Improved DNA Amplification of the Hallmark IS900 Element in Mycobacterium avium subsp. paratuberculosis: a Reexamination Based on Whole-Genome Sequence Analysis.

Amplification of the IS900 multicopy element is a hallmark nucleic acid-based diagnostic test for Mycobacterium avium subsp. paratuberculosis, which causes Johne's disease in ruminants. This assay is frequently used to determine the presence of the bacterium in feces of infected cattle and sheep. Two IS900 primer sets developed in the 1990s were widely used for decades, and their use has continued in current studies. However, these primers were developed prior to the availability of complete genome sequences. Recent sequence analysis of the binding locations for one primer pair (P90/P91) identified errors and binding inefficiencies that can be easily corrected to further increase detection sensitivity. The P90 primer is missing two nucleotides that should be present near the 3' end, and it does not bind all copies of IS900 due to 5' deletions at some IS900 loci. These IS900 primer pairs, along with newly developed primers, were tested by real-time PCR on purified genomic DNA to determine which primer set performed the best and how primer design errors affect amplification efficiencies. The newly designed PCR primer set (JB5) showed increased sensitivity by two to three quantification cycles using purified genomic DNA and was similar in efficiency to 150C/921. These tests were extended using DNA from feces and tissues of infected cows, which showed similar results. Finally, a 167-bp partial duplication of IS900 was found in type I strains. Although P90 and P91 primers successfully amplify M. avium subsp. paratuberculosis DNA, their use should be discontinued in favor of more efficient primer pairs in future studies. IMPORTANCE This study is an example of how applied genomic analysis can aid diagnostic test improvements. Detection of Mycobacterium avium subsp. paratuberculosis infection of livestock prior to the appearance of clinical disease signs is very difficult but essential for identifying animals shedding the bacterium to prevent transmission of Johne's disease. Total M. avium subsp. paratuberculosis quantity in the feces as determined by real-time PCR (qPCR) using the IS900 target indicates bacterial shedding status and potential for transmission of the pathogen. However, legacy primers designed prior to the availability of complete genome sequences that are used in these tests to detect M. avium subsp. paratuberculosis were based on data from only a single copy of IS900 and not considering all copies collectively as a group. This approach resulted in primer design errors which can be easily corrected to improve test sensitivities. We tested original primers that contain these errors and their corrected versions by qPCR and showed improved sensitivity on purified genomic DNA as well as fecal and tissue samples. These findings may help detect the organism from environmental samples on farms where sensitivity is currently lacking.

Prediction of Johne's disease state based on quantification of T cell markers and their interaction with macrophages in the bovine intestine.

Cell-mediated immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) are regulated by various types of T lymphocytes. The aim of this study was to quantitate T cell subsets in the mid-ileum of cows naturally infected with MAP to identify differences during different stages of infection, and to determine whether these subsets could be used as predictors of disease state. Immunofluorescent labeling of T cell subsets and macrophages was performed on frozen mid-ileal tissue sections archived from naturally infected dairy cows in either subclinical or clinical disease status, and noninfected control cows. Comprehensive IF staining for CD4, CD8α, TcR1-N24 (gamma delta), FoxP3, CXCR3 and CCR9 served to define T cell subsets and was correlated with macrophages present. Clinically affected cows demonstrated significantly higher numbers of CXCR3+ (Th1-type) and CCR9+ (total small intestinal lymphocytes) cells at the site of infection compared to the subclinical cows and noninfected controls. Further, predictive modeling indicated a significant interaction between CXCR3+ and AM3K+ (macrophages) cells, suggesting that progression to clinical disease state aligns with increased numbers of these cell types at the site of infection. The ability to predict disease state with this model was improved from previous modeling using immunofluorescent macrophage data. Predictive modelling indicated an interaction between CXCR3+ and AM3K+ cells, which could more sensitively detect subclinical cows compared to clinical cows. It may be possible to use this knowledge to improve and develop an assay to detect subclinically infected animals with more confidence during the early stages of the disease.

Bovine NK-lysin-derived peptides have bactericidal effects against Mycobacterium avium subspecies paratuberculosis.

Infection with Mycobacterium avium subspecies paratuberculosis (MAP) is complex, but little is known about the role that natural killer (NK) cells play. In the present study, four bovine NK-lysin peptides were synthesized to evaluate their bactericidal activity against MAP. The results demonstrated that bNK-lysin peptides were directly bactericidal against MAP, with bNK1 and bNK2A being more potent than bNK2B and bNK2C. Mechanistically, transmission electron microscopy revealed that the incubation of MAP with bNK2A resulted in extensive damage to cell membranes and cytosolic content leakage. Furthermore, the addition of bNK2A linked with a cell-penetrating peptide resulted in increased MAP killing in a macrophage model.

Quantification of Macrophages and Mycobacterium avium Subsp. paratuberculosis in Bovine Intestinal Tissue During Different Stages of Johne's Disease.

Johne's disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon ingestion of MAP, it is translocated across the intestinal epithelium and may be killed by intestinal macrophages, or depending on the bacterial burden and immunological status of the animal, MAP may thwart innate defense mechanisms and persist within the macrophage. This study aimed to determine the numbers of macrophages and MAP present in bovine midileal tissue during different stages of infection. Immunofluorescent (IF) labeling was performed on frozen bovine midileal intestinal tissue collected from 28 Holstein dairy cows. The number of macrophages in midileal tissue sections was higher for clinically affected cows, followed by subclinically affected cows and then uninfected control cows. Macrophages were present throughout the tissue sections in clinical cows, including the tunica muscularis, submucosa, and the lamina propria around the crypts and in the villous tips, with progressively fewer macrophages in subclinically affected and control cows. Clinically affected cows also demonstrated significantly higher numbers of MAP and higher numbers of macrophages with intracellular MAP compared to subclinically affected cows. MAP IF labeling was present within the submucosa and lamina propria around the crypts, progressing into the villous tips in some clinically affected cows. Our findings indicate that number of macrophages increases with progression of infection, but a significant number of the macrophages present in the midileum are not associated with MAP.

Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide.

Mycobacteria have a complex cell wall structure that includes many lipids; however, even within a single subspecies of Mycobacterium avium these lipids can differ. Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S-type) contained no identifiable glycopeptidolipids or lipopentapeptide (L5P), yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis as well as biochemical and physico-chemical approaches. This strategy showed that a nonribosomal peptide synthase, encoded by mps1, contains three amino acid specifying modules in ovine strains, compared to five modules in bovine strains (C-type). Sequence analysis predicted these modules would produce the tripeptide Phe-N-Methyl-Val-Ala with a lipid moiety, termed lipotripeptide (L3P). Comprehensive physico-chemical analysis of Map S397 extracts confirmed the structural formula of the native L3P as D-Phe-N-Methyl-L-Val-L-Ala-OMe attached in N-ter to a 20-carbon fatty acid chain. These data demonstrate that S-type strains, which are more adapted in sheep, produce a unique lipid. There is a dose-dependent effect observed for L3P on upregulation of CD25+ CD8 T cells from infected cows, while L5P effects were static. In contrast, L5P demonstrated a significantly stronger induction of CD25+ B cells from infected animals compared to L3P.

Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies for novel mutant discovery.

Mycobacterium avium subsp. paratuberculosis (MAP), the aetiological agent of Johne's disease, is one of the most important bacterial pathogens in ruminants. A thorough understanding of MAP pathogenesis is needed to develop new vaccines and diagnostic tests. The generation of comprehensive random transposon mutant libraries is a fundamental genetic technology to determine the role of genes in physiology and pathogenesis. In this study, whole MAP genome analysis compared the insertion sites for the mycobacterial transposon Tn5367 derived from the Mycobacterium smegmatis insertion sequence IS1096 and the mariner transposon MycoMarT7 carrying the Himar1 transposase. We determined that only MycoMarT7 provides a random representation of insertions in 99 % of all MAP genes. Analysis of the MAP K-10 genome indicated that 710 of all ORFs do not possess IS1096 recognition sites, while only 37 do not have the recognition site for MycoMarT7. Thus, a significant number of MAP genes remain underrepresented in insertion libraries from IS1096-derived transposons. Analysis of MycoMarT7 and Tn5367 mutants showed that Tn5367 has a predilection to insert within intergenic regions, suggesting that MycoMarT7 is the more adequate for generating a comprehensive library. However, we uncovered the novel finding that both transposons have loci-dependent biases, with Tn5367 being the most skewed. These loci-dependent transposition biases led to an underestimation of the number of independent mutants required to generate a comprehensive mutant library, leading to an overestimation of essential genes. Herein, we also demonstrated a useful platform for gene discovery and analysis by isolating three novel mutants for each transposon.

Comparison of methods to isolate peripheral blood mononuclear cells from cattle blood.

Peripheral blood mononuclear cells (PBMCs) are critical for assessment of host immune responses to infectious disease. The isolation of PBMCs from whole blood is a laborious process involving density gradients and multiple centrifugation steps. In the present study we compared a more traditional method of PBMC isolation used in our laboratory to two novel methods of cell isolation for efficiency, cell viability, and enumeration of cell subsets. Our laboratory method uses Histopaque-1077 density gradient in standard conical tubes and this was compared with isolation of cells using SepMate™ tubes, a novel conical tube containing an insert to separate the density gradient. Multiple experiments were performed to optimize the SepMate™ tubes for use with cattle blood. A final experiment was conducted to compare traditional methodology, the optimized SepMate™ method with a more novel method using cell preparation tubes (CPT-10 vacutainers containing density gradient). Results demonstrated that optimization of the SepMate™ tube methodology was necessary, including dilution of blood and addition of centrifugation steps to reduce platelet contamination. The CPT-10 tubes worked well but cell recovery was lower compared to other methods. Both of the newer methods were comparable to a modified version of our traditional laboratory method of PBMC isolation in terms of numbers of recovered viable cells and the frequency of immune cell subsets. Additionally, efficiency was improved, particularly with the SepMate™ tube method, resulting in reduced time in the laboratory as well as reduced usage of plasticware.

Stage of infection with Mycobacterium avium subsp. paratuberculosis impacts expression of Rab5, Rab7, and CYP27B1 in macrophages within the ileum of naturally infected cows.

Introduction: Macrophages are the preferential target of Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of ruminant paratuberculosis. Uptake of pathogens by intestinal macrophages results in their trafficking through endosomal compartments, ultimately leading to fusion with an acidic lysosome to destroy the pathogen. MAP possesses virulence factors which disrupt these endosomal pathways. Additionally, levels of serum vitamin D3 have proven relevant to host immunity. Dynamics of endosomal trafficking and vitamin D3 metabolism have been largely unexplored in bovine paratuberculosis. Methods: This study aimed to characterize expression of early and late endosomal markers Rab5 and Rab7, respectively, within CD68+ macrophages in frozen mid-ileum sections harvested from cows at different stages of natural paratuberculosis infection. Additionally, factors of vitamin D3 signaling and metabolism were characterized through expression of vitamin D3 activating enzyme 1α-hydroxylase (CYP27B1), vitamin D3 inactivating enzyme 24-hydroxylase (CYP24A1), and vitamin D3 receptor (VDR) within CD68+ ileal macrophages. Results and discussion: Cows with clinical paratuberculosis had significantly greater macrophage and MAP burden overall, as well as intracellular MAP. Total expression of Rab5 within macrophages was reduced in clinical cows; however, Rab5 and MAP colocalization was significantly greater in this group. Intracellular Rab7 colocalization with MAP was not detected in subclinical or Johne's Disease negative (JD-) control cows but was present in clinical cows. Additionally, macrophage CYP27B1 expression was significantly reduced in clinical cows. Taken together, the results from this study show disparate patterns of expression for key mediators in intracellular MAP trafficking and vitamin D metabolism for cows at different stages of paratuberculosis.

Mycobacterium avium subsp. paratuberculosis Candidate Vaccine Strains Are Pro-apoptotic in RAW 264.7 Murine Macrophages.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis. Two wild-type strains, a transposon mutant, and two deletion mutant MAP strains (MOI of 10 with 1.2 × 106 CFU) were tested in murine RAW 264.7 macrophages to determine if they induce apoptosis and/or necrosis. Both deletion mutants were previously shown to be attenuated and immunogenic in primary bovine macrophages. All strains had similar growth rates, but cell morphology indicated that both deletion mutants were elongated with cell wall bulging. Cell death kinetics were followed by a real-time cellular assay to measure luminescence (apoptosis) and fluorescence (necrosis). A 6 h infection period was the appropriate time to assess apoptosis that was followed by secondary necrosis. Apoptosis was also quantified via DAPI-stained nuclear morphology and validated via flow cytometry. The combined analysis confirmed the hypothesis that candidate vaccine deletion mutants are pro-apoptotic in RAW 264.7 cells. In conclusion, the increased apoptosis seen in the deletion mutants correlates with the attenuated phenotype and immunogenicity observed in bovine macrophages, a property associated with good vaccine candidates.

Immunological Evaluation of Goats Immunized with a Commercial Vaccine against Johne's Disease.

Johne's disease affects ruminants causing an economic burden to dairy, meat and wool industries. Vaccination against Mycobacterium avium subspecies paratuberculosis (Map), which causes Johne's disease, is a primary intervention for disease control in livestock. Previously, a comprehensive, multi-institutional vaccine trial for Johne's disease was conducted to test the efficacy of live attenuated Map strains. Here, we report the humoral and cell-mediated immune responses from kid goats enrolled in that trial. Both vaccinated and unvaccinated animals showed IFN-γ stimulation and proliferation of T cell subpopulations on challenge with Map. CD4+, CD25+ and γδ cells from cultured PBMCs in the vaccinated goats showed significantly greater proliferation responses on stimulation with Map antigens. The increase in CD44+ and decrease in CD62L+ cells suggest that vaccine administration reduced the inflammatory responses associated with Map infection. Overall, a stronger antibody response was observed in the infected goats as compared to vaccinated goats. Two independent experimental approaches were used to identify differences in the antibody responses of vaccinated and unvaccinated goats. The first approach involved screening a phage expression library with pooled serum from infected goats, identifying previously reported Map antigens, including MAP_1272c and MAP_1569. However, three specific antigens detected only by vaccinated goats were also identified in the library screens. A second approach using dot blot analysis identified two additional differentially reacting proteins in the vaccinated goats (MAP_4106 and MAP_4141). These immunological results, combined with the microbiological and pathological findings obtained previously, provide a more complete picture of Johne's disease control in goats vaccinated against Map.

Effects of 1,25-Dihydroxyvitamin D3 and 25-Hydroxyvitamin D3 on PBMCs From Dairy Cattle Naturally Infected With Mycobacterium avium subsp. paratuberculosis.

The role of vitamin D3 in modulating immune responses has been well-established for over two decades; however, its specific functions have not been extensively detailed in cattle, particularly cattle in different stages of infection with Mycobacterium avium subspecies paratuberculosis (MAP). Consistent with previous work in our lab, the present study showed that infected cattle in the clinical stage of disease have reduced serum 25-hydroxyvitamin D3 [25(OH)D3]. Additionally, effects of vitamin D3 on peripheral blood mononuclear cells (PBMCs) from naturally infected dairy cattle in subclinical (n = 8) or clinical (n = 8) stages of infection were compared to non-infected control cows (n = 8). Briefly, PBMCs were isolated and cultured in vitro with 4 ng/ml 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or 100 ng/ml 25(OH)D3. Treatment with 1,25(OH)2D3 resulted in decreased secretion for some pro-inflammatory cytokines in clinical animals, including IL-1ß, IL-6, and IFN-γ. Similar responses for IL-1ß and IL-6 were noted with the addition of 25(OH)D3. Additionally, pro-inflammatory cytokine gene expression tended to be upregulated in PBMCs from clinical animals after treatment with 1,25(OH)2D3. In contrast, PBMCs from clinical animals treated with 25(OH)D3 showed downregulation of pro-inflammatory cytokine gene expression, although only significant for IL1B. Following 25(OH)D3 treatment, clinical animals showed significant reduction in CD4+CD25+ T cells. CYP27B1 gene expression was notably decreased in clinical and control animals following 25(OH)D3 treatment but increased in subclinical cows. 1,25(OH)2D3 treatment reduced CYP24A1 gene expression in all groups, while 25(OH)D3 treatment only significantly reduced expression for control cows. Lastly, serum 25(OH)D3 levels were significantly lower in clinical animals. Taken together, these data show vitamin D3 modulates cytokine signaling in cattle at different stages of MAP infection and, therefore, may have implications on disease progression.

Bovine Immunity and Vitamin D3: An Emerging Association in Johne's Disease.

Mycobacterium avium subspecies paratuberculosis (MAP) is an environmentally hardy pathogen of ruminants that plagues the dairy industry. Hallmark clinical symptoms include granulomatous enteritis, watery diarrhea, and significant loss of body condition. Transition from subclinical to clinical infection is a dynamic process led by MAP which resides in host macrophages. Clinical stage disease is accompanied by dysfunctional immune responses and a reduction in circulating vitamin D3. The immunomodulatory role of vitamin D3 in infectious disease has been well established in humans, particularly in Mycobacterium tuberculosis infection. However, significant species differences exist between the immune system of humans and bovines, including effects induced by vitamin D3. This fact highlights the need for continued study of the relationship between vitamin D3 and bovine immunity, especially during different stages of paratuberculosis.

Vitamin D3 alters macrophage phenotype and endosomal trafficking markers in dairy cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

Macrophages are important host defense cells in ruminant paratuberculosis (Johne's Disease; JD), a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). Classical macrophage functions of pathogen trafficking, degradation, and antigen presentation are interrupted in mycobacterial infection. Immunologic stimulation by 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances bovine macrophage function. The present study aimed to investigate the role of vitamin D3 on macrophage phenotype and endosomal trafficking of MAP in monocyte-derived macrophages (MDMs) cultured from JD-, JD+ subclinical, and JD+ clinically infected cattle. MDMs were pre-treated 100 ng/ml 25(OH)D3 or 4 ng/ml 1,25(OH)2D3 and incubated 24 hrs with MAP at 10:1 multiplicity of infection (MOI). In vitro MAP infection upregulated pro-inflammatory (M1) CD80 and downregulated resolution/repair (M2) CD163. Vitamin D3 generally decreased CD80 and increased CD163 expression. Furthermore, early endosomal marker Rab5 was upregulated 140× across all stages of paratuberculosis infection following in vitro MAP infection; however, Rab5 was reduced in MAP-activated MDMs from JD+ subclinical and JD+ clinical cows compared to healthy controls. Rab7 expression decreased in control and clinical cows following MDM infection with MAP. Both forms of vitamin D3 reduced Rab5 expression in infected MDMs from JD- control cows, while 1,25(OH)2D3 decreased Rab7 expression in JD- and JD+ subclinical animals regardless of MAP infection in vitro. Vitamin D3 promoted phagocytosis in MDMs from JD- and JD+ clinical cows treated with either vitamin D3 analog. Results from this study show exogenous vitamin D3 influences macrophage M1/M2 polarization and Rab GTPase expression within MDM culture.

Corrigendum: Exogenous Vitamin D3 Modulates Response of Bovine Macrophages to Mycobacterium avium subsp. paratuberculosis Infection and Is Dependent Upon Stage of Johne's Disease.

[This corrects the article DOI: 10.3389/fcimb.2021.773938.].

Evaluation of the association between fecal excretion of Mycobacterium avium subsp paratuberculosis and detection in colostrum and on teat skin surfaces of dairy cows.

OBJECTIVE: To evaluate the association between fecal excretion of Mycobacterium avium subsp paratuberculosis (MAP) by dairy cows in the periparturient period and detection of MAP DNA in colostrum specimens and on teat skin surfaces. DESIGN: Cross-sectional study. ANIMALS: 112 Holstein cows. PROCEDURES: Fecal specimens were collected within 48 to 72 hours prior to parturition, and colostrum and teat swab specimens were collected immediately after parturition. Detection of MAP in fecal specimens was performed via microbial culture, and detection of MAP DNA in colostrum and teat swab specimens was achieved via a PCR assay targeting the genetic element ISMAP02. Logistic regression was used to model the relationship between MAP fecal shedding status and detection of MAP DNA in colostrum or teat swab specimens. Population attributable fractions for the proportion of colostrum and teat swab specimens containing MAP DNA were also calculated. RESULTS: The odds of detecting MAP DNA in colostrum or teat swab specimens in cows with MAP-positive (vs negative) fecal specimens were 2.02 and 1.87 respectively. Population attributable fractions estimates suggested that withholding colostrum from MAP-positive cows could reduce the odds of exposing calves to MAP in colostrum by 18.2%. Not permitting natural suckling by calves could reduce the odds of exposing calves to MAP on the teat surfaces of MAP-positive cows by 19.5%. CONCLUSIONS AND CLINICAL RELEVANCE: Results underscored the need for strict adherence to practices that limit contact of calves with adult cows from the time of birth and promote hygienic colostrum handling to avoid possible contamination with MAP during colostrum harvest, storage, or feeding.

Electrochemical Detection of Serum Antibodies Against Mycobacterium avium Subspecies paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) causes a chronic inflammatory intestinal disease, called Johne's disease (JD) in many ruminants. In the dairy industry, JD is responsible for significant economic losses due to decreased milk production and premature culling of infected animals. Test-and-cull strategy in conjunction with risk management is currently recommended for JD control in dairy herds. However, current diagnostic tests are labor-intensive, time-consuming, and/or too difficult to operate on site. In this study, we developed a new method for the detection of anti-M. paratuberculosis antibodies from sera of M. paratuberculosis-infected animals. M. paratuberculosis antigen-coated magnetic beads were sequentially reacted with bovine serum followed by a horseradish peroxidase (HRP)-labeled secondary antibody. The reaction of HRP with its substrate was then quantitatively measured electrochemically using a redox-active probe, ferrocyanide. After optimization of electrochemical conditions and concentration of the redox-active probe, we showed that the new electrochemical detection method could distinguish samples of M. paratuberculosis-infected cattle from those of uninfected cattle with greater separation between the two groups of samples when compared with a conventional colorimetric testing method. Since electrochemical detection can be conducted with an inexpensive, battery-operated portable device, this new method may form a basis for the development of an on-site diagnostic system for JD.

Exogenous Vitamin D3 Modulates Response of Bovine Macrophages to Mycobacterium avium subsp. paratuberculosis Infection and Is Dependent Upon Stage of Johne's Disease.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of ruminant enteritis, targets intestinal macrophages. During infection, macrophages contribute to mucosal inflammation and development of granulomas in the small intestine which worsens as disease progression occurs. Vitamin D3 is an immunomodulatory steroid hormone with beneficial roles in host-pathogen interactions. Few studies have investigated immunologic roles of 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in cattle, particularly cattle infected with MAP. This study examined the effects of exogenous vitamin D3 on immune responses of monocyte derived macrophages (MDMs) isolated from dairy cattle naturally infected with MAP. MDMs were pre-treated with ± 100 ng/ml 25(OH)D3 or ± 4 ng/ml 1,25(OH)2D3, then incubated 24 hrs with live MAP in the presence of their respective pre-treatment concentrations. Following treatment with either vitamin D3 analog, phagocytosis of MAP by MDMs was significantly greater in clinically infected animals, with a greater amount of live and dead bacteria. Clinical cows had significantly less CD40 surface expression on MDMs compared to subclinical cows and noninfected controls. 1,25(OH)2D3 also significantly increased nitrite production in MAP infected cows. 1,25(OH)2D3 treatment played a key role in upregulating secretion of pro-inflammatory cytokines IL-1ß and IL-12 while downregulating IL-10, IL-6, and IFN-γ. 1,25(OH)2D3 also negatively regulated transcripts of CYP24A1, CYP27B1, DEFB7, NOS2, and IL10. Results from this study demonstrate that vitamin D3 compounds, but mainly 1,25(OH)2D3, modulate both pro- and anti-inflammatory immune responses in dairy cattle infected with MAP, impacting the bacterial viability within the macrophage.

Comparison of a mycobacterial phage assay to detect viable Mycobacterium avium subspecies paratuberculosis with standard diagnostic modalities in cattle with naturally infected Johne disease.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP), the cause of Johne disease, is a slow growing mycobacterium. Viable MAP detection is difficult, inconstant and time-consuming. The purpose of this study was to compare a rapid phage/qPCR assay performed on peripheral blood mononuclear cells (PBMCs) with three standard methods of MAP detection: fecal MAP PCR; plasma antigen-specific IFN-γ & serum MAP ELISA hypothesizing that, if sensitive and specific, Johne animals would be positive and Control animals negative. We studied a well characterized herd of Holstein cattle that were naturally infected with MAP and their Controls. RESULTS: With phage/qPCR 72% (23/32) of Johne and 35% (6/17) of Controls were MAP positive. With fecal PCR 75% (24/32) of Johne and 0% (0/17) of Controls were MAP positive. With plasma antigen-specific IFN-γ 69% (22/32) of Johne and 12% (2/17) of Controls were MAP positive. With serum MAP ELISA, 31% (10/32) of Johne and 0% (0/17) of Controls were MAP positive. When phage / qPCR and fecal PCR results were combined, 100% (32/32) Johne and 35% (6/17) of Control animals were MAP positive. Younger Control animals (1-3 years) had significantly fewer plaques (25 ± 17 SEM) than older Controls (4-12 years) (309 ± 134 p = 0.04). The same trend was not observed in the Johne animals (p = 0.19). CONCLUSIONS: In contrast to our hypothesis, using the phage/qPCR assay we find that viable circulating MAP can rapidly be detected from the blood of animals infected with, as well as those in the Control group evidently colonized by MAP. These data indicate that the presence of viable MAP in blood does not necessarily signify that an animal must of necessity be demonstrably ill or be MAP positive by standard diagnostic methods.

Characteristics of an extensive Mycobacterium avium subspecies paratuberculosis recombinant protein set.

In the first step of a comprehensive large-scale antigen discovery project, 651 genes of Mycobacterium avium subspecies paratuberculosis were expressed in Escherichia coli. All of these were purified by affinity chromatography, dialyzed in phosphate buffered saline, and analyzed on SDS-PAGE gels. Collectively, these purified recombinant proteins represent 14.9% of the total M. avium subsp. paratuberculosis proteome. This volume of protein expression and purification has yielded unique observations that may be missed in smaller scale expression and purification projects. For example, the 252 putative membrane proteins predicted by PSORTb analysis, resulted in lower average expression yields (3.51mg/l culture) than the 176 predicted cytoplasmic proteins (7.27mg/l culture). A few proteins (MAP0107c, MAP3169c and MAP3640) appear to promote lysis of E. coli since there was a drop in optical density of the growth culture minutes after the inducing agent was added. Certain M. avium subsp. paratuberculosis proteins, when expressed in E. coli changed the color of the column resin or appearance of harvested cell pellets. Finally, 19 proteins showed an absorbance maximum at 260nm rather than 280nm that was attributed to binding of nucleic acid during purification. This extensive recombinant protein repository provides a powerful tool for proteome- and genome-scale research of this organism.

Experimental validation of a nested polymerase chain reaction targeting the genetic element ISMAP02 for detection of Mycobacterium avium subspecies paratuberculosis in bovine colostrum.

Colostrum samples experimentally inoculated with Mycobacterium avium subsp. paratuberculosis (MAP; strain K-10) at increasing concentrations between 1 x 10(1) and 1 x 10(9) cells/ml were tested for recovery of MAP DNA using a nested ISMAP02 target polymerase chain reaction initially developed for detecting MAP DNA in fecal samples. The following detection rates were achieved for sample replicates inoculated with unsonicated MAP pure stock: 100% between 1 x 10(7) and 1 x 10(9) cells/ml, 75% between 1 x 10(3) and 1 x 10(6) cells/ml, and 50% between 1 x 10(1)and 1 x 10(2) cells/ml replicates. Detection rates achieved for the colostrum sample replicates inoculated with sonicated MAP cell suspension were 75% for 1 x 10(9) cells/ml, 100% between 1 x 10(7) and 1 x 10(8) cells/ml, 75% for 1 x 10(6) cells/ml, 0 for 1 x 10(4) cells/ml, and 25% between 1 x 10(1) and 1 x 10(3) cells/ml. When negative control colostrum samples were tested, 16 of 18 (89%) samples were correctly detected as negative for MAP DNA using the current assay. In conclusion, the MAP DNA detection rates of the present assay improved with increasing concentrations of MAP in the colostrum sample replicates, although MAP DNA was also detected in 2 of 18 (11%) negative control samples, suggesting an undefined technical problem with the assay or, perhaps, sample contamination during preparation. Overall, the present findings suggest a potential role of the proposed polymerase chain reaction assay to detect MAP in colostrum. However, adoption of this test for use in routine screening of field colostrum for MAP awaits findings from an ongoing field validation study.