In vitro effects of uncarboxylated osteocalcin on buffalo Leydig cell steroidogenesis.

Uncarboxylated osteocalcin (UcOCN), a bone derived circulating protein, has been demonstrated to influence steroidogenesis in testicular Leydig cells of murine and human species. However, the role of UcOCN in testosterone biosynthesis remains unexplored in domestic animals. The present study aimed to investigate the impact of UcOCN on the expressions of steroidogenic genes (HSD3ß1, HSD3ß6, CYP17A1, CYP11A1), testosterone production and GPRC6A receptor localization in buffalo Leydig cells. Leydig cells from the testes of adult Murrah buffalo were isolated, with an average cell count and viability after digestion and Percoll enrichment of 1.43 × 106 cells/g of testes and 78.5%, respectively. Immunophenotyping of Percoll-enriched cell suspension by flow cytometry showed populations of Leydig cells ranging between 69 and 73.9%. Immunostaining confirmed the presence of GPRC6A receptors and CYP11A1 positive Leydig cells. When these cells were cultured and incubated with varying levels of UcOCN (6, 12, 24, and 48 ng/ml) and LH, there was a significant (P < 0.01) increase in testosterone production and up-regulation (P < 0.05) of CYP11A1, CYP17A1, HSD3ß1 and HSD3ß6 gene expression. In summary, the present study underscored the effects of UcOCN on testosterone biosynthesis, expression of crucial steroidogenic genes and interaction with GPRC6A receptors in buffalo Leydig cells, emphasizing its potential implications in andrology.

Molecular epidemiology of Orientia tsutsugamushi from outbreak regions, Madhya Pradesh, central India.

Scrub typhus is a seriously neglected life threatening disease in Asia and the islands of the Pacific and Indian oceans. The causative agent is Orientia tsutsugamushi, an obligate intra-cytosolic bacterium transmitted to vertebrate animals such as human or rodent by larval trombiculid mites (chiggers). Here, we conducted an outbreak investigation in Madhya Pradesh state in central India after sudden upsurge in scrub typhus cases and mortality in humans. Sixty-five live traps were placed for capturing the rodents. The captured rodent species were identified and the samples were subjected to nested PCR targeting 56kDa TSA gene. Analysis revealed for the first-time predominant circulation of Karp genotype in this region. The vector species involved in transmission is Leptotrobidium deliense. In conclusion, we reported the circulation of Karp strain in outbreak regions of Madhya Pradesh and suggest that public health monitoring and surveillance needs to be improved. Studies on antigenic diversity, association of strains with clinical spectrum and pathogenicity in the local setup will be essential for development of region-specific diagnostics and vaccines.

A Multi-copy Nucleic Acid-Based Diagnostic Test for Bovine Tropical Theileriosis.

BACKGROUND: Bovine tropical theileriosis (BTT) is a haemoprotozoan tick-borne disease that implicates huge losses to livestock in terms of considerable mortality and morbidity in tropical and subtropical regions of the globe. Currently available diagnostic methods have less specificity and sensitivity towards the detection of Theileria species. Therefore, an attempt was made to diagnose Theileria annulata by targeting a multi-copy gene, viz. mitochondrially encoded cytochrome b (MT-CYB) gene via polymerase chain reaction (PCR) in different agro-zones of India. METHODS AND RESULTS: 129 cattle blood samples were collected from major livestock rearing regions of India and processed for both molecular and microscopic techniques. Screening of Giemsa-stained thin blood smears was able to detect 14 samples (10.85%) as positive for T. annulata. However, the MT-CYB gene-based PCR assay detected 107 samples (82.94%) positive for T. annulata out of 129 samples. Furthermore, the MT-CYB gene-based PCR assay was standardized in terms of its sensitivity and specificity. Specificity of PCR assay was evaluated against other common haemoprotozoan parasites of tropical countries viz. Babesia bigemina, Anaplasma marginale and Trypanosoma evansi. The multi-copy MT-CYB gene-based PCR assay provided an optimum level of sensitivity (up to the level of 10 femtogram) and high specificity. Haematological examination (Hb, PCV and TLC) of 113 samples revealed significantly (p < 0.05) decreased Hb and PCV levels in positive animals in comparison with the control group of healthy animals. However, the control group had significantly higher (p < 0.001) TLC levels than the positive group. CONCLUSION: The MT-CYB gene-based PCR assay was found to be highly sensitive that can accurately detect the occurrence of T. annulata infection in carrier animals which are potential infection sources to healthier populations in naive demographic locations through infected ticks.

Screening for the "Achilles Heel" of Hyalomma anatolicum Ticks by RNA Interference Technology and an Update on Anti-Tick Vaccine Design.

Over the years, RNA interference (RNAi) has evolved as a valuable tool to study the tick gene function, screening and preliminary characterization of tick-protective antigens in a relatively short time, with a minimal use of laboratory animals before conducting expensive vaccine trials for the development of improved vaccine composition. In this process, a double-stranded RNA (dsRNA) of gene of interest is introduced into the tick system which specifically suppresses expression of a target gene. The results of RNAi-based gene silencing were interpreted by reduction in targeted gene transcript, changes in phenotypic data and anatomical/ biochemical changes in ticks; thereby, providing a clue to the probable role played by the gene in the tick biological system. Across the globe, various tick research groups applied RNAi technique for characterization and identification of new anti-tick vaccine targets. Herein, we used the RNAi tool in Hyalomma anatolicum ticks for identification and characterization of vaccine candidates.

Determination of cut-off of diagnostic ELISA for Scrub typhus in endemic setup: Central India.

Serology remains the mainstay for diagnosis of scrub typhus. In central India, diagnosis of this neglected emerging zoonotic disease suffers due to lack of standardized region-specific cutoff value and diagnostic test. Henceforth, standardized region-specific cutoff value for diagnostic ELISA play a significant role in initial treatment of patients and to differentiate other febrile diseases in endemic setup. A total of 144 patients of all age groups with acute undifferentiated febrile illness patients, forty healthy controls, dengue and chikungunya positive thirty-five samples in each category, respectively were enrolled in the study and subjected to IgM ELISA (InBioS, International, Inc.). Samples showing OD value more than 0.5 in IgM ELISA, were subjected to nested PCR. Both, receiver operating characteristic (ROC) curve and healthy volunteer samples mean with +3 SD were considered to generate region specific cutoff OD value. A total of 48 patients were diagnosed as cases of scrub typhus through IgM ELISA. Out of 48 samples, 30 were positive by nested PCR. The ROC curve analysis revealed a diagnostic ELISA cutoff value of 0.73 with sensitivity and specificity of 95% and 100%, respectively. The cut off arrived from healthy volunteer is mean OD + 3 SD is 0.72. Considering the significance of scrub typhus diagnosis for treatment and to understand disease dynamics in region wise, the cutoff value of >0.72 for diagnostic ELISA for Madhya Pradesh in central India can be used.

Prevalence of torch infections and its associated poor outcome in high-risk pregnant women of Central India: Time to think for prevention strategies.

Introduction: The TORCH infections during pregnancy are associated with adverse congenital abnormalities, poor foetal outcome and subsequent reproductive failures. The absence of baseline data on status of TORCH infections and associated foetal outcomes prompted us to conduct the current study in Central India. Materials and Methods: : A total of 144 high-risk pregnant women attending tertiary care unit, suspected for TORCH infections were enrolled from August 2017 to December 2018. All the participants were tested for the presence of IgM and IgG antibodies and followed up to record the foetal outcome. Results: The overall TORCH infection (IgM positivity) positivity rate was 61.1% (88/144). Rubella was the most prevalent infection (46.5%) followed by herpes simplex virus (HSV) 1 and 2 (41%) and cytomegalovirus (CMV) (34.7%). The highest IgG sero-positivity was recorded against CMV (88.6%), followed by Rubella (86.8%), HSV 1 and 2 (28.4%), and toxoplasmosis (15.2%). Follow-up of IgM TORCH positive pregnant women revealed that majority of the neonates/infants are having congenital cardiac abnormalities (39.2%), followed by microcephaly/hydrocephaly (25%), low birth weight (10.7%), and deafness (3.6%). Thirty-two percent of neonatal mortality was associated to multiple TORCH infections. Conclusion: A high prevalence of IgM seropositivity of TORCH infection was noted in the present study with the increased rate of poor foetal outcome warrants the need of proper prenatal counselling, universal immunisation and nutritional supplements during pregnancy.

Functional characterization of candidate antigens of Hyalomma anatolicum and evaluation of its cross-protective efficacy against Rhipicephalus microplus.

Hyalomma anatolicum and Rhipicephalus microplus seriously affect dairy animals and immunization of host is considered as a sustainable option for the management of the tick species. Identification and validation of protective molecules are the major challenges in developing a cross-protective vaccine. The subolesin (SUB), calreticulin (CRT) and cathepsin L-like cysteine proteinase (CathL) genes of H. anatolicum were cloned, sequenced and analysed for sequence homology. Both Ha-SUB and Ha-CRT genes showed very high level of homogeneity within the species (97.6-99.4% and 98.2-99.7%) and among the tick species (77.3-99.3% and 85.1-99.7%) while for Ha-CathL the homogeneity was lower among ticks (57.5-89.5%). Besides tick species, both Ha-SUB and Ha- CRT genes showed high level of homogeneity with dipterans (47.2-53.4% and 72.0-74.4%) and nematodes (64.0% by CRT). The level of expression of the conserved genes in different stages of the tick species was studied. The differences in fold change of expression (FCE) of the targeted genes in life stages of tick were not statistically significant except Ha-SUB in eggs and in frustrated females, Ha-CRT in fed male and Ha-CathL in unfed and frustrated females where highest FCE was recorded. The functional properties of the genes were studied by RNAi technology and a significant level of gene suppression (p<0.05) resulted in very low percentage of engorgement of treated ticks viz., 3.7%, 11.1% and 30.0% in Ha-SUB, Ha-CRT and Ha-CathL respectively, in comparison to control was recorded. The recombinant proteins rHa-SUB, rHa-CRT and rHa-CathL encoded by the genes were expressed in prokaryotic expression system. They were evaluated for cross-protective efficacy and found to be respectively, 65.4%, 41.3% and 30.2% protective against H. anatolicum and 54.0%, 37.6% and 22.2%, against R. microplus infestations.

Characterization and establishment of a reference deltamethrin and cypermethrin resistant tick line (IVRI-IV) of Rhipicephalus (Boophilus) microplus.

The problem of ticks and tick borne diseases is a global threat and growing reports of resistance to commonly used insecticides further aggravated the condition and demands for country specific resistance monitoring tools and possible solutions of the problem. Establishment of standard reference is prerequisite for development of monitoring tools. For studying possible role of different mechanisms involved in development of resistance in Rhipicephalus (Boophilus) microplus population and to develop newer drug to manage the problem of resistance, a deltamethrin exposed and selected tick colony, referred to as IVRI-IV, was characterized using reference susceptible IVRI-I tick line as control. The RF values of IVRI-IV ticks against deltamethrin, cypermethrin and diazinon were determined as 194.0, 26.6, 2.86, respectively, against adults. The esterase enzyme ratios of 2.60 and 5.83 was observed using α-naphthyl and ß-naphthyl acetate while glutathione S-transferase (GST) ratio was 3.77. Comparative analysis of IVRI-I and IVRI-IV carboxylesterase gene sequences revealed 13 synonymous and 5 non synonymous mutations, reported for the first time. The C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to leucine to isoleucine (L64I) amino acid substitution was also detected in the IVRI-IV population. In the present study, monitorable indicators for the maintenance of the reference IVRI-IV colony, the first established deltamethrin and cypermethrin resistant tick line of India, were identified.

Nasal Oestrosis in a Jamunapari goat.

Oestrus ovis is a well-known parasite of the nasal cavities and adjoining sinuses in sheep and goats. A 3-year-old female Jamunapari goat was presented with a history of anorexia, weight loss, in-coordinated movements, nasal discharge since 10 days and two nasal bots expelled during sneezing 3 days back. Upon clinical examination the animal was dull, dysphonic, unsteady gait and having blocked nostrils with thick mucoid discharge along with an increased respiratory rate. Parasitiological examination revealed it as an O. ovis (nasal bots). The occurrence of nasal bots in a goat and its successful therapeutic management and public health significance have been reported and discussed in the present communication.

Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle.

BACKGROUND: Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level. OBJECTIVE: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle. METHODS: LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR). RESULTS: The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye. CONCLUSION: The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.