Ratna B. Gurung

In silico identification of epitopes in Mycobacterium avium subsp. paratuberculosis proteins that were upregulated under stress conditions.

ABSTRACT Johnes disease in ruminants is caused by Mycobacterium avium subsp. paratuberculosis. Diagnosis of M. avium subsp. paratuberculosis infection is difficult, especially in the early stages. To date, ideal antigen candidates are not available for efficient immunization or immunodiagnosis. This study reports the in silico selection and subsequent analysis of epitopes of M. avium subsp. paratuberculosis proteins that were found to be upregulated under stress conditions as a means to identify immunogenic candidate proteins. Previous studies have reported differential regulation of proteins when M. avium subsp. paratuberculosis is exposed to stressors which induce a response similar to dormancy. Dormancy may be involved in evading host defense mechanisms, and the host may also mount an immune response against these proteins. Twenty-five M. avium subsp. paratuberculosis proteins that were previously identified as being upregulated under in vitro stress conditions were analyzed for B and T cell epitopes by use of the prediction tools at the Immune Epitope Database and Analysis Resource. Major histocompatibility complex class I T cell epitopes were predicted using an artificial neural network method, and class II T cell epitopes were predicted using the consensus method. Conformational B cell epitopes were predicted from the relevant three-dimensional structure template for each protein. Based on the greatest number of predicted epitopes, eight proteins (MAP2698c [encoded by desA2], MAP2312c [encoded by fadE19], MAP3651c [encoded by fadE3_2], MAP2872c [encoded by fabG5_2], MAP3523c [encoded by oxcA], MAP0187c [encoded by sodA], and the hypothetical proteins MAP3567 and MAP1168c) were identified as potential candidates for study of antibody- and cell-mediated immune responses within infected hosts.

In silico screened Mycobacterium avium subsp. paratuberculosis (MAP) recombinant proteins upregulated under stress conditions are immunogenic in sheep

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johnes disease (JD) in ruminants. MAP is known to enter a dormant phase outside the host, typically on soil. In vitro experiments have reported regulation of certain MAP genes when exposed to stressors similar to what is thought to produce dormancy. It is believed that in vivo regulation of dormancy genes and associated proteins by MAP may play a role in evading the host defence mechanisms and induce the host immune response against these dormancy-related proteins. Five proteins encoded by dormancy-related genes that were previously found to be upregulated under stress conditions and predicted through in silico analysis to possess immune epitopes (three hypothetical proteins and two proteins involved in fatty acid metabolism) were selected. Recombinant proteins were produced, purified and evaluated by indirect enzyme-linked immunosorbent assay (ELISA) for immunogenicity using a panel of sera obtained from sheep unexposed and exposed to MAP. The antibody levels of the exposed group were significantly higher than the unexposed group (P<0.001). Individually, the five proteins were found to discriminate between sera from sheep exposed to MAP compared to unexposed sheep. At 91% diagnostic specificity, the diagnostic sensitivity of the recombinant antigen ELISA ranged from 24% to 42% and AUC(ROC) from 0.7015 to 0.8405.

Cellular and humoral immune responses in sheep vaccinated with candidate antigens MAP2698c and MAP3567 from Mycobacterium avium subspecies paratuberculosis

Control of Johnes disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP) in ruminants using commercially available vaccine reduces production losses, mortality, fecal shedding and histopathological lesions but does not provide complete protection from infection and interferes with serological diagnosis of Johnes disease and bovine tuberculosis. At this time no recombinant antigens have been found to provide superior protection compared to whole killed or live-attenuated MAP vaccines. Therefore, there is a need to evaluate more candidate MAP antigens. In this study recombinant MAP antigens MAP2698c and MAP3567 were formulated with four different MONTANIDE™ (ISA 50V2, 61VG, 71VG, and 201VG) adjuvants and evaluated for their ability to produce specific immune responses in vaccinated sheep. The cellular immune response was measured with an interferon-gamma (IFN-γ) release assay and the humoral immune response was measured by antibody detection enzyme linked immunosorbent assay. Recombinant vaccine formulation with the antigen MAP2698c and MONTANIDE™ ISA 201VG adjuvant produced strong whole-MAP as well as MAP2698c-specific IFN-γ responses in a high proportion of the vaccinated sheep. The formulation caused less severe injection site lesions in comparison to other formulations. The findings from this study suggest that the MAP2698c + 201VG should be evaluated in a challenge trial to determine the efficacy of this vaccine candidate.

Lymphoproliferative and Gamma Interferon Responses to Stress-Regulated Mycobacterium avium subsp. paratuberculosis Recombinant Proteins

ABSTRACT Johnes disease in ruminants is a chronic infection of the intestines caused by Mycobacterium avium subsp. paratuberculosis. An important strategy to control disease is early detection, and a potentially efficient method for early detection is measurement of cell-mediated immune responses developed by the host in response to exposure or infection. One method is to measure lymphoproliferation and cytokine release from the host cells when exposed to the organism or parts of the organism. In this study, 10 recombinant M. avium subsp. paratuberculosis proteins known to be upregulated under in vitro stress conditions were evaluated by examining their ability to evoke memory as a result of exposure by vaccination or oral challenge with live Mycobacterium avium subsp. paratuberculosis. Out of 10 proteins, MAP2698c was found to induce higher cell-mediated immune responses in vaccinated and challenged sheep in comparison to healthy controls. The findings suggest that not all stress-regulated proteins have the diagnostic potential to detect cell-mediated immune responses in ovine paratuberculosis.

Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

ABSTRACT Mycobacterium avium subsp. paratuberculosis causes Johnes disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced.

Immunoreactivity of protein tyrosine phosphatase A (PtpA) in sera from sheep infected with Mycobacterium avium subspecies paratuberculosis

Evasion of host defense mechanisms and survival inside infected host macrophages are features of pathogenic mycobacteria including Mycobacterium avium subspecies paratuberculosis, the causative agent of Johnes disease in ruminants. Protein tyrosine phosphatase A (PtpA) has been identified as a secreted protein critical for survival of mycobacteria within infected macrophages. The host may mount an immune response to such secreted proteins. In this study, the humoral immune response to purified recombinant M. avium subsp. paratuberculosis PtpA was investigated using sera from a cohort of sheep infected with M. avium subsp. paratuberculosis and compared with uninfected healthy controls. A significantly higher level of reactivity to PtpA was observed in sera collected from M. avium subspecies paratuberculosis infected sheep when compared to those from uninfected healthy controls. PtpA could be a potential candidate antigen for detection of humoral immune responses in sheep infected with M. avium subspecies paratuberculosis.

Immunopathological changes and apparent recovery from infection revealed in cattle in an experimental model of Johne’s disease using a lyophilised culture of Mycobacterium avium subspecies paratuberculosis

Johnes disease (JD) or paratuberculosis is an economically significant, chronic enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Experimental models of JD in cattle are logistically challenging due to the need for long term monitoring, because the clinical disease can take years to manifest. Three trials were undertaken, the largest involving 20 cattle exposed orally to a low dose of C strain MAP and 10 controls studied for 4.75 years. Frequent blood and faecal sampling was used to monitor immunological and infection parameters, and intestinal biopsies were performed at two time points during the subclinical disease phase. Although clinical disease was not seen, there was evidence of infection in 35% of the animals and at necropsy 10% had histopathological lesions consistent with JD, similar to the proportions expected in naturally infected herds. Faecal shedding occurred in two distinct phases: firstly there was intermittent shedding <∼9 months post-exposure that did not correlate with disease outcomes; secondly, in a smaller cohort of animals, this was followed by more consistent shedding of increasing quantities of MAP, associated with intestinal pathology. There was evidence of regression of histopathological lesions in the ileum of one animal, which therefore had apparently recovered from the disease. Both cattle with histopathological lesions of paratuberculosis at necropsy had low MAP-specific interferon-gamma responses at 4 months post-exposure and later had consistently shed viable MAP; they also had the highest loads of MAP DNA in faeces 4.75 year s post-exposure. In a trial using a higher dose of MAP, a higher proportion of cattle developed paratuberculosis. The information derived from these trials provides greater understanding of the changes that occur during the course of paratuberculosis in cattle.

Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens

Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research.

A national serosurvey to determine the prevalence of paratuberculosis in cattle in Bhutan following detection of clinical cases

Abstract Johnes disease is an economically important ruminant disease predominantly affecting cattle, sheep and goats. The economic losses are due to early culling, reduced growth rate, progressive weight loss and reduced production. It is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Johnes disease was reported in cattle in Bhutan, based on clinical signs and histopathology; in the late 1990s samples from one mithun that was suspected to have died due to this disease was confirmed by molecular testing at the Faculty of Veterinary Science, University of Sydney, Australia. However, no detailed study on prevalence of JD has been attempted in Bhutan. Objective of this study was to conduct serosurveillance to determine the national prevalence of Johnes disease in cattle for the period 2013–2014 to provide the basis for planning a future control strategy. A national serosurvey was conducted wherein a two‐stage sampling procedure was used with 95% confidence and an error level of ±0.05. The sample size required for the survey was calculated using the software‐Survey Toolbox for Livestock Diseases, available as Epitools at http://www.ausvet.com.au. A total of 1123 serum samples were collected from an administrative structure of 52 villages, 40 sub‐districts and 15 districts. Serum samples were tested using commercially available antibody enzyme linked immunosorbent assay. Statistical analysis was performed using GraphPad Prism 5.0. Illustration such as maps was produced using QGIS version 2.18 ‘Las Palmas. The mean national apparent prevalence of Johnes disease was found to be 2.31 (26/1123) (95% CI: 0.80–4.50) with an estimated true prevalence was found to be 8.00 (95% CI: 2.00–17.00). Trongsa district had the highest prevalence (12.96) followed by Zhemgang (4.34), Lhuntse (4.25), Sarpang (3.89), Bumthang (3.60), Trashigang (2.67) and Haa (2.63). Prevalence for all other districts was 2.00 or below. Seropositive samples were reported from all over the country with varying levels of sero‐positivity. In the recent past many more cattle were imported from India to boost dairy production. Nevertheless, the wide distribution of seroreactive JD cattle all over the country is a concern for future control. Therefore, in future, a detailed study on the impact of cattle import with regard to disease incursion such as Johnes disease and other diseases should be undertaken.