First report on molecular characterization of Oestrus ovis in sheep from India.

The objective of the present study is to characterize the dipteran larvae species infesting the sheep being maintained at SRRC, Mannavanur, by means of COI gene based PCR. During the last week of May 2021, post mortem examination of the skull of an Avikalin male sheep (20 months old) revealed the presence of larvae in its nasal sinuses. The larvae were washed in PBS (pH 7.2) and preserved in 70% alcohol. Total genomic DNA was isolated from the larvae using an initial step of grinding with liquid Nitrogen in a sterile mortar and pestle. Using the isolated genomic DNA from the larvae as a template, Cytochrome c oxidase subunit I (COI) gene based PCR was employed using the primers designed based on the COI gene of reference isolate of Oestrus ovis available in the GenBank. Full length COI gene (1534 bp) gene of Oestrus ovis in sheep from South India was targeted in the PCR experiment. The pTZ57R/T vector was used for the cloning of the PCR amplified fragment and the confirmed recombinant plasmid was subjected to sequencing experiments. In addition to morphological examination, based on COI gene based PCR, eventual sequencing experiments and BLAST analysis, it was confirmed that the larvae in the nasal sinuses of sheep from South India were Oestrus ovis. The South Indian isolate of Oestrus ovis is sharing 100% sequence identity both at nucleotide and amino acid levels with that of O. ovis from Spain. The North Indian isolate of O. ovis (from Jammu) exhibited 92% and 99% identity at respective nucleotide and amino acid levels with South Indian isolate. With other members of the subfamily Oestrinae, the share of per cent nucleotide and amino acid identities of South Indian O. ovis ranged from 85-86% to 95-96%, respectively. O. ovis from South India was grouped with the other members of Oestrinae from different geographical areas of the globe in the analysis of phylogenetic tree based on COI amino acid sequences. Based on the research findings, it is concluded that Oestrus ovis is the dipteran species infesting the sheep at Mannavanur, Tamil Nadu, India. To our knowledge, this is the first report on full length nucleotide sequences of COI gene of O. ovis in sheep from Indian subcontinent. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-024-01666-2.

First Report on Molecular Identification of Moniezia expansa in Sheep from Mannavanur, Palani Hills, Tamil Nadu, India.

PURPOSE: Tape worm infection is common among sheep at SRRC, Mannavanur, Palani hills, Tamil Nadu, India. The aim of the present study is to find out the cestode species infecting the sheep being maintained at SRRC, Mannavanur, by means of molecular method. METHODS: During the second week of June 2021, the hogget flock of sheep (comprising both Bharat Merino and Avikalin sheep breeds) was drenched on empty stomach with commercial preparation of anthelmintic drug containing Niclosamide plus Albendazole, as per the standard dose specified by the manufacturer (Niclozole™: each 5 ml contains 500 mg of Niclosamide and 150 mg of Albendazole: dose for sheep-10 ml/15 kg body weight). The tapeworms expelled in dung by the drug-treated sheep were collected, washed in PBS (pH 7.2), and fixed in between two glass slides using 10% formalin. Furthermore, cytochrome c oxidase subunit I (Cox-I) gene-based PCR was carried out. Only partial sequence (1593 bp) of Cox-I gene of Moniezia expansa from Sheep at SRRC, Mannavanur, Tamil Nadu, India was obtained by PCR. The PCR amplified fragment was cloned into pGEM-T vector and the recombinant plasmid was sequenced. The obtained nucleotide sequences of Cox-I gene of the M. expansa from Indian sheep were analysed with that of 27 more cestode species from different mammalian species (available in GenBank) using bioinformatics tools. RESULTS: The species of the tapeworm was identified as Moniezia species by the Department of Veterinary Parasitology, VC& RI, Orathanadu, TANUVAS by the standard Acidic alum carmine staining method. Due to the ambiguity in the conventional method, Cox-I gene-based PCR and subsequent gene sequencing protocols were used for the identification of the species of cestode infecting sheep at SRRC, Mannavanur, and it was confirmed as M. expansa upon BLAST analysis. Moniezia expansa from SRRC, Mannavanur is having 100% sequence identity at nucleotide level with that of M. expansa from Sengal/Ethiopia. M. benedeni shared 87-88% nucleotide identity with Indian M. expansa. With taenids, the share of percent nucleotide identity of Indian M. expansa ranged from 79 to 81%. M. expansa from Indian sheep was clustering with other anaplocephalids from various mammalian species in the analysis of phylogenetic tree based on Cox-I nucleotide sequences. CONCLUSION: From the present study, it is concluded that M. expansa is the anoplocephalid cestode infecting the sheep at Mannavanur, Tamil Nadu, India. To our knowledge, this is the first report on partial nucleotide sequences of Cox-I gene of M. expansa from Sheep of Indian peninsula. An investigation on the involvement of oribatid mites as the vector in the transmission of M. expansa among sheep at SRRC, Mannavanur needs to be carried out.

Identification of important miRNAs in bubaline mammary gland at heifer stage - An in-silico approach.

The present study was undertaken to identify the microRNAs (miRNAs) expressed in the mammary tissue of a bubaline heifer. Small RNAs were isolated from the mammary gland tissue and enriched for miRNA fraction. The linker-ligated small RNAs were reverse transcribed to synthesize cDNA and amplified by PCR. The PCR products were ligated to the pGEM-T Easy vector; cloned into DH5 alpha cells and sequenced. Sequencing of 40 clones, randomly chosen from this library, produced 115 concatemerized short sequences. The short sequences were checked for their matches with the help of refseq_rna database, EST database (NCBI) and Ensembl. The analysis was performed for more than 90% identity with miRNAs across different species in miRBase. Alignment of putative small RNA sequences with the bovine genome was attempted in GenBank (NCBI) and Ensembl using BLAST. The small RNA sequences with a partial matches within the bovine genome and/or with flanking sequences (upstream or downstream) were analyzed for hairpin structures using the Mfold web server. Reverse complements were also assessed for the homology search. miRNA sequences showing only a partial match with already reported sequences were considered as a putative bubaline miRNAs. Six developmentally important putative miRNA precursors were identified from this study using cloning and sequencing followed by the Bioinformatics approach. This study will help in the elucidation of pathways involving miRNAs in bubaline species at the heifer stage.

Molecular characterization of Orf virus isolates from Kodai hills, Tamil Nadu, India.

AIM: The present study was carried out to find out the causative agent of exanthematous skin lesions in sheep maintained by Southern Regional Research Centre, Mannavanur, Kodai hills, Tamil Nadu. MATERIALS AND METHODS: Polymerase chain reaction (PCR) with Orf virus (ORFV) B2L gene-specific primers was carried out by employing the total genomic DNA isolated from the scabs as the template. The ORFV isolates from Kodai hills were characterized by the use of bioinformatics tools. RESULTS: The amino acid identity of ORFV isolate 1 from Kodai hills is having 98.14%, 96.29%, and 83.59% identity with reference strains of ORFV, Pseudocowpox virus, and bovine papular stomatitis virus, respectively. Phylogenetic analysis revealed that ORFV isolates from Kodai hills clustered with the other ORFV isolates from different geographical areas of India. CONCLUSION: The etiological agent of exanthematous skin lesion among sheep of Kodai hills is ORFV.

Repertoire of noncoding RNAs in corpus luteum of early pregnancy in buffalo (Bubalus bubalis).

AIM: The present study was designed to identify other noncoding RNAs (ncRNAs) in the corpus luteum (CL) during early pregnancy in buffalo. MATERIALS AND METHODS: For this study, CL (n=2) from two buffalo gravid uteri, obtained from the slaughter house, was transported to laboratory after snap freezing in liquid nitrogen (-196°C). The stage of pregnancy was determined by measuring the crown-rump region of the fetus. This was followed by isolation of RNA and deep sequencing. Post-deep sequencing, the obtained reads were checked and aligned against various ncRNA databases (GtRNA, RFAM, and deep guide). Various parameters, namely, frequency of specific ncRNAs, length, mismatch, and genomic location target in several model species were deciphered. RESULTS: Frequency of piwi-interacting RNAs (piwi-RNAs), having target location in rodents and human genomes, were significantly higher compared to other piwi-RNAs and ncRNAs. Ribosomal RNAs (rRNAs) deduced had nucleotides (nts) ranging from 17 to 50 nts, but the occurrence of small length rRNAs was more than lengthier fragments. The target on 16S rRNA species confirms the conservation of 16S rRNA across species. With respect to transfer RNA (tRNA), the abundantly occurring tRNAs were unique with no duplication. Small nucleolar RNAs (snoRNAs), identified in this study, showed a strong tendency for coding box C/D snoRNAs in comparison to H/ACA snoRNAs. Regulatory and evolutionary implications of these identified ncRNAs are yet to be delineated in many species, including buffaloes. CONCLUSION: This is the first report of identification of other ncRNAs in CL of early pregnancy in buffalo.

Identification of microRNAs in corpus luteum of pregnancy in buffalo (Bubalus bubalis) by deep sequencing.

This study was aimed to identify miRNAs of corpus luteum (CL) in buffaloes during pregnancy. For this study, CL (n=2) were collected from gravid uteri of buffalo and RNA was isolated. Following this, the purity and integrity of RNA was checked and used for deep sequencing using Illumina Hiseq 2500 platform. The reads' quality was checked prior to in silico analyses viz. identification of conserved, novel and target of miRNAs. In this study, out of identified miRNAs (3018), 3013 were known and 5 were novel miRNAs on alignment with reference genomes. In addition, prediction of putative target genes for identified abundant miRNAs revealed several genes viz. HOX, KLF4, NCOR2, CDKN2Z, MAPK7, COX2, PPARA, PTEN, ASS3A, ELK1, CASP3, BCL211, MCL1, CCND2, Cyclin A2 and CDC25A during early pregnancy in buffalo. These predicted target genes have been associated with various cellular house-keeping processes including apoptosis. In conclusion, this study reports the identification of conserved and novel microRNAs (miRNAs) in CL during pregnancy in buffalo by deep sequencing.

Genetic trend for growth and wool performance in a closed flock of Bharat Merino sheep at sub temperate region of Kodai hills, Tamil Nadu.

AIM: The study was conducted at Southern Regional Research Center, ICAR-Central Sheep and Wool Research Institute (CSWRI), Mannavanur, Kodaikanal, Tamil Nadu to estimate genetic trends for birth weight (BWT), weaning weight (3WT), 6 months weight (6WT), and greasy fleece weight (GFY) in a Bharat Merino (BM) flock, where selection was practiced for 6WT and GFY. MATERIALS AND METHODS: The data for this study represents a total of 1652 BM lambs; progeny of 144 sires spread over 15 years starting from 2000 to 2014, obtained from the BM flock of ICAR-SRRC (CSWRI), Mannavanur, Kodaikanal, Tamil Nadu, India. The genetic trends were calculated by regression of average predicted breeding values using software WOMBAT for the traits BWT, 3WT, 6WT and GFY versus the animal's birth year. RESULTS: The least square means were 3.28±0.02 kg, 19.08±0.23 kg, 25.00±0.35 kg and 2.13±0.07 kg for BWT, 3WT, 6WT and GFY, respectively. Genetic trends were positive and highly significant (p<0.01) for BWT, while the values for 3WT, 6WT and GFY though positive, were not significant. The estimates of genetic trends in BWT, 3WT, 6WT and GFY were 5 g, 0.8 g, 7 g and 0.3 g/year gain and the fit of the regression shows 55%, 22%, 42% and 12% coefficient of determination with the regressed value, respectively. In this study, estimated mean predicted breeding value (kg) in BWT and 3WT, 6WT and GFY were 0.067, 0.008, 0.036 and -0.003, respectively. CONCLUSION: Estimates of genetic trends indicated that there was a positive genetic improvement in all studied traits and selection would be effective for the improvement of body weight traits and GFY of BM sheep.