Unveiling the role of NK cells, NKT-like cells, and γδ cells in pathogenesis of type 1 reactions in leprosy.

Leprosy is a disease with spectral clinical manifestations along with two types of reactions, type 1 reaction (T1R) and type 2 reaction (T2R). T1R especially occurs because of the defensive upgradation of cell-mediated immunity (CMI) to M. leprae antigens. T1R is the main cause of disability in leprosy. The role of conventional adaptive T cells has been well studied to understand T1R. A comprehensive understanding of the role of unconventional T cells in the manifestation of inflammation during T1R is crucial and has not been studied. In our study, we found significantly higher plasma levels of TNFα, IL1ß, IL17, and IP10 in T1R when compared to non-reaction (NR). Gene expression for cytokines in blood circulation by qPCR showed significantly higher expression of IFNγ, IP10, TNFα, IL6, IL17A and chemokines CCL3, CCR1, CCR5, and CXCR3 in T1R as compared to NR. Frequencies of NKT-like cells (48.7 %) and NK cells (22.3 %) were found significantly higher in T1R in comparison to NR (36.9 %, 18.3 %, respectively) (p = 0.0001). Significantly lower levels of γδT cells (3.32 %) were observed in T1R in comparison to NR (5.16 %). The present study has provided evidence for the first time on the role of plausible unconventional T cells in the immunopathogenesis of T1R in leprosy.

Dominant negative biologics normalise the tumour necrosis factor (TNF-α) induced angiogenesis which exploits the Mycobacterium tuberculosis dissemination.

BACKGROUND: Tumor necrosis factor (TNF) is known to promote T cell migration and increase the expression of vascular endothelial growth factor (VEGF) and chemokines. The administration of Xpro-1595, a dominant-negative TNF (DN-TNF) engineered to selectively inactivate soluble TNF (solTNF), has been extensively studied and proven effective in reducing TNF production without suppressing innate immunity during infection. The literature also supports the involvement of glutamic acid-leucine-arginine (ELR+) chemokines and VEGF in angiogenesis and the spread of infections. MATERIALS AND METHODS: In this study, we administered Xpro-1595 to guinea pigs to selectively inhibit solTNF, aiming to assess its impact on Mycobacterium tuberculosis (M.tb) dissemination, bacterial growth attenuation, and immunological responses. We conducted immunohistochemical analyses, immunological assays, and colony enumeration to comprehensively study the effects of Xpro-1595 by comparing with anti-TB drugs treated M.tb infected guinea pigs. Throughout the infection and treatment period, we measured the levels of Interleukin-12 subunit alpha (IL-12), Interferon-gamma (IFN-γ), TNF, Tumor growth factor (TGF), and T lymphocytes using ELISA. RESULTS: Our findings revealed a reduction in M.tb dissemination and inflammation without compromising the immune response during Xpro-1595 treatment. Notably, Xpro-1595 therapy effectively regulated the expression of VEGFA and ELR + chemokines, which emerged as key factors contributing to infection dissemination. Furthermore, this treatment influenced the migration of CD4 T cells in the early stages of infection, subsequently leading to a reduced T cell response and controlled proinflammatory signalling, thus mitigating inflammation. CONCLUSION: Our study underscores the pivotal role of solTNF in the dissemination of M.tb to other organs. This preliminary investigation sheds light on the involvement of solTNF in the mechanisms underlying M.tb dissemination, although further in-depth research is warranted to fully elucidate its role in this process.

Immuno-reactivity evaluation of Mce-truncated subunit candidate vaccine against Mycobacterium avium subspecies paratuberculosis challenge in the goat models.

BACKGROUND: Detection of an appropriate antigen with high immunogenicity can be a big step in the production of an effective vaccine for control of Johne's disease (JD). The aim of this study was to evaluate the efficacy of Mce-truncated protein as a subunit vaccine candidate for the control of JD in experimentally challenged goats. MATERIALS AND METHODS: Six healthy goat kids were immunized with Mce-truncated protein, and two goats were kept as controls. All kids were twice challenged orally with live Mycobacterium avium subspecies paratuberculosis(MAP) strain and half the goats from both the categories were sacrificed at 7 and 10 months after start of challenge study. Culture of MAP was performed from all the necropsied tissues to determine the true JD infection status. RESULTS: Mce-truncated protein only reacted with pooled vaccinated goat sera in western-blot. A significant increase in humoral immune response against Mce protein was also observed in vaccinated goats. Compared to the control group, vaccinated goats gained higher body weights and none of them shed MAP or showed histopatological lesions or colonization of MAP in their necropsy tissues. CONCLUSIONS: The new Mce protein based vaccine provided significant immunity in goats as they could meet the challenge with live MAP bacilli. Although the vaccine used in this study showed the high potential as a new effective vaccine for the control of JD, further validation study is still required to successfully implement the vaccine for JD control program.

Bacterial dissemination in Mycobacterium tuberculosis by CD+ T-cells & proinflammatory cytokines.

Background & objectives: As CD4+ and CD8+ T lymphocyte numbers decline, the conventional, localized forms of tuberculosis shift to the atypical, disseminated forms. Variations in lymphocyte and immune cell expression levels affect how tuberculosis manifests in disseminated forms. Understanding the relationship between lymphocyte counts (CD4+ and CD8+) and pro-inflammatory cytokines such as tumour necrosis factor-alpha, interleukin-12 and interferon, we may therefore be able to shed light on how infections spread and suggest potential biomarkers for these immune factors. Methods: In this study, 15 guinea pigs were infected with Mycobacterium tuberculosis (M.tb) H37Rv strain and grouped into three groups of five each for further investigation. Serum samples and bronchoalveolar lavage (BAL) fluid were examined for the expression of pro-inflammatory cytokines and T-cell subsets in guinea pigs infected with pulmonary tuberculosis and disseminated tuberculosis. Results: We found that M.tb escapes macrophages due to pro-inflammatory cytokine dysregulation. Despite the protective immunity created by T-cells and cytokines, M.tb bacilli may spread to other organs due to inflammation induced by these immune components. A high number of T-cells and stimulated cytokine production are involved in triggering inflammation after necrotic tissue develops and tuberculosis spreads. Interpretation & conclusions: Our findings imply that increased bacilli in the spleen at the 8th wk of infection may be caused by the overexpression of CD4+ T-cell lymphocyte subsets and cytokines that generated inflammation during the 4th wk of infection. This is a pilot study with a small sample size and less assertive inference. Larger studies would be helpful to validate the results of the present investigation.

Bioprospective Role of Ocimum sanctum and Solanum xanthocarpum against Emerging Pathogen: Mycobacterium avium Subspecies paratuberculosis: A Review.

Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic, contagious, and typically life-threatening enteric disease of ruminants caused by a bacterium of the genus Mycobacterium, but it can also affect non-ruminant animals. MAP transmission occurs through the fecal-oral pathway in neonates and young animals. After infection, animals generate IL-4, IL-5, and IL-10, resulting in a Th2 response. Early detection of the disease is necessary to avoid its spread. Many detection methods, viz., staining, culture, and molecular methods, are available, and numerous vaccines and anti-tuberculosis drugs are used to control the disease. However, the prolonged use of anti-tuberculosis drugs leads to the development of resistance. Whereas vaccines hamper the differentiation between infected and vaccinated animals in an endemic herd. This leads to the identification of plant-based bioactive compounds to treat the disease. Bioactive compounds of Ocimum sanctum and Solanum xanthocarpum have been evaluated for their anti-MAP activity. Based on the MIC50 values, Ursolic acid (12 µg/mL) and Solasodine (60 µg/mL) were found to be suitable for anti-MAP activity.

Computational Analysis to Predict Drug Targets for the Therapeutic Management of Mycobacterium avium sub. Paratuberculosis.

BACKGROUND: Mycobacterium avium sp. paratuberculosis (MAP) is a pathogen, which causes paratuberculosis in animals; it has also been found to be associated with a number of autoimmune disorders in humans. The emergence of drug resistance has also been found in this bacillus during disease management. OBJECTIVE: The present study's focus was to identify potential therapeutic targets for the therapeutic management of Mycobacterium avium sp. paratuberculosis infection by in silico analysis. METHODS: Differentially-expressed genes (DEGs) can be good drug targets, which can be identified from microarray studies. We used gene expression profile GSE43645 to identify differentiallyexpressed genes. An integrated network of upregulated DEGs was constructed with the STRING database and the constructed network was analyzed and visualized by Cytoscape. Clusters in the proteinprotein interaction (PPI) network were identified by the Cytoscape app ClusterViz. MAP proteins predicted in clusters were analyzed for their non-homology with the human proteins, and homologous proteins were excluded. Essential proteins and cellular localization analysis and the physicochemical characteristics prediction were also done. Finally, the druggability of the target proteins and drugs that can block the targets was predicted using the DrugBank database and confirmed by molecular docking. Structural prediction and verification of drug target proteins were also carried out. RESULTS: Two drug targets, MAP_1210 (inhA) and MAP_3961 (aceA), encoding enoyl acyl carrier protein reductase and isocitrate lyase enzymes, respectively, were finally predicted as potential drug targets. CONCLUSION: Both of these proteins have been predicted as drug targets in other mycobacterial species also, supporting our results. However, further experiments are required to confirm these results.

Atorvastatin Potentially Reduces Mycobacterial Severity through Its Action on Lipoarabinomannan and Drug Permeability in Granulomas.

The majority of preclinical research has shown that Mycobacterium tuberculosis can modify host lipids in various ways. To boost its intramacrophage survival, M. tuberculosis causes host lipids to build up, resulting in the development of lipid-laden foam cells. M. tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration. Here, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on the bacterial burden by increasing the drug concentration at the infection site. We looked into how atorvastatin could be used in conjunction with first-line drugs to promote drug permeation. In this study, we detected an accumulation of drugs at the peripheral sites of the lungs and impaired drug distribution to the diseased sites. The efficacy of antituberculosis drugs, with atorvastatin as an adjunct, on the viability of M. tuberculosis cells was demonstrated. A nontoxic statin dosage established phenotypic and normal granuloma vasculature and showed an additive effect with rifampicin and isoniazid. Our data show that statins help to reduce the tuberculosis bacterial burden. Our findings reveal that the bacterial load is connected with impaired drug permeability resulting from lipid accumulation in the bacterial cell wall. Statin therapy combined with antituberculosis medications have the potential to improve treatment in tuberculosis patients. IMPORTANCE Mycobacterium tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. M. tuberculosis limits phagosomal maturation and activation without engaging in phagocytosis. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in the vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration, which can be increased through a reduction in cholesterol and vascularity. Herein, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on bacterial burden through increasing the drug concentration at the infection site. Our main research goal is to diminish mycobacterial dissemination and attenuate bacterial growth by increasing drug permeability.

Toll-like receptor 2 (-196 to -174) del and TLR1 743 A > G gene polymorphism-a possible association with drug-resistant tuberculosis in the north Indian population.

Objectives: The objective of this study is to analyze the association between TLR2 deletion (-196 to -174) and TLR1 743 A > G gene polymorphism with drug resistant tuberculosis (PTB, MDR-TB, and XDR-TB) in a population from Agra, Uttar Pradesh. Methods: The present case-control study included 101 pulmonary TB patients, 104 multidrug-resistant TB patients, 48 extremely drug-resistant TB patients, and 130 healthy and unrelated controls residing in the same locality. The genotyping method for TLR2 deletion (-196 to -174) was carried out by allele-specific polymerase chain reaction (PCR), and TLR1 743 A > G gene polymorphism was performed by hybridization probe chemistry in Roche Real-Time PCR. Genotype and allele frequencies were analyzed by the chi-square test. Cytokine levels were measured by ELISA and compared using Mann-Whitney and Kruskal-Wallis tests. Results: The frequency of heterozygous (Ins/del) genotypes for TLR2 (-196 to -174) polymorphism was predominant in XDR-TB patients (0.57), whereas heterozygous A/G genotype for TLR1 743 A > G single nucleotide polymorphism (SNP) was predominant in healthy controls (0.57) for TLR1 743 A > G gene polymorphism. The heterozygous genotype of TLR2 deletion polymorphism was found to be significantly higher in XDR-TB (p = 0.0001). TLR1 743 A > G SNP, AG genotypes were found to be significantly associated with healthy controls than PTB (p = 0.047). The level of serum cytokines (IL-6, TNF-α, and IFN-γ) was also found to be significantly different among TB patients and healthy controls. Conclusion: The findings suggested that in the present population, the heterozygous (Ins/Del) genotype and deletion allele of TLR2 deletion (-196 to -174) polymorphism are associated with the risk for the development of drug-resistant TB. Furthermore, for TLR1 743 A > G gene polymorphism, A/G genotype, and G allele are found associated with healthy controls, suggesting the protective role against TB.

Mycobacterium bovis induced human tuberculosis in India: Current status, challenges & opportunities.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a leading cause of human deaths due to any infectious disease worldwide. However, infection of Mycobacterium bovis, primarily an animal pathogen, also leads to the development of 'human tuberculosis'. Infected animals have been considered the major source of M. bovis infection and humans get exposed to M. bovis through close contact with infected animals or consumption of contaminated milk, unpasteurized dairy products and improperly cooked contaminated meat. The information on the global distribution of bovine TB (bTB) is limited, but the disease has been reported from all the livestock-producing middle- and low-income countries of the world. In recent years, there is a renewed interest for the control of bTB to minimize human infection worldwide. In India, while the sporadic presence of M. bovis has been reported in domestic animals, animal-derived food products and human beings from different geographical regions of the country, the information on the national prevalence of bTB and transmission dynamics of zoonotic TB is, however, not available. The present article reviewed published information on the status of M. bovis-induced zoonotic TB to highlight the key challenges and opportunities for intervention to minimize the risk of M. bovis infection in humans and secure optimum animal productivity in India.

Corroboration of cross-reactivity between Mycobacterium leprae and hosts' salivary and cutaneous proteins: A hope for prognostic biomarkers for the pathogenesis of reactions in leprosy.

Introduction: Immunological reactions are frequent complications that may occur either before, during, or after treatment and affect 30-50% of leprosy patients. The presence of autoantibodies like rheumatoid factor, antinuclear factor, and antibodies to host collagen, keratin, actin, myosin, endothelial cells, and myelin basic protein (MBP) has been earlier reported in leprosy patients. The purpose of this study was to identify cross-reactive proteins in clinical samples such as saliva and slit skin scrapings (SSS) of leprosy patients which could be utilised as prognostic biomarkers for Type 1 Reaction (T1R) in leprosy. Method: A total of 10 leprosy patients in T1R and 5 healthy volunteers were recruited. The protein was extracted from their SSS and saliva samples, thereafter, isoelectric focusing (IEF) and two-dimensional PAGE were performed to analyse the proteins. Furthermore, the cross-reactivity was identified by western blotting host proteins in gel against purified IgG from Mycobacterium leprae soluble antigen (MLSA)- hyperimmunized rabbit sera, thereafter, cross-reactive proteins were identified by MS/MS. The cross-reactive host proteins were analysed for homologous bacterial proteins and B cell epitopes (BCEs) were predicted by using bioinformatic tools. Results: A total of five spots of salivary proteins namely S100-A9, 35.3 kDa, and 41.5 kDa proteins, Serpin peptidase inhibitor (clade A), Cystatin SA-III, and four spots of SSS namely 41.4 kDa protein, Alpha-1 antitrypsin, vimentin, and keratin 1, were identified as cross-reactive. Further, a total of 22 BCEs of cross-reactive host proteins were predicted and visualised. Discussion: This data provides strong evidence of cross-reactivity/molecular mimicry between host and pathogen in leprosy patients with reaction. These BCEs of cross-reactive proteins could be further studied to predict reactions and may be utilised as an early diagnostic biomarker for T1R in leprosy.

Exploration of Solanum xanthocarpum Schrad. & Wendl. against Mycobacterium avium Subspecies paratuberculosis and Assessment of Its Immunomodulatory and Anti-Inflammatory Potential.

Mycobacterium avium subspecies paratuberculosis (MAP), being a dairy-borne pathogen, resistant of pasteurization and other sterilization techniques, is a major cause for development of inflammatory bowel disorders such as Johne's disease (JD) in dairy animals and Crohn's Disease (CD) in humans, for which no therapy is available to date. In the absence of effective therapy or a vaccine, management of CD has been accomplished by removal of the affected intestines. However, usually, even after removal of 2/3 of the intestine, CD reoccurs. Hence, there exists a need to develop an alternative therapy for such infection. The potential of herbals remains unexplored against MAP and related infections. Therefore, the conducted study is a novel initiative for the evaluation of anti-mycobacterial activity of bioactive extracts of Solanum xanthocarpum Schrad. & Wendl. against MAP infection. The said plant was authenticated according to the Ayurvedic Pharmacopoeia of India. Qualitative and quantitative evaluation of the extracts were done using chromatographic and spectroscopic techniques. Preliminary in vitro pharmacological assessments revealed the immunomodulatory and anti-inflammatory potential of the extracts. REMA assay was conducted to determine their anti-MAP activity along with determination of the best active extract. The hydro-alcoholic extract showed the best inhibition of MAP, providing a potential ray of hope against this emerging major pathogen of animals, and associated with Crohn's disease and other autoimmune disorders in human beings.

Stimulated expression of ELR+ chemokines, VEGFA and TNF-AIP3 promote mycobacterial dissemination in extrapulmonary tuberculosis patients and Cavia porcellus model of tuberculosis.

Pathogenic mycobacteria induce and accelerate blood vessel formation driven by extensive inflammation during granuloma formation, which is a central feature of mycobacterial pathogenesis. Tumor necrosis factor-alpha (TNF-α) enhances the expression of vascular endothelial growth factor (VEGF) and glutamic acid-leucine-arginine (ELR+) chemokines, which are potent inducers of vascularization. Most of the reported research work contends that VEGF growth factor induces neovascularization in human tuberculosis (TB) patients, but the evidence is inconclusive. Considerable ambiguity exists concerning the factors responsible for miliary tuberculosis. To identify such factors, we proposed an alternative explanation that could be found in miliary tuberculosis (MTB) cases. We performed a comparative analysis of angiogenic factors TNF-α, VEGF, and angiogenic ELR+ CXC and CC chemokine ligands in extrapulmonary tuberculosis (EPTB) and pulmonary tuberculosis (PTB) patients. To observe the relationship of these factors with the severity of bacterial burden, guinea pigs were infected with Mycobacterium tuberculosis (M.tb) and levels of the angiogenic factors were examined at different time intervals. Expression of these factors also exhibited a significant positive correlation with bacterial burden in other organs like the spleen, liver, and lymph nodes. We demonstrated statistical data on bacterial burden at different time points following the dissemination of infection in guinea pigs. In this study, we observed that there was a stimulated increase in the expression of ELR+ chemokines and VEGF in EPTB patients as compared to PTB patients. Following increased dissemination, the host immune response clears bacteria from the lungs during disease progression in guinea pigs.

Application of Bayesian modeling for diagnostic assays of Mycobacterium avium subsp. paratuberculosis in sheep and goats flocks.

BACKGROUND: This study aimed to screen the sera of goats and sheep from flocks suspected of Mycobacterium avium subsp. paratuberculosis (MAP) infection by a newly standardized Mce-truncated ELISA (Mt-ELISA) kit for the detection of antibodies against MAP. Four diagnostic applied tests were evaluated including Indigenous plate-ELISA (IP-ELISA), Mt-ELISA, fecal Polymerase Chain Reaction (f-PCR) and fecal culture (FC). MATERIALS AND METHODS: Assuming the absence of a gold standard, latent-class models in a Bayesian framework were used to estimate the diagnostic accuracy of the four tests for MAP. RESULTS: Mt-ELISA had higher Sensitivity (Se) in sheep (posterior median: 0.68 (95% Probability Interval (PI): 0.43-0.95), while IP-ELISA recorded the highest Se in goats as 0.83 (95% PI, 0.61-0.97). The f-PCR Se estimate slightly differed between species [sheep 0.36 (0.19-0.58), goats 0.19 (0.08-0.35)], while the Se of FC was similar between species [sheep 0.29 (0.15-0.51), goats 0.27 (0.13-0.45)]. The specificity estimates for all tests were high, close to unity, and similar between species. CONCLUSION: Overall, the results showed that the Mt-ELISA method can be used for MAP detection in small ruminants' flocks.

Ursolic Acid and Solasodine as Potent Anti-Mycobacterial Agents for Combating Paratuberculosis: An Anti-Inflammatory and In Silico Analysis.

Mycobacterium avium subspecies paratuberculosis (MAP) infection in domestic livestock causes persistent diarrhea, weight loss, and death and is also a potential cause of Crohn's disease (CD) in humans; notably, treatments against MAP are insufficient, costly, and can cause adverse reactions. Hence, plant-derived bioactive constituents have been taken into consideration in this regard. Herein, we present the results of two bioactive constituents (Solasodine and Ursolic acid) that were evaluated for their safety and efficacy against MAP protein (Dephospho-Coenzyme A kinase (DPCK) by utilizing in vitro assays and different tools of in silico biology. The ADME/t-test, the drug-likeness property test, pharmacophore modelling, and PASS prediction have proven that both the constituents have better binding capacities than the available antibiotic drugs used to target protein inhibition pathways. Through our observations, it can be inferred that these two phytochemicals can be adequately used to treat paratuberculosis, thereby combating inflammatory bowel disorders (IBD) of an autoimmune nature.

Association of genetic variability in CD209 gene with bovine paratuberculosis disease: a case-control study in the Indian cattle population.

This study aimed to explore the association of single nucleotide polymorphisms (SNPs) in CD209 gene with the occurrence of bovine paratuberculosis (PTB) disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) in Indian cattle. A total of 213 animals were preliminarily selected on the basis of physical body condition score, which was then screened by a panel of diagnostic tests viz. Johnin, ELISA, fecal microscopy, and fecal culture, for the establishment of a case-control resource population. A total of four SNPs viz. rs208222804, rs211654540, rs208814257, and rs210748127 in CD209 gene were genotyped by PCR-RFLP. All SNPs, except rs210748127, were polymorphic in our population. Genotypic-phenotypic associations were assessed by the PROCLOGISTIC procedure of SAS 9.3. The SNP rs208814257 yielded three genotypes viz. CC, CG, and GG, which were significantly (p < 0.05) different in case as compared to the control population. The odds of CC and CG in comparison to GG genotype were 1.21 and 0.40, respectively. The CG genotype was significantly higher in control population, indicating that this genotype may provide resistance against PTB in our resource population. Upon validation in an independent, larger test population and following biological characterization, SNP rs208814257 can be incorporated in marker panel for selection of animals with greater resistance to MAP infection.

Mimicking B and T cell epitopes between Mycobacterium leprae and host as predictive biomarkers in type 1 reaction in leprosy.

Several Mycobacterial infections including leprosy and tuberculosis are known to evoke autoimmune responses by modulating homeostatic mechanism of the host. Presence of autoantibodies like, rheumatoid factor, anti-nuclear factor and antibodies to host, collagen, keratin, myelin basic protein (MBP) and myosin, have been earlier reported in leprosy patients. In the present study, we detected the role of mimicking epitopes between Mycobacterium leprae and host components in the induction of autoimmune response in leprosy. Based on our previous findings, we predicted and synthesized a total of 15 mimicking linear B cell epitopes (BCE) and 9 mimicking linear T cell epitopes (TCE) of keratin and MBP. Humoral and cell-mediated immune responses against these epitopes were investigated in Non-reaction (NR), Type 1 reaction (T1R) leprosy patients, and healthy controls. We observed significantly higher levels of antibodies against 8 BCE in T1R in comparison to NR leprosy patients. Further, we also found 5 TCE significantly associated with lymphocyte proliferation in the T1R group. Our results indicated that these epitopes play a key role in the induction of autoimmune response in leprosy and are also strongly associated with the inflammatory episodes of T1R. Conclusively, these molecules may be employed as a biomarker to predict the inflammatory episodes of T1R.

In vivo kinetics of peripheral cellular immune responses in Mycobacterium avium subspecies paratuberculosis (MAP) infected and vaccinated goats.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (ParaTB) also known as Johne's disease (JD) in ruminants, which is characterized by chronic intestinal inflammation. A similar counterpart has been observed in the form of Crohn's disease in humans. The present study is the first trail in goats to understand the peripheral cellular immune responses following experimental MAP infection and vaccination. Fifteen apparently healthy male kids (3-6 months old) of Barbari breed were included in this study. In the experimental study, 5 kids were infected with 'S 5' strain of MAP ("Indian Bison Type"), 5 were vaccinated (Indigenous Vaccine) against MAP infection (Singh et al., 2007) and the remaining 5 kids were uninfected and non-vaccinated controls. Kids were observed for a period of 180 days post exposure (infection and vaccination) and were tested for development of infection. Cellular immune responses (in blood) were recorded post-exposure by three assays. We measured the frequencies of CD4 and CD8T cells, estimated plasma IFNγ and TNα and in the third assay, in vitro cytokine production by peripheral blood mononuclear cells (PBMCs) from vaccinated, infected and controls were examined in response to polyclonal stimulation. The frequencies of peripheral CD4 and CD8T cells were comparable in control, infected and vaccinated animals except around day 49 post-infection where MAP infected animals showed a trend towards significantly reduced frequencies of CD4 T cells compared to apparently healthy controls. Significantly reduced plasma TNFα levels were also observed in infected animals compared to vaccinated animals,during the course of infection. Diminished levels (although non significant) of TNFα were observed in the supernatants from polyclonally stimulated PBMCs at around day 49 post infection. It is conceivable that the diminished cellular immune responses may coincide with an impairment (immune exhaustion) of perhaps antigen-specific CD4T cells that might, in the course of infection, contribute to the progressive nature of caprine paratuberculosis.

Therapeutic management of Mycobacterium avium subspecies paratuberculosis infection with complete resolution of symptoms and disease in a patient with advanced inflammatory bowel syndrome.

BACKGROUND: A 26-year-old male had a history of frequent bowel movements, mushy stool with mucus and loss of 25 kg body weight in 6 months was diagnosed as a case of inflammatory bowel disease (IBD). The patient did not respond to routine and standard treatment for IBD. His condition was steadily deteriorating, and he was in a very precarious state when he reported to us. METHODS: Upon laboratory investigation by using IS900 specific PCR [which is specific for Mycobacterium avium subspecies paratuberculosis (MAP)], the blood and stool samples were found negative. However, the presence of low titer MAP-antibodies by indigenous ELISA were found followed by detection of the typical acid-fast MAP bacilli (with 3 + or 4 + grade) microscopically. The MAP stool culture was positive after 6 months incubation. The biotyping by IS1311 specific polymerase chain reaction restriction enzyme (PCR-RE) confirmed infection with 'Indian Bison Type Genotype', a dominant biotype infecting the domestic livestock population of India. Standard anti-MAP therapy was initiated under supervision of the treating physician. The drug of choice in prescribed treatment regimen included Isoniazid (5 mg/kg), Rifampicin (10 mg/kg), Ethambutol (15-25 mg/kg) once a day for 24 weeks and Clarithromycin (250 mg)/Levofloxacin (250 mg) twice a day for 6 weeks. RESULTS: Following treatment, the patient started improving progressively with reduction in bowel movement frequency and gained body weight with an enhanced appetite propensity. Upon follow-up of the patient after 1 year of treatment, stool-microscopy and stool-culture were found negative for MAP. Till the recent past, the patient was further monitored for disease relapse, if any. CONCLUSIONS: This patient has experienced a complete resolution of IBD using a combination of anti-MAP antibiotics. The initial detection of heavy shedding of acid-fast MAP bacilli and typical colony morphology with its characterization obtained from culturing of stool sample indicated the infection of MAP. Interestingly, the present case is one more example of the linkage of demonstrable MAP infection treated with anti-MAP therapy in the presence and then absence of disease in the human host.

Bio-typing of Mycobacterium avium subspecies paratuberculosis isolates recovered from the Himalayan sheep and goats.

Information on bio-type profile of Mycobacterium avium subspecies paratubeculosis (MAP) in sheep flocks and goat herds of Himalayan region is not reported earlier. The aim of our study was to determine the bio-type of MAP infecting livestock of this region. A total of 71 faecal samples (sheep-57, goats-14) were screened by Ziehl-Neelsen (ZN) staining and IS900 PCR, and then processed for culture on Herrold's egg yolk medium (HEYM) having mycobactin J (MJ). Out of 71 faecal samples, MAP colonies were seen only in four samples (sheep-3 and goat-1). Isolates were confirmed as MAP on the basis of slow growth, acid fastness, MJ dependency, IS900 and IS1311 PCR. All the IS900 and IS1311 PCR positive samples were bio-typed by IS1311 PCR-REA (restriction endonuclease analysis), which confirmed all four isolates as 'bison type.' In IS1311 based phylogeny of MAP isolates by ClustalW method of the MegAlign program of DNASTAR Lasergene software, the four sequences of MAP isolates (NCBI sequence nos. MH988763, MH988765, MH988766 and MH988764) did not show any distinct clustering/grouping pattern. However, these four isolates showed a bit of closeness to the MAP sequences (KC990353.1 and KC990352.1) of 'bison type' isolated from wood bison in Canada. In conclusion, this is the first report on isolation and bio-type profile of MAP infecting sheep and goats of Himalayan region. Study will help in devising prevention and control strategies against spread of MAP infection in livestock population of Himalayan region.