No Evidence of Neutrophil Response Modulation in Goats after Immunization against Paratuberculosis with a Heat-Inactivated Vaccine.

Neutrophils are believed to play a role in the initial stages of paratuberculosis, and it has recently been demonstrated that vaccination can modulate their function via priming or through epigenetic and metabolic reprogramming (training). Modulation of the neutrophil response against Mycobacterium avium subspecies paratuberculosis (Map) through vaccination has been demonstrated in a rabbit model but not in ruminants. Therefore, in the present work, the effect of vaccination on the response of caprine neutrophils against Map was studied. Neutrophils were isolated from non-vaccinated (n = 7) and Gudair®-vaccinated goat kids (n = 7), before vaccination and 30 days post-vaccination. Then, several neutrophil functions were quantified ex vivo: cell-free and anchored neutrophil extracellular trap (NET) release, phagocytosis, and the differential expression of several cytokines and TLR2. The induction of cell-free NETosis and TLR2 expression by Map is reported for the first time. However, vaccination showed no significant effect on any of the functions studied. This suggests that the protection conferred by Gudair® vaccination is based on mechanisms that are independent of the neutrophil function modulation. Further research into the impact of alternative vaccination strategies or the paratuberculosis infection stage on ruminant neutrophil function could provide valuable insights into its role in paratuberculosis.

Probiotic bacteria can modulate immune responses to paratuberculosis vaccination.

Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis (PTB), a chronic intestinal inflammatory disease that causes high economical losses in dairy livestock worldwide. Due to the absence of widely available preventive or therapeutical treatments, new alternative therapies are needed. In this study, the effect of a probiotic alone or in combination with a commercial vaccine has been evaluated in a rabbit model. Vaccination enhanced the humoral response, exerted a training effect of peripheral polymorphonuclear neutrophils (PMNs) against homologous and heterologous stimuli, stimulated the release of pro-inflammatory cytokines by gut-associated lymphoid tissue (GALT) macrophages, and reduced the bacterial burden in GALT as well. However, the administration of the probiotic after vaccination did not affect the PMN activity, increased metabolic demand, and supressed pro-inflammatory cytokines, although humoral response and bacterial burden decrease in GALT was maintained similar to vaccination alone. The administration of the probiotic alone did not enhance the humoral response or PMN activity, and the bacterial burden in GALT was further increased compared to the only challenged group. In conclusion, the probiotic was able to modulate the immune response hampering the clearance of the infection and was also able to affect the response of innate immune cells after vaccination. This study shows that the administration of a probiotic can modulate the immune response pathways triggered by vaccination and/or infection and even exacerbate the outcome of the disease, bringing forward the importance of verifying treatment combinations in the context of each particular infectious agent.

Modeling Paratuberculosis in Laboratory Animals, Cells, or Tissues: A Focus on Their Applications for Pathogenesis, Diagnosis, Vaccines, and Therapy Studies.

Paratuberculosis is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. Paratuberculosis that affects a wide variety of domestic and wild animals. It is considered one of the diseases with the highest economic impact on the ruminant industry. Despite many efforts and intensive research, paratuberculosis control still remains controversial, and the existing diagnostic and immunoprophylactic tools have great limitations. Thus, models play a crucial role in understanding the pathogenesis of infection and disease, and in testing novel vaccine candidates. Ruminant animal models can be restricted by several reasons, related to space requirements, the cost of the animals, and the maintenance of the facilities. Therefore, we review the potential and limitations of the different experimental approaches currently used in paratuberculosis research, focusing on laboratory animals and cell-based models. The aim of this review is to offer a vision of the models that have been used, and what has been achieved or discovered with each one, so that the reader can choose the best model to answer their scientific questions and prove their hypotheses. Also, we bring forward new approaches that we consider worth exploring in the near future.

Evaluation of the innate immune response of caprine neutrophils against Mycobacterium avium subspecies paratuberculosis in vitro.

Neutrophils constitute an essential component of the innate immune response, readily killing most bacteria through phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs) among other mechanisms. These cells play an unclear role in mycobacterial infections such as Mycobacterium avium subspecies paratuberculosis (Map), the etiological agent of paratuberculosis, and its response is particularly understudied in ruminants. Herein, a wide set of techniques were adapted, or newly developed, to study the in vitro response of caprine neutrophils after Map infection. Immunofluorescence was used to demonstrate, simultaneously, chemotaxis, phagocytosis, degranulation, and NETs. The quantification of neutrophil phagocytic activity against Map at a 1:10 multiplicity of infection (MOI), through flow cytometry, showed values that varied from 4.54 to 5.63% of phagocyting neutrophils. By immunofluorescence, a 73.3 ± 14.5% of the fields showed NETs, and the mean release of DNA, attributable to NETosis, calculated through a fluorometric method, was 16.2 ± 3.5%. In addition, the RNA expression of TGF-ß, TNF and IL-1ß cytokines, measured through reverse transcription qPCR, was significantly higher in the two latter. Overall, neutrophil response was proportional to the number of bacteria. This work confirms that the simultaneous study of several neutrophil mechanisms, and the combination of different methodologies, are essential to reach a comprehensive understanding of neutrophil response against pathogens, demonstrates that, in vitro, caprine neutrophils display a strong innate response against Map, using their entire repertoire of effector functions, and sets the basis for further in vitro and in vivo studies on the role of neutrophils in paratuberculosis.

The outcome of boosting mitochondrial activity in alcohol-associated liver disease is organ-dependent.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS: C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS: Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.

Oral vaccination stimulates neutrophil functionality and exerts protection in a Mycobacterium avium subsp. paratuberculosis infection model.

Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis (PTB), a granulomatous enteritis in ruminants that exerts high economic impact on the dairy industry worldwide. Current vaccines have shown to be cost-effective against Map and in some cases confer beneficial non-specific effects against other pathogens suggesting the existence of trained immunity. Although Map infection is mainly transmitted by the fecal-oral route, oral vaccination has not been deeply studied. Therefore, the aim of this study was to compare the oral route with a set of mycobacterial and non-mycobacterial vaccines with a subcutaneously administered commercially available vaccine. Training effects on polymorphonuclear neutrophils (PMNs) and homologous and heterologous in vivo protection against Map were investigated in the rabbit infection model. Oral vaccination with inactivated or live vaccines was able to activate mucosal immunity as seen by elevation of serum IgA and the expression of IL4 in peripheral blood mononuclear cells (PBMCs). In addition, peripheral PMN phagocytosis against Map was enhanced by vaccination and extracellular trap release against Map and non-related pathogens was modified by both, vaccination and Map-challenge, indicating trained immunity. Finally, PBMCs from vaccinated animals stimulated in vitro with Map antigens showed a rapid innate activation cytokine profile. In conclusion, our data show that oral vaccination against PTB can stimulate neutrophil activity and both innate and adaptive immune responses that correlate with protection.

Early response of monocyte-derived macrophages from vaccinated and non-vaccinated goats against in vitro infection with Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis is a disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Vaccination is the most cost-effective control method. However, despite the fact that macrophages are the main target cells for this pathogen, the precise mechanisms behind the response of the macrophage to Map infection and how it is modified by vaccination are yet poorly understood. The aim of this study was to investigate the effect of Silirum® vaccination in the early immune response of caprine monocyte-derived macrophages (CaMØs). Peripheral blood mononuclear cells (PBMCs) were obtained from vaccinated and non-vaccinated goats, cultured in vitro until differentiation to macrophages and infected with Map. After a 24 h incubation, Map viability and DNA were assessed in culture by viable colony count and real time quantitative polymerase chain reaction (qPCR). In addition, Map phagocytosis and expression of IL-10, IL-12, IFN-γ, TNF-α, IL-17A, IL-1ß, iNOS, IL-6 and MIP-1ß were also evaluated through immunofluorescence labelling and reverse transcriptase qPCR (RT-qPCR), respectively. A significant reduction of Map viability was observed in both supernatants (P < 0.05) and CaMØs (P < 0.001) from the vaccinated group. Similarly, the percentage of infected CaMØs and the number of internalized Map by CaMØs (P < 0.0001) was higher in the vaccinated group. Finally, iNOS (P < 0.01) and IL-10 were significantly up-regulated in CaMØs from vaccinated goats, whereas only MIP-1ß was up-regulated in non-vaccinated animals (P < 0.05). These results show that vaccination modifies the immune response of CaMØs, suggesting that the phagocytosis and microbiocidal activity of macrophages against Map is enhanced after vaccination.

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury.

BACKGROUND & AIMS: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. METHODS: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. RESULTS: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. CONCLUSIONS: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. LAY SUMMARY: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.

Bovine Neutrophils Release Extracellular Traps and Cooperate With Macrophages in Mycobacterium avium subsp. paratuberculosis clearance In Vitro.

Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guérin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1ß and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1ß and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection site.

Influence of Heterologous and Homologous Vaccines, and Their Components, on the Host Immune Response and Protection Against Experimental Caprine Paratuberculosis.

Vaccination against paratuberculosis, a chronic disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), has been considered as the most effective control method. However, protection is incomplete, and the mechanisms operating in the response of the animals to vaccination are not fully understood. Therefore, this study analyzed the immune response and the effects on protection against Map infection, elicited by paratuberculosis (Silirum®) and tuberculosis (heat-inactivated M. bovis [HIMB]) vaccines and their components in a caprine experimental model. Fifty goat kids were divided into 10 groups (n = 5) according to their vaccination (Silirum®, HIMB and nonvaccinated), immunization (inactivated bacteria or adjuvant), and/or infection. Oral challenge with Map was performed 45 days postvaccination/immunization (dpv), and animals were euthanized at 190 dpv. Peripheral immune response and proportion of lymphocyte subpopulations were assessed monthly by enzyme-linked immunosorbent assay and flow cytometry analysis, respectively. Local immune response, proportion of tissue lymphocyte subpopulations, Map detection (polymerase chain reaction), and histological examination were conducted in gut-associated lymphoid tissues. All infected groups developed paratuberculosis granulomatous lesions despite vaccination or immunization. The Silirum® and HIMB-vaccinated groups showed a considerable lesion reduction consistent with a significant peripheral cellular and humoral immune response. Besides, a lower number of granulomas were observed in groups immunized with inactivated bacteria and adjuvants in comparison to nonvaccinated and infected group. However, despite not being significant, this reduction was even higher in adjuvant immunized groups, which developed milder granulomatous lesion with no detectable peripheral immune responses associated with immunization. No changes in the peripheral and local proportion of lymphocyte subsets or local immune response were detected in relation to either vaccination/immunization or infection. Despite that paratuberculosis and tuberculosis vaccination showed a partial and cross-protection against Map infection, respectively, only histological examination could assess the progression of infection in these animals. In addition, the pattern observed in the reduction of the lesions in adjuvant immunized groups suggests the possible involvement of a nonspecific immune response that reduces the development of granulomatous lesions.

Alternative Vaccination Routes against Paratuberculosis Modulate Local Immune Response and Interference with Tuberculosis Diagnosis in Laboratory Animal Models.

Paratuberculosis (PTB) is an enteric granulomatous disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that mainly affects ruminants. Current vaccines have shown to be cost-effective control reagents, although they are restricted due to cross-interference with bovine tuberculosis (bTB). Therefore, novel vaccination strategies are needed and this study is focused on evaluating alternative vaccination routes and their effect on the local immune response. The MAP oral challenge rabbit model was used to evaluate and compare an experimental inactivated MAP vaccine through oral (VOR) and intradermal (VID) routes. The VID group presented the highest proportion of animals with no visible lesions and the lowest proportion of animals with MAP positive tissues. Immunohistochemistry analysis revealed that the VID group presented a dominantly M1 polarized response indicating an ability to control MAP infection. In general, all vaccinated groups showed lower calprotectin levels compared to the non-vaccinated challenged group suggesting less active granulomatous lesions. The VID group showed some degree of skin test reactivity, whereas the same vaccine through oral administration was completely negative. These data show that PTB vaccination has an effect on macrophage polarization and that the route influences infection outcome and can also have an impact on bTB diagnosis. Future evaluation of new immunological products against mycobacterial diseases should consider assaying different vaccination routes.

Tuberculosis outbreak caused by Mycobacterium caprae in a rabbit farm in Spain.

Animal tuberculosis remains a great source of socioeconomic and health concern worldwide. Its main causative agents, Mycobacterium bovis and Mycobacterium caprae, have been isolated from many different domestic and wild animals. Naturally, occurring tuberculosis is extremely rare in rabbits, and implication of M. caprae has never been reported earlier. This study describes a severe tuberculosis outbreak caused by M. caprae in a Spanish farm of rabbits raised for meat for human consumption. The disease was first identified in a cachectic dam, and then it was confirmed in ten does with similar clinical signs. Subsequently, a depopulation operation was ordered for public health, animal welfare and environmental reasons. To broaden knowledge of spontaneous tuberculosis in rabbits, a study focused on pathological, epidemiological and diagnostic aspects was carried out on 51 does and 16 kittens after receiving the necessary authorizations. These animals were subjected to a modified intradermal test. After being euthanized, rabbits were examined for the presence of visible tuberculosis-compatible lesions. Lung, kidney, caecal appendix and sacculus rotundus samples underwent microbiological and anatomopathological analysis. Infection was revealed by at least one of the methods used in 71% of dams and in 44% of kittens. The intradermal test was shown to be a good indicator of infection. Lung was the tissue for which more animals were positive but renal and intestinal tissues were also affected in many cases. Apparently, M. caprae spread mainly through the aerogenous route. Infection was pathologically characterized by the absence of evident fibrous capsules surrounding granulomas. A spoligotype (SB0415) frequently found in this area was considered responsible for the outbreak but the source could not be established. Regardless of the exceptional nature of animal tuberculosis in this host, rabbit industry might not escape from its effects and therefore, current biosafety and surveillance strategies should also consider this disease.

Diet induced changes in the microbiota and cell composition of rabbit gut associated lymphoid tissue (GALT).

The gut associated lymphoid tissue (GALT) is the largest immune organ of the body. Although the gut transient and mucosa-associated microbiota have been largely studied, the microbiota that colonizes the GALT has received less attention. The gut microbiome plays an important role in competitive exclusion of pathogens and in development and maturation of immunity. Diet is a key factor affecting the microbiota composition in the digestive tract. To investigate the relation between diet, microbiota and GALT, microbial and cell composition of vermiform appendix (VA) and sacculus rotundus (SR) were studied in two groups of New Zealand white rabbits on different diets. Diet shifted the lymphoid tissue microbiota affecting the presence and/or absence of certain taxa and their abundances. Immunohistochemistry revealed that a higher fibre content diet resulted in M cell hyperplasia and an increase of recently recruited macrophages, whereas T-cell levels remained unaltered in animals on both high fibre and standard diets. These findings indicate that diet has an impact on the microbiota and cell composition of the GALT, which could act as an important microbial recognition site where interactions with beneficial bacteria can take place favouring microbiota replacement after digestive dysregulations.

Different lesion distribution in calves orally or intratracheally challenged with Mycobacterium bovis: implications for diagnosis.

Animal tuberculosis (TB) remains a major problem in some countries despite the existence of control programmes focused mainly on cattle. In this species, aerogenous transmission is accepted as the most frequent infection route, affecting mainly the respiratory system. Under the hypothesis that the oral route could be playing a more relevant role in transmission, diagnosis and disease persistence than previously thought, this study was performed to assess the course of TB infection in cattle and its effects on diagnosis depending on the route of entry of Mycobacterium bovis. Two groups of five calves each were either endotracheally (EC) or orally (OC) challenged. Necropsies were carried out 12 weeks after challenge except for three OC calves slaughtered 8 weeks later. All animals reacted to the tuberculin skin test and the entire EC group was positive to the interferon-gamma release assay (IGRA) 2 weeks after challenge and thereafter. The first positive IGRA results for OC calves (3/5) were recorded 4 weeks after challenge. Group comparison revealed significant differences in lesion and positive culture location and scoring. TB-compatible gross lesions and positive cultures were more frequently found in the thorax (p < 0.001) and lung (p < 0.05) of EC animals, whereas OC animals presented lesions (p = 0.23) and positive cultures (p < 0.05) mainly located in the abdomen. These results indicate that the infection route seems to be a determining factor for both the distribution and the time needed for the development of visible lesions. Our study suggests that confirmation of TB infection in some skin reactor animals can be problematic if current post-mortem examination and diagnostics are not improved.

Preliminary Results Indicate That Inactivated Vaccine against Paratuberculosis Could Modify the Course of Experimental Mycobacterium bovis Infection in Calves.

Although paratuberculosis (PTB) vaccination has been recognized as an effective tool to control the disease, its use has been limited in countries undergoing bovine tuberculosis (bTB) eradication programs because of its interference with the diagnostic techniques. Due to this restraint, little is known about the effect of vaccinating against PTB on the progression of bTB infection. To assess this topic, an experimental infection was carried out including the following three groups of five calves each: non-vaccinated infected with Mycobacterium bovis (NVI), vaccinated against PTB infected with M. bovis (VI), and vaccinated against PTB non-infected (VNI). The level of infection attending to pathological and bacteriological parameters was evaluated at necropsy in collected tissue samples. Infection was confirmed in all challenged animals being the lung and thoracic regions most affected for all studied parameters. The VI group presented 15.62% less gross lesions in the thoracic region than the NVI, although no significant differences were found. Only one vaccinated animal presented gross lesions in the lung, compared to three non-vaccinated calves. NVI animals showed an average of 1.8 lung lobes with gross lesions whereas in the vaccinated group the average number of affected lobes was 0.2, representing an 89% reduction. Significant differences were not found, although a tendency was observed (p = 0.126). Pathological and culture scores showed the same tendency. Vaccination induced a 71.42 and 60% reduction in lesion and culture scores in the lung as well as a 23.75 and 26.66% decline, respectively, in the thoracic region. The VI group showed lower positivity in the rest of the areas for all measured criteria except for the head. In order to reinforce our results, further research on a larger sample size is needed, but the results from this study suggest that PTB vaccination could confer certain degree of protection against bTB infection, supporting the view that PTB vaccination could increase resistance to the main mycobacterioses that affect animals.

Detection of Mycobacteria by Culture and DNA-Based Methods in Animal-Derived Food Products Purchased at Spanish Supermarkets.

Mycobacteria include obligate and opportunistic pathogens that cause significant human and animal disease. The burden of tuberculosis has been largely reduced in developed territories but remains a huge problem worldwide. The significance of nontuberculous mycobacteria is growing considerably, especially in developed regions with higher life expectancy and more therapy-related immunosuppressed individuals. Due to their robustness mycobacteria can contaminate animal products by direct transmission from infected individuals or by environmental contamination during processing. The situation at market level is poorly known. Most studies analyzing commercially available foods are limited to a small or local scale and mainly focused on a particular mycobacterial species. There is a need to investigate if animal products that have passed the established controls to be for sale at main supermarkets could represent a route of contact with any mycobacteria. Thus, our goal was to study the prevalence of mycobacteria in these foods to assess if this could represent a source of human exposure. Five stores from the main supermarket chains in Spain were selected. 138 dairy and 119 meat products were purchased. All were processed using culture and multiplex real-time PCR methods. Additional molecular methods were used to specifically identify any positive result. Mycobacterium avium subsp. hominissuis (2), M. avium subsp. avium (1), and M. fortuitum (1) were isolated from powdered infant formula and ground beef, chicken sausage, and mortadella cold cut, respectively. Mycobacterial DNA (M. avium, M. tuberculosis complex and other nontuberculous mycobacteria) was detected in 15% of dairy products and 2% of meat products. These results show that the prevalence of viable mycobacteria in foods of animal origin obtained at the supermarket was not substantial although a considerable proportion of them contained mycobacterial DNA. Contact with mycobacteria through this route could be ensured over time. Further investigation is necessary to determine the real impact of foodborne mycobacterial exposure on human health and identify critical points in the food production system to enable setting up more stringent control measures.

Immunohistochemical expression of interferon-γ in different types of granulomatous lesions associated with bovine paratuberculosis.

Animals infected with Mycobacterium avium subsp. paratuberculosis (Map) show a variety of lesions, from focal forms, seen in subclinical stages to diffuse lesions in clinical cases. The purpose of this study was to evaluate the local expression of IFN-γ by immunohistochemistry in relation with the type of lesion in naturally Map-infected cows. The number of immunolabelled cells, -the majority morphologically consistent with lymphocytes-, was higher in focal and diffuse paucibacillary forms than in diffuse multibacillary lesions, where they appeared closely related to epithelioid cells. Diffuse multibacillary lesions had the lowest numbers, but higher than controls, and positive cells were intermingled among the macrophages. The peripheral IFN-γ production was higher in all Map infected cows and a positive correlation was found with the number of immunolabelled cells in the intestine. The findings of this study show that IFN-γ would play a role in the development of the different types of lesions in paratuberculosis, and also points out the importance of adequate sampling of lymphoid tissue containing samples when studying the local immune response in which IFN-γ expression may be involved, especially in cases where focal lesions are present.

Tuberculosis Detection in Paratuberculosis Vaccinated Calves: New Alternatives against Interference.

Paratuberculosis vaccination in cattle has been restricted due to its possible interference with the official diagnostic methods used in tuberculosis eradication programs. To overcome this drawback, new possibilities to detect Mycobacterium bovis infected cattle in paratuberculosis vaccinated animals were studied under experimental conditions. Three groups of 5 calves each were included in the experiment: one paratuberculosis vaccinated group, one paratuberculosis vaccinated and M. bovis infected group and one M. bovis infected group. The performance of the IFN-gamma release assay (IGRA) and the skin test using conventional avian and bovine tuberculins (A- and B-PPD) but also other more specific antigens (ESAT-6/CFP10 and Rv3615c) was studied under official and new diagnostic criteria. Regarding the IGRA of vaccinated groups, when A- and B-PPD were used the sensitivity reached 100% at the first post-challenge sampling, dropping down to 40-80% in subsequent samplings. The sensitivity for the specific antigens was 80-100% and the specificity was also improved. After adapting the diagnostic criteria for the conventional antigens in the skin test, the ability to differentiate between M. bovis infected and non-infected animals included in paratuberculosis vaccinated groups was enhanced. Taking for positive a relative skin thickness increase of at least 100%, the single intradermal test specificity and sensitivity yielded 100%. The comparative intradermal test was equally accurate considering a B-PPD relative skin increase of at least 100% and greater than or equal to that produced by A-PPD. Using the specific antigens as a proteic cocktail, the specificity and sensitivity reached 100% considering the new relative and absolute cut-offs in all experimental groups (Δ≥30% and Δmm ≥ 2, respectively). Results suggest that the interference caused by paratuberculosis vaccination in cattle could be completely overcome by applying new approaches to the official tuberculosis diagnostic tests.

Vaccination sequence effects on immunological response and tissue bacterial burden in paratuberculosis infection in a rabbit model.

Paratuberculosis (PTB), a chronic granulomatous enteritis produced by Mycobacterium avium subspecies paratuberculosis (MAP), is considered as one of the diseases with the highest economic impact in the ruminant industry. Vaccination against MAP is recommended during the first months after birth on the basis that protection would be conferred before the first contact with mycobacteria. However, little is known about the therapeutic effect of MAP vaccination in controlled experimental conditions. The current study was designed to evaluate the efficacy of vaccination before and after challenge with MAP in a rabbit infection model. The rabbits were divided into four groups: non-infected control (NIC, n = 4), infected control challenged with MAP (IC, n = 5), vaccinated and challenged 1 month after with MAP (VSI, n = 5) and challenged with MAP and vaccinated 2 months later (IVS, n = 5). The results from this study show a quick increase in IFN-γ release upon stimulation with bovine, avian and johnin PPD in animals vaccinated before MAP challenge. All vaccinated animals show an increased humoral response as seen by western blot and ELISA. The final bacteriology index (considering tissue culture and qPCR) shows that the IC group was the most affected. Vaccination after infection (IVS) produced the lowest bacteriology index showing significant differences with the IC group (p = 0.034). In conclusion, vaccination against MAP shows positive effects in a rabbit model. However, vaccination after infection shows a slightly stronger protective effect compared to vaccination before infection, suggesting a therapeutic effect. This feature could be applied to previously infected adult animals under field conditions.

Mycobacterium avium Subspecies paratuberculosis Infection Modifies Gut Microbiota under Different Dietary Conditions in a Rabbit Model.

Mycobacterium avium subspecies paratuberculosis (MAP) the causative agent of paratuberculosis, produces a chronic granulomatous inflammation of the gastrointestinal tract of ruminants. It has been recently suggested that MAP infection may be associated with dysbiosis of intestinal microbiota in ruminants. Since diet is one of the key factors affecting the balance of microbial populations in the digestive tract, we intended to evaluate the effect of MAP infection in a rabbit model fed a regular or high fiber diet during challenge. The composition of microbiota of the cecal content and the sacculus rotundus was studied in 20 New Zealand white female rabbits. The extracted DNA was subjected to paired-end Illumina sequencing of the V3-V4 hypervariable region of the 16S rRNA gene for microbiota analysis. Microbial richness (Chao1) in the cecal content was significantly increased by MAP infection in regular diet rabbits (p = 0.0043) and marginally increased (p = 0.0503) in the high fiber group. Analysis of beta-diversity showed that MAP infection produces deeper changes in the microbiota of sacculus rotundus than in the cecal content. A lower abundance of Proteobacteria in the cecal content of infected animals fed the high fiber diet and also lower abundance of Bacteroidetes in the sacculus rotundus of infected animals fed the regular diet were observed. Based on OPLS-DA analysis, we observed that some bacteria repeatedly appear to be positively associated with infection in different samples under different diets (families Dehalobacteriaceae, Coriobacteriaceae, and Mogibacteriaceae; genus Anaerofustis). The same phenomenon was observed with some of the bacteria negatively associated with MAP infection (genera Anaerostipes and Coprobacillus). However, other groups of bacteria (Enterobacteriaceae family and ML615J-28 order) were positively associated with infection in some circumstances and negatively associated with infection in others. Data demonstrate that MAP infection and diet changes do interact and result in shifts in the microbiota of the cecal content and sacculus rotundus of rabbits.