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Biomarkers for Early Stages of Johne's Disease Infection and Immunization in Goats.

ABSTRACT: Background: Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a chronic enteric infection of ruminants. Infection occurs within the first few months of life but remains subclinical for an average of 2-5 years. Current diagnostics to detect early subclinical infections lack diagnostic sensitivity, which hinders disease control resulting in significant economic losses to the dairy industry worldwide. The pathophysiology of early infection with M. paratuberculosis is still not well understood and represents a key hurdle toward the development of better diagnostics. Methods: The present study employed a large-scale RNA-Sequencing technology to better understand early stages of M. paratuberculosis infection and immunization. Specifically, gene expression profiles of peripheral blood mononuclear cells (PBMCs) from infected or vaccinated goats were compared to controls. Results: When compared to the naïve control goats, we identified a large number of transcripts (N = 226, 1018, 1714) that were differentially expressed in the M. paratuberculosis-infected goats, goats vaccinated with live attenuated or inactivated vaccines. There were also 1133 differentially expressed (DE) transcripts between vaccinated goats and infected ones. Bioinformatics evaluation of the DE genes indicated the regulation of a large number of genes with immunity and inflammatory functions including IL-18BP, IFN-?, IL-17A, NOS2, LIPG, and IL-22. Interestingly, a large number of goat genes (N = 667) were regulated whether live or inactivated vaccine were used. Some of the regulated genes (e.g., IL-17A, IFN-?) continued its unique transcriptional profile up to 12 months post-challenge. Conclusion: Overall, transcriptome analysis of infected and/or immunized goats identified potential targets for developing early diagnostics for Johne's disease and a potential approach to differentiate infected from vaccinated animals. A similar approach could be used to analyze later stages of Johne's disease or other chronic infections.

SUBMITTER: Berry A

PROVIDER: S-EPMC6172484 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Biomarkers for Early Stages of Johne's Disease Infection and Immunization in Goats.

Berry Aubrey A Wu Chia-Wei CW Venturino Amanda J AJ Talaat Adel M AM

Frontiers in microbiology 20180928

Background: Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a chronic enteric infection of ruminants. Infection occurs within the first few months of life but remains subclinical for an average of 2-5 years. Current diagnostics to detect early subclinical infections lack diagnostic sensitivity, which hinders disease control resulting in significant economic losses to the dairy industry worldwide. The pathoph ...[more]

PMID: 30323794

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Project description:Background: Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease, a chronic enteric infection of ruminants. Infection occurs within the first few months of life but remains subclinical for an average of 2-5 years. Current diagnostics to detect early subclinical infections lack diagnostic sensitivity, which hinders disease control resulting in significant economic losses to the dairy industry worldwide. The pathophysiology of early infection with M. paratuberculosis is still not well understood and represents a key hurdle towards the development of better diagnostics. Methods: The present study employed a large-scale RNA-Sequencing technology to better understand early stages of M. paratuberculosis infection and immunization. Specifically, gene expression profiles of peripheral blood mononuclear cells (PBMCs) from infected and vaccinated goats were compared to controls. Results: The transcriptome of the control goats revealed a large number of genes (N = 226, 1018, 1714, 1133) that were differentially expressed compared to the M. paratuberculosis-infected goats, goats vaccinated with live attenuated or inactivated vaccines, and goats both vaccinated and infected. Bioinformatics evaluation of the differentially expressed genes indicated the regulation of a large number of genes with immunity and inflammatory functions including IL-18BP, IFN-γ, IL-17A, NOS2, LIPG and IL-22. Interestingly, a large number of goat genes (N=667) were regulated whether live or inactivated vaccine were used. Some of the regulated genes (e.g. IL-17A, IFN-g) continued its unique transcriptional profile up to 12 months post-challenge. Conclusions: Overall, transcriptome analysis of infected and/or immunized goats identified potential targets for developing early diagnostics for Johne’s disease and a potential approach to differentiate infected from vaccinated animals. A similar approach could be used to analyze later stages of Johne’s disease or other chronic infections.

Project description:Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic intestinal inflammatory disease of cattle and other ruminants. JD has a high herd prevalence and causes serious animal health problems and significant economic loss in domesticated ruminants throughout the world. Since serological detection of MAP infected animals during the early stages of infection remains challenging due to the low sensitivity of extant assays, we screened 180 well-characterized serum samples using a whole proteome microarray from Mycobacterium tuberculosis (MTB), a close relative of MAP. Based on extensive testing of serum and milk samples, fecal culture and qPCR for direct detection of MAP, the samples were previously assigned to one of 4 groups: negative low exposure (n = 30, NL); negative high exposure (n = 30, NH); fecal positive, ELISA negative (n = 60, F+E-); and fecal positive, ELISA positive (n = 60, F+E+). Of the 740 reactive proteins, several antigens were serologically recognized early but not late in infection, suggesting a complex and dynamic evolution of the MAP humoral immune response during disease progression. Ordinal logistic regression models identified a subset of 47 candidate proteins with significantly different normalized intensity values (p<0.05), including 12 in the NH and 23 in F+E- groups, suggesting potential utility for the early detection of MAP infected animals. Next, the diagnostic utility of four MAP orthologs (MAP1569, MAP2942c, MAP2609, and MAP1272c) was assessed and reveal moderate to high diagnostic sensitivities (range 48.3% to 76.7%) and specificity (range 96.7% to 100%), with a combined 88.3% sensitivity and 96.7% specificity. Taken together, the results of our analyses have identified several candidate MAP proteins of potential utility for the early detection of MAP infection, as well individual MAP proteins that may serve as the foundation for the next generation of well-defined serological diagnosis of JD in cattle.

Project description:Considerable effort has been directed toward controlling Johne's disease (JD), a chronic granulomatous intestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in cattle and other ruminants. However, progress in controlling the spread of MAP infection has been impeded by the lack of reliable diagnostic tests that can identify animals early in the infection process and help break the transmission chain. To identify reliable antigens for early diagnosis of MAP infection, we constructed a MAP protein array with 868 purified recombinant MAP proteins, and screened a total of 180 well-characterized serum samples from cows assigned to 4 groups based on previous serological and fecal test results: negative low exposure (NL, n?=?30); negative high exposure (NH, n?=?30); fecal-positive, ELISA-negative (F?+?E-, n?=?60); and both fecal- and ELISA-positive (F?+?E+, n?=?60). The analyses identified a total of 49 candidate antigens in the NH, F?+?E-, and F?+?E+ with reactivity compared with the NL group (p?<?0.01), a majority of which have not been previously identified. While some of the antigens were identified as reactive in only one of the groups, others showed reactivity in multiple groups, including NH (n?=?28), F?+?E- (n?=?26), and F?+?E+ (n?=?17) groups. Using combinations of top reactive antigens in each group, the results reveal sensitivities of 60.0%, 73.3%, and 81.7% in the NH, F?+?E-, and F?+?E+, respectively at 90% specificity, suggesting that early detection of infection in animals may be possible and enable better opportunities to reduce within herd transmission that may be otherwise missed by traditional serological assays that are biased towards more heavily infected animals. Together, the results suggest that several of the novel candidate antigens identified in this study, particularly those that were reactive in the NH and F?+?E- groups, have potential utility for the early sero-diagnosis of MAP infection.

Project description:Mastitis is an inflammation of the mammary gland (MG), usually due to bacterial infection. Although considerable attention has been paid to this economically important disease, the early stages of the host response remain poorly defined. In particular, it is unclear how mammary epithelial cells (MEC), a first barrier against pathogens, respond to infection. Indeed, it is difficult to differentiate between the contributions of MEC and infiltrating immune cells to gene expression profiles of mammary tissue during mastitis. The current investigation examines the response of MEC at the early stage of infection using a non invasive RNA sampling method taking advantage of the presence of cytoplasmic crescents contained in milk fat globules. We have recently shown that, in goats, Milk Fat Globules (MFG) provide a unique source of RNA to study the in vivo regulation of gene expression in MEC. This non invasive RNA sampling method was used during the time course of an experimental intra mammary infection (IMI) with S. aureus. Experiments were performed using ovine microarrays (Agilent) to compare gene expression patterns before infection, at 12h, 18h and 24h post-infection (PI). Furthermore, we combined this approach with laser capture microdissection of MEC isolated from frozen slides of mammary tissue to study some specific genes at the late stage of infection (30h PI). We show that at 18h PI, before the burst of somatic cells in milk, MEC play an important role in the recruitment and activation of inflammatory cells through the IL-8 signaling pathways. Then, at the late stage of infection (30h PI), the contribution of MEC in immune response changes to produce different acute phase proteins, including SAA3, serpin A1 and PTX3. These cells also express factors that contribute directly to fighting infection, such as S100A12. In summary, we demonstrate for the first time in vivo how MEC orchestrate innate immune response during an IMI with S. aureus in the goat species. We report here new opportunities to assess the dynamics of gene expression in the mammary gland, thus providing significant advances in the understanding of MEC immune capacity.M-bM-^@M-^BFurthermore, the production of some molecules by MEC, in the early stages of IMI, could provide sensitive biomarkers for early detection and therefore, treatment of mastitis. Experiments were performed using ovine microarrays (Agilent) to compare gene expression patterns before infection, at 12h, 18h and 24h post-infection (PI). 20 sample records; mono-colour experimental design

Project description:Mastitis is an inflammation of the mammary gland (MG), usually due to bacterial infection. Although considerable attention has been paid to this economically important disease, the early stages of the host response remain poorly defined. In particular, it is unclear how mammary epithelial cells (MEC), a first barrier against pathogens, respond to infection. Indeed, it is difficult to differentiate between the contributions of MEC and infiltrating immune cells to gene expression profiles of mammary tissue during mastitis. The current investigation examines the response of MEC at the early stage of infection using a non invasive RNA sampling method taking advantage of the presence of cytoplasmic crescents contained in milk fat globules. We have recently shown that, in goats, Milk Fat Globules (MFG) provide a unique source of RNA to study the in vivo regulation of gene expression in MEC. This non invasive RNA sampling method was used during the time course of an experimental intra mammary infection (IMI) with S. aureus. Experiments were performed using ovine microarrays (Agilent) to compare gene expression patterns before infection, at 12h, 18h and 24h post-infection (PI). Furthermore, we combined this approach with laser capture microdissection of MEC isolated from frozen slides of mammary tissue to study some specific genes at the late stage of infection (30h PI). We show that at 18h PI, before the burst of somatic cells in milk, MEC play an important role in the recruitment and activation of inflammatory cells through the IL-8 signaling pathways. Then, at the late stage of infection (30h PI), the contribution of MEC in immune response changes to produce different acute phase proteins, including SAA3, serpin A1 and PTX3. These cells also express factors that contribute directly to fighting infection, such as S100A12. In summary, we demonstrate for the first time in vivo how MEC orchestrate innate immune response during an IMI with S. aureus in the goat species. We report here new opportunities to assess the dynamics of gene expression in the mammary gland, thus providing significant advances in the understanding of MEC immune capacity. Furthermore, the production of some molecules by MEC, in the early stages of IMI, could provide sensitive biomarkers for early detection and therefore, treatment of mastitis.

Dataset Information

Biomarkers for Early Stages of Johne's Disease Infection and Immunization in Goats.

Publications

Biomarkers for Early Stages of Johne's Disease Infection and Immunization in Goats.

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