correlated with daylight exposure and supplemental food intake. Ad libitum supplementation with VitD-enriched food was insufficient to prevent VitD deficiencies in wild boar from November to April, probably because food consumption is lower during this period.

The expression of Indoleamine 2, 3-dioxygenase (IDO) is reduced in granulomas from BCG vaccinated cattle compared to granulomas from unvaccinated controls after experimental challenge with Mycobacterium bovis.

Bovine tuberculosis (bTB), mainly caused by Mycobacterium bovis (M. bovis), is a major economic disease of livestock worldwide. Vaccination is considered as a potentially sustainable adjunct to the current control strategy. Cattle vaccination with the live attenuated M. bovis bacillus Calmette-Guerin (BCG) confers variable protection; the reasons for this variability are not understood. Indoleamine 2, 3-dioxygenase (IDO), through the catalysis of tryptophan, is thought to have an immunoregulatory role in the immune response to Mycobacterium tuberculosis (M. tuberculosis). In this work, we used immunohistochemistry and digital image analysis to evaluate the presence of IDO in granulomas at different stages of development in cattle that had been BCG-vaccinated or not and then challenged with M. bovis. Our results show that the expression of IDO in granulomas from non-vaccinated M. bovis challenged animals is higher than in granulomas from BCG-vaccinated M. bovis challenged animals. Thus, it is possible that vaccination with BCG prevents the induction of what are thought to be host immunosuppressive pathways by M. bovis, which contribute to pathology during the disease.

Vaccination Against Porcine Circovirus-2 Reduces Severity of Tuberculosis in Wild Boar.

Tuberculosis (TB) in wild boar (Sus scrofa) may be affected by coinfections with other pathogens, such as porcine circovirus type 2 (PCV2). Therefore, sanitary measures focused on controlling PCV2 could be useful in reducing the impact of TB in this wild suid. The aim of this study was to explore whether vaccination against PCV2 targeting young animals affects TB prevalence and TB severity in wild boar. The study was conducted on a game estate in mid-western Spain. Seventy animals of ages ranging from 4 to 8 months were captured, individually identified, vaccinated against PCV2 and released, forming a vaccinated group. Not-captured animals cohabiting with the vaccinated wild boar constituted the control group. Animals from both groups were hunted between 2013 and 2016 and a TB diagnosis based on pathological assessment and microbiological culture was made in all of them. The effect of PCV2 vaccination on TB prevalence and severity was explored using generalized lineal models. Whereas TB prevalence was similar in vaccinated and control groups (54.55 vs. 57.78%), vaccinated animals showed less probabilities to develop generalized TB lesions. Furthermore, mean TB severity score was significantly lower in vaccinated animals (1.55 vs. 2.42) suggesting a positive effect of PCV2 vaccination.

Histopathological and immunohistochemical characterisation of hepatic granulomas in Leishmania donovani-infected BALB/c mice: a time-course study.

BACKGROUND: Visceral leishmaniasis (VL) is a neglected tropical disease (NTD), caused by the intracellular protozoan parasites Leishmania donovani and Leishmania infantum. Symptomatic VL is considered fatal when left untreated. At present, there is no effective vaccine licensed for human use and available chemotherapies have limitations. Understanding the local immune mechanisms required for the control of infection is a key factor for developing effective vaccines and therapeutics. METHODS: We have investigated the development of the typical granulomatous lesions in the liver in experimental VL over time, together with the local immune responses. BALB/c mice were infected intravenously with a dose of 2 × 107 L. donovani amastigotes (MHOM/ET/67/HU3) and sacrificed at 15, 35 and 63 days post-infection (dpi). Histopathology and immunohistochemical techniques were used for the detection of Leishmania antigen, selected cell types including B and T lymphocytes, macrophages and neutrophils (CD45R-B220+, CD3+, F4/80+ and Ly-6G+) and iNOS. RESULTS: Granulomatous lesions were identified as early as 15 dpi in the livers of all infected animals. Three categories were used to classify liver granulomas (immature, mature and clear). Clear granulomas were exclusively detected from 35 dpi onwards. Kupffer cells (F4/80+) were predominant in immature granulomas, regardless of the dpi. Nonetheless, the highest expression was found 63 dpi. Positive staining for iNOS was mainly observed in the cytoplasm of fused Kupffer cells and the highest expression observed at 35 dpi. T cells (CD3+) and B cells (CD45R-B220+) were predominant in more advanced granuloma stages, probably related to the establishment of acquired immunity. Neutrophils (Ly-6G+) were predominantly observed in mature granulomas with the highest expression at 15 dpi. Neutrophils were lower in numbers compared to other cell types, particularly at later time points. CONCLUSIONS: Our results reflect the role of macrophages during the early stage of infection and the establishment of a lymphocytic response to control the infection in more advanced stages.

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

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.

Histopathological and immunohistochemical characterization of Burkholderia pseudomallei lesions in an acute model of infection with BALB/c mice.

Organ tissue damage is a key contributor to host morbidity and mortality following infection with microbial agents. Severe immune responses, excessive cellular recruitment and necrosis of cells all play a role in disease pathology. Understanding the pathogenesis of disease can aid in identifying potential new therapeutic targets or simply act as a diagnostic tool. Burkholderia pseudomallei is a Gram-negative bacterium that can cause acute and chronic diseases. The BALB/c mouse has been shown to be highly susceptible to aerosol challenge with B. pseudomallei and hence acts as a good model to study the acute and potentially lethal form of the disease melioidosis. In our study, BALB/c mice were challenged and culled at predetermined time points to generate a pathological time course of infection. Lung, liver and spleen were subjected to pathological and immunohistochemical analysis. The number and type of microscopic lesions within each organ, as well as the location and the mean percentage of neutrophils, B cells, T cells and Burkholderia capsule antigen within the lesions, were all characterized during the time course. Neutrophils were determined as the key player in tissue pathology and generation of lesions, with B cells playing an insignificant role. This detailed pathological assessment increases our understanding of B. pseudomallei disease.

Spoligotype diversity and 5-year trends of bovine tuberculosis in Extremadura, southern Spain.

Bovine tuberculosis (bTB) causes significant losses to farming economies worldwide. A better understanding on the epidemiology of this disease and the role that the different hosts develop in the maintenance and spread of bTB is vital to control this zoonotic disease. This study reports the spoligotype diversity and temporal evolution of Mycobacterium tuberculosis Complex (MTBC) isolates obtained from Extremadura (southern Spain). Genotyping data of Mycobacterium bovis (n = 2102) and Mycobacterium caprae (n = 96) isolates from cattle and wildlife species, collected between 2008 and 2012, were used in this study. The isolates resulted clustered into 88 spoligotypes which varied largely in frequency and occurrence in the three hosts. The 20 most frequent patterns represented 91.99 % of the isolates, the spoligotype SB0121 being the clearly predominant and most widely dispersed geographically. The major variety of the spoligotype patterns (78 out of 88) was isolated from the cattle, in fact 50 (56.83 %) of the patterns were found only in this species. Within the spoligotypes shared between the cattle and wildlife species, 17 patterns (1747 isolates) were shared with wild boar and Iberian red deer, 10 patterns (308 isolates) were exclusively shared with wild boar, and only one pattern (two isolates) was shared exclusively with Iberian red deer. The significant number of spoligotypes shared between the three hosts (79.49 %) highlights the components of the multi-host system that allows the bTB maintenance in our study area. The greater percentage of isolates shared by the wild boar and cattle (93.50 %) supports the role of wild boar as main maintenance host for bTB in cattle. These results could be extrapolated to areas with a similar epidemiological scenario and could be helpful for other countries where wild reservoirs represent a handicap for the successful eradication of bTB from livestock.

FATAL CASE OF STREPTOCOCCUS SUIS INFECTION IN A YOUNG WILD BOAR (SUS SCROFA) FROM SOUTHWESTERN SPAIN.

Streptococcus suis is a recognized pathogen that may cause important diseases in pigs and humans. This microorganism has been repeatedly isolated from wild boar (Sus scrofa). However, its health implications for this wild species are still unknown. This article reports a detailed description of a fatal case of septicemia by S. suis affecting a young wild boar. The affected animal, about 15 days old, was found near death and exhibiting neurologic signs at a wild boar estate in southwestern Spain. Postmortem examination showed generalized congestion, brain hemorrhages and lobular pneumonia. Histopathological evaluation demonstrated the presence of meningitis and encephalitis with marked congestion and suppurative bronchopneumonia. Streptococcus suis serotype 2 isolates exhibiting important virulence factors (extracellular factor, muramidase-released protein, and suylisin) were isolated from the affected animal. This study confirms the presence of potentially virulent and zoonotic strains of S. suis in wild boar from Spain.

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.

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.

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.

Fatal infection due to Haemophilus parasuis in a young wild boar (Sus scrofa).

Haemophilus parasuis is a recognized pathogen in domestic pigs; the pathogen has been also isolated from healthy wild boar (Sus scrofa). In the current report, a case of fatal H. parasuis infection in a wild boar piglet from central Spain is described. The affected animal presented severe pneumonic lesions, inflammation in tarsal joints with presence of fibrinous deposits, and epidural hemorrhage in the atlanto-occipital joint. Pure growth of H. parasuis was obtained from lungs and tarsal joints. The current case illustrates the susceptibility of wild boar to this agent. The gross pathology results were similar to that described in domestic pigs, but there were no fibrinous deposits on serosal surfaces.