Long-term outcomes of the Italian Mycobacterium avium subspecies paratuberculosis control programme for dairy cattle.

BACKGROUND: The considerable epidemiological and economic implications of paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), have placed importance on control efforts aimed at preventing MAP transmission. In this context, Italy issued national guidelines for the control and status certification of MAP in dairy cattle in 2013. METHODS: We assessed the long-term outcomes of the Italian MAP control programme for 14 dairy farms located in northern Italy by retrospectively reviewing the results of yearly serological tests, presence of clinical cases, MAP faecal shedding in serologically positive animals, farm management and health ranking as indicators of herd health between 2014 and 2021. RESULTS: A significantly higher number of serologically positive animals were observed between 2014 and 2016 than between 2017 and 2021, as well as an improving trend in the paratuberculosis health ranking for nine of the 14 farms. No clinical cases were reported. MAP shedding was detected in 9.4% of serologically positive animals. Discarding colostrum and prioritised culling of seropositive animals assisted by adoption of standardised serological testing were presumed to have a key role in MAP control, despite the reluctance of some farmers to address hygienic issues and improve the separation of calves from adult animals. LIMITATIONS: The small number of farms included in this study and the fact that these were not randomly selected may limit the generalisability of the findings. CONCLUSIONS: The Italian paratuberculosis control plan has provided measures to limit the uncontrolled spread of MAP infection within and between herds by promoting animal trading between farms certified as negative or low risk.

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.

Selection of vaccine-candidate peptides from Mycobacterium avium subsp. paratuberculosis by in silico prediction, in vitro T-cell line proliferation, and in vivo immunogenicity.

Mycobacterium avium subspecies paratuberculosis (MAP) is a global concern in modern livestock production worldwide. The available vaccines against paratuberculosis do not offer optimal protection and interfere with the diagnosis of bovine tuberculosis. The aim of this study was to identify immunogenic MAP-specific peptides that do not interfere with the diagnosis of bovine tuberculosis. Initially, 119 peptides were selected by either (1) identifying unique MAP peptides that were predicted to bind to bovine major histocompatibility complex class II (MHC-predicted peptides) or (2) selecting hydrophobic peptides unique to MAP within proteins previously shown to be immunogenic (hydrophobic peptides). Subsequent testing of peptide-specific CD4+ T-cell lines from MAP-infected, adult goats vaccinated with peptides in cationic liposome adjuvant pointed to 23 peptides as being most immunogenic. These peptides were included in a second vaccine trial where three groups of eight healthy goat kids were vaccinated with 14 MHC-predicted peptides, nine hydrophobic peptides, or no peptides in o/w emulsion adjuvant. The majority of the MHC-predicted (93%) and hydrophobic peptides (67%) induced interferon-gamma (IFN-γ) responses in at least one animal. Similarly, 86% of the MHC-predicted and 89% of the hydrophobic peptides induced antibody responses in at least one goat. The immunization of eight healthy heifers with all 119 peptides formulated in emulsion adjuvant identified more peptides as immunogenic, as peptide specific IFN-γ and antibody responses in at least one heifer was found toward 84% and 24% of the peptides, respectively. No peptide-induced reactivity was found with commercial ELISAs for detecting antibodies against Mycobacterium bovis or MAP or when performing tuberculin skin testing for bovine tuberculosis. The vaccinated animals experienced adverse reactions at the injection site; thus, it is recommend that future studies make improvements to the vaccine formulation. In conclusion, immunogenic MAP-specific peptides that appeared promising for use in a vaccine against paratuberculosis without interfering with surveillance and trade tests for bovine tuberculosis were identified by in silico analysis and ex vivo generation of CD4+ T-cell lines and validated by the immunization of goats and cattle. Future studies should test different peptide combinations in challenge trials to determine their protective effect and identify the most MHC-promiscuous vaccine candidates.

Association between major histocompatibility complex haplotypes and susceptibility of unvaccinated and vaccinated cattle to paratuberculosis.

Bovine Johne's disease (BJD) or paratuberculosis is caused by Mycobacterium avium spp. paratuberculosis (MAP) and is a worldwide problem among domestic and wild ruminants. While vaccines are available, natural differences in background immunity between breeds within species and between individuals within herds suggest that genetic differences may be able to be exploited in marker-assisted selection as an aid to disease control. The major histocompatibility complex (MHC) is an important component in immune recognition with considerable genetic variability. In this study, associations between the MHC and resistance to BJD were explored in dairy cattle across two herds in which some of the cattle had been vaccinated with Silirum® (n = 540 cows). A BJD susceptible animal was exposed to MAP and became infected, while a resistant animal was exposed but did not become infected. There are different ways to define both exposure and infection, with different levels of stringency, therefore many classifications of the same set of animals are possible and were included in the analysis. The polymorphic regions of major histocompatibility complex class I (MHC I) and class II (MHC II) genes were amplified from the genomic DNA by PCR and sequenced, targeting exons 2 and 3 of the classical and non-classical MHC I genes and exon 2 from the DRB3, DQA1, DQA2 + 3 and DQB MHC II genes. The frequencies of MHC I and MHC II haplotypes and alleles were determined in susceptible and resistant populations. In unvaccinated animals, seven MHC I haplotypes and seven MHC II haplotypes were associated with susceptibility while two MHC I and six MHC II haplotypes were associated with resistance (P < 0.05). In vaccinated animals, two MHC I and three MHC II haplotypes were associated with susceptibility, while one MHC I and two MHC II haplotypes were associated with resistance (P < 0.05). The alleles in significant haplotypes were also identified. Case definitions with higher stringency resulted in fewer animals being included in the analyses, but the power to detect an association was not reduced and there was an increase in strength and consistency of associations. Consistent use of stringent case definitions is likely to improve agreement in future association studies.

Immuno-reactivity evaluation of Mce-truncated subunit candidate vaccine against Mycobacterium avium subspecies paratuberculosis challenge in the goat models.

BACKGROUND: Detection of an appropriate antigen with high immunogenicity can be a big step in the production of an effective vaccine for control of Johne's disease (JD). The aim of this study was to evaluate the efficacy of Mce-truncated protein as a subunit vaccine candidate for the control of JD in experimentally challenged goats. MATERIALS AND METHODS: Six healthy goat kids were immunized with Mce-truncated protein, and two goats were kept as controls. All kids were twice challenged orally with live Mycobacterium avium subspecies paratuberculosis(MAP) strain and half the goats from both the categories were sacrificed at 7 and 10 months after start of challenge study. Culture of MAP was performed from all the necropsied tissues to determine the true JD infection status. RESULTS: Mce-truncated protein only reacted with pooled vaccinated goat sera in western-blot. A significant increase in humoral immune response against Mce protein was also observed in vaccinated goats. Compared to the control group, vaccinated goats gained higher body weights and none of them shed MAP or showed histopatological lesions or colonization of MAP in their necropsy tissues. CONCLUSIONS: The new Mce protein based vaccine provided significant immunity in goats as they could meet the challenge with live MAP bacilli. Although the vaccine used in this study showed the high potential as a new effective vaccine for the control of JD, further validation study is still required to successfully implement the vaccine for JD control program.

An observational study of ear-tagged calf mortality (1 to 100 days) on Irish dairy farms and associations between biosecurity practices and calf mortality on farms participating in a Johne's disease control program.

Postnatal mortality among replacement stock has a detrimental effect on the social, economic, and environmental sustainability of dairy production. Calf mortality rates vary between countries and show differences in temporal trends; most, however, are characterized by high levels of between-farm variability. Explaining this variation can be difficult because herd-level information on management practices relevant to calf health is often not available. The Irish Johne's Control Programme (IJCP) contains a substantial on-farm monitoring program called the Veterinary Risk Assessment and Management Plan (VRAMP). Although this risk assessment is largely focused on factors relevant to the transmission of paratuberculosis, many of its principles are good practice biocontainment policies that are also advocated for the protection of calf health. The objectives of this study were (1) to quantify mortality in ear-tagged Irish dairy calves between 2016 and 2020 using both survival and risk approaches, (2) to determine risk factors for 100-d cumulative mortality hazard in ear-tagged Irish dairy calves between 2016 and 2020, (3) to determine whether 100-d cumulative mortality hazard was higher in ear-tagged calves within herds registered in the IJCP versus those that were not registered in the IJCP and whether there were differences between these cohorts over time, and (4) within IJCP herds, to determine whether VRAMP score or changes in VRAMP score were associated with 100-d cumulative mortality hazard. Excluding perinatal mortality, the overall 100-d cumulative mortality hazard was 4.1%. Calf mortality was consistently underestimated using risk approaches that did not account for calf censoring. Cox proportional hazards models showed that cumulative mortality hazard was greater in male calves; particularly, calves born to Jersey breed dams and those with a beef breed sire. Mortality hazard increased with increasing herd size, was highest in calves born in herds that contract-reared heifers, and lowest in those born in mixed dairy-beef enterprises. Mortality hazard decreased over time with the mortality hazard in 2020 being 0.83 times that of 2016. Mortality hazard was higher in IJCP-registered herds than nonregistered herds (hazard ratio 1.06, 95% CI 1.01-1.12), likely reflecting differences in herds that enrolled in the national program. However, we detected a significant interaction between IJCP status (enrolled vs. not enrolled) and year (hazard ratio 0.96, 95% CI 0.92-1.00), indicating that the decrease in mortality hazard between 2016 and 2020 was greater in IJCP herds versus non-IJCP herds. Finally, increasing VRAMP scores (indicating higher risk for paratuberculosis transmission) were positively associated with increased calf mortality hazard. Postnatal calf mortality rates in Irish dairy herds declined between 2016 and 2020. Our study suggests that implementation of recommended biocontainment practices to control paratuberculosis in IJCP herds was associated with a reduction in calf mortality hazard.

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.

Bovine paratuberculosis: results of a control plan in 64 dairy farms in a 4-year period.

Paratuberculosis is considered one of the most economically devastating infectious diseases of domestic livestock, and the most effective control strategy is a combination of 'test-and-cull' and on-farm biosecurity measures. In Italy, a Voluntary National Control Plan (VNCP) and guidelines have been introduced to reduce the impact of the disease, and farmers can voluntarily enroll in the control plan. The main aims of this study were: i) the description of the trend over a 4-year period on total, within-herd (WH) and between herd (BH) apparent seroprevalences observed in 64 dairy herds members of a mutual company located in Italy after the introduction of a proposed "Customized Control Plan" (CCP); ii) the evaluation of its effectiveness in terms of percentage of participating farms that decided to join the VNCP. Analyses on serum samples were performed with Enzyme-Linked Immuno Sorbent Assay (ELISA) method and revealed a general decrease in both total, WH and BH apparent seroprevalence. Total average apparent seroprevalence decreased from 2.39% in 2017 to 1% in 2020. Negative herds raised from 51.9% in 2017 to 71.1% in 2020, while farms with WH apparent seroprevalence > 5% decreased from 17.3% in 2017 to 4.4% in 2020. BH apparent seroprevalence decreased from 51.2% in 2017 to 29.2% in 2020. Among the 52 out of 64 herds that accepted to continue the proposed CCP after the first year, 41 (78.8%) joined in 2020 the VNCP, that assessed the health ranking of the herds. The results provide evidence that a control plan based on a farm-specific strategy and a subsidized testing process can effectively reduce the impact of paratuberculosis in dairy herds, especially in convincing farmers to continue in paratuberculosis control by joining the VNCP, including them in a National context and increasing their awareness of the disease.

Estimation of the specificity of an antibody ELISA for paratuberculosis generated from a sector of the UK cattle population using results from a paratuberculosis control programme.

In the United Kingdom (UK) a voluntary programme to control paratuberculosis in cattle based on herd management and serological screening has been operating since 1998. The programme assigns a risk level to each participating herd according to the within herd seroprevalence and the confirmation of the presence of infection with Mycobacterium avium subspecies paratuberculosis (MAP) by faecal culture or polymerase chain reaction (PCR). From the outset a general concern over the specificity of the paratuberculosis antibody enzyme-linked immunosorbent assay (ELISA) resulted in the use of a faecal screen for the causal organism to negate or confirm infection in individual seropositive animals. Progress in improving the diagnostic tests has been gradual throughout the life of the programme and the under-pinning approach to using tests to determine the risk of paratuberculosis for a herd required to be re-examined. This study used a large data set of more than 143,000 test results over five years from the lowest paratuberculosis risk level category of herds to estimate the specificity of a commercially available paratuberculosis antibody ELISA for cattle. In each year of the study the estimated specificity reached or exceeded 0.998. We also examined the apparent impact that annual or more frequent application of the single intradermal comparative cervical tuberculin (SICCT) test for tuberculosis (TB), using purified protein derivatives of Mycobacterium bovis and Mycobacterium avium subspecies avium, had on specificity of the antibody ELISA for paratuberculosis. We found a statistically significant difference in three of the five years with herds that were officially tuberculosis free and not subject to frequent SICCT testing. This difference was small and considered to be of little practical importance for the paratuberculosis assurance programme. We concluded that, in the UK the mandatory TB surveillance programme of cattle herds is not a limiting factor in the use of serological testing to support herd-level assurance schemes for paratuberculosis. Furthermore, in paratuberculosis, where shedding of MAP is intermittent and the sensitivity of the commercially available PCR tests for detection MAP is highly variable, faecal screening of seropositive animals is an unreliable method for negating infection in seropositive cattle.

IP10 is a predictor of successful vaccine protection against paratuberculosis infection in sheep.

The cell mediated immune response and ability of immune cells to migrate to the site of infection are both key aspects of protection against many pathogens. Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular pathogen and the causative agent of paratuberculosis, a chronic wasting disease of ruminants. Current commercial vaccines for paratuberculosis reduce the occurrence of clinical disease but not all animals are protected from infection. Therefore, there is a need to understand the immune responses triggered by these vaccines at the site of infection, in circulating immune cells and their relationships to vaccine-mediated protection. The magnitude and location of gene expression related to the cell mediated immune response and cellular migration were studied in the ileum of sheep. In addition, longitudinal IP10 (also known as IP10) secretion by circulating immune cells was examined in the same sheep. Animals were grouped based on vaccination status (vaccinated vs non-vaccinated) and MAP exposure (experimentally exposed vs unexposed). Vaccination of unexposed sheep increased the expression of IP10, CCL5 and COR1c. Sheep that were successfully protected by vaccination (uninfected following experimental exposure) had significantly reduced expression of IP10 in the ileum at 12 months post exposure compared to vaccine non-responders (those that became infected) and non-vaccinated infected sheep. Successfully protected sheep also had significantly increased secretion of IP10 in in vitro stimulated immune cells from whole blood compared to vaccine non responders at 4 months post exposure. Therefore, the IP10 recall response has the potential to be used as marker for infection status in vaccinated sheep and could be a biomarker for a DIVA test in sheep.

The effect of risk-based trading and within-herd measures on Mycobacterium avium subspecies paratuberculosis spread within and between Irish dairy herds.

Johne's disease (bovine paratuberculosis) is an endemic disease caused by Mycobacterium avium subspecies paratuberculosis (Map). Map is transmitted between herds primarily through movement of infected but undetected animals. Within infected herds, possible control strategies include improving herd hygiene by reducing calf exposure to faeces from cows, reducing stress in cows resulting in a longer latently infected period where shedding is minimal, or culling highly test-positive cows soon after detection. Risk-based trading can be a strategy to reduce the risk that Map spreads between herds. Our objective was to assess whether within-herd measures combined with risk-based trading could effectively control Map spread within and between dairy cattle herds in Ireland. We used a stochastic individual-based and between-herd mechanistic epidemiological model to simulate Map transmission. Movement and herd demographic data were available from 1st January 2009-31st December 2018. In total, 13,353 herds, with 4,494,768 dairy female animals, and 72,991 bulls were included in our dataset. The movement dataset consisted of 2,304,149 animal movements. For each herd, a weekly indicator was calculated that reflected the probability that the herd was free from infection. The indicator value increased when a herd tested negative, decreased when animals were introduced into a herd, and became 0 when a herd tested positive. Based on this indicator value, four Johne's assurance statuses were distinguished: A) ≥ 0.7 - 1.0, B) ≥ 0.3 - < 0.7, C) > 0.0 - < 0.3, and D) 0.0. A is the highest and D the lowest Johne's assurance status. With risk-based trading some of the observed movements between herds were redirected based on Johne's assurance status with the aim of reducing the risk that a non-infected herd acquired an infected animal. Risk-based trading effectively reduced the increase in herd prevalence over a 10-year-period in Ireland: from 50% without risk-based trading to 42% with risk-based trading in the metapopulation only, and 26% when external purchases were risk-based as well. However, for risk-based trading to be effective, a high percentage of dairy herds had to participate. The most important within-herd measures were improved herd hygiene and early culling of highly infectious cows. These measures reduced both herd and within-herd prevalence compared to the reference scenario. Combining risk-based trading with within-herd measures reduced within-herd prevalence even more effectively.

Descriptive analysis of the changes in Johne's disease management practices on Ontario dairy farms through repeat risk assessment.

Johne's disease (JD) control is often based on the culling of positive animals and the adoption of management practices that minimize exposure of calves to the pathogen Mycobacterium avium ssp. paratuberculosis (MAP). From 2010 to 2013, Ontario, Canada, instituted a voluntary Johne's control program consisting of whole-herd testing and a Risk Assessment and Management Plan (RAMP). The RAMP consisted of 38 questions that evaluated 5 different management areas to characterize herd risk for MAP introduction and within-herd spread. The RAMP produced a numerical score for each area, with higher scores associated with higher risk. The RAMP focused on animal purchases, calving management, calf management, and heifer and cow cleanliness and management. In the summer of 2019, the RAMP was repeated on 180 farms that had participated in the JD program of 2010 to 2013 and had bulk tank milk ELISA results from 2013 and 2017. This cross-sectional study demonstrated that many producers changed management practices over the 4- to 7-year period. Producers changed their cattle buying practices, with a reduction in purchasing from multiple sources and more herds refraining from buying in animals. However, overall scores were higher in 2019 than in 2013. The 2019 RAMP indicated that fewer farms were utilizing individual calving pens in 2019 than in 2013 (13% vs. 26%), yet more farms had policies in place to deal with sick or suspect JD cows entering the maternity area (92% vs. 74%). Management changes occurred over time, some of which represent increased risk (crowded maternity pens) and others decreased risk (closed herd, protocols in place for JD-positive cows) for MAP introduction and transmission. These results highlight the importance of frequent risk assessments and the documentation of changes to management practices on-farm as a means to assess herd disease risk more accurately.

Association between calf rearing technology and farm-level paratuberculosis infection in Hungarian dairy farms.

The ruminant paratuberculosis (PTB) is a global problem. The disease causes significant economic loss, primarily in dairy farms, due to premature culling, reduced slaughter value, and production losses. Newborn and young calves are the most susceptible to being infected, and the chance of infection decreases with increasing age. Animals are most commonly infected orally. The study aimed to assess the factors that have a greater association with PTB prevalence in Hungarian cattle farms and could play a major role in the eradication process. We visited 26 large-scale Hungarian dairy herds (having preliminary data on PTB and testing positive during the last two years). We assessed 10 management aspects that may influence the spread of PTB. Farms were evaluated, and answers were dichotomised based on whether the given management practice was advantageous or disadvantageous in controlling the spread of MAP. By summing up the obtained values, a 'Hygiene score' was achieved that would rank the farms based on their awareness in MAP control. In the visited herds, the average apparent animal-level PTB prevalence was 8.3 % (min. 2.0 %, max. 19.5 %). There was a significant negative correlation between the 'Hygiene score' and the apparent MAP seropositivity of the given farm. Taking the calf away immediately after birth was associated with an average 55 % decrease in the odds of seropositivity as compared to the longer time spent with the dam. Discarding of colostrum from previously PTB+ cows after calving was associated with a 48 % decrease in the odds of seropositivity as compared to farms where the colostrum or milk of PTB+ cows was not discarded but fed to the calves. In conclusion, calf management is key in controlling paratuberculosis where immediate removal of the calf from its mother and the feeding of MAP-free colostrum and milk are essential elements.

Dairy producer satisfaction and knowledge transfer with the veterinary-administered risk assessment and management plan in a voluntary Johne's disease control program.

The Atlantic Johne's Disease Initiative (AJDI) aims to control Mycobacterium avium ssp. paratuberculosis infection by using veterinary-administered risk assessments to identify high-risk management practices and prompt changes in management behavior. Objectives for this study were to measure producer satisfaction with the veterinary-administered risk assessment and management plan (RAMP) process in a voluntary Johne's disease (JD) control program, compare RAMP-specific satisfaction results based on herd JD status, and measure knowledge transfer from certified veterinarians to producers during the RAMP. A satisfaction questionnaire was adapted to the RAMP process in the AJDI to measure producer satisfaction. The questionnaire included 9 RAMP-specific producer satisfaction items, 1 global RAMP satisfaction item, and 16 questions to assess producer knowledge and knowledge translation about JD, bovine viral diarrhea (BVD), and bovine leukosis virus (BLV) during the RAMP (BVD and BLV used for comparison purposes). A total of 133 dairy producers in the AJDI (79.6% response rate) completed the questionnaire by telephone. The RAMP-specific satisfaction was high among the AJDI producers surveyed, and these results were not found to differ based on herd JD status. The lowest satisfaction scores and the highest number of "unable to assess" responses were for the item relating to cost. Factors that contributed to RAMP-specific producer satisfaction were not identified from the demographic and herd information available in this study. The knowledge scores indicated moderate knowledge about JD and fair knowledge about BVD and BLV. Evidence of knowledge translation from the RAMP was mixed in this study. Bovine viral diarrhea knowledge scores were not found to differ based on whether or not the certified veterinarian discussed BVD during the preceding RAMP, but BLV knowledge scores were higher among dairy producers that discussed BLV during the preceding RAMP. Strengths and gaps in producer knowledge about these 3 infectious diseases were identified. By using this producer questionnaire, interventions aimed at improving the content, delivery, and satisfaction of RAMP in JD control programs, such as the AJDI, can be developed.

Use of a Ferret Model to Test Efficacy and Immunogenicity of Live Attenuated Mycobacterium avium Subspecies paratuberculosis Vaccines.

Native hosts for the bacterial agent that causes Johne's disease are ruminants, which include cattle, sheep and goats among others. These large animals are often too costly to be used in testing experimental vaccines. In this chapter, we provide detailed methods to use an inexpensive and more manageable animal host, the ferret, to test efficacy and immunogenicity of live-attenuated Mycobacterium avium subspecies paratuberculosis (MAP) mutant strains prior to consideration as vaccine candidates.

Rewiring cattle trade movements helps to control bovine paratuberculosis at a regional scale.

Paratuberculosis is a worldwide disease mainly introduced through trade. Due to the low sensitivity of diagnostic tests, it is difficult to protect herds from purchasing infected animals. Our objective was to assess if rewiring trade networks to promote risk-based movements could reduce the spread of Mycobacterium avium subsp. paratuberculosis (MAP) between dairy cattle herds at a regional scale. Two levels of control strategies were assessed. At the between-herd scale, trade rewiring aimed to prevent animals from high-risk herds moving into low-risk herds. At the within-herd scale, complementary additional measures were considered based on the herd infection status, aiming to limit the within-herd spread by reducing calf exposure to adult faeces and culling more rapidly after positive test results. We used a stochastic individual-based and between-herd mechanistic epidemiological model adapted to the 12,857 dairy cattle herds located in Brittany, western France. We compared the regional spread of MAP using observed trade movements against a rewiring algorithm rendering trade movements risk-based. All females over two years old were tested. Based on the results, and taking into account the low test sensitivity, herds were annually assigned one of three statuses: A if the estimated true prevalence was below 7%, B if it ranged from 7 to 21 %, C otherwise. We also identified herds with a high probability of being MAP-free (AAA herds that had obtained an A status over three consecutive years) to assess the effect of decreasing their risk of purchasing infected animals on MAP regional spread. We showed that movement rewiring to prevent the sale of animals from high to low-prevalence herds reduces MAP regional spread. Targeting AAA herds made it possible to minimize the control effort to decrease MAP regional spread. However, animals purchased by AAA herds should have a moderate to high probability of being MAP-free, especially if the risk of purchasing animals from herds of unknown status cannot be managed. Improved hygiene and early culling of positive animals were relevant complementary on-farm control options to further decrease MAP spread. Future studies should identify how to define herd statuses to target optimal control measure combinations that could reduce the spread of MAP on a regional scale most effectively.

An Embedded Multiscale Modelling to Guide Control and Elimination of Paratuberculosis in Ruminants.

In recent years, multiscale modelling approach has begun to receive an overwhelming appreciation as an appropriate technique to characterize the complexity of infectious disease systems. In this study, we develop an embedded multiscale model of paratuberculosis in ruminants at host level that integrates the within-host scale and the between-host. A key feature of embedded multiscale models developed at host level of organization of an infectious disease system is that the within-host scale and the between-host scale influence each other in a reciprocal (i.e., both) way through superinfection, that is, through repeated infection before the host recovers from the initial infectious episode. This key feature is demonstrated in this study through a multiscale model of paratuberculosis in ruminants. The results of this study, through numerical analysis of the multiscale model, show that superinfection influences the dynamics of paratuberculosis only at the start of the infection, while the MAP bacteria replication continuously influences paratuberculosis dynamics throughout the infection until the host recovers from the initial infectious episode. This is largely because the replication of MAP bacteria at the within-host scale sustains the dynamics of paratuberculosis at this scale domain. We further use the embedded multiscale model developed in this study to evaluate the comparative effectiveness of paratuberculosis health interventions that influence the disease dynamics at different scales from efficacy data.

Evaluation of a virulent strain of Mycobacterium avium subsp. Paratuberculosis used as a heat-killed vaccine.

Bovine paratuberculosis is one of the most important chronic infectious diseases in livestock. This disease is difficult to control because of its inefficient management (test and cull strategy and inadequate biosecurity). Thus, the development of an effective vaccine is essential. In this study, we evaluated a local virulent strain (6611) of Mycobacterium avium subsp. paratuberculosis as an inactivated vaccine in comparison with the Silirum vaccine in mouse model and cattle. Regarding the mice model, only the groups vaccinated with 6611 showed lower colony forming unit (CFU) counts with a lower lesion score in the liver in comparison to the control group at 6 and 12 weeks post-challenge (wpc). The immune response was predominantly humoral (IgG1), although both vaccinated groups presented a cellular response with IFNγ production as well, but the 6611 group had also significant production of IL-2, IL-6, IL-17a, TNF, and IL-10. In cattle, the 6611 vaccinated group was the only one that maintained significant antibody values at the end of the trial, with significant production of IgG2 and IFNγ. No PPDb reactor was detected in the vaccinated animals, according to the intradermal caudal fold tuberculin test. Our results indicate that the 6611 local strain protected mice from challenge with a virulent strain, by inducing a humoral and cellular immune response. In the bovine, the natural host, the evaluated vaccine also induced humoral and cellular immune responses, with higher levels of CD4 + CD25+ and CD8 + CD25+ T cells populations than the commercial vaccine. Despite the encouraging results obtained in this study, an experimental challenge trial in cattle is mandatory to evaluate the efficacy of our candidate vaccine in the main host.

An Escherichia coli carrier vaccine with surface-displayed protein MAP3061c elicits protective immunity against Mycobacterium paratuberculosis in mice.

Johne's disease, or paratuberculosis, is a chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This disease occurs worldwide and results in considerable economic losses in the livestock industry. There are no effective treatments for Johne's disease, so there is an urgent need to develop an efficient, economical, and stable vaccine for MAP control. Here, a live Escherichia coli (E. coli) surface display vaccine harboring the MAP3061c gene was developed through an ice nucleation protein (INP) surface display system. The experimental data demonstrated that MAP3061c has strong immunogenicity and that the surface displayed vaccine can stimulate mice to produce high levels of antibodies. Both CD4+ and CD8+ T cell counts as well as several cytokines - including IFN-γ, IL-4, IL-10, IL-17A and IL-23 - were significantly increased in the display vaccine group. Post-vaccination challenge with MAP in mice resulted in improved fitness of the mice as demonstrated by a lack of weight loss. Pathological results revealed that the surface display vaccine could reduce the degree of pathological damage and slowed the course of disease. Taken together, our data suggests that the E. coli carrier vaccine with surface-displayed MAP3061c elicits protective immunity against MAP, providing new insights into the development of a MAP vaccine.

In vivo kinetics of peripheral cellular immune responses in Mycobacterium avium subspecies paratuberculosis (MAP) infected and vaccinated goats.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (ParaTB) also known as Johne's disease (JD) in ruminants, which is characterized by chronic intestinal inflammation. A similar counterpart has been observed in the form of Crohn's disease in humans. The present study is the first trail in goats to understand the peripheral cellular immune responses following experimental MAP infection and vaccination. Fifteen apparently healthy male kids (3-6 months old) of Barbari breed were included in this study. In the experimental study, 5 kids were infected with 'S 5' strain of MAP ("Indian Bison Type"), 5 were vaccinated (Indigenous Vaccine) against MAP infection (Singh et al., 2007) and the remaining 5 kids were uninfected and non-vaccinated controls. Kids were observed for a period of 180 days post exposure (infection and vaccination) and were tested for development of infection. Cellular immune responses (in blood) were recorded post-exposure by three assays. We measured the frequencies of CD4 and CD8T cells, estimated plasma IFNγ and TNα and in the third assay, in vitro cytokine production by peripheral blood mononuclear cells (PBMCs) from vaccinated, infected and controls were examined in response to polyclonal stimulation. The frequencies of peripheral CD4 and CD8T cells were comparable in control, infected and vaccinated animals except around day 49 post-infection where MAP infected animals showed a trend towards significantly reduced frequencies of CD4 T cells compared to apparently healthy controls. Significantly reduced plasma TNFα levels were also observed in infected animals compared to vaccinated animals,during the course of infection. Diminished levels (although non significant) of TNFα were observed in the supernatants from polyclonally stimulated PBMCs at around day 49 post infection. It is conceivable that the diminished cellular immune responses may coincide with an impairment (immune exhaustion) of perhaps antigen-specific CD4T cells that might, in the course of infection, contribute to the progressive nature of caprine paratuberculosis.