Multicomponent Pathogen-Mimicking Nanoparticles Induce Intestinal Immune Responses against Paratuberculosis.

Given the worldwide problem posed by enteric pathogens, the discovery of safe and efficient intestinal adjuvants combined with novel antigen delivery techniques is essential to the design of mucosal vaccines. In this work, we designed poly (lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) to codeliver all-trans retinoic acid (atRA), novel antigens, and CpG. To address the insolubility of the intestinal adjuvant atRA, we utilized PLGA to encapsulate atRA and form a "nanocapsid" with polydopamine. By leveraging polydopamine, we adsorbed the water-soluble antigens and the TLR9 agonist CpG onto the NPs' surface, resulting in the pathogen-mimicking PLPCa NPs. In this study, the novel fusion protein (HBf), consisting of the Mycobacterium avium subspecies paratuberculosis antigens HBHA, Ag85B, and Bfra, was coloaded onto the NPs. In vitro, PLPCa NPs were shown to promote the activation and maturation of bone marrow-derived dendritic cells. Additionally, we found that PLPCa NPs created an immune-rich microenvironment at the injection site following intramuscular administration. From the results, the PLPCa NPs induced strong IgA levels in the gut in addition to enhancing powerful systemic immune responses. Consequently, significant declines in the bacterial burden and inflammatory score were noted in PLPCa NPs-treated mice. In summary, PLPCa can serve as a novel and safe vaccine delivery platform against gut pathogens, such as paratuberculosis, capable of activating both systemic and intestinal immunity.

Comparative performance of different antigens on the lateral flow assay (LFA) platform for the rapid serodiagnosis of paratuberculosis.

Paratuberculosis is a globally prevalent disease, that adversely affects the economy of livestock farming. Control is largely based on early detection followed by 'Test and Cull' or 'Test and Segregate' Policy. Implementation of paratuberculosis control is a special challenge due to the non-availability of point of care diagnostics (PoCD). Therefore, the present study aimed to optimize and evaluate a lateral flow assay (LFA) for the rapid serodiagnosis of paratuberculosis in ruminant species, especially in the view of the resource-limited areas. Performance of three different antigenic preparations including native purified protoplasmic antigen (nPPA-LFA), commercial purified protoplasmic antigen (cPPA-LFA), and a cocktail of recombinant secretory proteins (RP-LFA) was evaluated as detection reagents for coating LFA strips. Comparative performance of the optimized LFA was also evaluated with gold standard tissue culture, fecal PCR (polymerase chain reaction), and plate ELISA. In addition, the onsite testing of animals belonging to different farms (endemic), species, and regions using optimized LFA was also done to highlight the on-farm testing approach. Findings revealed recombinant secretory proteins based LFA (RP-LFA) had a higher sensitivity of detection compared to other antigens. RP-LFA had a sensitivity of 77.7%, 75.44%, and 75.16% in comparison to gold standard tissue culture, fecal PCR, and plate ELISA, respectively. The specificity of RP-LFA was 100% with all reference tests. In comparison to plate ELISA, RP-LFA had a detection limit of 100% when the S/P ratio of the serum sample is ≥1.0 and 80% when the S/P ratio range of 0.8-1.0. Using RP-LFA, on-farm testing of 608 animals was done and 283 (46.5%) were found positive. Kappa analysis of present RP-LFA revealed 'good strength of agreement' with gold standard tissue culture, fecal PCR, and plate ELISA. Optimized RP-LFA had no cross-reactivity with bovine tuberculosis (bovine TB). The RP-LFA was found reproducible, user-friendly and test results can be interpreted within five minutes. In conclusion, the findings of the present study advocate the huge potential of LFA-based PoCD in the rapid diagnosis and control of paratuberculosis.

Infections and Multiple Sclerosis: From the World to Sardinia, From Sardinia to the World.

Multiple Sclerosis (MS) is an inflammatory disease of the central nervous system. Sardinia, an Italian island, is one of the areas with the highest global prevalence of MS. Genetic factors have been widely explored to explain this greater prevalence among some populations; the genetic makeup of the Sardinians appears to make them more likely to develop autoimmune diseases. A strong association between MS and some infections have been reported globally. The most robust evidence indicating the role of infections is MS development concerns the Epstein-Barr virus (EBV). Anti-EBV antibodies in patients once infected by EBV are associated with the development of MS years later. These features have also been noted in Sardinian patients with MS. Many groups have found an increased expression of the Human endogenous retroviruses (HERV) family in patients with MS. A role in pathogenesis, prognosis, and prediction of treatment response has been proposed for HERV. A European multi-centre study has shown that their presence was variable among populations, ranging from 59% to 100% of patients, with higher HERV expression noted in Sardinian patients with MS. The mycobacterium avium subspecies paratuberculosis (MAP) DNA and antibodies against MAP2694 protein were found to be associated with MS in Sardinian patients. More recently, this association has also been reported in Japanese patients with MS. In this study, we analysed the role of infectious factors in Sardinian patients with MS and compared it with the findings reported in other populations.

Elevated mycobacterium avium subsp. paratuberculosis (MAP) antibody titer in Japanese multiple sclerosis.

To investigate whether antibody production against mycobacterium avium subsp. paratuberculosis (MAP) is related to clinical characteristics of multiple sclerosis (MS) and human leukocyte antigen (HLA) alleles, IgG antibody against three MAP peptides and two human peptides homologous to MAP were measured in sera from 103 MS patients and 50 healthy controls (HCs). MS patients had higher IgG levels against MAP2694295-303 (MAP2694-IgG) than HCs, while the other antibodies were comparable. Multivariate analysis demonstrated that higher MAP2694-IgG titers were associated with higher EDSS scores, but not with HLA alleles or dairy product consumption. Immune response against MAP may worsen MS disability.

Revealing immune responses in the Mycobacterium avium subsp. paratuberculosis-infected THP-1 cells using single cell RNA-sequencing.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease, which is a chronic and debilitating disease in ruminants. MAP is also considered to be a possible cause of Crohn's disease in humans. However, few studies have focused on the interactions between MAP and human macrophages to elucidate the pathogenesis of Crohn's disease. We sought to determine the initial responses of human THP-1 cells against MAP infection using single-cell RNA-seq analysis. Clustering analysis showed that THP-1 cells were divided into seven different clusters in response to phorbol-12-myristate-13-acetate (PMA) treatment. The characteristics of each cluster were investigated by identifying cluster-specific marker genes. From the results, we found that classically differentiated cells express CD14, CD36, and TLR2, and that this cell type showed the most active responses against MAP infection. The responses included the expression of proinflammatory cytokines and chemokines such as CCL4, CCL3, IL1B, IL8, and CCL20. In addition, the Mreg cell type, a novel cell type differentiated from THP-1 cells, was discovered. Thus, it is suggested that different cell types arise even when the same cell line is treated under the same conditions. Overall, analyzing gene expression patterns via scRNA-seq classification allows a more detailed observation of the response to infection by each cell type.

Development of rELISA using novel markers for the diagnosis of paratuberculosis.

Paratuberculosis is one of the complex livestock infections whose control has largely been hampered due to the lack of efficacious diagnostics. Present study optimized plate ELISA assay for the diagnosis and screening of paratuberculosis using recombinant secretory proteins. Five secretory antigens (2677c, 3547c, 4308c, 1693c, and 2168c) were produced in the recombinant system using the E. coli host and used for the optimization of the assay. These proteins were selected because of their prior proven specificity and antigenicity as humoral immunity markers. The assay was first optimized using traditional ELISA reader and then the performance was evaluated using a handheld ELISA reader. Findings were identical in both traditional ELISA reader as well as handheld ELISA reader. Optimized ELISA was found reproducible using different batches of the recombinant antigens as well as in terms of the inter and intra assay %CV values. The present ELISA has a sensitivity and specificity of 91.6% and 100%, respectively. Also, rELISA revealed AUCROC and Youden index J of 0.95 and 0.91, respectively. In conclusion, assay conditions of MAP-recombinant protein-based ELISA were optimized and the optimized ELISA ODs can be read using portable handheld ELISA reader. Thereby, opening a future window to develop assay for onsite testing.

New and rapid strategies for the diagnosis of bovine paratuberculosis "in situ" using latex particles.

The chemical coupling of a protoplasmatic antigen from Mycobacterium avium subsp. paratubeculosis onto core-shell carboxylated particles was investigated with the aim of producing latex-protein complexes to be used in immunoagglutination assays capable of detecting bovine paratuberculosis disease. For this purpose, sensitizations were carried out using both colored and not colored carboxylated latexes as well as the protoplasmatic antigen at pH close to its isoelectric point to favor the antigenic protein to approach the particle surface. In all cases, higher fractions of proteins were chemically-bound to carboxyl groups on the surface of the particles. The assessment of the performance of the visual immunoagglutination assays consisted of evaluating 111 sera from healthy and infected bovines with Mycobacterium avium subsp. paratuberculosis. Complexes obtained from the colored latex allowed an acceptable visual discrimination between the studied positive and negative sera. Most of the positive samples showed strong to very strong agglutination and only a few samples reacted weakly, i.e. a sensitivity of 70%. The specificity of the assay, on the other hand, was 86%. Therefore, this rapid detection technique allows an easy and inexpensive identification of animals possibly infected with paratuberculosis "in situ" in the herds.

Is vaccination a viable method to control Johne's disease caused by Mycobacterium avium subsp. paratuberculosis? Data from 12 million ovine vaccinations and 7.6 million carcass examinations in New South Wales, Australia from 1999-2009.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (or paratuberculosis), a chronic wasting disease of ruminants and other animals resulting from granulomatous enteritis. There are increasing concerns that MAP is zoonotic. The prevalence of Johne's disease is increasing worldwide. In an attempt to control an epidemic of ovine Johne's disease (OJD) in New South Wales (NSW), a government/industry sponsored voluntary vaccination/on-farm management program commenced in 2000. We report herein an observational study of changes in disease prevalence as vaccination progressed, based on abattoir surveillance data for OJD from 1999 to 2009. We also discuss the epidemiological, policy, regulatory, research, economic and sociological elements that contributed to the development of a mature control program, whose aim was to halt the epidemic spread of OJD in a naïve sheep population. METHODS: NSW was divided into areas of "High" (HPA), "Medium" (MPA) and "Low" (LPA) OJD prevalence. A killed whole cell vaccine (Gudair®) was administered to sheep from 2000 to 2009. Trained examiners evaluated the viscera of adult sheep carcasses at slaughter for gross evidence of OJD. MAP infection was confirmed by histopathology. PRINCIPAL FINDINGS: From 2000-2009, 12 million vaccine doses were administered in NSW (91%; 10.9 million in the HPA). Many of the vaccinated flocks were suffering > 5% annual mortality in adult sheep, with some individual flocks with 10-15% losses attributable to OJD. A total of 7.6 million carcasses were examined (38%; 2.9 million from the HPA). Overall, 16% of slaughter consignments (sheep consigned to the abattoir from a single vendor) were positive for OJD, of which 94% were from the HPA. In the HPA, the percentage of animals with lesions attributable to OJD at slaughter fell progressively from 2.4% (10,406/432,860) at commencement of vaccination in 2000 to 0.8% (1,573/189,564) by 2009. Herd immunity from vaccination in the HPA was estimated at 70% by 2009, the target commonly espoused for an effective control program based on vaccination. This coincided with a progressive decrease in reports of clinical disease and mortalities in vaccinated flocks. SIGNIFICANCE: We show a decrease in the prevalence of lesions attributable to OJD in NSW concomitant with initiation of voluntary vaccination, on-farm management plans, abattoir monitoring and feedback of animal prevalence data to sheep producers. We conclude that a target of ≤ 1% regional prevalence of OJD affected sheep at slaughter is achievable using these interventions.

Factors influencing the effectiveness of the Gudair vaccine for controlling Johne's disease in sheep flocks in Australia.

Ovine Johne's disease is a chronic debilitating disease of sheep caused by Mycobacterium avium subsp. paratuberculosis (Mptb) which results in diarrhoea, emaciation and mortalities in infected animals. Vaccination with Gudair® has been a key strategy for controlling the disease in Australia since its approval in 2002. Previous research conducted in Australia has demonstrated that the vaccine is quite effective in reducing sheep mortalities. While some farms have also been successful in reducing the prevalence of the disease in their flocks to undetectable levels, sheep in other flocks continue to shed Mptb in faeces even after an ongoing vaccination program . This study was conducted to investigate management, husbandry and biosecurity factors associated with paratuberculosis infection in Gudair® vaccinated sheep flocks in Australia. We enrolled 64 sheep farmers and interviewed them to obtain information about their management and biosecurity practices. Pooled faecal samples were collected from sheep at each farm and cultured to create two outcome variables: Mptb positive (yes/no) and disease prevalence level (nil, < 1 %, ≥ 1 %). Binary and ordinal logistic regression analyses were conducted to evaluate the association of management, husbandry and biosecurity factors with these outcome variables. Farms were more likely to have Mptb positive sheep and a higher disease prevalence in their flocks if they: (a) provided supplementary feed on the ground (instead of in a trough); (b) had a greater number of neighbours with sheep; and (c) had introduced rams from a greater number of sources. The results suggest the effectiveness of Gudair® vaccination to control OJD can be improved if sheep producers maintain other risk management strategies and biosecurity practices. Extension agencies should advise farmers not to relax their biosecurity practices and to purchase rams from only low-risk sources, even if they are continuing to vaccinate their flocks.

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.

Comparative cellular immune responses in calves after infection with Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis.

In the present study, calves were infected with Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. avium (M. avium), Mycobacterium kansasii (M. kansasii), or Mycobacterium bovis (M. bovis) to determine differences in cellular immunity. Comparative cellular responses were assessed upon stimulation of cells with mycobacterial whole cell sonicates respective of each infection group. Antigen-specific whole blood interferon gamma (IFN-γ) responses were observed in all infection groups compared to noninfected control calves, however, responses were more robust for M. bovis calves. Upon antigen stimulation of PBMCs, secretion of IFN-γ and IL-10 was higher for M. bovis calves compared to other infection groups. In contrast, IL-12 secretion was lower for M. bovis calves compared to MAP infected calves. Within the total PBMC population, higher numbers of CD4+, CD8+, and γδ TCR + T cells were observed for MAP and M. avium calves compared to M. bovis calves. This aligned with higher expression of CD26 on these subpopulations for MAP and M. avium calves, as well. In contrast, greater expression of CD25 was observed on CD4+ and γδ TCR + T cells and natural killer cells for M. bovis calves. Overall, similarities in cellular immune responses were observed between the closely related MAP and M. avium during infection of calves. In contrast, significant differences were noted between calves infected with MAP and M. bovis. This suggests that host immune responses to different mycobacteria may impact interpretation of diagnostic tools based upon their cellular immunity.

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.

SEROPREVALENCE OF ANTIBODIES AGAINST MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS AND ITS RELATIONSHIP TO AGE AND SEX OF TEXAS WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) IN COAHUILA, MEXICO.

Paratuberculosis (PTB) is a disease that affects cattle (Bos taurus), goats (Capra aegagrus hircus), sheep (Ovis aries), and wild animals, such as white-tailed deer (Odocoileus virginianus), since all ruminants are susceptible. The causal agent is Mycobacterium avium subsp. paratuberculosis (MAP). The disease is chronic, consumptive, and incurable; it causes chronic granulomatous gastroenteritis with lymphangiectasis and lymphangitis leading to a syndrome of malnutrition and eventually to death. Mycobacterium avium subsp. paratuberculosis is transmitted in feces mainly orally; however, it can also be transmitted vertically. Thus, the objective of this study was to determine the seroprevalence of MAP antibodies and its relationship to age and sex of Texas white-tailed deer in the subclinical stage of PTB in Coahuila, Mexico. The entire population (n=99) belonging to the Wildlife Management and Conservation Unit (WMCU) San Juan, Monclova, Coahuila, Mexico was captured. Mycobacterium avium subsp. paratuberculosis was diagnosed using an enzyme-linked immunosorbent assay by serologic test. Seroprevalence variables of adult vs. young females and males vs. females were compared. The treatments were assigned at random. For the analysis of data, the chi-square test was used. Total seroprevalence in an intensive WMCU was 16% (16/99). Total seroprevalence by sex was 5.0% (5/99) for males and 11% (11/99) for females, and total seroprevalence by age was 7% (7/99) for young and 9% (9/99) for adult. Within sex, the seroprevalence in males was 16% (5/31) and 16% (11/68) in females. There were no statistical differences for any of the comparisons. Total seroprevalence of the white-tailed deer population in the WMCU was 16%, and PTB seroprevalence was independent of sex or age of the sampled individuals of this population.

Alfalfa Plants (Medicago sativa L.) Expressing the 85B (MAP1609c) Antigen of Mycobacterium avium subsp. paratuberculosis Elicit Long-Lasting Immunity in Mice.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Paratuberculosis, a contagious, untreatable, and chronic granulomatous enteritis that results in diarrhea, emaciation, and death in farmed ruminants (i.e., cattle, sheep, and goats). In this study, the Ag85B antigen from MAP was expressed in transgenic alfalfa as an attractive vaccine candidate. Agrobacterium-mediated transformation allowed the rescue of 56 putative transformed plants and transgenesis was confirmed in 19 lines by detection of the Ag85B gene (MAP1609c) by PCR. Line number 20 showed the highest Ag85B expression [840 ng Ag85B per gram of dry weight leaf tissue, 0.062% Total Soluble Protein (TSP)]. Antigenicity of the plant-made Ag85B was evidenced by its reactivity with a panel of sera from naturally MAP-infected animals, whereas immunogenicity was assessed in mice immunized by either oral or subcutaneous routes. The plant-made Ag85B antigen elicited humoral responses by the oral route when co-administered with cholera toxin as adjuvant; significant levels of anti-85B antibodies were induced in serum (IgG) and feces (IgA). Long-lasting immunity was evidenced at day 180 days post-first oral immunization. The obtained alfalfa lines expressing Ag85B constitute the first model of a plant-based vaccine targeting MAP. The initial immunogenicity assessment conducted in this study opens the path for a detailed characterization of the properties of this vaccine candidate.

A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne's Disease.

Tuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

Antibody detection and molecular analysis for Mycobacterium avium subspecies paratuberculosis (MAP) in goat milk: Systematic review and meta-analysis.

Paratuberculosis is an incurable infectious disease that affects several species, including goat (Capra hircus). The etiologic agent is Mycobacterium avium subspecies paratuberculosis (MAP) that has tropism for the intestine, causing anorexia, progressive weight loss and death. In goats, the main transmission route is the ingestion of water and food contaminated by infected feces. Affected animals also eliminate the agent through milk, with a potential biological risk to public health. Thus, the aim of this study was to conduct a research of the literature available in electronic media for a systematic review, followed by a meta-analysis of the results found on prevalence and diagnostic tests adopted in the detection of MAP antibodies and DNA in goat milk. The following search parameters were used: "Mycobacterium avium subsp. paratuberculosis" AND (goat OR small ruminant) AND (milk OR pasteurized milk). Strictly obeying pre-established criteria, 437 articles were selected from the respective electronic databases of scientific content: ScienceDirect (285), PubMed (68), Web of Science (60) and Scopus (24), of which nine papers were elected to the construction of the systematic review and meta-analysis. The prevalence of MAP antibodies in milk detected by milk-ELISA ranged from 1.1 to 67.7% and the prevalence of MAP DNA in goat milk detected by MAP-specific polymerase chain reaction (PCR) ranged from 1.94 to 37.74%. A meta-analysis indicated a combined MAP infection prevalence of 8.24%, but with high heterogeneity among study findings (I2 = 98.7%). The identification of the MAP in goat milk implies the need for surveillance of the agent in order to prevent economic losses and impact on public health.

A mucosal immune response induced by oral administration of heat-killed Mycobacterium avium subsp. paratuberculosis exacerbates EAE.

Findings in humans and animals have demonstrated a potential role for Mycobacterium avium subsp. paratuberculosis (MAP) antigenic components in encephalitogenic T cell activation. Here we reported that oral administration of MAP activates the mucosal immunity and exacerbates active experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice, modulating the immune cell traffic from secondary lymphoid organs to central nervous system. The detection of antigenic mycobacterial components by intestinal antigen-presenting cells may modulate the immune system and the subsequent inflammatory status through various signaling mechanisms, including the synthesis of pro-inflammatory cytokines involved in EAE pathogenesis.

Influence of vaccination against paratuberculosis on the diagnosis of caprine tuberculosis during official eradication programmes in Castilla y León (Spain).

The information generated from the official eradication programmes of caprine tuberculosis (TB) in Castilla y León, Spain, during 2018, has been used to assess the effect of vaccination against paratuberculosis (PTB) and the presence of this infection, on the single intradermal tuberculin (SIT) test results. Data from 121,665 goats belonging to 1936 different herds were analysed using generalized linear models. An epidemiological survey was conducted to know the herd immunization status against PTB and the date of last vaccination. All SIT test-positive animals were further investigated in order to confirm the diagnosis of TB, through bacterial culture, and PTB, by histopathological and qPCR analyses. SIT positivity was found in 39 (2.01%) herds and 507 (0.41%) goats. TB was confirmed by M. caprae or M. bovis isolation in 10 (0.51%) herds and 46 (0.038%) goats. PTB was diagnosed in 13 (33.33%) and 55 (10.84%) of the SIT test-positive herds and goats, respectively. Vaccination against PTB showed a significant influence on the results of the SIT test at herd level, with higher positivity detected among those herds vaccinated. However, this effect was not observed when the total number of animals was considered, where the highest positivity was found in unvaccinated goats. The time elapsed between vaccination and SIT test performance also influenced the results. The strongest effect was found when less than eight months elapsed between performing both activities, and to a lesser extent between 8 and 12 months. Conversely, no positive herds or animals were found when the time elapsed was higher than one year. No significant effect of the presence of PTB was observed. These findings demonstrate that the use of PTB vaccine does not result in false positives to a SIT test at individual level, provided that the time elapsed between the performance of both practices is higher than 12 months.

Isotype-specific Antibody Responses to Mycobacterium avium paratuberculosis Antigens Are Associated With the Use of Biologic Therapy in Inflammatory Bowel Disease.

BACKGROUND: The role of Mycobacterium avium paratuberculosis [MAP] in inflammatory bowel disease [IBD], especially Crohn's disease [CD] is controversial due conflicting results and lack of reproducibility and standardised tests. The current study focuses on the role of MAP in disease progression and genetic susceptibility, as MAP is likely one of many factors involved in the complex pathogenesis of IBD, potentially affecting a subgroup depending on genetic susceptibility. METHODS: Serum from 812 patients was evaluated with seven immunoglobulin [Ig] isotype-specific serology tests assessing humoral response to three different MAP antigens. For each of these in total 21 tests, the intra-assay and inter-assay coefficients were used to evaluate test accuracy. Reliable assays were subsequently analysed in relation to disease characteristics and need for biologic therapy/surgery. Genome-wide genotyping was available for all participants. Genetic determinants of humoral response to MAP antigens were evaluated using genome-wide association analysis and polygenic risk scores [PRS]. RESULTS: High IgA or IgM response to MAP2609 was associated with increased use of biologic therapy in CD and ulcerative colitis [UC] [odds ratios 2.69; 95% confidence interval 1.44-5.01; and 2.60, 1.46-4.64, respectively]. No associations were seen for risk of surgery [p-values > 0.29]. We could not identify genetic determinants nor polygenic risk scores for MAP response with genome-wide significance. CONCLUSIONS: Extensive assays for serological response to MAP were evaluated using stringent criteria for reliability. Increased IgA and IgM response to MAP antigens was seen in patients exposed to biologic therapy, but no genetic determinants underlying this humoral response were found.

The enhancement of Th1 immune response by anti-PD-L1 antibody in cattle infected with Mycobacterium avium subsp. paratuberculosis.

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis of ruminants. Previous studies have shown that programmed death-ligand 1 (PD-L1) is associated with the disease progression, and PD-L1 blockade activates MAP-specific Th1 responses in vitro. Here, we performed anti-PD-L1 antibody administration using 2 MAP-infected cattle at the late subclinical stage of infection. After administration, bacterial shedding was reduced or maintained at a low level. Additionally, MAP-specific Th1 cytokine production was upregulated, and CD69 expression was increased in T cells. Collectively, the treatment has a potential as a novel control method against Johne's disease.