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Little is known about the role that B cells play in immune responses to infection with the intracellular pathogen, Mycobacterium avium subsp. paratuberculosis (MAP). Traditionally, the role of B cells has been constrained to their function as antibody-producing cells, however, antibodies are not thought to play a protective role in mycobacterial infections. The present study was designed to characterize B cell subpopulations as well as activation/maturation states in cattle with paratuberculosis. Peripheral blood mononuclear cells (PBMCs) were isolated from noninfected control cows (n = 8); as well cattle naturally infected with MAP in the subclinical (n = 8) and clinical (n = 7) stage of infection and stimulated with MAP antigen for 6 days. MAP infection resulted in greater numbers of total B cells for clinical cows compared to control noninfected cows. The major subpopulation in freshly isolated PBMCs in clinical cows was B-1a B cells, but this shifted to a composite of both B-1a and B-2 B cells upon stimulation of PBMCs with either MAP antigen or pokeweed mitogen, with higher numbers of B-2 B cells. Early B cells were observed to predominate the population of B cells in PBMCs, with lesser populations of germinal B cells, memory B cells and plasma cells. These subpopulations were elevated in clinical cows upon stimulation of PBMCs with MAP antigen, except for plasma cells which were lower compared to control noninfected cows. Increased numbers of B cells in clinical cows aligned with higher expression of B cell markers such as MAPK1/3, BTG1, Bcl2, CD79A and SWAP70, depending upon in vitro stimulation with either mitogen or antigen. This would indicate that the B cells were capable of activation but were anti-apoptotic in nature. The shift to B-2 B cells in the periphery of clinical cows seems to be indicative of an expansion of memory B cells, rather than plasma cells. This may be a last attempt by the host to control the rampant inflammatory state associated with advanced clinical disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

An increasing prevalence of paratuberculosis supports the need for new efficacious vaccines as an essential management tool. Two separate studies were performed in neonatal calves to evaluate the effectiveness of pooled recombinant Mycobacterium avium subsp. paratuberculosis (MAP) proteins (MAP1087, MAP1204, MAP1272c, MAP2077c) as a potential vaccine. In the first study vaccinated calves were immunized with 400 µg protein cocktail per dose, whereas the second study compared doses of 400 µg and 800 µg of protein cocktail, followed by challenge with live MAP for both vaccinated and nonvaccinated control calves 28 days post-vaccination. At the end of 12 months, tissue colonization with MAP was significantly reduced for the vaccinated calves compared to control animals. A higher dose of vaccine improved protection, with further reductions of MAP burden. Antigen-specific IFN-γ responses and serum antibody responses were similar regardless of vaccination, indicating exposure to MAP invoked conventional host immune responses. Host immunity differed due to vaccination, resulting in increased percentages of CD4+ T cells and B cells after stimulation of PBMCs with antigen. Interestingly, gene expression in PBMCs was similar for both control and vaccinated calves except for significant increases in IFN-γ, IL-12, and IL-17 expression observed in vaccinated calves. Vaccination with a cocktail of immunogenic recombinant MAP proteins was efficacious in reducing the level of infection and fecal shedding of neonatal calves and may be a potential tool for curtailing the spread of Johne's disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Mycobacteria are difficult to kill due to the complexity of their cell wall. Further, Mycobacterium avium subsp. paratuberculosis (MAP) has one of the more elaborate cell wall compositions of all the mycobacteria. As a working pathogen within a research laboratory setting or as an environmental contaminant shed in the manure from infected animals, MAP is highly resistant to typical disinfectants. In the past, the most successful disinfectants to kill mycobacteria were based upon phenolics, harsh compounds that can break down the lipids within the cell wall. New disinfectants have been developed that are less toxic to the environment, however, it is unknown how well they perform compared to more traditional disinfectants. In the present study, we present comparative data on the utility of a commercial eco-friendly disinfectant, Benefect®, compared to Amphyl®, a phenolic-based disinfectant, and Lysol®, a quaternary ammonium-based disinfectant, to kill MAP in pure culture, tissues, and manure. Results demonstrated that Benefect was highly effective with up to 100% kill of MAP within 30 min in all experiments, paralleling results obtained with Amphyl. Lysol performed the most poorly, requiring longer contact times to kill MAP. These results suggest that natural, nontoxic ingredients can be used to disinfect even hearty pathogens such as MAP effectively, both within the laboratory and on-farm., Competing Interests: Declaration of Competing Interest Authors declare that there are no competing interests to this research., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Infection of the host with Mycobacterium avium subsp. paratuberculosis results in chronic and progressive enteritis that traverses both subclinical and clinical stages. The mechanism(s) for the shift from an asymptomatic subclinical disease state to advanced clinical disease is not fully understood. In the present study, naturally infected dairy cattle were divided into subclinical and clinical infection groups, along with noninfected control cows of similar parity, to study host immune responses in different stages of infection. Both infection groups had higher levels of secretion of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) than control cows, whereas only clinical cows had increased secretion of IL-10, IL-12, and IL-18 upon stimulation of peripheral blood mononuclear cells (PBMCs) with antigen. Conversely, secretion of IL-17Α was decreased for clinical cows compared to subclinical and control cows. Proinflammatory cytokine genes were upregulated only for subclinical cows, whereas increased IL-10 and IL-17 gene expression levels were observed for both infection groups. Increased CD4 + , CD8 + , and γδ T cell receptor-positive (TCR + ) T cells were observed for subclinical cows compared to clinical cows. Although clinical cows expressed antigen-specific immune responses, the profile for subclinical cows was one of a dominant proinflammatory response to infection. We reason that a complex coordination of immune responses occurs during M. avium subsp. paratuberculosis infection, with these responses shifting as the host transitions through the different stages of infection and disease (subclinical to clinical). A further understanding of the series of events characterized by Th1/Th2/Th17 responses will provide mechanisms for disease progression and may direct insightful intervention strategies., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

In the present study, we evaluated expression of IFN-γ, IL-17, TNF-α, IL-10 and TGF-β by mucosal cells, including WC1 + γδ T cells, in ileal tissues taken from non-infected cattle and cattle naturally infected with Mycobacterium avium subsp paratuberculosis (MAP). Infected cattle were either in the subclinical or clinical stage of infection. We hypothesized that the cytokine profile of the WC1 + γδ T cell subset would be different between subclinical and clinical cattle. Our data indicate a significant increase in the numbers of WC1 + γδ T cells expressing IL-10 in clinical cattle compared to subclinical and non-infected cattle. We observed a significant increase in TGF-β expression by non-WC1 + cells in clinically infected cattle. Expression of IFN-γ, IL-17 and TNF-α in mucosal cells, including the WC1 + γδ T cell subset, was identified in all examined groups. However, our data indicate that the stage of infection did not significantly influence expression of these proinflammatory cytokines. This study demonstrates changes in the cytokine mRNA expression profile of mucosal cells in the ileum, and specifically WC1 + γδ T cells, as cattle progress to the clinical disease. The change is characterized by an increase in expression of anti-inflammatory cytokines., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A role for γδ T cells in protection against mycobacterial infections including Johne's disease (JD) has been suggested. In neonatal calves where the risk to infection with Mycobacterium avium subsp. paratuberculosis (MAP) is high, the majority of circulating CD3 + lymphocytes are γδ TCR + . Bovine γδ T cells are divided into two major subsets based on the surface expression of workshop cluster 1 (WC1). The WC1 + subset, the predominant subset in periphery, is further divided into WC1.1 + and WC1.2 + subpopulations. The ability of γδ T cells to produce IFN-γ prior to CD4 + αβ T cell activation could be crucial to the outcome of MAP infection. In the current study, cattle were naturally infected with MAP and were classified as either in the subclinical or clinical stage of infection. Compared to the control non-infected group, γδ T cell frequency in circulating lymphocytes was significantly lower in the clinical group. The observed decline in frequency was restricted to the WC1.2 + subset, and was not associated with preferential migration to infection sites (distal-ileum). γδ T cells proliferated significantly in recall responses to stimulation with purified protein derivative from MAP (PPD-J) only in subclinically infected cattle. These responses were a heterogeneous mixture of WC1.1 and WC1.2 subsets. Proliferation and IFN-γ production by the WC1.1 + γδ T cell subset was significantly higher in the subclinical group compared to the control and clinical groups. Our data indicates differences in MAP-specific ex-vivo responses of peripheral WC1 + γδ T cells of cattle with the subclinical or clinical form of JD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Peripheral blood mononuclear cells (PBMC) and mesenteric node lymphocytes (MNL) were obtained from 30 calves that were assigned randomly at birth to 1 of 6 treatment groups with 5 calves per treatment in a 14-d study: (1) colostrum-deprived (CD), no vitamins; (2) colostrum-replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D 3 ; (5) CR, vitamin E; (6) CR, vitamins A, D 3 , E. Calves were injected with appropriate vitamin supplements and fed pasteurized whole milk (CD calves) or fractionated colostrum replacer (CR calves) at birth. Thereafter, all calves were fed pasteurized whole milk fortified with vitamins according to treatment group. Calves were orally inoculated with 10 8 cfu of Mycobacterium avium ssp. paratuberculosis (MAP) on d 1 and 3. The PBMC and MNL harvested on d 13 were analyzed by flow cytometry as fresh cells, after 3-d culture with phytohemagglutinin (PHA), and after 6-d culture with a whole-cell sonicate of MAP (MPS). Peripheral γδ T cells were a predominant lymphocyte subset in neonatal calves, with a decreased percentage noted in CD calves compared with CR calves. As well, CD25 expression was higher in γδ T cells compared with other cell subsets, regardless of treatment group. Stimulation of PBMC with PHA resulted in increased CD4 + and CD8 + subsets, whereas MNL response was dominated by expansion of B-cell subpopulations. Stimulation with PHA and MPS decreased the relative abundance of PBMC γδ T cells, but MNL γδ T cells increased upon stimulation with MPS. These results identify γδ T cells as key early responders to intracellular infection in neonatal calves and suggest that colostrum may be an important mediator of this response., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and colostrum of infected cows was affected by severity of infection as well as the number of days in milk (DIM). Milk was collected over the 305-d lactation period from naturally infected cows in the asymptomatic subclinical (n=39) and symptomatic clinical (n=29) stages of disease, as well as 8 noninfected control cows. All milk samples were assayed for MAP by culture on Herrold's egg yolk medium and either BACTEC 12B (Becton Dickinson, Franklin Lakes, NJ) or para-JEM (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH) liquid medium, and by direct PCR for the IS900 target gene. Mycobacterium avium ssp. paratuberculosis was detected in 3.8, 4.1, and 12.6% of milk samples collected from cows with subclinical JD after culture in Herrold's egg yolk medium, liquid medium, and direct PCR, respectively. The frequency of MAP positivity increased to 12.9, 18.4, and 49.2% of milk samples collected from cows with clinical JD by these same methods, respectively. None of the milk samples collected from control cows was positive for MAP by any detection method. Viable MAP was primarily isolated from milk and colostrum of subclinically and clinically infected cows collected in early lactation (DIM 0-60), with negligible positive samples observed in mid (DIM 60-240) and late (DIM 240-305) lactation. This study demonstrates that shedding of MAP into milk is affected by infection status of the cow as well as stage of lactation, providing useful information to producers to help break the cycle of infection within a herd., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mycobacterium avium ssp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne's disease and, therefore, is a route of transmission for infection in young stock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of MAP from individual bovine milk samples from known infected herds. Decontamination of milk samples (n = 17) with either 0.75% hexadecylpyridinium chloride for 5h or N-acetyl-L-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15 min before culture in BACTEC 12 B (Becton Dickinson, Franklin, NJ), para-JEM [Thermo Fisher Scientific (TREK Diagnostic Systems, Inc.), Cleveland, OH], and Herrold's egg yolk (HEY; Becton Dickinson) media was compared. Treatment with NALC-NaOH resulted in a lower percentage (6%) of contaminated samples than did treatment with hexadecylpyridinium chloride (47%), regardless of culture medium. The decontamination protocol (NALC-1.5% NaOH) was then applied to milk samples (n = 144) collected from cows at 7 US dairies. Recovery of viable MAP from the milk samples was low, regardless of culture medium, with recovery from 2 samples cultured in BACTEC 12 B medium, 1 sample cultured in para-JEM medium, and no viable MAP recovered on HEY medium. However, 32 cows were fecal culture positive and 13 milk samples were positive by direct PCR, suggesting that several cows were actively shedding MAP at the time of milk collection. Contamination rates were similar across media, with 39.6, 34.7, and 41.7% of samples contaminated after culture in BACTEC 12 B, para-JEM, and HEY media, respectively. Herd-to-herd variation had a major effect on sample contamination, with the percentage of contaminated samples ranging from 4 to 83%. It was concluded that decontamination of milk with NALC-1.5% NaOH before culture in BACTEC 12 B medium was the most efficacious method for the recovery of viable MAP from milk, although the ability to suppress the growth of contaminating microorganisms varied greatly between herds., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

CD40 and CD40 ligand (CD40L) have costimulatory effects as part of a complex series of events in host immunity. In this study, the expression of CD40 and CD40L on peripheral blood mononuclear cells (PBMCs) isolated from cattle with Johne's disease were measured on freshly isolated PBMCs and on cells cultured for 8, 24, and 72 h in the presence or absence of live Mycobacterium avium subsp. paratuberculosis and exogenous gamma interferon, interleukin 10, and transforming growth factor β. Results demonstrated greater CD40 and CD40L expression on fresh PBMCs obtained from animals in the clinical stage of disease (symptomatic) than those from healthy control animals or cows in the subclinical stage of disease (asymptomatic). A similar expression profile with greater magnitude was noted for cultured PBMCs, with increased CD40 expression after 8 and 24 h of culture and increased CD40L expression between 24 and 72 h on PBMCs obtained from clinically infected animals. The addition of live M. avium subsp. paratuberculosis to cell cultures resulted in downregulation of CD40L expression in naturally infected cows, regardless of the disease stage. In contrast, the addition of live M. avium subsp. paratuberculosis to cultures resulted in upregulation of CD40 expression on cells obtained from clinically infected animals, while a decrease in expression was noted for healthy and subclinically infected cows. No effects of exogenous cytokines on CD40 or CD40L expression were observed. These results clearly point for the first time to a disparity in the expression of these costimulatory molecules on immune cells from cattle in different stages of Johne's disease and suggest further investigation into their roles in paratuberculosis pathogenesis.

Mycobacterium avium subsp. paratuberculosis is shed into the milk and feces of cows with advanced Johne's disease, allowing the transmission of M. avium subsp. paratuberculosis between animals. The objective of this study was to formulate an optimized protocol for the isolation of M. avium subsp. paratuberculosis in milk. The parameters investigated included chemical decontamination with N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and the efficacy of solid (Herrold's egg yolk medium [HEY]) and liquid (Bactec 12B and para-JEM) culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 10(2) to 10(8) CFU/ml of live M. avium subsp. paratuberculosis organisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. avium subsp. paratuberculosis. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. avium subsp. paratuberculosis cells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. avium subsp. paratuberculosis from milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. avium subsp. paratuberculosis cells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. avium subsp. paratuberculosis can be detected.

The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratuberculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with either M. avium subsp. paratuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO(+) CD25(+) T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.

A protocol was optimized for the isolation of Mycobacterium avium subsp. paratuberculosis (MAP) from milk and colostrum, with parameters including chemical decontamination, antibiotics, and different culture media. This study demonstrates that the efficiency of MAP recovery from milk is highly dependent upon the culturing protocol, and such protocols should be optimized to ensure that low concentrations of MAP in milk can be detected.

A calf model was used to determine if the depletion of CD4 T cells prior to inoculation of Mycobacterium avium subsp. paratuberculosis (Map) would delay development of an immune response to Map and accelerate disease progression. Ileal cannulas were surgically implanted in 5 bull calves at 2 months of age. Two calves were depleted of CD4 T cells by intravenous injection of anti-bovine CD4 antibody administered 24h prior to inoculation with Map. The two CD4-depleted calves and one non-depleted calf were inoculated via ileal cannula with 1 × 10(8)cfu live Map every 3 days for a total of 4 inoculations. Two additional calves served as non-depleted and uninfected controls. Injection with the anti-CD4 mAb reduced the frequency of CD4 T cells from a pre-depletion average of 15% to less than 1% in PBMC at 24h. However, a consistent proliferative response dominated by CD4 T cells, developed in both treated and untreated calves over the course of the 6-month study period. Recovery of Map from serial biopsies obtained from the CD4-depleted and non-depleted calves after Map infection did not differ. In addition, CD4 depletion did not increase the level of Map shed in the feces over the non-depleted animal., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis is the interference with diagnostic tests for bovine tuberculosis (TB) and paratuberculosis. The current study was designed to explore the effects of immunization with a heat-killed whole-cell vaccine (Mycopar) on diagnostic test performance and to characterize host immune responses to vaccination over a 12-month period. Neonatal dairy calves were assigned to treatment groups consisting of (i) controls, not vaccinated (n = 5), and (ii) vaccinates, vaccinated with Mycopar vaccine (n = 5). The results from this study demonstrated a rapid initiation of M. avium subsp. paratuberculosis-specific gamma interferon (IFN-γ) in vaccinated calves by 7 days, with robust responses throughout the study. Vaccinated calves also had responses to M. bovis purified protein derivative tuberculin (BoPPD) but minimal reactivity to ESAT-6/CFP-10, an M. bovis recombinant fusion protein. The levels of antigen-specific interleukin-4 (IL-4) and IL-10 were markedly decreased in vaccinated calves between days 7 and 90 of the study but thereafter were similar to the levels in controls. Vaccinated calves began to seroconvert at 4 months, with 4/5 calves having detectable M. avium subsp. paratuberculosis antibody by 6 months. The responses in test platforms for bovine TB were negligible in the vaccinate group, as only one calf had a response, which was in the suspect range of the comparative cervical skin test. Serum antibody responses to M. bovis antigens ESAT-6, CFP-10, and MPB83 were negative on the Vet TB STAT-PAK, DPP VetTB, and DPP BovidTB tests. These results suggest that the Mycopar vaccine will interfere with diagnostic tools for paratuberculosis but result in low interference with the comparative cervical skin test and emerging serologic tests for M. bovis.

The objective of this study was to determine if experimental infection of neonatal calves with Mycobacterium avium subsp. paratuberculosis would invoke changes in the percentages of total B cells in the peripheral blood mononuclear cell population and of subpopulations of B cells as determined by CD5, CD25, and CD45RO markers during a 12-month period. Experimental infection groups included control (noninfected), oral (infected with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), i.p. (intraperitoneal inoculation), and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. Over the course of the study, the percentages of total B cells in nonstimulated and antigen-stimulated cell cultures increased for oral and i.p. group calves, with the highest percentages noted at 3 and 6 months. Oral/M group calves had increased percentages of activated B cells, as determined by CD5(dim) and CD5(bright) markers, at 9 and 12 months. Experimental infection by all methods resulted in increased expression of CD25(+) and CD45RO(+) B cells early in the study, but the most significant results were observed at 12 months for oral/DXM and oral/M group calves. Immunoblot analyses with a whole-cell sonicate of M. avium subsp. paratuberculosis demonstrated the most reactivity with sera from i.p. group calves and the least reactivity with sera from oral group calves. Further evidence of M. avium subsp. paratuberculosis-specific antibody responses in the i.p. group calves was demonstrated using the ethanol vortex enzyme-linked immunosorbent assay (EvELISA) method. In summary, an induction of B cell responses was noted after experimental infection with M. avium subsp. paratuberculosis, with differences in responses noted according to the method of experimental inoculation.

The objective of this study was to observe early markers of cell-mediated immunity in naïve calves infected with Mycobacterium avium subsp. paratuberculosis and how expression of these markers evolved over the 12-month period of infection. Groups for experimental infection included control (noninfected), oral (infected orally with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), intraperitoneal (i.p.) inoculation, and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. One of the earliest markers to emerge was antigen-specific gamma interferon (IFN-γ). Only i.p. inoculated calves had detectable antigen-specific IFN-γ responses at 7 days, with responses of the other infection groups becoming detectable at 90 and 120 days. All infection groups maintained robust IFN-γ responses for the remainder of the study. At 1 month, calves in the oral and oral/M groups had higher antigen-stimulated interleukin-10 (IL-10) levels than calves in the other treatment groups, but IL-10 secretion declined by 12 months for all calves. T-cell activation markers such as CD25, CD26, CD45RO, and CD5 were significantly upregulated in infected calves compared to noninfected controls. Oral inoculation of calves resulted in significantly increased antigen-specific lymphocyte proliferation at 9 and 12 months, as well as inducible nitric oxide synthase (iNOS) secretion at 6 and 12 months. These results demonstrate that infection of naïve calves with M. avium subsp. paratuberculosis invoked early immunologic responses characterized by robust antigen-specific IFN-γ responses and induction of CD25 and CD45RO expression on T-cell subsets. These were followed by antigen-specific lymphocyte proliferation, iNOS secretion, and expression of CD26 and CD5(bright) markers in the latter part of the 12-month study.

Understanding the host response to Mycobacterium avium subsp. paratuberculosis is critical to the development of effective vaccines and therapeutics for the control of this disease in the field. The current study compared the effectiveness of oral and intraperitoneal (IP) methods of experimental inoculation and two strains of M. avium subsp. paratuberculosis (strain K-10 and clinical isolate 509) on the level of infection and lesion development. Calves were inoculated with 4x10(11) to 8x10(12)cfu live bacteria, depending upon treatment group. Fecal shedding of M. avium subsp. paratuberculosis was minimal and infrequent over the course of the study for calves that received strain K-10 (oral and IP), however, calves orally inoculated with the clinical isolate shed high numbers of bacteria in their feces up to 4 months post-inoculation. Colonization was present in a number of intestinal tissues and lymph nodes with the lowest number of affected tissues in the IP calves and the highest for calves receiving the clinical isolate via oral inoculation. Microscopic lesions were predominantly found in the ileal and jejunal sections of small intestine and their associated lymph nodes, as well as the ileocecal valve and node. These data suggest that a variety of experimental infection regimes can be effective but oral inoculation with a clinical isolate may result in greater colonization of tissues and fecal shedding of M. avium subsp. paratuberculosis.

CD5 is a cell surface molecule involved in antigen recognition and is present on all T lymphocytes and a subset of B lymphocytes. The purpose of this study was to examine CD5+ expression on peripheral blood B cells from healthy, noninfected cattle and cattle with subclinical and clinical paratuberculosis. Peripheral blood mononuclear cells (PBMC) were freshly isolated or cultured for 7 days in the presence or absence of live Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis), and then analyzed by flow cytometry for CD5 expression within the B cell subpopulation. Analysis demonstrated a significant increase (P<0.01) in B cells in clinical animals as compared to healthy control cows and subclinically infected cows. In addition, three subpopulations within the CD5+ B cell population were identified: CD5dim, CD5bright, and a minor population that was characterized as CD5extra bright. A decrease in the CD5dim B cell population along with a concomitant increase in CD5bright B cells was observed in infected cows, an effect that was highly significant (P<0.01) for subclinically infected cows in cultured PBMC. In vitro infection with live M. avium subsp. paratuberculosis did not affect CD5+ expression patterns on B cells, regardless of animal infection status. Addition of exogenous IL-10 to PBMC cultures resulted in decreased numbers of CD5(bright) B cells for healthy control cows, whereas, a synergistic effect of IL-10 and infection with live M. avium subsp. paratuberculosis resulted in increased CD5bright B cells for subclinically infected cows. These results suggest that differential expression of CD5bright and CD5dim subpopulations on B cells in animals with paratuberculosis may reflect a shift in host immunity during the disease process.

Osteopontin (Opn), an important mediator of the cell-mediated immune response, enhances the host immune response against mycobacterial infections. Infections caused by Mycobacterium avium ssp. paratuberculosis (MAP) have a devastating effect on the dairy industry. We sought to characterize Opn at the level of gene and protein expression in periparturient dairy cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMC) were isolated from control, subclinical, and clinical periparturient dairy cows naturally infected with MAP beginning 3 wk precalving to 5 wk postcalving and incubated with medium alone (non-stimulated: NS), concanavalin A (ConA), or a whole-cell sonicate of MAP (MPS). Real-time PCR was performed to evaluate expression of Opn and classical Th1 and Th2 cytokines. Results demonstrated greater Opn expression in nonstimulated PBMC isolated from subclinical cows compared with control and clinical cows. For clinical cows, there was a strong correlation between Opn expression and expression of the Th1 cytokines IFN-gamma and IL-1 alpha for nonstimulated PBMC and IFN-gamma and IL-12 for PBMC stimulated with MPS. Expression of tumor necrosis factor-alpha was greater in clinical cows than the other groups. Nonstimulated, ConA, and MPS-stimulated PBMC from subclinical cows secreted more IFN-gamma, and MPS-stimulated PBMC from clinical cows secreted more IL-4 compared with the other groups. Immunoblot analysis of PBMC detected 4 Opn proteins at 60, 52, 34, and 27 kDa. This is the first study to evaluate the role of Opn on the immune response of dairy cows naturally infected with MAP, and results suggest Opn may be a key regulator against MAP infection.

Johne's disease (JD) is characterized by a protracted period of subclinical infection. Infected cows may remain in the subclinical state until stressors such as parturition and lactation invoke more clinical signs of disease. The objective of this study was to evaluate changes in the percentages of CD4(+), CD8(+), and gammadelta T-cells, B-cells, monocytes, as well as the expression of the activation marker, CD5, on these cell subpopulations in the peripheral blood of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) during the periparturient period. Peripheral blood mononuclear cells (PBMCs) were collected from 3 wk pre- to 4 wk post-calving and freshly isolated or cultured for 7d. Day 7 cultures were infected with live MAP at a 10:1 MOI (bacteria to adherent PBMC), and cultures were incubated for an additional 24h. Fluorescent antibody labeling of lymphocyte subsets and monocytes was conducted and analyzed with flow cytometry. Freshly isolated PBMCs from subclinical cows expressed a greater (P<0.05) percentage of CD8(+) and gammadelta T-cells compared with clinical cows. The percentage of CD4(+) T-cells increased (P<0.08) in clinical cows as parturition approached. During the postpartum period, clinical cows had greater (P<0.05) CD4:CD8 ratios compared with subclinical and control cows. After 8d, uninfected PBMCs from clinical cows had greater (P<0.05) percentages of CD14(+) cells compared with subclinical cows. When infected with live MAP, there was no effect of infection group or parturition on cell subpopulations. In fresh PBMCs, clinical cows expressed lower percentages of CD4(+)CD5(bright) and CD8(+)CD5(bright) compared with control cows, but greater percentages of CD5(dim) cells for all lymphocyte subsets. These results suggest changes in the percentages of lymphocyte subsets, monocytes, and CD5 markers are modulated by both infection status and the periparturient period.

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is estimated to infect more than 22% of US dairy herds. Periods of immunosuppression may contribute to the transition from the subclinical to the clinical stage of infection. Understanding the effects of stressors such as parturition on the escalation of disease may provide information that will help to manage JD. The objective of this study was to characterize cytokine gene expression and secretion in periparturient dairy cows naturally infected with MAP. Blood was collected from the jugular vein of healthy noninfected, and subclinically and clinically infected dairy cows for 3 weeks pre-calving to 4 weeks post-calving. Real-time PCR was performed to evaluate the expression of the following cytokine genes by peripheral blood mononuclear cells: IFN-gamma, TNF-alpha, IL-12p35, IL-10, TGF-beta, and IL-4. To assess the effects of parturient immunosuppression on cytokine gene expression, RT-PCR data were analyzed by using 2(-ddCt) values calibrated to dCt value at +1 day relative to calving for each animal. Overall, cytokine gene expression was not influenced by infection status of the cows in this study. However, significant effects in cytokine gene expression were noted across sampling days within the periparturient period. Expression of IFN-gamma by NS and ConA-stimulated PBMCs declined at calving compared with prepartum values in both control and infected cows. Similarly, a decline in expression of IL-4 and IL-10 was observed for cells isolated from subclinically infected cows after stimulation with ConA. ConA-stimulated PBMCs isolated from infected cows secreted higher concentrations of IFN-gamma compared with the controls. A significant decline in IFN-gamma secretion was noted for MPS-stimulated cells for clinical cows from -21 days to +1 day. Stimulating cells with MPS resulted in greater secretion of IL-10 by infected cows during the postpartum period. A trend was also observed for higher TGF-beta secretion by NS PBMCs isolated from clinical cows in the postpartum period. Cells isolated from clinically infected cows and stimulated with MPS secreted higher levels of nitric oxide throughout the periparturient period when compared to control or subclinically infected cows. These data suggest that parturition is a very dynamic time period for host immunity, with potential for altered immunity to hinder the ability of dairy cows to thwart infectious diseases.

Our objective was to evaluate the effects of time and temperature on whole blood used in the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) for paratuberculosis along with evaluating four potential positive controls, and four different mycobacterial antigens for the ELISA. Nine adult Holstein cattle naturally infected with Mycobacterium avium ssp. paratuberculosis were used in a randomized complete block design. Forty-nine blood tubes were collected from each animal and held at 48.9, 37.8, 26.7, 21.1, 15.6 and 4.4 degrees C for 0, 4, 8, 12, 18, 24, 32, 48 and 72 h. Each blood tube was tested with four mycobacterial antigens (two johnin PPDs, an avain PPD and a whole cell sonicate) and four potential positive controls [concanavalin A (conA), phytohaemagglutinin A (PHA), pokeweed mitogen (PWM) and Staphylococcus enterotoxin A (SEA)]. After incubation for 24 h, the plasma was assayed with a commercial IFN-gamma ELISA. Blood stored at 21.1 and 15.6 degrees C maintained the highest ELISA optical densities (OD) over time with severe reduction in OD values at or above 37.8 degrees C. None of the potential positive controls exactly mimicked the antigen response. SEA and PWM were able to elicit a response after the whole blood quit responding to the antigen and conA underestimated the responsiveness. Phytohemagglutinin A was similar to the antigens on an average, but there was significant disagreement among samples. The PPDs were more potent at stimulating IFN-gamma production than the whole cell sonicate. In conclusion, whole blood should be stored/transported at ambient room temperature and stimulated within 12 h of collection.

The immune system is not always successful in recognizing and destroying pathogens it may encounter. Host immunity to mycobacteria is characterized by a very complex series of events, designed to clear the infection. The first line of defense is uptake and processing of the pathogen by macrophages, followed by the initiation of cell-mediated immunity. The secretion of pro-inflammatory cytokines such as IFN-gamma is credited with containment of mycobacterial infections. Yet it is clear that activated T-cells may contain but fail to clear the infection in some hosts. Further, it is recognized that if infection progresses to a more clinical state, the production of pro-inflammatory cytokines is suppressed and expression of anti-inflammatory cytokines is increased. It is unclear what defines a host that can successfully contain the infection versus one that succumbs to severe immunopathologic disease. This review will address some of the key elements in host immunity to mycobacterial pathogens, with an emphasis on Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), in an attempt to understand the dialogue between immune cells and their mediators during infection and what causes this discourse to go awry.

Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization.

This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis that is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 10(2) CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 10(2) CFU/g for the IS900 element and 10(3) CFU/g for the ISMap02 element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.

Tissues and fecal material were collected from 14 North American bison (Bison bison) that were suspected of having Johne's disease and analyzed for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis). Sections of ileum, ileal-cecal lymph node, and three sequential sections of jejunum with their associated mesenteric lymph nodes were taken from each animal. Fecal culture indicated that 5 of 14 (35.7%) animals were infected, whereas cultures from tissues detected 12 of 14 (85.7%) animals as infected and 59 of 111 (53.2%) of the tissues as positive for M. paratuberculosis. Polymerase chain reaction analysis identified infection in 14 of 14 (100%) animals and in 91 of 112 (81.2%) tissues. In addition, tissues were processed for Ziehl-Neelsen acid-fast staining, auramine O/acridine orange fluorescent staining, and immunohistochemical staining. Ziehl-Neelsen and auramine O staining identified 7 of 14 (50%) and 5 of 14 (35.7%) animals as infected and 24 of 112 (21.4%) and 28 of 112 (25%) tissues as positive, respectively. Immunohistochemical analyses of bison tissues, using antisera collected from rabbits immunized with four different preparations of M. paratuberculosis, identified a greater percentage of infected animals (ranging from 57 to 93%) and positive tissues (ranging from 28 to 46%). Collectively, these data indicate that DNA-based detection of M. paratuberculosis was more sensitive than bacterial culture or staining, identified infection in all the bison, and detected the greatest number of positive tissues within each animal.

Mycobacterium avium subsp. paratuberculosis, the causative agent of a chronic enteritis in ruminants (Johne's disease), has been linked to Crohn's disease in humans. This microorganism is shed by infected animals primarily in the feces but is also shed in the milk at much lower levels. Therefore, dairy products from infected animals may be one mode of transmission of this animal pathogen. This study was designed to evaluate the effectiveness of the holder and high-temperature short-time pasteurization standards on the destruction of M. paratuberculosis. One hundred eighty experiments were conducted in this study using a slug-flow pasteurizer unit and a laboratory scale pasteurizer unit. Ultrahigh-temperature milk was inoculated at two concentrations, 10(8) and 10(5) CFU/ml, with three different field strains of M. paratuberculosis. Five different time-temperature combinations were evaluated: 62.7 degrees C for 30 min, 65.5 degrees C for 16 s, 71.7 degrees C for 15 s, 71.7 degrees C for 20 s, and 74.4 degrees C for 15 s. Three replicates of each experiment were run for the pasteurizer unit, time-temperature combination, and strain of M. paratuberculosis. Treatment of milk regardless of bacterial strain or pasteurizer unit resulted in an average 5.0- and 7.7-log kill for the low and high concentrations of inoculum, respectively. Milk treated for cheese production (65.5 degrees C for 16 s) resulted in a much lower and more variable kill. Results from this study indicate that the current U.S. minimum standards for batch and high-temperature short-time pasteurization of grade A milk significantly reduced the survivability of M. paratuberculosis, but some bacteria survived subpasteurization heat treatment of milk used for cheese manufacture.

The 2002 NAHM's Dairy Survey indicated that 87.2% of dairy farms in the United States feed waste milk to their neonatal calves. Although cost-effective, this practice can lead to increased calf morbidity and mortality due to ingestion of pathogenic agents. In an effort to reduce the risk of infection, dairy producers are implementing on-farm pasteurization of the waste milk as a control procedure before feeding the milk to calves. In the present study, the efficacy of a commercial high-temperature, short-time (HTST) on-farm pasteurizer unit to destroy Mycobacterium paratuberculosis, Salmonella enterica spp., and Mycoplasma spp. in raw milk was evaluated. Replicate experiments were run for 3 isolates of M. paratuberculosis, 3 serovars of Salmonella (derby, dublin, typhimurium); and 4 species of Mycoplasma (bovis, californicum, canadense, serogroup 7) at 2 different levels of experimental inoculation. In addition, HTST pasteurization experiments were performed on colostrum experimentally inoculated with M. paratuberculosis. After culture of the pasteurized milk samples, no viable M. paratuberculosis, Salmonella, or Mycoplasma were recovered, regardless of species, strain, or isolate. Pasteurization of colostrum was also effective in the destruction of M. paratuberculosis but resulted in an average 25% reduction in colostral immunoglobulin. These results suggest that HTST pasteurization is effective in generating a safer product to feed to young calves.

Johne's disease progresses through distinct stages including a protracted subclinical stage in which the infection appears to be controlled; followed by a more acute stage in which the host animal demonstrates clinical signs such as diarrhea and weight loss. Little is known about the dynamics of the host immune response during these two phases of disease, however, it is possible that immune modulation in the early stages of disease may play an important role in disease progression. We hypothesized that the clinical stage of Johne's disease is mediated by the expression of cytokines such as transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) that may be accompanied by the downregulation of IFN-gamma gene expression. In the present study, tissue samples were collected from the ileum, ileocecal junction, ileocecal lymph node, and mesenteric lymph nodes of healthy, subclinically or clinically infected cows. The expression of TGF-beta, IL-10, and IFN-gamma genes in these tissues was determined by quantitative competitive RT-PCR. The results demonstrate that TGF-beta and IL-10 mRNA levels are higher in cows that have progressed to the clinical stage of disease compared to subclinically infected or healthy cows. In contrast, IFN-gamma gene expression was significantly higher in subclinically infected cows. These results suggest that a change in the balance of cytokines at the site of infection may contribute to the ability of the host to control Mycobacterium avium subsp. paratuberculosis infection., (Copyright 2004 Elsevier B.V.)

Gamma interferon (IFN-gamma) plays a significant role in the control of mycobacterial infections, including Mycobacterium avium subsp. paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection with M. avium subsp. paratuberculosis on the production of IFN-gamma, IL-10, and TGF-beta by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-gamma, IL-10, and TGF-beta on M. avium subsp. paratuberculosis survival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-beta in culture supernatants in response to stimulation with live M. avium subsp. paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-beta, but after stimulation with live M. avium subsp. paratuberculosis, TGF-beta levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viable M. avium subsp. paratuberculosis recovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection with M. avium subsp. paratuberculosis. The addition of exogenous IL-10 and TGF-beta to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. avium subsp. paratuberculosis recovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-beta during infection with M. avium subsp. paratuberculosis that may be directly related to their effects on macrophage activation and killing of M. avium subsp. paratuberculosis.

Diagnosis of paratuberculosis (Johne's disease) is stymied by the lack of 1 diagnostic tool that can be used to detect both subclinically and clinically infected animals. At present, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals actively shed Mycobacterium paratuberculosis in their feces. Yet, fecal culture has a disadvantage associated with the protracted incubation period of 8-16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosis in fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple, and effective method to extract DNA from fecal samples and modification of a polymerase chain reaction assay for optimal sensitivity of detection. An evaluation of 1,000 well-characterized fecal samples was performed by the Colorado Department of Agriculture (Denver, CO) and the National Animal Disease Center (Ames, IA) to determine the sensitivity, specificity, and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent on the load of bacteria in the fecal sample with 81% detection of samples containing >70 colony-forming units (cfu)/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the 2 laboratories (n = 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosis and reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple, and inexpensive compared with other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g.

Johne's disease (paratuberculosis) of cattle is widespread and causes significant economic losses for producers due to decreased production and poor health of affected animals. The chronic nature of the disease and the lack of a reproducible model of infection hinder research efforts. In the present study, instillation of Mycobacterium avium subsp. paratuberculosis into the tonsillar crypts of neonatal calves resulted in peripheral colonization as detected by antemortem culture of feces and postmortem (320 days postchallenge) culture of intestinal tissues. Antigen-specific blastogenic, gamma interferon (IFN-gamma), and nitric oxide responses by blood mononuclear cells from infected calves exceeded prechallenge responses beginning 194 days postchallenge. Upon in vitro stimulation with paratuberculosis antigens, CD4(+) cells from infected calves proliferated, produced IFN-gamma, and increased expression of CD26 and CD45RO (indicative of an activated memory phenotype). Utilizing a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum immunoglobulin was detected as early as 134 days postchallenge and generally increased after this time point. Two antigens of approximately 50 and approximately 60 kDa were particularly immunodominant early in infection, as shown by immunoblot with serum collected within 2 weeks postchallenge. Findings indicate that the intratonsillar inoculation route will prove useful as an experimental model for paratuberculosis infection. Additionally, this study confirms that mycobacteria-specific antibody is detectable early in the course of experimental Johne's disease, even preceding the development of specific cell-mediated responses.

Paratuberculosis is endemic in domestic and wild ruminants worldwide. We designed the following study to compare host immune responses and pathologic changes in beef calves and bison calves after challenge with either a cattle or bison (Bison bison) strain of Mycobacterium avium subsp. paratuberculosis. In the first part of the study, six bison and six beef calves were orally inoculated with a cattle isolate of M. avium subsp. paratuberculosis over a 2 wk period. In the second part, an additional six bison and six beef calves were similarly inoculated with a bison strain of M. avium subsp. paratuberculosis. Throughout each of the studies, blood and fecal samples were taken monthly for a 6 mo infection period. Tissue samples were obtained at necropsy for culture and histopathologic analyses. Results from this study demonstrated that bison calves were more susceptible to tissue colonization than beef calves after challenge with the cattle isolate and, conversely, that beef calves were more susceptible to the bison strain of M. avium subsp. paratuberculosis. Although lesions were minimal they were most apparent in the jejunum and distal ileum. Interferon-gamma (IFN-gamma) responses were noted in some calves by 1 mo postinoculation and were sustained longer in beef calves after challenge with the bison isolate. Antibody was not detected in either beef or bison calves during the 6 mo infection period. These results indicate that the host response to strains of M. avium subsp. paratuberculosis may differ between ruminant species.

Mycobacterium paratuberculosis is the causative agent of paratuberculosis (Johne's disease), a chronic inflammation of the terminal portion of the ileum in ruminants. The predominance of cell-mediated immunity in early stages of the disease suggests that T lymphocytes are essential to protect the host from infection with M. paratuberculosis. In this study, we investigated the role of alphabeta and gammadelta T cells in resistance to M. paratuberculosis infection using a T-cell receptor (TCR) knockout mouse model. Weanling TCR-alpha-deficient, TCR-delta-deficient, and C57BL/6 control mice (5-6 weeks of age) were acclimated for 2 weeks and then inoculated intraperitoneally with 10(8)CFU/ml of M. paratuberculosis (either strain 19698 or strain Ben). Groups of mice within each treatment group were euthanized at 1, 3 and 6 months post-inoculation. Sections of spleen, liver, ileum and mesenteric lymph node were prepared for bacterial culture and histologic examination. At all time points of infection and regardless of bacterial strain, TCR-alpha-deficient mice had higher levels of M. paratuberculosis colonization in their tissues compared to TCR-delta-deficient mice or C57BL/6 control mice. Lesions were located predominately in the liver and the ileum, depending upon period of infection, and lesion scores were higher for TCR-alpha-deficient mice compared to the other treatment groups. These results suggest that alphabeta T cells play a major role in resistance to infection with M. paratuberculosis and that gammadelta T cells may play a lesser role and potentially confound protective immune responses., (Copyright 2002 Elsevier Science B.V.)

A commercially available kit consisting of twenty 10-mer random primers was evaluated to allow selection of a suitable primer that would permit identification and sub-typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium by randomly amplified polymorphic DNA (RAPD). A primer OPE-20 (5'-AAC-GGT-GAC-C-3') was identified to be the most suitable primer when tested with four ATCC reference strains of M. paratuberculosis and eight well characterized field strains each of M. paratuberculosis and M. avium. Primer OPE-20 was further tested for its ability to identify and subtype 200 field isolates of M. paratuberculosis. The fingerprint patterns of M. paratuberculosis (n=212) consisted of five unique common fragments (620, 450, 310, 230, 180bp) and nine variable fragments resulting in six distinct genotypes. The DNA fingerprints of M. avium (n=8) consisted of a single common fragment of 620bp, and 15 variable fragments resulting in six different genotypes. The cattle, human and goat isolates of M. paratuberculosis were genetically similar, but a sheep isolate had a different RAPD profile as compared to RAPD profiles from other species. RAPD was observed to be a rapid, reproducible and reliable technique for identification and sub-typing of M. paratuberculosis.

Objective: To evaluate sensitivity and specificity of a new ELISA for antibodies against Mycobacterium avium subsp paratuberculosis., Design: Cross-sectional observational survey., Sample Population: Serum samples from 590 cattle that were infected with M avium subsp paratuberculosis and 723 cattle that were not infected., Procedure: Serum samples were tested by use of an ELISA for antibodies against M avium subsp paratuberculosis., Results: Sensitivity of the test varied from 15.4 to 88.1%, depending on the clinical stage and bacterial shedding status of the cattle., Conclusions and Clinical Relevance: Results obtained with use of the new ELISA agreed favorably with those of a previous ELISA. Practitioners must be aware of variability in the sensitivity of the test, which depends on the clinical and shedding status of the cattle, because this may affect interpretation of test results.

A recent dairy survey conducted in 1996 by the National Animal Health Monitoring System suggests between 20 and 40% of dairy herds in the United States have some level of Johne's disease. This figure will continue to increase unless producers implement management regimes that will help control the spread of this disease within their herds. The neonatal calf is the target for infection with Mycobacterium paratuberculosis, the causative agent of Johne's disease. Calves become infected via exposure to the bacterium through contaminated feces, bedding, colostrum, and milk. Shedding of viable M. paratuberculosis has been documented in the colostrum and milk of infected dams. This study evaluated the efficacy of on-farm pasteurization to destroy M. paratuberculosis in waste milk fed to calves to circumvent this mode of transmission. In three replicate experiments, waste milk was experimentally inoculated with M. paratuberculosis and heated at 65.5 degrees C for 30 min. No viable bacteria were recovered after 28 wk of incubation. These results suggest that batch pasteurization of waste milk contaminated with M. paratuberculosis was effective at generating a clean product to feed to young calves.

The host immune response to infection with Mycobacterium paratuberculosis is paradoxical, with strong cell-mediated immune responses during the early, subclinical stages of infection and strong humoral responses during the late clinical stages of the disease. Cell-mediated immune responses modulated by various T cell subsets are essential to provide protective immunity and prevent progression of the disease. Secretion of cytokines by T cell populations serves to activate macrophages to kill ingested M. paratuberculosis as well as activate other T cell subsets to contain the infection. This paper reviews the current knowledge of T cell immune responses in M. paratuberculosis infection based upon clinical studies and research using mouse models.

A portion of the gene encoding HspX has been previously identified as a sequence specific to Mycobacterium avium subspecies paratuberculosis (hereafter referred to as M. paratuberculosis) based on DNA hybridization experiments. In this study, rabbit antisera were raised against a recombinant protein of HspX fused to the Escherichia coli maltose binding protein (MBP/HspX). Immunoblots of lysates of M. paratuberculosis-infected macrophages probed with the rabbit antisera showed that HspX was present within infected macrophages of bovine and murine origin. This observation was confirmed by immunofluorescence microscopy of infected macrophages. Lysates of E. coli expressing HspX without the MBP fusion partner were loaded onto preparative SDS-PAGE gels and used to determine whether infected cattle generated a humoral immune response to the antigen. Sera from four of 24 paratuberculous cows (17%) detected HspX. No reactivity was present in sera from control cows. While HspX may be immunogenic during infection in some cows, the protein is not secreted and it does not stimulate cell-mediated immunity. Collectively, these data give a preliminary characterization of the first described M. paratuberculosis protein identified within infected macrophages.

Objective: To compare cytokine secretion patterns of peripheral blood mononuclear cells (PBMC) from healthy cows and cows subclinically and clinically infected with Mycobacterium paratuberculosis., Animals: 5 noninfected cows, 6 cows with subclinical paratuberculosis, and 4 cows with clinical paratuberculosis., Procedure: PBMC were isolated, and concentrations or activities of secreted interleukin (IL)-1, IL-2, IL-6, tumor necrosis factor (TNF), and interferon-gamma (IFN-gamma) were measured after in vitro stimulation of cells with concanavalin A (ConA), lipopolysaccharide (LPS), or a whole-cell sonicate of M paratuberculosis (MpS). Proliferative responses of PBMC were also determined after stimulation with ConA, phytohemagglutinin, pokeweed mitogen (PWM), or MpS., Results: After stimulation with ConA, cells from subclinically infected cows secreted significantly more, and cells from clinically infected cows secreted significantly less, IFN-gamma, compared with cells from control cows. Cells from cows with subclinical paratuberculosis produced significantly more TNF and IFN-gamma in response to MpS than cells from the other 2 groups. Stimulation of PBMC from subclinically infected cows with ConA or MpS resulted in significantly higher proliferative responses, compared with cells from control and clinically infected cows. In contrast, clinically infected cows had significantly higher proliferative responses to PWM than cells from the other 2 groups., Conclusions and Clinical Relevance: A decrease in T-cell responses to mitogens or MpS was observed in cows clinically infected with M paratuberculosis, compared with subclinically infected cows, suggesting that activated T cells may delay the progression of paratuberculosis.

Mycobacterium paratuberculosis, an acid-fast bacillus that causes enteritis in ruminants, has been suggested as an etiological agent of Crohn's disease in humans. The mode of transmission is unclear; however, some evidence suggests that humans may become infected via contaminated milk. Currently, it is not known whether commercial pasteurization effectively kills M. paratuberculosis in contaminated raw milk. Using a laboratory-scale pasteurizer unit designed to simulate the high-temperature, short-time method (72 degrees C, 15 sec) currently used by commercial dairies, we previously demonstrated that treatment of raw milk inoculated with 10(4) to 10(6) cfu of M. paratuberculosis/ml reduced numbers to an undetectable level. However, M. paratuberculosis is an intracellular pathogen that resides within the macrophages of the host and evades destruction. We subsequently performed further experiments examining heat treatment of milk inoculated with mammary gland macrophages containing ingested M. paratuberculosis. Heat treatment of these samples under high-temperature, short-time conditions demonstrated that the macrophage does not protect the organism because we were unable to recover any viable M. paratuberculosis from the samples. Conversely, other researchers have demonstrated that a residual population of M. paratuberculosis may survive heat treatment of milk. In addition, a recent news report stated that viable M. paratuberculosis organisms have been cultured from retail-ready milk in Ireland. A summary of past and current studies concerning this issue along with a discussion of methodologies used to recover M. paratuberculosis from experimentally inoculated milk will be presented in this paper.

Mycobacterium avium subsp. paratuberculosis infection of cattle results in a chronic granulomatous enteritis. Clinical disease (i.e., cachexia, diarrhea, and high fecal bacterial counts) is preceded by a lengthy subclinical stage of disease. The immunologic mechanisms associated with the progression of infected cattle from subclinical to clinical disease are unclear. In this study, a cell proliferation assay was used in combination with flow cytometry to compare peripheral blood lymphocyte responses of cattle with subclinical paratuberculosis to responses of cattle with clinical paratuberculosis. B cells from cattle with subclinical disease proliferated vigorously upon stimulation with M. avium subsp. paratuberculosis antigen, with up to 12.4% of the total B cells responding. However, B cells from cattle with clinical disease did not proliferate upon antigen stimulation despite good proliferation in response to concanavalin A stimulation. In addition, these animals had high percentages of peripheral blood B cells. B cells from noninfected animals did not proliferate upon M. avium subsp. paratuberculosis antigen stimulation. Thus, it appears that B-cell proliferation is a sensitive indicator of subclinical Johne's disease. Furthermore, the immunologic mechanisms responsible for the antigen-specific unresponsiveness of peripheral blood B cells may be significant in the eventual progression from subclinical to clinical Johne's disease in cattle.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the etiologic agent of paratuberculosis (Johne's disease), a chronic granulomatous enteritis in ruminants. Currently, there is a need for improved diagnostic tests because of the lack of methods for accurate, rapid and reliable detection of M. paratuberculosis infection. A M. paratuberculosis gene (hspX) was cloned, sequenced, and a 30 bp species-specific oligonucleotide was synthesized. As an internal control to identify mycobacterial strains, a 33 bp Mycobacterium genus-specific oligonucleotide was synthesized based on the conserved 5' terminus of the mycobacterial recA gene. Dioligonucleotide hybridization (dOH) analysis identified 28/28 (100%) mycobacterial strains and specifically identified 14/14 (100%) reference (ATCC 19698), bovine, ovine and human isolates of M. paratuberculosis. The M. paratuberculosis-specific oligonucleotide distinguished M. paratuberculosis isolates from related mycobacteria, including all closely related members of the Mycobacterium avium complex (MAC) tested in this study. The members of MAC tested in this study included Mycobacterium avium subspecies avium (M. paratuberculosis, Mycobacterium avium subspecies silvaticum (M. silvaticum) and Mycobacterium intracellulare strains. Hybridization was not observed with DNA extracted from a selected group of other bacterial pathogens. The experiments indicate that the dOH analysis is a useful diagnostic tool to detect mycobacterial infection, specifically M. paratuberculosis. The dOH method could be a good alternative to existing assays and will be adapted for specific identification of M. paratuberculosis from faecal samples, mixed bacteriologic cultures, tissue specimens and whole blood.

Currently, it is not known whether commercial pasteurization effectively kills Mycobacterium paratuberculosis in contaminated raw milk. Results from holder test tube experiments indicated that a residual population of viable bacteria remained after treatment at 65, 72, 74, or 76 degrees C for 0 to 30 min. Use of a laboratory-scale pasteurizer unit demonstrated that treatment of raw milk at 72 degrees C for 15 s effectively killed all M. paratuberculosis.

A new method (NADC) for isolation of Mycobacterium paratuberculosis from fecal samples is described and evaluated using fecal samples from a known paratuberculosis-infected herd of cattle. The NADC method includes centrifugation of the total fecal sample supernatant and use of a 2-step decontamination protocol. The growth rate of M. paratuberculosis and contamination rate of cultures when using this method are compared to 3 other published methods: sedimentation, centrifugation, and Cornell. Sensitivity was lowest for the Cornell method precluding detection of some low shedders; however, contamination was not observed for this method. Contamination was the most severe in samples processed by the centrifugation method but was also high for the sedimentation method, resulting in unreadable culture tubes for some fecal samples. The NADC method was 10-fold more sensitive for detection of M. paratuberculosis colonies and contamination was significantly reduced compared to other 3 methods.

Polyclonal antisera were raised in rabbits against preparations of live and heat-killed Mycobacterium paratuberculosis and cell-wall proteins of M. paratuberculosis and were evaluated as diagnostic tools in immunohistochemical staining of bovine tissue. Live preparations of M. paratuberculosis (LMp) were inoculated intraperitoneally or intravenously at 10(9)/ml. Heat-killed M. paratuberculosis (HKMp) was prepared by treatment of bacteria at 85 C for 10 minutes. Cell-wall proteins were isolated from M. paratuberculosis and conjugated to keyhole limpet hemocyanin to improve antigenicity (KLH-CWPMp). The HKMp and KLH-CWPMp preparations were emulsified in incomplete Freund's adjuvant before subcutaneous inoculation of rabbits. Antibody titers in the terminal blood sample were higher for HKMp and KLH-CWPMp than for LMp rabbits (1:1,024 vs. 1:64). The KLH-CWPMp antibody did not cross-react with M. bovis-infected tissues. Sensitivity and specificity of immunohistochemical detection of Johne's disease (paratuberculosis) from bovine tissues was much higher for the KLH-CWPMp polyclonal antibody. Immunoreactivity of the antibody resulted in staining of bacteria in the cytoplasm of macrophages, mononuclear giant cells, and extracellular bacteria in both intestine and lymph node.

The causative agent in Johne's disease is Mycobacterium paratuberculosis, an intracellular pathogen which causes enteritis in ruminants. Little is known about interactions between the host cell (macrophage) and M. paratuberculosis; however, this bacterium is able to evade normal host immune defenses and cause a chronic infective state. In the present study, we evaluated whether activation of a murine macrophage cell line (J774.16) by pretreatment with recombinant murine tumor necrosis factor alpha (TNF) prior to infection with M. paratuberculosis would affect their ability to restrict growth and kill the ingested bacteria. A murine cell line was utilized owing to difficulty in obtaining bovine reagents and lack of a continuous bovine macrophage cell line for repeated experimentation. After 4 h of infection, numbers of viable bacteria in cell lysates were significantly lower for macrophages pretreated with 1000 IU TNF ml-1. The rate of bacterial growth as assessed by BACTEC radiometric culture system was also reduced at this time point. Upon further extension of the infection period to 72 h, we observed that moderate doses of TNF (10-1000 IU ml-1) significantly increased the number of viable M. paratuberculosis recovered whereas the highest dose of TNF (4000 IU ml-1) effectively reduced bacterial numbers. These data indicate that TNF can either enhance or reduce macrophage mycobactericidal and mycobacteriostatic activity depending upon both the level of TNF to which cells are exposed and the duration of infection.

Protein expression profiles of 10 isolates of Mycobacterium paratuberculosis, M avium 18 (formerly M paratuberculosis 18), and 1 isolate each of M avium serotype 2, M avium serotype 8, and M bovis BCG were examined. Protein expression profiles of M paratuberculosis and M avium were similar. However, two-dimensional gel analysis of [35S]methionine-labeled cellular proteins resolved 4 proteins, with molecular mass of 28,000, 32,000, 32,000, and 42,000 daltons, which were expressed in greater amounts in M paratuberculosis than in M avium. Two proteins, with molecular mass of 43,000 and 60,000 daltons, were identified, which were expressed in greater amounts in M avium than in M paratuberculosis. Immuno (western)-blot analysis, using antiserum from 2 cows clinically infected with M paratuberculosis as the primary antibodies, suggested that the 42,000-dalton protein may be specific for M paratuberculosis. Comparison of protein expression profiles may be useful as a tool for differentiating isolates of M paratuberculosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled extracellular proteins revealed variability among the isolates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled cellular proteins divided the M paratuberculosis isolates into 2 groups on the basis of a difference in the amount of expression of a 28,000-dalton protein. This information may be useful in epidemiologic studies.