Simple method for isolation and culture of primary buffalo (Bubalus bubalis) endometrial epithelial cells (pBuEECs) and its characterization using high throughput proteomics approach.

Early embryonic mortality resulting from insufficient interaction between the embryo and the uterus leads to the failure of pregnancy in livestock animals. Thus, it is imperative to comprehend the multifaceted process of implantation at molecular levels, which requires synchronized feto-maternal interaction. The in-vitro models serve as valuable tools to investigate the specific stages of implantation. The present study was undertaken to develop a simple method to isolate and culture the primary buffalo endometrial epithelial cells (pBuEECs), followed by proteome profiling of the proliferating cells. Collagenase I was used to separate uterine epithelial cells (UECs) from the ipsilateral uterine horn, and then the cells were separated using a cell strainer. After being seeded on culture plates, UECs developed colonies with characteristic epithelial shape and expressed important markers such as cytokeratin 18 (KRT18), progesterone receptor (PGR), ß-estrogen receptor (ESR1), and leukemia inhibitory factor (LIF), which were confirmed by PCR. The purity of epithelial cells was assessed using cytokeratin 18 immunostaining, which indicated approximately 99% purity in cultured cells. The proteome profiling of pBuEECs via high-throughput tandem mass spectrometry (MS), identified a total of 3383 proteins. Bioinformatics analysis revealed enrichment in various biological processes, including cellular processes, metabolic processes, biological regulation, localization, signaling, and developmental processes. Moreover, the KEGG pathway analysis highlighted associations with the ribosome, proteosome, oxidative phosphorylation, spliceosome, and cytoskeleton regulation pathways. In conclusion, these well characterized cells offer valuable in-vitro model to enhance the understanding of implantation and uterine pathophysiology in livestock animals, particularly buffaloes.

Antigenic characterization of 52-55kDa protein isolated from Trypanosoma evansi and its application in detection of equine trypanosomosis.

Trypanosoma evansi is a haemo-protozoan parasite responsible for the disease surra, an economically important disease of wide range of domestic and wild animals. The present diagnostic methods using soluble antigens have inherent problems like lack of standardized and reproducible antigens, as well as ethical issues. This entails further efforts for search of defined antigenic molecules with satisfying sensitivity and specificity for sero-epidemiology of trypanosomosis. In present investigation, we have identified and purified 52-55kDa immuno-dominant protein cluster in molecular mass ranges by preparatory SDS-PAGE methods from T. evansi proteome. The purified protein was further characterized by hyper immune serum raised in rabbits and also further evaluated for its immunodiagnostic potential using experimentally infected horse serum samples by different immunological tests. The immunoblot, ELISA and dot blot assay using purified cluster in infected pooled serum samples showed detection of infection early as 10th days post infection till termination of experiment. The observations revealed that purified cluster is expressed not only at early stage but also persisted and detected throughout course of infection. Further, whole cell lysate antigen separated out and detected 141 spots by 2-D gel electrophoresis. The isoelectric focussing (PI) of 52-55kDa was determined in pH range between 6.9 and 7.5 along with two other cluster of proteins recognised by immune sera of ponies infected with T. evansi. MS/MS analysis of the purified protein cluster identified five proteins i.e. pyruvate kinase 1, beta tubulin, paraflagellar rod protein, alanine aminotransferase and variable surface glycoprotein showing homology to protein present in Trypanosome database. These identified proteins may be useful for development of vaccines and diagnostic targets against animal trypanosomosis.

Dataset on the importation of the exotic shrimp Penaeus vannamei broodstock (Boone, 1931) to India.

Penaeus vannamei is an exotic shrimp species that has gained high culture momentum, since its introduction to India [1]. Currently, the culture of the species in the Country is being done by the shrimp farmers by importation of Specific Pathogen Free (SPF) vannamei broodstock from approved suppliers, which are located overseas. The value of one brooder normally ranges from 50 to 61 US $, excluding the custom duty, processing fee and other charges for the transboundary shipment of the stock to India. The P. vannamei stock are permitted to be imported to the Country by the hatchery operators only through the single declared port of entry, i.e. Chennai in Tamil Nadu in the Country. The imported parent shrimps are then to be quarantined at the Aquatic Quarantine Facility before being transported to the vannamei hatcheries [2]. This article reports the data available on import of vannamei broodstock to India since its importation to India in 2009. The dataset presented here contains information on transit and quarantine mortality of the brooders following the shipment of the stock by the various broodstock suppliers from the overseas.

Comparative evaluation of recombinant HSP70 (N & C-terminal) fragments in the detection of equine trypanosomosis.

Trypanosomosis (Surra) is an economically important disease caused by Trypanosoma evansi which is an extracellular parasite present in the plasma, tissues and other body fluids of a wide range of hosts including domesticated animals. Currently, serological reports are based on detection of antibodies by ELISA using whole cell lysate (WCL) antigen, which has a limitation of persistence of anti-trypanosomal antibodies after successful treatment of the disease. Moreover, it has some ethical issues also like requirement of mice for in vivo maintenance of parasite for preparing the antigen. Therefore, in the present study, an attempt was made to evaluate the in vitro production of recombinant heat shock protein 70 (HSP70) for detection of antibodies in experimentally infected ponies. The amino acid sequence analysis of HSP70 revealed that N-terminal region of the protein was highly conserved while the C-terminal region was most divergent. The four different regions of HSP70 protein viz. HSP-1, HSP-2, HSP-3 and HSP-4 were cloned and expressed, among which HSP-1 (N-terminal region) & HSP-2 (C-terminal region) were truncated while HSP-3 & HSP-4 were complete C-terminal proteins. The recombinant fragments were probed with sequentially pooled experimental serum samples where antibodies were detected in these fragments from 10(th) day post infection till the termination of the experiment. Further, these recombinant fragments were also comparatively evaluated with WCL antigen in ELISA using experimental as well as field serum samples. It was observed that after successful treatment of infected ponies, there was a sharp fall in antibodies (within 90 days) when tested with recombinant HSP's fragments, while antibodies persisted even after 469 days when tested against WCL antigen. The sensitivity and specificity of all HSP70 fragments were also estimated from field serum samples with reference to WCL antigen ELISA. The HSP-1 showed minimum sensitivity (41.03%) among all the recombinant fragments. Among the C-terminal fragments, maximum sensitivity was observed with the HSP-2 (61.54%) while minimum was observed with HSP-4 (48.72%). The specificity increases for recombinant fragments from N-terminal to C-terminal region of protein and maximum specificity was observed with HSP-4 fragment (91.3%).

Effect of preservatives, temperature and storage duration on stability of nucleic acids of pleopod tissues of Penaeus vannamei (Boone) and screening of viral infections.

In shrimp farming, screening for economically significant viral pathogens in nucleic acids of shrimps is vital for disease surveillance programmes and further, to take necessary precautions to ensure the sustainability of the farms and thereby the shrimp industry. Different preservatives, temperature and storage durations of the pleopod tissues of Penaeus vannamei broodstock were tested to investigate its effect on the quality and quantity of the nucleic acids. The pleopods were subjected to two preservation regimes and the yield and stability of the extracted nucleic acids were monitored over a time period of 12 months. Stability of the nucleic acids was assessed with nested polymerase chain reaction, and the yield was checked spectrophotometrically. Data was analysed by performing two way ANOVA and Tukeys Paired test. Preservation treatments included storage at -20 degrees C and 5 degrees C in RNAlater and in 70% ethanol. Significant variation (P < 0.05) was observed in both DNA and RNA yield and stability from ethanol and RNAlater stored pleopods at 5 degrees C. However, the yield and stability did not differ (P > 0.05) in both the preservatives at -20 degrees C. The RNA was degraded and yielded lesser quantity when pleopod tissues were stored in ethanol at -20 degrees C than when stored in RNAlater during storage duration of 9 months. This study would help the shrimp farmers and researchers to adopt better preservation strategy, vital for shrimp disease surveillance programmes and for traceability studies in the event of any disease outbreak.

Production and preliminary evaluation of Trypanosoma evansi HSP70 for antibody detection in Equids.

The present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most closely related to T. brucei brucei and T. brucei gambiense, whereas T. congolense HSP70 laid in separate clade. The two partial HSP70 sequences (HSP-1 from N-terminal region and HSP-2 from C-terminal region) were expressed and evaluated as diagnostic antigens using experimentally infected equine serum samples. Both recombinant proteins detected antibody in immunoblot using serum samples from experimental infected donkeys with T. evansi. Recombinant HSP-2 showed comparable antibody response to Whole cell lysate (WCL) antigen in immunoblot and ELISA. The initial results indicated that HSP70 has potential to detect the T. evansi infection and needs further validation on large set of equine serum samples.

Knowledge level of Ayurveda practitioner on public health.

BACKGROUND: Looking at the current scenario of shortage of public health professionals on one hand and intense demand of community health services on the other it is imperative that the contribution of Ayurveda practitioners is increased in the field of public health. However, the updating of the knowledge of public health issues and concepts will ultimately decide whether they can be successfully integrated into the community health arena or not. AIM: This study was conducted to assess the knowledge level of Ayurveda practitioners about public health Issues with the aim find out the competence of Ayurveda practitioners regarding knowledge of public health issues. MATERIALS AND METHODS: Cross-sectional study was conducted in the union territory, Chandigarh and two districts each of the states of Haryana and Punjab. Public health knowledge assessment tool comprising a questionnaire was used to collect information from the respondents who were registered Ayurveda doctors and interns. The data was analyzed with the help of IBM SPSS (Statistical Product and Service Solutions). RESULTS: The respondents scored between 5 and 17 points out of a total of 19 points and majority (82%) of the respondents fell in the category of "having average knowledge". The mean score was 8.42 ± 2. CONCLUSION: Curriculum and training of Ayurveda education need to have more public health related inputs and hence that the Ayurveda practitioners are well-versed with the public health concepts and could contribute in the public health field meaningfully.