Mycobacteria in rabbits: Searching for an efficient paratuberculosis infection model

238 p.La paratuberculosis es una enfermedad intestinal granulomatosa crónica, producida por Mycobacterium avium subsp paratuberculosis (Map) perteneciente al complejo mycobacterium avium (MAC). Esta enfermedad que afecta principalmente a los rumiantes causa cuantiosas pérdidas económicas y podría estar relacionada con la enfermedad de Crohn en humanos. El avance en el estudio de la paratuberculosis se ha visto limitado debido a la falta de un modelo de laboratorio adecuado.En esta Tesis Doctoral se ha avanzado en el estudio de la susceptibilidad del conejo a la infección por MAC, así como en la utilización del conejo como modelo animal de laboratorio, lo que ha permitido progresar en el conocimiento de la patogenia y la vacunación frente a Map. Así, se ha demostrado que la utilización de diferentes estrategias alimentarias a través de la modificación de la microbiota intestinal modula la infección por Map. Además se ha observado que la vacunación frente a Map disminuye los índices de infección a través del aumento de la respuesta inmune humoral y celular. Por otra parte, la vacunación tras el desafío ha mostrado cierto grado de efecto terapéutico. Estos hallazgos sugieren que estas estrategias deberían ensayarse con un enfoque holístico, teniendo en cuenta las posibles interacciones y/o efectos aditivos. En el presente trabajo hemos demostrado que el conejo es un modelo animal de infección de Map útil, que permite avanzar en el estudio de la infección subclínica y por ende podría ser de utilidad en el estudio de otras enfermedades inflamatorias intestinales en humanos.Neiker/Tecnali

Evaluation of Mycobacterium avium subsp. paratuberculosis isocitrate lyase (IcL) and ABC transporter (BacA) knockout mutants as vaccine candidates

There has been little success in controlling Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis, due to suboptimal diagnostics and the ineffectiveness of available vaccines. By knocking out BacA and IcL, genes required for MAP survival in dairy calves, two live-attenuated vaccine candidates were created. This study evaluated the host-specific attenuation of MAP IcL and BacA mutants in mouse and calf models, as well as the elicited immune responses. Deletion mutants were generated in MAP strain A1-157 through specialized transduction and found viable in vitro. First, the mutants’ attenuation and elicited cytokine secretion were assessed in a mouse model, 3 weeks after intraperitoneal inoculation with MAP strains. Later, vaccine strains were assessed in a natural host infection model where calves received 109CFU oral dose of MAP wild-type or mutant strains at 2 weeks old. Transcription levels of cytokines in PBMCs were evaluated at 12-, 14-, and 16-weeks post-inoculation (WPI) and MAP colonization in tissue was assessed at 4.5 months after inoculation. Whereas both vaccine candidates colonized mouse tissues similarly to wild-type strain, both failed to persist in calf tissues. In either mouse or calf models, gene deletion did not reduce immunogenicity. Instead, inoculation with ΔBacA induced a greater upregulation of proinflammatory cytokines than ΔIcL and wild-type in both models and a greater expansion of cytotoxic and memory T-cells than uninfected control in calves. ΔBacA and wild-type strains significantly increased secretion of IP-10, MIG, TNFα, and RANTES in mice serum compared to uninfected control. This agreed with upregulation of IL-12, IL-17, and TNFα in calves inoculated with ΔBacA at all time points. The ΔBacA also gave rise to greater populations of CD4+CD45RO+, and CD8+ cells than uninfected control calves at 16 WPI. Low survival rate of MAP in macrophages co-incubated with PBMCs isolated from the ΔBacA group indicated that these cell populations are capable of killing MAP. Overall, the immune response elicited by ΔBacA is stronger compared to ΔIcL and it is maintained over two different models and over time in calves. Further investigation is warranted to evaluate the BacA mutant's protection against MAP infection as a live attenuated vaccine candidate

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

[EN] 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 dysregulationsSIAuthors thank Félix Blanco, Sergio Ayuso and Fidel Goiri for animal care and handling. The research was funded by grant (AGL2012-39818-C02-02) from the Spanish Ministry of Economy and Competiveness (MINECO), and by the Department of Economy and Infrastructures (DEI) of the Basque Government. RA held a pre-doctoral fellowship (BFI-2012-237) and a visiting fellowship (EP_2015_1_53) from the Department of Education, Universities and Research of the Basque Governmen

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

[EN] 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 conditionsSIThe authors thank Felix Blanco from the animal facilities (NEIKER-Tecnalia). This study was supported with funds from the Ministerio de Economía y Competitividad from the Spanish Government (AGL 2012-39818-C02-02). RA acknowledges support from the Departamento de Educación y Universidades of the Basque Government (BFI-2012-237

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

[EN] Paratuberculosis (PTB) is an enteric granulomatous disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that mainly aects ruminants. Current vaccines have shown to be cost-eective 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 eect 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 eect 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 dierent vaccination routesSIThe research was funded by the Department of Economy and Infrastructures (DEI) of the Basque Government and partially by grant RTA 2017-00089-00-00 of the National Institute for Agronomic Research (INIA). R.A. held a pre-doctoral fellowship (BFI-2012-237) from the Department of Education, Universities and Research of the Basque Government and IL is recipient of a pre-doctoral fellowship from the DEI of the Basque Governmen

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

[EN] 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 protectionSIThe research was funded by the Department of Economy, Sustainability and Environment of the Basque Government and by grant RTA 2017-00089-00-00 of the National Institute for Agronomic Research (INIA) to N.E. I.L.-A. and M.O. both held predoctoral fellowships from the DEI of the Basque Government. This research was also partly supported by the Agriculture Funding Consortium members Alberta Agriculture and Forestry and Alberta Milk (2018F019R) to J.D.B. CIC bioGUNE thanks the Ministry of Science and Innovation for the Severo Ochoa excellence award (SEV- 2016-0644). The authors thank Félix Blanco, Sergio Ayuso, and Fidel Goiri for animal care and handling and Ainara Badiola for technical support at NEIKER. We thank Joseba Garrido for critical reading of the manuscript. Also, thanks to the technical and human support provided by SGIker of the UPV/EHU and European Funding (ERDF and ESF) in the analysis of the expression of mRNAs with the Fluidigm BioMark HD Nanofluidic qPCR syste

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

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

Abstract 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.The research was funded by the Department of Economy, Sustainability and Environment of the Basque Government and by grant RTA 2017-00089-00-00 of the National Institute for Agronomic Research (INIA) to N.E. I.L.-A. and M.O. both held predoctoral fellowships from the DEI of the Basque Government. This research was also partly supported by the Agriculture Funding Consortium members Alberta Agriculture and Forestry and Alberta Milk (2018F019R) to J.D.B. CIC bioGUNE thanks the Ministry of Science and Innovation for the Severo Ochoa excellence award (SEV-2016-0644

A Differential Innate Immune Response in Active and Chronic Stages of Bovine Infectious Digital Dermatitis

Digital dermatitis (DD) commonly associated with Treponema spp. infection is a prevalent infectious bovine foot disease characterized by ulcerative and necrotic lesions. Lesions associated with DD are often classified using the M-stage scoring system, with M0 indicating healthy heel skin and M4 indicating chronic lesions. Current treatments utilizing antimicrobials or chemical footbaths are often ineffective and rarely cure DD lesions. Understanding the function of the innate immune response in the pathogenesis of DD will help to identify novel therapeutic approaches. In this study, the expression of the local innate host defense peptides cathelicidins and β-defensins was investigated in cows with DD and associated with the presence of treponemes and inflammatory reactions. Samples from active ulcerative DD lesions (M2) had considerable epidermal neutrophilic infiltration and increased gene expression of β-defensin tracheal antimicrobial peptides compared to control skin. Samples from acute lesions also had elevated local Cxcl-8 and TLR4 gene expression and abundant treponemes as identified by direct visualization, immunohistochemistry, and culture. Conversely, the anti-inflammatory peptide IL-10 was elevated in skin from chronic (M4) lesions, whereas bovine cathelicidin myeloid antimicrobial peptide 28 (Bmap-28) was increased in skin from oxytetracycline-treated M2 lesions. Experiments using cultured human keratinocytes challenged with Treponema spp. isolated from clinical cases of bovine DD showed that structural products from treponemes are able to initiate the innate immune response, in part through TLR2 signaling. These findings indicate that neutrophil influx, Cxcl-8, and β-defensin are key markers of active DD. Cathelicidins and IL-10 seem important in response to treatment or during the chronic proliferative stages of the disease