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Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne's disease, a chronic granulomatous enteritis of ruminants. We have previously used naturally MAP-infected heifer calves to document metabolomic changes occurring in MAP infections. Herein, we used experimentally MAP-inoculated heifer calves to identify biomarkers for MAP infections. At 2-weeks of age, 20 Holstein-Friesian (HF) calves were experimentally inoculated with MAP. These calves, along with 20 control calves, were sampled biweekly up to 13-months of age and then monthly up to 19-months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares-discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) discriminated between MAP-inoculated and control heifer calves. Out of 34 identified metabolites, six fatty acyls were able to differentiate between experimental groups throughout the study, including 8, 11, 14-eicosatrienoic acid and cis-8, 11, 14, 17-eicosatetraenoic acid which were also detected in our previous study and so further suggested their value as biomarkers for MAP infection. Pathway analysis highlighted the role of the alpha-linoleic acid and linoleic acid metabolism. Within these pathways, two broad types of response, with a rapid increase in some saturated fatty acids and some n-3 polyunsaturated fatty acids (PUFAs) and later n-6 PUFAs, became predominant. This could indicate an initial anti-inflammatory colonisation phase, followed by an inflammatory phase. This study demonstrates the validity of the metabolomic approach in studying MAP infections. Nevertheless, further work is required to define further key events, particularly at a cell-specific level., (© 2022. The Author(s).)

Countries survey wildlife for bovine tuberculosis (bTB) to ensure case detection or to ascertain a high probability of freedom from bTB in wildlife. The Eurasian badger (Meles meles) is a potential bTB reservoir host. Between 2008 and 2019, 282 badgers were examined post-mortem in the context of general wildlife disease and targeted bTB surveillance programmes in the Netherlands, and no bTB cases were detected. However, it was unclear how effective this surveillance effort was to demonstrate freedom from Mycobacterium bovis infection in the badger population of ±6000 or to detect cases if present. Therefore, surveillance effectiveness was assessed using scenario tree modelling. For lack of standards for wildlife, the models were run against three assumed levels of disease in the population called design prevalence P*: 0.1%, 0.5%, and 3%. A small risk of introduction (0.015/year) was applied, because the Netherlands are officially free from bTB in cattle, with rare import of bTB-infected cattle and no bTB-infected wildlife reported along the Belgian and German borders with the Netherlands. Surveillance more readily picks up bTB presence in badgers when case detection sensitivity tends towards 100% and demonstrates freedom best when the probability of freedom tends towards 100%. For P* 0.1%, 0.5% and 3%, respectively, maximum case detection sensitivity during 2008-2019 was 8%, 35% and 94% and the probability of freedom in 2019 was 46%, 67%, and 95%. At P* = 3%, performing targeted surveillance on 300 badgers in a year would make it extremely unlikely to miss a case (case detection sensitivity > 99.9%); and if no cases are detected, the adjusted probability of freedom would then reach nearly 98.5%. Stakeholders should be made aware that at P* = 3%, one case detected implies around 3% infected badgers. Additional surveillance system components to assess bTB in wildlife and its economics are to be explored further., (© 2021 Utrecht University. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Background: Staphylococcus aureus, a leading cause of mastitis in dairy cattle, causes severe mastitis and/or chronic persistent infections with detrimental effects on the cows' wellbeing, lifespan and milk production. Despite years of research there is no effective vaccine against S. aureus mastitis. Boosting of non-protective pre-existing immunity to S. aureus, induced by natural exposure to S. aureus, by vaccination may interfere with vaccine efficacy. The aim was to assess whether experimental immunization of S. aureus naïve animals results in an immune response that differs from immunity following natural exposure to S. aureus., Results: First, to define the period during which calves are immunologically naïve for S. aureus, Efb, LukM, and whole-cell S. aureus specific serum antibodies were measured in a cohort of newborn calves by ELISA. Rising S. aureus specific antibodies indicated that from week 12 onward calves mounted an immune response to S. aureus due to natural exposure. Next, an experimental immunization trial was set up using 8-week-old heifer calves (n = 16), half of which were immunized with the immune evasion molecules Efb and LukM. Immunization was repeated after one year and before parturition and humoral and cellular immunity specific for Efb and LukM was determined throughout the study. Post-partum, antibody levels against LukM and EfB were significantly higher in serum, colostrum and milk in the experimentally immunized animals compared to animals naturally exposed to S. aureus. LukM specific IL17a responses were also significantly higher in the immunized cows post-partum., Conclusions: Experimental immunization with staphylococcal immune evasion molecules starting before natural exposure resulted in significantly higher antibody levels against Efb and LukM around parturition in serum as well as the site of infection, i.e. in colostrum and milk, compared to natural exposure to S. aureus. This study showed that it is practically feasible to vaccinate S. aureus naïve cattle and that experimental immunization induced a humoral immune response that differed from that after natural exposure only.

Although maternal vaccination is generally considered to be safe, the occurrence of Bovine Neonatal Pancytopenia (BNP) in cattle shows that maternal vaccination may pose a risk to the offspring. Pregsure BVD-induced maternal alloantibodies cause BNP in newborn calves. The occurrence of BNP years after last Pregsure BVD vaccination indicates that alloantibody levels may remain high in dams. Since pregnancy induces alloantibodies we hypothesized that pregnancy boosts the vaccine-induced alloantibody response. Alloantibody levels in Pregsure BVD-vaccinated dams increased from conception towards the end of gestation and declined after parturition. In parallel, BVDV-antibody levels remained constant, indicating that there is specific boosting of alloantibodies. Since the rise in alloantibodies coincides with pregnancy and other alloantigen sources were excluded, we concluded that fetal alloantigens expressed during pregnancy boost the alloimmune response in the dam. These results help explain why BNP cases occur even years after Pregsure BVD has been taken off the market., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Johne's disease (JD) is a chronic disease in ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). At late stages of the disease, MAP bacilli are shed via feces excretion and in turn create the potential for oral-fecal transmission. The role of the host immune response in MAP bacteria shedding patterns at different stages of JD is still unclear. We employed mathematical modeling to predict if the variation in MAP shedding could be correlated to the immune response in infected animals. We used a novel inverse modeling approach that assumed biological interactions among the antigen-specific lymphocyte proliferation response (cell-mediated response), antibody/humoral immune responses, and MAP bacteria. The modeling framework was used to predict and test possible biological interactions between the measured variables and returns only the essential interactions that are relevant in explaining the observed cattle MAP experimental infection data. Through confronting the models with data, we predicted observed effects (enhancement or suppression) and extents of interactions among the three variables. This analysis enabled classification of the infected cattle into three different groups that correspond to the unique predicted immune responses that are essential to explain the data from cattle within these groups. Our analysis highlights the strong and weak points of the modeling approach, as well as the key immune mechanisms predicted to be expressed in all animals and those that were different between animals, hence giving insight into how animals exhibit different disease dynamics and bacteria shedding patterns.

Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF' on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF', shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.

Bovine Neonatal Pancytopenia (BNP), a fatal bleeding syndrome of neonatal calves, is caused by maternal alloantibodies absorbed from colostrum and is characterized by lymphocytopenia, thrombocytopenia and bone marrow hypoplasia. An inactivated viral vaccine is the likely source of alloantigens inducing BNP-associated alloantibodies in the dam. In this study the specificity of BNP alloantibodies was assessed and was linked to the pathology of BNP. We demonstrated that Major Histocompatibility Complex class I (MHC I) and Very Late Antigen-3, an integrin α3/β1 heterodimer, were the major targets of BNP alloantibodies. However, alloantibody binding to various bovine cell types correlated with MHC I expression, rather than integrin β1 or α3 expression. Likewise, alloantibody-dependent complement-mediated cell lysis correlated strongly with MHC I expression. Examination of several tissues of third trimester bovine foetuses revealed that cells, shown to be affected in calves with BNP, were characterized by high MHC class I expression and high levels of alloantibody binding. We conclude that in spite of the heterogeneous specificity of BNP associated maternal alloantibodies, MHC I-specific antibodies mediate the pathogenicity of BNP in the calf and that cells with high MHC I expression were preferentially affected in BNP.

Mycobacterium avium ssp. paratuberculosis (MAP) is an infection of the ruminant intestine. In cows, a long subclinical phase with no or low intermittent shedding precedes the clinical phase with high shedding. It is generally considered that an adaptive cell-mediated immune response controls the infection during the subclinical phase, followed by unprotective antibodies later in life. Based on recent observations, we challenge the importance of adaptive immunity and instead suggest a role of the structural organization of infected macrophages in localized granulomatous lesions. We investigated this hypothesis by mathematical modelling. Our first model describes infection in a villus, assuming a constant lesion volume. This model shows the existence of two threshold parameters, the MAP reproduction ratio R MAP determining if a lesion can develop, and the macrophage replacement ratio R MF determining if recruitment of macrophages is sufficient for unlimited growth. We show that changes in R MF during a cow's life - i.e. changes in the innate immune response - can cause intermittent shedding. Our second model describes infection in a granuloma, assuming a growing lesion volume. This model confirms the results of the villus model, and can explain early slow granuloma development: small granulomas grow slower because bacteria leave the granuloma quickly through the relatively large surface area. In conclusion, our models show that the long subclinical period of MAP infection can result from the structural organization of the infection in granulomatous lesions with an important role for innate rather than adaptive immunity. It thus provides a reasonable hypothesis that needs further investigation.

Uptake of Mycobacterium avium subsp. paratuberculosis (MAP) by calves in the first days of life from colostrum, milk and faeces is regarded an important moment of transmission. The objective of this study was to quantify the association between the MAP status of dams as determined by the presence of MAP DNA and antibody in colostrum and that of DNA in faeces and the environment with subsequent MAP shedding of their daughters. A cohort of 117 dam-daughter pairs giving birth/being born on eight commercial dairy farms with endemic paratuberculosis was followed where colostrum, faecal and environmental samples (dust) were analysed for the presence of MAP using an IS900 real-time PCR. Antibodies in colostrum were measured by ELISA. Analysis of dust samples showed that on all farms environmental MAP exposure occurred continuously. In significantly more colostrum samples (48%) MAP DNA was detected compared to faecal samples (37%). MAP specific antibodies were present in 34% of the colostrum samples. In total MAP DNA was present in faecal samples of 41% of the daughters at least once during the sampling period. The association between faecal shedding in the offspring and the dam MAP status defined by MAP PCR on colostrum, MAP PCR on faeces or ELISA on colostrum was determined by an exact cox regression analysis for discrete data. The model indicated that the hazard for faecal shedding in daughters born to MAP positive dams was not significantly different compared to daughters born to MAP negative dams. When born to a dam with DNA positive faeces the HR was 1.05 (CI 0.6; 1.8) and with DNA positive colostrum the HR was 1.17 (CI 0.6; 2.3). When dam status was defined by a combination of both PCR outcomes (faeces and colostrum) and the ELISA outcome the HR was 1.26 (CI 0.9; 1.9). Therefore, this study indicates that neither the presence of MAP DNA in colostrum, MAP DNA in faeces nor the presence of MAP antibodies in colostrum of the dam significantly influences the hazard of MAP shedding in their subsequent daughters up to the age of two years when raised in a contaminated environment.

The objective of this paper is to study shedding patterns of cows infected with Mycobacterium avium subsp. paratuberculosis (MAP). While multiple single farm studies of MAP dynamics were reported, there is not large scale meta-analysis of both natural and experimental infections. Large difference in shedding patterns between experimentally and naturally infected cows were observed. Experimental infections are thus probably driven by different pathological mechanisms. For further evaluations of shedding patterns only natural infections were used. Within such infections, the transition to high shedding was studied as a proxy to the development of a clinical disease. The majority of studied cows never developed high shedding levels. Those that do, typically never reduced their shedding level to low or no shedding. Cows that eventually became high shedders showed a pattern of continuous shedding. In contrast, cows with an intermittent shedding pattern had a low probability to ever become high shedders. In addition, cows that start shedding at a younger age (less than three years of age) have a lower hazard of becoming high shedders compared to cows starting to shed at an older age. These data suggest the presence of three categories of immune control. Cows that are intermittent shedders have the infection process under control (no progressive infection). Cows that start shedding persistently at a young age partially control the infection, but eventually will be high shedders (slow progressive infection), while cows that start shedding persistently at an older age cannot effectively control the infection and become high shedders rapidly.

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.

Johne's disease or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), occurs in domestic and wild animals worldwide, causing a significant economic loss to livestock industries. After a prolonged incubation time, infected cattle shed MAP bacilli into feces and spread the disease to an uninfected animal population. It is largely unknown how (or whether) the interplay between the pathogen and the host immunity determines timing of shedding after the long incubation time. Such information would provide an understanding of pathogenesis in individual animals and the epidemiology of MAP infection in animal populations. In this review, we summarize current knowledge of bovine Johne's disease pathology, pathogenesis, immunology and genetics. We discuss knowledge gaps that direly need to be addressed to provide a science-based approach to diagnostics and (immuno)prophylaxis. These knowledge gaps are related to anatomical/clinical manifestation of MAP invasion, interaction of bacteria with phagocytes, granuloma formation, shedding, establishment and kinetics of adaptive immune responses in the pathogenesis of the disease. These topics are discussed at the molecular, cellular and tissue levels with special attention to the within host dynamics including the temporal and the spatial context relevant for the various host-pathogen interactions.

This study investigated the metabolic effects of glucocorticoids when administered to propylene glycol-treated cows with clinical ketosis. Clinical ketosis was defined by depressed feed intake and milk production, and a maximal score for acetoacetate in urine. All cows received 250 mL oral propylene glycol twice daily for 3 days and were randomly assigned to a single intramuscular injection with sterile isotonic saline solution (n = 14) or dexamethasone-21-isonicotinate (n = 17). Metabolic blood variables were monitored for 6 days and adipose tissue variables for 3 days. β-Hydroxybutyrate (BHBA) concentrations in blood decreased in all cows during treatment, but were lower in glucocorticoid-treated cows. Cows treated with glucocorticoids had higher plasma glucose and insulin concentrations, whereas concentrations of non-esterified fatty acids, 3-methylhistidine and growth hormone were unaffected. mRNA expression of hormone-sensitive lipase, BHBA receptor and peroxisome proliferator-activated receptor type γ in adipose tissue was not affected. This shows that lipolytic effects do not appear to be important in ketotic cows when glucocorticoids are combined with PG. Plasma 3-methyl histidine concentrations were similar in both groups, suggesting that glucocorticoids did not increase muscle breakdown and that the greater rise in plasma glucose in glucocorticoid-treated cows may not be due to increased supply of glucogenic amino acids from muscle., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bovine Neonatal Pancytopenia (BNP), a bleeding syndrome of neonatal calves, is caused by alloantibodies absorbed from the colostrum of particular cows. A commercial BVD vaccine is the likely source of alloantigens eliciting BNP associated alloantibodies. We hypothesized that the rare occurrence of BNP in calves born to vaccinated dams could be associated with genetic differences within dams and calves. We found that the development of BNP within calves was a heritable trait for dams, not for calves and had a high heritability of 19%. To elucidate which genes play a role in the development of BNP we sequenced candidate genes and characterized BNP alloantibodies. Alloantigens present in the vaccine have to be presented to the dam's immune system via MHC class II, however sequencing of DRB3 showed no differences in MHC class II haplotype between BNP and non-BNP dams. MHC class I, a highly polymorphic alloantigen, is an important target of BNP alloantibodies. Using a novel sequence based MHC class I typing method, we found no association of BNP with MHC class I haplotype distribution in dams or calves. Alloantibodies were detected in both vaccinated BNP and non-BNP dams and we found no differences in alloantibody characteristics between these groups, but alloantibody levels were significantly higher in BNP dams. We concluded that the development of BNP in calves is a heritable trait of the dam rather than the calf and genetic differences between BNP and non-BNP dams are likely due to genes controlling the quantitative alloantibody response following vaccination.

The control of chronic bacterial diseases with high prevalence in areas of endemicity would strongly benefit from availability of postexposure vaccines. The development of these vaccines against mycobacterial infections, such as (para)tuberculosis, is hampered by lack of experience in natural hosts. Paratuberculosis in cattle is both a mycobacterial disease of worldwide importance and a natural host model for mycobacterial infections in general. The present study showed beneficial effects of therapeutic heat shock protein 70 (Hsp70) vaccination in cattle with naturally acquired chronic infection with Mycobacterium avium subsp. paratuberculosis. Vaccination-induced protection was associated with antibody responses, rather than with induction of specific T helper 1 cells. Targeted therapeutic postexposure vaccination complementary to selective use of antibiotics could be an effective approach for control of chronic mycobacterial infections.

Problem: In cattle, retained placenta (RP) is suggested to arise from failure of immune-mediated rejection of the fetal membranes by the maternal immune system and is associated with major histocompatibility (MHC) class I compatibility between calf and dam., Method of Study: To study the association between RP and different MHC class I compatibilities between calf-dam-granddam combinations, massively parallel pyrosequencing was used to determine the MHC class I haplotypes of cows with and without RP., Results: Two-way calf to dam MHC class I compatibility gave a high risk for RP. There was a tendency for a higher risk for RP with calf to dam MHC class I compatibility., Conclusions: We concluded that in two-way compatible pregnancies, the maternal immune system fails to reject the fetal membranes, and the fetal immune system does not mount an immune response against maternal MHC class I antigens that could influence the immune-mediated rejection of the fetal membranes by the maternal immune system. The lack of immune-mediated rejection of the fetal membranes by the maternal immune system increases the risk of occurrence of RP., (© 2011 John Wiley & Sons A/S.)

A challenge experiment was performed to investigate whether administration of Mycobacterium avium subsp. paratuberculosis (MAP) via the respiratory route leads to MAP infection in calves. Eighteen calves from test negative dams were randomly allocated to four groups. Six calves were challenged with MAP nasally and six calves were challenged by transtracheal injection; three orally challenged calves served as positive controls, and three non challenged calves as negative controls. The challenge was performed as a nine-fold trickle dose, 10(7) CFU in total. Blood and faecal samples were collected frequently. Calves were euthanized three months post-challenge and extensively sampled. Blood samples were tested for the presence of antibodies and interferon gamma producing cells by ELISA. Faecal and tissue samples were cultured in a liquid culture system and the presence of MAP was confirmed by IS900 realtime PCR. Fourteen out of fifteen calves had no MAP antibody response. The negative controls remained negative; all positive controls became infected. Two nasally challenged calves showed a Purified Protein Derivative Avian (PPDA) specific interferon gamma response. In all nasally challenged calves, MAP positive intestinal samples were detected. In three calves of the nasal group MAP positive retropharyngeal lymph nodes or tonsils were detected. In all calves of the transtracheal group MAP positive intestinal tissues were detected as well and three had a MAP positive tracheobronchial lymph node. These findings indicate that inhalation of MAP aerosols can result in infection. These experimental results may be relevant for transmission under field conditions since viable MAP has been detected in dust on commercial dairy farms.

Bovine paratuberculosis is a highly prevalent chronic infection of the small intestine in cattle, caused by Mycobacterium avium subspecies paratuberculosis. Current control strategies based on test-and-cull and biosecurity measures do not suffice in lowering the prevalence of paratuberculosis in an adequate manner. Therefore, control programmes are in need of an effective vaccine, but at the moment no vaccine is registered for use in cattle in the European Union. This review provides a brief overview of the microbiology, epidemiology and immunology of bovine paratuberculosis, and focuses on recent advances in the development of vaccines against paratuberculosis.

Strong anti glycolipid IgG responses can occur in humans and animals, but contrary to anti protein responses and anti glycoprotein responses, the exact mechanism of induction is unknown. We have previously shown that experimental immunization with the glycolipid glucose monomycolate (GMM) causes the development of specific T cell responses, but not of anti GMM antibodies. However, cattle naturally infected with Mycobacterium avium ssp. paratuberculosis produce high levels of anti GMM IgG. In the present study, we tested whether vaccination with GMM conjugated to a protein mimics natural infection in its capacity to induce the production of antibodies against GMM. Cattle were immunized (n=5 per group) with GMM conjugated to a protein, or GMM and protein non-conjugated and administered at contralateral locations, or carrier only. Although immunization with the GMM-protein conjugate vaccine and the non-conjugated vaccine induced protein specific antibody responses, GMM specific antibodies were not detected in either of the groups. In conclusion, the generation of isotype-switched anti lipid antibodies appears to require more than providing peptide epitopes for T helper cells to support glycolipid specific B cells in antibody production., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The role of beef in human infections with Toxoplasma gondii is not clear. To get a better understanding of the value of seroprevalence as an indication of the role of beef in human infections with T. gondii we studied the seroprevalence of T. gondii in Dutch cattle and analysed the correlation between detection of antibodies and parasitic DNA. An indirect ELISA was developed and used to test a sample of the Dutch cattle population. Since validation of the ELISA was hampered by a lack of sufficient bovine reference sera, the results were analysed in two different ways: using a cut-off value that was based on the course of the OD in 27 calves followed from birth until 16 months of age, and by fitting a mixture of two normal distributions (binormal mixture model) to the log-transformed ODs observed for the different groups of cattle in the study population. Using the cut-off value, the seroprevalence was estimated at 0.5% for white veal, 6.4% for rosé veal and 25.0% for cattle. However, using the frequency distributions the prevalences were higher: 1.9% for white veal, 15.6% for rosé veal and 54.5% for cattle. Next, for 100 cattle the results with two different serological assays (ELISA and Toxo-Screen DA) were compared with detection of parasites by our recently developed sensitive magnetic capture PCR. Toxoplasma gondii DNA was detected in only two seronegative cattle. This discordance demonstrates that seroprevalence cannot be used as an indicator of the number of cattle carrying infectious parasites. Demonstrating parasitic DNA in seronegative cattle and not in seropositive cattle suggests that only recent infections are detectable. Whether beef from these PCR-positive cattle is infectious to humans remains to be studied., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The recombinant 70kDa heat-shock protein of Mycobacterium avium subspecies paratuberculosis (MAP Hsp70) has been shown to be an immunodominant antigen and a subunit vaccine candidate for bovine paratuberculosis. The aim of the present study was to define MAP Hsp70 specific T cell epitopes in cows immunized with MAP Hsp70 and cows experimentally infected with MAP. Nine peptides were shown to induce proliferation and interferon-gamma production by lymphocytes from MAP Hsp70 immunized cattle. From 28 calves experimentally infected with MAP 82% responded to at least one of the 5 most immunodominant peptides, indicating relevance of the epitopes during infection. In these 28 animals 15 different BoLA class II haplotypes were present indicating that the peptides were presented by multiple BoLA class II DRB3 alleles. These findings indicate the potential of the MAP Hsp70 subunit vaccine as a tool to control paratuberculosis in outbred cattle populations.

Accurate immunodiagnosis of bovine paratuberculosis is among others hampered by the lack of specific antigens. One of the most frequently used antigen preparations is purified protein derivative (PPD), also known as tuberculin. This crude extract has limitations when used in diagnostic assays due to the presence of cross-reactive antigens. The aim of the current study was to systematically analyze the qualitative protein composition of PPD of the major mycobacterial pathogens. One-dimensional gel electrophoresis followed by tandem mass spectrometry analysis of PPD from Mycobacterium avium subspecies paratuberculosis (MAP), Mycobacterium avium subspecies avium (MAA) and Mycobacterium bovis (MB) identified 156, 95 and 132 proteins, respectively. Comparative sequence analysis led to the selection of a MAP-specific protein (MAP1718c), and finally heterologous expression in Escherichia coli of this and other diagnostic candidate proteins (MAP3515c and MAP1138c (LprG)) enabled evaluation of their immunogenicity. Lymphocyte proliferation responses did not indicate substantial diagnostic potential of the antigens tested. In contrast serum antibody levels for MAP1138c in paratuberculosis infected cows (N=20) were significantly higher (p<0.01) than in control animals (N=20), despite the conserved nature of this protein. In conclusion, this study showed that a combination of proteomics and genomics, starting from complex protein mixtures, present in tuberculins, can reveal novel proteins aiding the development of immunodiagnostics for mycobacterial diseases.

Bovine paratuberculosis is caused by the infection of young calves with Mycobacterium avium subsp. paratuberculosis, resulting in a chronic granulomatous infection of predominantly the ileum. After an incubation period of 2 to 5 years, the disease becomes progressive in some of the chronically infected, but asymptomatic cows. This results in a protein-losing enteropathy that will ultimately be fatal. A loss of cell-mediated immune responses in symptomatic animals has been described, but no information is available concerning immune reactivity in the intestine. We sought to investigate putative disease status-associated lymphocyte subset distributions and antigen-specific functional characteristics of mononuclear cells isolated from blood, gut-associated lymphoid tissue, and the intestinal walls of 22 cows in different stages of disease and in control animals. The results demonstrated a significant decrease in CD4(+) T-cell frequency and a significant increase in TcR1-N12(+) gamma delta T-cell frequency in ileum lamina propria lymphocytes of symptomatic animals compared to the asymptomatic shedders. Immunohistology revealed that there was also an absolute decrease in the number of CD4(+) T cells in sections of the lesional ileum. Our findings also indicated that both peripheral and intestinal cell-mediated responses are decreased in symptomatic animals compared to asymptomatic animals. We conclude that the decrease in cell-mediated responses is likely related to a loss of antigen-specific CD4(+) T cells, which is most prominent in the lesional ileum from symptomatic animals, thus contributing to the progressive nature of bovine paratuberculosis.