Ashok Kumar Bhatia

Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock.

ABSTRACT Paratuberculosis (pTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP) in a wide variety of domestic and wild animals. Control of pTB is difficult due to the lack of sensitive, efficacious and cost-effective diagnostics and marker vaccines. Microscopy, culture, and PCR have been used for the screening of MAP infection in animals for quite a long time. Besides, giving variable sensitivity and specificity, these tests have not been considered ideal for large-scale screening of domestic livestock. Serological tests like ELISA easily detects anti-MAP antibodies. However, it cannot differentiate between the vaccinated and infected animals. Nanotechnology-based diagnostic tests are underway to improve the sensitivity and specificity. Newer generation diagnostic tests based on recombinant MAP secretory proteins would open new paradigm for the differentiation between infected and vaccinated animals and for early detection of the infection. Due to higher seroreactivity of secretory proteins vis-à-vis cellular proteins, the secretory proteins may be used as marker vaccine, which may aid in the control of pTB infection in animals. Secretory proteins can be potentially used to develop future diagnostics, surveillance and monitoring of the disease progression in animals and the marker vaccine for the control and eradication of pTB.

Immunomodulating property of Ocimum sanctum by regulating the IL-2 production and its mRNA expression using rat's splenocytes

Objective: To investigate the regulatory effect of aqueous extract of leaves of Ocimum sanctum on IL-2 cytokine production in vivo and in vitro, and the effect of leaves extract on general blood picture including T& B lymphocytes. Methods: For in vivo studies albino Wistar rats were treated with aqueous crude leaves extract of Ocimum sanctum for 20 consecutive days. Spleen cells were harvested and assayed for IL-2 production by using sandwich enzyme-linked immunosorbent assay (ELISA) and mRNA expression methods. For in vitro study aqueous Ocimum sanctum leaves extract= in different concentrations (25-500 滋g/mL) was added into culture plates containing ConA stimulated splenocytes. To study the overall effect on blood picture, density gradient purified lymphocytes analysis and conventional methodology for total and differential leukocyte count and hemoglobin level were also done. Results: It indicated that the rats treated with Ocimum sanctum leaves extract had significantly enhanced (P<0.001) ability of spleen cells to secrete IL-2. Investigation in vitro also showed regulation of IL-2 production. Blood study exhibited leucocytosis and augmentation of T& B lymphocytes by 25% approximately. 4-5% increase in Hemoglobin value was also noticed. Conclusion: Aqueous Ocimum sanctum leaves extract may have stimulatory effect on T & B lymphocytes particularly on Th1 subset of lymphocytes as shown by enhancement in IL-2 production.

Effect of Ocimum Sanctum on the Development of Protective Immunity against Salmonella typhimurium Infection through Cytokines

To investigate the protective role of Ocimum sanctum (O. sanctum) leaves against Salmonella typhimurium (S. typhimurium) infection in rats by inducing TNF-α, IFN-γ & IL-2 cytokines. Methods: Wistar albino rats were fed with aqueous extract of O. sanctum leaves using 250mg/kg body weight dose once a day for 20 consecutive days. Control rats were fed with placebo. Rats were infected with LD50 dose of S. typhimurium infection and monitored for their survival. Bacterial blood burden in both the groups was compared and numbers of activated peritoneal macrophages were counted. Concentration of TNF-α, IFN-γ and IL-2 cytokines in serum during different time intervals was assayed by sandwich ELISA. Results: Rats of control group showed a high mortality rate and had higher bacterial blood burden when compared with O. sanctum extract fed rats. There was a significant increase in the number of S. typhimurium engulfed peritoneal macrophages in the peritoneal fluid of O. sanctum fed animals. The protective control against bacterial infection in O. sanctum fed rats was associated with elevated level of TNF-α, IFN-γ and IL-2 cytokines in serum. Conclusions: These findings suggest that orally administered O. sanctum leaves extract effectively enhanced activation in macrophage and lymphocytes, depicted by the elevated serum concentration of TNF-α, IFN-γ and IL-2 cytokines, leading to induce a protective resistance against Salmonella typhimurium infection.

Green remediation. Tool for safe and sustainable environment: a review

Nowadays, the bioremediation of toxic pollutants is a subject of interest in terms of health issues and environmental cleaning. In the present review, an eco-friendly, cost-effective approach is discussed for the detoxification of environmental pollutants by the means of natural purifier, i.e., blue–green algae over the conventional methods. Industrial wastes having toxic pollutants are not able to eliminate completely by existing the conventional techniques; in fact, these methods can only change their form rather than the entire degradation. These pollutants have an adverse effect on aquatic life, such as fauna and flora, and finally harm human life directly or indirectly. Cyanobacterial approach for the removal of this contaminant is an efficient tool for sustainable development and pollution control. Cyanobacteria are the primary consumers of food chain which absorbed complex toxic compounds from environments and convert them to simple nontoxic compounds which finally protect higher food chain consumer and eliminate risk of pollution. In addition, these organisms have capability to solve secondary pollution, as they can remediate radioactive compound, petroleum waste and degrade toxins from pesticides.

Efficacy of T Regulatory Cells, Th17 Cells and the Associated Markers in Monitoring Tuberculosis Treatment Response

Treatment monitoring is an essential aspect for tuberculosis (TB) disease management. Sputum smear microscopy is the only available tool for monitoring, but it suffers from demerits. Therefore, we sought to evaluate markers and cellular subsets of T regulatory (Treg) cells and T helper (Th) 17 cells in pulmonary TB patients (PTB) for TB treatment monitoring. Peripheral blood mononuclear cells (PBMCs) were stimulated in vitro (with purified protein derivative (PPD)) overnight which was followed by a polychromatic flow cytometry approach to study Treg and Th17 markers and cellular subsets in PTB (n = 12) undergoing antituberculous treatment (ATT). The baseline levels of these markers and cellular subsets were evaluated in normal healthy subjects (NHS). We observed a significant decrease in the expression of CD25 (p<0.01) marker and percentage of T-cell subsets like CD4+CD25+ (p<0.001) and CD4+CD25+CD39+ (p<0.05) at the end of intensive phase (IP) as well as in the continuation phase (CP) of ATT. A decrease in CD25 marker expression and percentage of CD4+CD25+ T cell subset showed a positive correlation to sputum conversion both in high and low sputum positive PTB. In eight PTB with cavitary lesions, only CD4+CD25+FoxP3 Treg subset manifested a significant decrease at the end of CP. Thus, results of this study show that CD25 marker and CD4+CD25+ T cells can serve as better markers for monitoring TB treatment efficacy. The Treg subset CD4+CD25+FoxP3 may be useful for prediction of favorable response in PTB with extensive lung lesions. However, these findings have to be evaluated in a larger patient cohort.

Immunomodulatory activity of aqueous extract of Nyctanthes arbor-tristis flowers with particular reference to splenocytes proliferation and cytokines induction

Objectives: To investigate the immunomodulatory activity of aqueous extract of Nyctanthes arbor-tristis flowers (NAFE) with particular reference to splenocytes proliferation and induction of cytokines. Materials and Methods: Antibody titer was determined by tube agglutination and indirect ELISA assay in four groups of mice-control, antigen alone, and NAFE-treated (400 and 800 mg/kg for 21 days) after immunization with Salmonella antigen while cellular immunity was studied in three groups of rats (control and NAFE-treated - 400 and 800 mg/kg) following DNCB application. Splenocytes from untreated and NAFE-treated rats were stimulated using concanavalin-A (Con-A) and optical density (OD) and stimulation index were determined. Splenocytes from control rats were also treated in vitro with NAFE (50–1600 μg/ml) and Con-A to determine the effect on splenocytes proliferation. Interleukin-2 (IL-2) and IL-6 levels in splenocytes supernatant from control and NAFE-treated rats and following in vitro treatment of splenocytes with NAFE (50–1600 μg/ml) were determined using ELISA kits. Results: Marked to a significant increase in antibody titer by both the methods in NAFE-treated mice and a significant increase in skin thickness in rats after challenge with DNCB, respectively suggested humoral and cell-mediated immunostimulant potential of NAFE. Significant increase in OD and stimulation index following e x vivo and in vitro exposure of splenocytes and sensitization with Con-A and significant elevation in IL-2 and IL-6 levels in splenocytes supernantant was also observed after their ex vivo and in vitro exposure to NAFE. Conclusion: Humoral and cell-mediated immunostimulant activity of NAFE seems to be mediated through splenocytes proliferation and increased production of cytokines, especially IL-2 and IL-6.

Mycobacterium avium subspecies paratuberculosis – an important food borne pathogen of high public health significance with special reference to India: an update

ABSTRACT This review underlines the public health significance of ‘Indian Bison Type’ of Mycobacterium avium subspecies paratuberculosis (MAP) and also its potential as ‘zoonotic infection’. In the absence of control programs, bio-load of MAP is increasing and if we take total population of animals (500 million plus) and human beings (1.23 billion plus) into account, the number of infected animals and human beings will run into millions in India. Our research on screening of over 26,000 domestic livestock for MAP infection using 4 different diagnostic tests (microscopy, culture, ELISA and PCR), during last 31 years has shown that the average bio-load of MAP in the livestock population of India is very high (cattle 43%, buffaloes 36%, goats 23% and sheep 41%). ‘Mass screening’ of 28,291 human samples between 2008–2016 revealed also high bio-load of MAP. It has been proved that MAP is not in-activated during pasteurization and therefore live bacilli are continuously reaching human population by consumption of even pasteurized milk and other milk products. Live bacilli have also been recovered from meat products and the environment thus illustrating the potential of MAP as pathogen of public health concern. However, at present, there is inadequate scientific evidence to confirm a conclusive link between MAP infection and Johnes disease in ruminants and some cases of Crohns disease in human beings.

Antiepileptic Effect of Nux vomica, Homeopathic Remedy, Against Strychnine-Induced Seizers

Objective: To investigate the antiepileptic effect of homeopathic remedy Nux vomica on mice and its comparison with standard therapeutic diazepam. Methods: BALB-c mice were taken and divided into three groups comprising ten mice in each group. The first group was treated as control; the second group received standard therapeutics (diazepam, i.p.) and the third group received Nux vomica CH7. All groups were treated with strychnine intra peritoneally. Following parameters were observed; start time of convulsions, the number of animals had convulsions, and survival time until death. Results: Nux vomica CH7 homeopathic preparation was found effective in suspending onset of convulsions (P˂ 0.01), and extending survival time until death (P˂ 0.01) in comparison to control mice. It also increased percentage survival in comparison to control as well as diazepam treated animals. Conclusion: Our study demonstrated efficacy of Nux vomica in epilepsy management.

The role of T regulatory cell-associated markers in monitoring tuberculosis treatment completion and failure

Monitoring tuberculosis (TB) treatment success is crucial for clinical decision-making. The only available tool in this regard is sputum microscopy, but it has demerits. Moreover, in case of smear negatives and extrapulmonary TB, an efficient tool is still sought for. Therefore, we evaluated T regulatory cell (Treg)-associated markers (CD25, CD39, and FoxP3) and cellular subsets in monitoring treatment success in treatment-completed groups. Expression profile of various markers and subsets were compared real time among treatment-naive pulmonary TB patients (TN-PTB), followed-up treatment-completed (TC-fu) cohort, and a not followed-up (TC-nfu) cohort. Peripheral blood mononuclear cells from various groups were incubated overnight and were stained with antibodies for specific markers and studied by flow cytometry. In both the treatment-completed groups, a decline in frequencies of CD25+ marker and CD4+CD25+, CD4+CD25+FoxP3, CD4+CD25+CD39+ Treg was observed with clearance of infection, indicating their potential in monitoring treatment success. However, in the case of treatment failure patient (Tfp), a drastic increase in frequency of CD4+CD25+FoxP3+ Treg subset was found, indicating its usefulness in predicting treatment failure. Although the investigation unveils markers useful in predicting treatment success or failure, the findings from this study needs to be validated in a larger cohort.

Immunomodulating potential of Argemone mexicana through cytokine regulation

Biofilm formation on indwelling urinary catheters is a leading cause of Urinary tract infection (UTI). Presence of biofilm is associated with increased bacterial resistance to antimicrobial therapy and resultant treatment failure. The study detected a reliable method for diagnosis of biofilm formation by comparing scanning electron microscopy (SEM) and tissue culture plate method (TCP). The work was conducted on 20 urinary catheters from patients ranging from 1.5 to 85 years with catheters that remained in situ for a period of 3 to 20 days. Samples of catheters for culture and SEM and samples of urine were taken at the same time. The correlation between renal conditions and biofilm formation was not significant (p=0.336). No significant correlation (p =0.836, 0.163 respectively) was found between predisposing conditions (DM, renal insufficiency, diarrhea and impaired immunity) and development of Catheter associated urinary tract infection ( CAUTI )and biofilm formation. Biofilm formation increased with duration of catheter in situ, but no significant.correlation was found (p=0.095). This could be due to small number of specimens. 9/20(45%) urine samples, 12/20(60%) catheter samples were positive by culture and 14/20(70%) catheters showed biofilm on SEM. 4/12(33.33%) organisms isolated from catheter culture produced biofilm by TCP method. 9 isolates were recovered from 9 positive urine cultures. The microorganisms isolated were non Candida albicans (3/9), E. coli (2/9), C. albicans (2/9) and Acenitobacter (2/9). 14 isolates were recovered from 12 culture- positive catheters. The organisms isolated were E.- coli (3/14), non-Candida albicans (3/14), C. albicans (2/14), C tropicalis (2/14), Acenitobacter (2/14), Klebsiella (1/14) and Enterococcus (1/14). Reduction in microbial diversity with antimicrobial use was noticed but the correlation was insignificant (p=0.317). The correlation between urine culture results as well as catheter culture results and biofilm formation by SEM were both significant (p = 0.008 & 0.000 respectively). The correlation between urine culture and TCP assay was insignificant (p =0.237). Using SEM as the gold standard method for the detection of biofilm, the sensitivity, specificity; total accuracy, PPV & NPV of urine culture and catheter culture were, 64.30%, 100%, 75%, 100%, 54% & 85.70%, 100%, 90%, 100%, 75% respectively.