Genome-wide epitope mapping across multiple host species reveals significant diversity in antibody responses to Coxiella burnetii vaccination and infection.

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next-generation Q fever vaccines and diagnostic tests.

Metabolomic changes in polyunsaturated fatty acids and eicosanoids as diagnostic biomarkers in Mycobacterium avium ssp. paratuberculosis (MAP)-inoculated Holstein-Friesian heifers.

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

Isotype-specific Antibody Responses to Mycobacterium avium paratuberculosis Antigens Are Associated With the Use of Biologic Therapy in Inflammatory Bowel Disease.

BACKGROUND: The role of Mycobacterium avium paratuberculosis [MAP] in inflammatory bowel disease [IBD], especially Crohn's disease [CD] is controversial due conflicting results and lack of reproducibility and standardised tests. The current study focuses on the role of MAP in disease progression and genetic susceptibility, as MAP is likely one of many factors involved in the complex pathogenesis of IBD, potentially affecting a subgroup depending on genetic susceptibility. METHODS: Serum from 812 patients was evaluated with seven immunoglobulin [Ig] isotype-specific serology tests assessing humoral response to three different MAP antigens. For each of these in total 21 tests, the intra-assay and inter-assay coefficients were used to evaluate test accuracy. Reliable assays were subsequently analysed in relation to disease characteristics and need for biologic therapy/surgery. Genome-wide genotyping was available for all participants. Genetic determinants of humoral response to MAP antigens were evaluated using genome-wide association analysis and polygenic risk scores [PRS]. RESULTS: High IgA or IgM response to MAP2609 was associated with increased use of biologic therapy in CD and ulcerative colitis [UC] [odds ratios 2.69; 95% confidence interval 1.44-5.01; and 2.60, 1.46-4.64, respectively]. No associations were seen for risk of surgery [p-values > 0.29]. We could not identify genetic determinants nor polygenic risk scores for MAP response with genome-wide significance. CONCLUSIONS: Extensive assays for serological response to MAP were evaluated using stringent criteria for reliability. Increased IgA and IgM response to MAP antigens was seen in patients exposed to biologic therapy, but no genetic determinants underlying this humoral response were found.

Application of Transcriptomics to Enhance Early Diagnostics of Mycobacterial Infections, with an Emphasis on Mycobacterium avium ssp. paratuberculosis.

Mycobacteria cause a wide variety of disease in human and animals. Species that infect ruminants include M. bovis and M. avium ssp. paratuberculosis (MAP). MAP is the causative agent of Johne's disease in ruminants, which is a chronic granulomatous enteric infection that leads to severe economic losses worldwide. Characteristic of MAP infection is the long, latent phase in which intermittent shedding can take place, while diagnostic tests are unable to reliably detect an infection in this stage. This leads to unnoticed dissemination within herds and the presence of many undetected, silent carriers, which makes the eradication of Johne's disease difficult. To improve the control of MAP infection, research is aimed at improving early diagnosis. Transcriptomic approaches can be applied to characterize host-pathogen interactions during infection, and to develop novel biomarkers using transcriptional profiles. Studies have focused on the identification of specific RNAs that are expressed in different infection stages, which will assist in the development and clinical implementation of early diagnostic tests.

Viable Coxiella burnetii Induces Differential Cytokine Responses in Chronic Q Fever Patients Compared to Heat-Killed Coxiella burnetii.

Cytokine responses of chronic Q fever patients to the intracellular bacterium Coxiella burnetii have mostly been studied using ex vivo stimulation of immune cells with heat-killed C. burnetii due to the extensive measures needed to work with viable biosafety level 3 agents. Whether research with heat-killed C. burnetii can be translated to immune responses to viable C. burnetii is imperative for the interpretation of previous and future studies with heat-killed C. burnetii Peripheral blood mononuclear cells (PBMCs) of chronic Q fever patients (n = 10) and healthy controls (n = 10) were stimulated with heat-killed or viable C. burnetii of two strains, Nine Mile and the Dutch outbreak strain 3262, for 24 h, 48 h, and 7 days in the absence or presence of serum containing anti-C. burnetii antibodies. When stimulated with viable C. burnetii, PBMCs of chronic Q fever patients and controls produced fewer proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha, and IL-1ß) after 24 h than after stimulation with heat-killed C. burnetii In the presence of Q fever seronegative serum, IL-10 production was higher after stimulation with viable rather than heat-killed C. burnetii; however, when incubating with anti-C. burnetii antibody serum, the effect on IL-10 production was reduced. Levels of adaptive, merely T-cell-derived cytokine (gamma interferon, IL-17, and IL-22) and CXCL9 production were not different between heat-killed and viable C. burnetii stimulatory conditions. Results from previous and future research with heat-killed C. burnetii should be interpreted with caution for innate cytokines, but heat-killed C. burnetii-induced adaptive cytokine production is representative of stimulation with viable bacteria.

The within host dynamics of Mycobacterium avium ssp. paratuberculosis infection in cattle: where time and place matter.

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

Cytokine gene expression profiles of bovine dendritic cells after interaction with Mycobacterium avium ssp. paratuberculosis (M.a.p.), Escherichia coli (E. coli) or recombinant M.a.p. heat shock protein 70.

Mycobacterium avium paratuberculosis (M.a.p.) resides and replicates in macrophages. Many of the of immune mechanisms aiding M.a.p. survival in the host's cells are known. However, little is known about interactions of M.a.p. with dendritic cells (DC). As DC are important for the induction of protective immunity against infectious diseases, we investigated the interaction of M.a.p. with these cells. Quantitative real-time PCR (RT-PCR) was used to analyse differential expression of cytokine genes after 6 h and 24 h of incubation by immature DC that phagocytosed either M.a.p. or Escherichia coli (E. coli). We hypothesized that phagocytosis of E. coli would induce pro-inflammatory cytokines due to abundant presence of lipopolysaccharide (LPS) and that the cytokine expression profile induced by phagocytosis of live M.a.p. would differ. In addition we hypothesized that incubation of immature DC with rHsp70, an immunodominant antigen of M.a.p., would induce a similar profile of cytokine gene expression as phagocytosis of intact M.a.p. However, phagocytosis of both E. coli and M.a.p. resulted in a cytokine gene expression pattern representative of a (pro-)inflammatory reaction, dominated by strong induction of IL-12 gene expression, that was higher after 24 h than after 6 h of incubation, although the response to M.a.p. was less vigorous than to E. coli. Incubation with rHsp70 resulted in a more inhibitory type of cytokine gene expression, with delayed IL-12 gene expression and downregulation of the genes for IL-1beta and IL-6 after 24 h of incubation. We conclude that bovine DC produce an immuno-stimulatory, anti-mycobacterial response to infection with M.a.p., while Hsp70 potentially contributes to pathogen virulence by allowing the bacteria to invade the host cell.