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.     

From mouth to macrophage: mechanisms of innate immune subversion by Mycobacterium avium subsp. paratuberculosis

Johne’s disease (JD) is a chronic enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). The high economic cost and potential zoonotic threat of JD have driven efforts to develop tools and approaches to effectively manage this disease within livestock herds. Efforts to control JD through traditional animal management practices are complicated by MAP’s ability to cause long-term environmental contamination as well as difficulties associated with diagnosis of JD in the pre-clinical stages. As such, there is particular emphasis on the development of an effective vaccine. This is a daunting challenge, in large part due to MAP’s ability to subvert protective host immune responses. Accordingly, there is a priority to understand MAP’s interaction with the bovine host: this may inform rational targets and approaches for therapeutic intervention. Here we review the early host defenses encountered by MAP and the strategies employed by the pathogen to avert or subvert these responses, during the critical period between ingestion and the establishment of persistent infection in macrophages.     

Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in cattle and other farm ruminants, and is also a suspected pathogen of Crohn’s disease in humans. Development of diagnostic methods for MAP infection has been a challenge over the last few decades. The objective of this study was to investigate the relationship between different methods for detection of MAP in milk and fecal samples. A total of 134 milk samples and 110 feces samples were collected from 146 individual cows in 14 MAP-infected herds in southwestern Ontario. Culture, IS900 polymerase chain reaction (PCR) and nested PCR methods were used for detecting MAP in milk; results were compared with those of fecal culture. A significant relationship was found between milk culture, direct PCR, and nested PCR (P < 0.05). The fecal culture results were not related to any of the 3 assay methods used for the milk samples (P > 0.10). Although fecal culture showed a higher sensitivity than the milk culture method, the difference was not significant (P = 0.2473). The number of MAP colony-forming units (CFU) isolated by culture from fecal samples was, on average, higher than that isolated from milk samples (P = 0.0083). There was no significant correlation between the number of CFU cultured from milk and from feces (Pearson correlation coefficient = 0.1957, N = 63, P = 0.1243). The animals with high numbers of CFU in milk culture may not be detected by fecal culture at all, and vise versa. A significant proportion (29% to 41%) of the positive animals would be missed if only 1 culture method, instead of both milk and feces, were to be used for diagnosis. This suggests that the shedding of MAP in feces and milk is not synchronized. Most of the infected cows were low-level shedders. The proportion of low-level shedders may even be underestimated because MAP is killed during decontamination, thus reducing the chance of detection. Therefore, to identify suspected Johne’s-infected animals using the tests in this study, both milk and feces samples should be collected in duplicate to enhance the diagnostic rate. The high MAP kill rate identified in the culture methods during decontamination may be compensated for by using the nested PCR method, which had a higher sensitivity than the IS900 PCR method used.     

An immuno-epidemiological model for Johne’s disease in cattle

To better understand the mechanisms involved in the dynamics of Johne’s disease in dairy cattle, this paper illustrates a novel way to link a within-host model for Mycobacterium avium ssp. paratuberculosis with an epidemiological model. The underlying variable in the within-host model is the time since infection. Two compartments, infected macrophages and T cells, of the within-host model feed into the epidemiological model through the direct transmission rate, disease-induced mortality rate, the vertical transmission rate, and the shedding of MAP into the environment. The epidemiological reproduction number depends on the within-host bacteria load in a complex way, exhibiting multiple peaks. A possible mechanism to account for the switch in shedding patterns of the bacteria in this disease is included in the within-host model, and its effect can be seen in the epidemiological reproduction model.     

Shedding patterns of dairy calves experimentally infected with Mycobacterium avium subspecies paratuberculosis

Although substantial fecal shedding is expected to start years after initial infection with Mycobacterium avium subspecies paratuberculosis (MAP), the potential for shedding by calves and therefore calf-to-calf transmission is underestimated in current Johne’s disease (JD) control programs. Shedding patterns were determined in this study in experimentally infected calves. Fifty calves were challenged at 2 weeks or at 3, 6, 9 or 12 months of age (6 calves served as a control group). In each age group, 5 calves were inoculated with a low and 5 with a high dose of MAP. Fecal culture was performed monthly until necropsy at 17 months of age. Overall, 61% of inoculated calves, representing all age and dose groups, shed MAP in their feces at least once during the follow-up period. Although most calves shed sporadically, 4 calves in the 2-week and 3-month high dose groups shed at every sampling. In general, shedding peaked 2 months after inoculation. Calves inoculated at 2 weeks or 3 months with a high dose of MAP shed more frequently than those inoculated with a low dose. Calves shedding frequently had more culture-positive tissue locations and more severe gross and histological lesions at necropsy. In conclusion, calves inoculated up to 1 year of age shed MAP in their feces shortly after inoculation. Consequently, there is potential for MAP transfer between calves (especially if they are group housed) and therefore, JD control programs should consider young calves as a source of infection.     

Longitudinal evaluation of diagnostics in experimentally infected young calves during subclinical and clinical paratuberculosis

Five calves were inoculated orally at 2 weeks of age with a dose of 5 × 10⁹ colony-forming units of Mycobacterium avium subspecies paratuberculosis (MAP) on 2 consecutive days. Two calves developed clinical Johne’s disease at 12 and 16 months of age after being consistently positive for MAP on fecal culture and antibody enzyme-linked immunosorbent assay (ELISA), starting 2 to 3 weeks and 4 to 5 months after inoculation, respectively.     

Immunity,safety and protection of an Adenovirus 5 prime - Modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves

Vaccination is the most cost effective control measure for Johne’s disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4⁺, CD8⁺ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1β and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0112-9) contains supplementary material, which is available to authorized users.     

Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review

Mycobacterium avium subspecies paratuberculosis (Map) is an important pathogen that causes a chronic, progressive granulomatous enteritis known as Johne’s disease or paratuberculosis. The disease is endemic in many parts of the world and responsible for considerable losses to the livestock and associated industries. Diagnosis and control are problematic, due mostly to the long incubation period of the disease when infected animals show no clinical signs and are difficult to detect, and the ability of the organism to survive and persist in the environment. The existence of phenotypically distinct strains of Map has been known since the 1930s but the genetic differentiation of Map strain types has been challenging and only recent technologies have proven sufficiently discriminative for strain comparisons, tracing the sources of infection and epidemiological studies. It is important to understand the differences that exist between Map strains and how they influence both development and transmission of disease. This information is required to develop improved diagnostics and effective vaccines for controlling Johne’s disease. Here I review the current classification of Map strain types, the sources of the genetic variability within strains, growth characteristics and epidemiological traits associated with strain type and the influence of strain type on infection and pathogenicity.     

Agent-based model for Johne’s disease dynamics in a dairy herd

Johne’s disease is an infectious gastrointestinal disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis that causes diarrhea, emaciation, decreased milk production and eventually death. The disease is transmitted in utero and via milk and colostrums to calves, and fecal-orally to all age classes. Financial losses due to the disease are estimated to be over $200 million in the US dairy industry. The goal of this study was to evaluate the cost effectiveness of control measures based on diagnosis with a sensitive ELISA, EVELISA. An agent-based, discrete time model was developed to simulate Johne’s disease dynamics in a US dairy herd. Spatial aspects of disease transmission were taken into account by using six spatial compartments. The effects on disease prevalence were studied with and without transmission routes included in the model. Further, using the model, cost effectiveness of ELISA-based Johne’s disease control was evaluated. Using the parameters we collected and assumed, our model showed the initial prevalence of Johne’s disease (33.1 ± 0.2%) in the farm increased to 87.7 ± 1.7% in a 10 year-simulation. When ELISA-based control measures were included in the simulation, the increase in prevalence was significantly slowed down, especially when EVELISA was used. However, the level of the prevalence was still higher than the initial level after 10 year simulation even with the ELISA-based diagnostic intervention. The prevalence was further reduced when quarterly ELISA testing was included. The cost analysis showed that the quarterly ELISA and EVELISA testing could bring $44.8 and $51.5/animal/year more revenues, respectively, to a dairy farm.

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The online version of this article (doi:10.1186/s13567-015-0195-y) contains supplementary material, which is available to authorized users.     

Longitudinal study of the distribution of Mycobacterium avium subsp. paratuberculosis in the environment of dairy herds in the Michigan Johne’s disease control demonstration herd project

The objective of this study was to describe the distribution of Mycobacterium avium subsp. paratuberculosis (MAP) in the environment of infected dairy farms over time. Johne’s disease (JD) prevalence was monitored annually in 7 Michigan dairy herds. Environmental samples were collected bi-annually and cultured for MAP. Of 731 environmental samples that were cultured, 81 (11%) were positive. The lactating cow floor and manure storage areas were the areas most commonly contaminated, representing 30% and 33% of positive samples, respectively. When herd prevalence was > 2%, MAP was cultured from the lactating cow floor and/or manure storage area 75% of the time. When herd prevalence was ≤ 2%, MAP was never cultured from samples collected. For every 1 unit increase in number of positive environmental samples, within herd JD prevalence increased 1.62%. Environmental contamination with MAP is consistent over time on infected dairy farms, and management practices to reduce environmental contamination are warranted.     

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.     

Impact of imperfect Mycobacterium avium subsp. paratuberculosis vaccines in dairy herds: A mathematical modeling approach

The objective of this study was to investigate the potential impacts of imperfect Mycobacterium avium subsp. paratuberculosis (MAP) vaccines on the dynamics of MAP infection in US dairy herds using a mathematical modeling approach. Vaccine-based control programs have been implemented to reduce the prevalence of MAP infection in some dairy herds; however, MAP vaccines are imperfect. Vaccines can provide partial protection for susceptible calves, reduce the infectiousness of animals shedding MAP, lengthen the latent period of infected animals, slow the progression from low shedding to high shedding in infectious animals, and reduce clinical disease. To quantitatively study the impacts of imperfect MAP vaccines, we developed a deterministic multi-group vaccination model and performed global sensitivity analyses. Our results explain why MAP vaccination might have a beneficial, negligible, or detrimental effect in the reduction of prevalence and show that vaccines that are beneficial to individual animals may not be useful for a herd-level control plan. The study suggests that high efficacy vaccines that are aimed at reducing the susceptibility of the host are the most effective in controlling MAP transmission. This work indicates that MAP vaccination should be integrated into a comprehensive control program that includes test-and-cull intervention and improved calf rearing management.     

A high-morbidity outbreak of Johne’s disease in game-ranched elk

Following an outbreak of Johne’s disease on an elk farm in northern Alberta, Canada, fecal culture, fecal polymerase chain reaction (PCR), and serum enzyme-linked immunosorbent assay (ELISA) tests were performed on individual animals. The magnitude of the outbreak is described and the challenges associated with poor test agreement, as well as herd management options, are discussed.     

Evaluating contribution of the cellular and humoral immune responses to the control of shedding of Mycobacterium avium spp. paratuberculosis in cattle

Mycobacterium avium spp. paratuberculosis (MAP) causes a persistent infection and chronic inflammation of the gut in ruminants leading to bacterial shedding in feces in many infected animals. Although there are often strong MAP-specific immune responses in infected animals, immunological correlates of protection against progression to disease remain poorly defined. Analysis of cross-sectional data has suggested that the cellular immune response observed early in infection is effective at containing bacterial growth and shedding, in contrast to humoral immune responses. In this study, 20 MAP-infected calves were followed for nearly 5 years during which MAP shedding, antigen-specific cellular (LPT) and humoral (ELISA) immune responses were measured. We found that MAP-specific cellular immune response developed slowly, with the peak of the immune response occurring one year post infection. MAP-specific humoral immunity expanded only in a subset of animals. Only in a subset of animals there was a statistically significant negative correlation between the amount of MAP shedding and magnitude of the MAP-specific cellular immune response. Direct fitting of simple mechanistic mathematical models to the shedding data suggested that MAP-specific immune responses contributed significantly to the kinetics of MAP shedding in most animals. However, whereas the MAP-specific cellular immune response was predicted to suppress shedding in some animals, in other animals it was predicted to increase shedding. In contrast, MAP-specific humoral response was always predicted to increase shedding. Our results illustrate the use of mathematical methods to understand relationships between mycobacteria and immunity in vivo but also highlight problems with establishing cause-effect links from observational data.

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Gene-expression profiling of calves 6 and 9?months after inoculation with Mycobacterium avium subspecies paratuberculosis

Early detection of Johne’s disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is essential to reduce transmission; consequently, new diagnostic techniques and approaches to detect MAP or markers of early MAP infection are being explored. The objective was to identify biomarkers associated with MAP infection at 6 and 9 months after oral inoculation. Therefore, gene expression analysis was done using whole blood cells obtained from MAP-infected calves. All MAP-inoculated calves had a cell-mediated immune response (IFN-γ) to Johnin PPD specific antigens, and 60% had an antibody response to MAP antigens. Gene expression analysis at 6 months after inoculation revealed downregulation of chemoattractants, namely neutrophil beta-defensin-9 like peptide (BNBD9-Like), S100 calcium binding protein A9 (s100A9) and G protein coupled receptor 77 (GPR77) or C5a anaphylatoxin chemotactic receptor (C5a2). Furthermore, BOLA/MHC-1 intracellular antigen presentation gene was downregulated 9 months after inoculation. In parallel, qPCR experiments to evaluate the robustness of some differentially expressed genes revealed consistent downregulation of BOLA/MHC-I, BNBD9-Like and upregulation of CD46 at 3, 6, 9, 12, and 15 months after inoculation. In conclusion, measuring the expression of these genes has potential for implementation in a diagnostic tool for the early detection of MAP infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0096-5) contains supplementary material, which is available to authorized users.     

Veterinarians’ perspective on a voluntary Johne’s disease prevention program in Ontario and western Canada

A survey was conducted to assess the beliefs of veterinarians on Johne’s disease (JD) and their attitudes towards the Canadian, risk assessment based, JD prevention program. The veterinarians surveyed believed Mycobacterium avium subsp. paratuberculosis may have zoonotic potential, liked the risk assessment based program, and thought it could lead to the prevention of other on-farm diseases.     

Molecular detection and typing of Mycobacterium avium subspecies paratuberculosis from milk samples of dairy animals

Genotyping of Mycobacterium avium subspecies paratuberculosis (MAP) is important for precise classification of bacterium and for understanding the molecular epidemiology. The present study reports detection and typing of the MAP from milk. On the basis of clinical signs of diarrhea and/or weakness, the dairy animals suspected for Johne’s disease were screened by Ziehl–Neelsen staining of fecal samples. The milk samples from 13 selected animals were processed for DNA extraction and direct IS900 polymerase chain reaction (PCR). MAP identified by IS900 PCR was genotyped using IS1311 PCR-restriction endonuclease analysis (REA). IS900 milk PCR revealed 30.8% animals positive for MAP, including 40% of the moderate and 50% of the heavy fecal shedders. All infected animals showed Bison type MAP in IS1311 PCR-REA. IS900 PCR can be used for screening of milk for MAP; however, the method needs to be evaluated for subclinical cases. IS1311 PCR-REA results indicated the predominance of Bison type MAP in the dairy animals of this region.     

Calves shedding Mycobacterium avium subspecies paratuberculosis are common on infected dairy farms

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease, a chronic progressive enteritis. It is generally assumed that calves rarely shed MAP bacteria and that calf-to-calf transmission is of minor importance. The objectives were 1) to estimate the prevalence of MAP-shedding young stock in MAP-infected dairy herds, and identify predictors for test-positive young stock; and 2) to estimate proportions of MAP-contaminated young stock group housing pens and air spaces, and furthermore, identify predictors for test-positive pens. Fecal samples were collected from 2606 young stock on 18 MAP-infected dairy farms. Environmental fecal samples were collected from all group-housing pens and dust samples were collected from all barns. All individual samples were analysed using IS900 and F57 qPCR; fecal samples positive by either PCR and all environmental and dust samples were cultured. Overall, 8.1, 1.2 and 2.0% of cattle were positive on IS900 qPCR, F57 qPCR and bacterial culture, respectively. Young stock housed on farms with culture-positive environmental samples collected from adult cow housing and manure storage had higher odds of testing IS900 qPCR-positive than young stock housed on farms with only negative environmental samples. Furthermore, 14% of collected environmental samples, but no dust samples, were test-positive. Age of cattle in the pen was a significant predictor for environmental sample results. Young stock excreted MAP bacteria in their feces which provided strong evidence for calves as sources of within-herd transmission of MAP on dairy farms known to be infected with this organism.

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The online version of this article (doi:10.1186/s13567-015-0192-1) contains supplementary material, which is available to authorized users.     

Culture of Mycobacterium avium subspecies paratuberculosis (MAP) from blood and extra-intestinal tissues in experimentally infected sheep

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or paratuberculosis, a chronic enteritis of ruminants, and has been suggested to play a role in Crohn's disease in humans. While Johne's disease is primarily expressed in the gastrointestinal tract, isolation of MAP from extra-intestinal tissues indicates that microbial dissemination via the haematogenous route may occur during the infection. Consequently, the occurrence of mycobacteraemia and dissemination to the liver and hepatic lymph node was investigated in 111 sheep. Disseminated infection was detected in 18 of the 53 sheep that were confirmed to be infected following oral exposure to MAP while the bacterium was isolated from the blood of only 4 of these animals. Disseminated infection was detected more frequently from animals with a positive compared to a negative faecal culture result, multibacillary compared to paucibacillary lesions, and clinical compared to subclinical disease. Detection of MAP in blood by culture was significantly associated with increased time post-exposure and clinical disease, with trends for increased detection in animals with multibacillary lesions and positive faecal culture results. Isolation of MAP from blood was difficult in the early stages of the disease and in paucibacillary animals as the bacteraemia may be intermittent, below the limit of detection or MAP may be present in a dormant non-culturable form. Prolonged incubation periods prior to growth in BACTEC were consistent with inhibition of growth or dormancy in some blood cultures.     

Biosecurity practices in western Canadian cow-calf herds and their association with animal health

Biosecurity practices of beef cow-calf herds in western Canada have not been studied extensively nor is there a good understanding of their association with herd health. A survey was sent to 103 cow-calf producers of the Western Canadian Cow-Calf Surveillance Network. Eighty completed questionnaires were returned. Bulls were purchased for all herds during the 2014 to 2017 study period; 54% of herds purchased heifers and 42% purchased cows. The use of standard biosecurity practices was generally low with 30% of producers keeping purchased animals separate and 30% vaccinating new additions. None of the evaluated biosecurity practices were associated with reporting Johne’s disease. The purchase of > 10 bulls, the purchase of cows, not vaccinating animals bought into the herd, and use of community pasture were associated with a bovine respiratory disease outbreak. Outbreaks of calf diarrhea were associated with the purchase of 10 or more bulls, the use of a community pasture, and leasing or sharing bulls.