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Rohu, Labeo rohita, is one of the most important aquaculture species in the Indian subcontinent. Understanding the molecular-level physiological responses to thermal stress or climate change is essential. In the present work, transcriptome sequencing was carried out in the muscle tissue of the rohu in response to heat stress (35 °C) in comparison with the control (28 °C). A total of 125 Gb of sequence data was generated, and the raw-reads were filtered and trimmed, which resulted in 484 million quality reads. Reference-based assembly of reads was performed using L. rohita genome, and a total of 90.17% of reads were successfully mapped. A total of 37,462 contigs were assembled with an N50 value of 1854. The differential expression analysis revealed a total of 107 differentially expressed genes (DEGs) (15 up-, 37 down-, and 55 neutrally regulated) as compared to the control group (Log2FC > 2, P < 0.05). Gene enrichment analysis of DEGs indicates that transcripts were associated with molecular, biological, and cellular activities. The randomly selected differentially expressed transcripts were validated by RT-qPCR and found consistent expression patterns in line with the RNA-seq data. Several transcripts such as SERPINE1(HSP47), HSP70, HSP90alpha, Rano class II histocompatibility A beta, PGC-1 and ERR-induced regulator, proto-oncogene c-Fos, myozenin2, alpha-crystallin B chain-like protein, angiopoietin-like protein 8, and acetyl-CoA carboxylases have been identified in muscle tissue of rohu that are associated with stress/immunity. This study identified the key biomarker SERPINE1 (HSP47), which showed significant upregulation (~ 2- to threefold) in muscle tissue of rohu exposed to high temperature. This study can pave a path for the identification of stress-responsive biomarkers linked with thermal adaptations in the farmed carps. [ABSTRACT FROM AUTHOR]

Paratuberculosis (PTB) is a chronic infectious enteritis of ruminants, caused by Mycobacterium avium subspecies paratuberculosis (MAP) that brings huge economic loss to the dairy farmers. The study was conducted to explore the association of selected SNPs in IFNG, SLC11A1, ANKRA2 and PGLYRP1 genes with resistance to PTB disease in Indian cattle population. A case-control resource population was established based on the results of diagnostic tests used for detection of MAP infection status viz. ELISA, Johnin PPD test, faecal microscopy and IS900 blood PCR. The PCR-RFLP method was used for genotyping of SNPs. SNPs rs109453173 in SLC11A1, rs110853455 in IFNG and rs41933863 in ANKRA2 genes were significantly (P<0.05) associated with resistance to MAP infection. For SNP rs109453173, GG genotype and G allele was found to be associated with resistance against MAP infection than CC and CG genotypes and C allele, respectively. For SNP rs110853455, AG genotype was found to be associated with susceptibility to MAP infection than AA and GG genotype. For SNP rs41933863, the AG genotype provided three and six times more resistance against MAP infection than GG and AA genotype. The results of this study are suggestive of SNPs rs109453173, rs110853455 and rs41933863 as potential markers for screening MAP resistant cattle and a breeding programme favouring GG genotype and G allele for rs109453173, AG genotype for rs41933863 and against AG genotype for rs110853455 might confer resistance against MAP infection in Indian cattle. However, investigation of these SNPs in an independent and larger population will warrant the strength of association for resistance against MAP infection in cattle. [ABSTRACT FROM AUTHOR]

The EGR family comprises of EGR 1, EGR 2, EGR 3 and EGR 4 which are involved in the transactivation of several genes. A broad range of extracellular stimuli by growth factors is capable of activating EGR mediated transactivation of genes involved in angiogenesis and cell proliferation. However, their role in controlling VEGF A and FGF 2 signaling in the CL of water buffalo is not known. The present study was conducted to understand the role of EGR mediated regulation of VEGF A and FGF 2 signaling in buffalo luteal cells. Towards this goal, luteal cells were cultured and treated with VEGF A and FGF 2 and the mRNA expression pattern of EGR family members were documented. The EGR 1 message was found to be up-regulated in luteal cells of buffalo at 72 hours of culture. The functional validation of EGR 1 gene was accomplished by knocking out (KO) of EGR 1 in cultured luteal cells by CRISPR/Cas9 mediated gene editing technology. The EGR 1 KO cells were then cultured and stimulated with VEGF A and FGF 2. It was observed that VEGF A and FGF 2 induced angiogenesis, cell proliferation and steroidogenesis in wild type luteal cells, whereas the response of the growth factors was attenuated in the EGR 1 KO cells. Taken together our study provides evidence convincingly that both VEGF and FGF mediate their biological action through a common intermediate, EGR 1, to regulate corpus luteum function of buffalo. [ABSTRACT FROM AUTHOR]

Genetic susceptibility to brucellosis is multifactorial, and it is known that impairment of the immune system could contribute to risk for getting brucellosis. The aim of the study was to find association of bovine brucellosis with 20 SNPs pertaining to bovine cytokine ( IFNG, IFNGR1, IFNGR2, TNFA) and innate immunity ( SLC11A1, TLR1, TLR4, and TLR9) genes using PCR-RFLP genotyping technique and it was observed that SLC11A1 (+ 1066 C/G), TLR1 (+ 1446 C/A), TLR1 (+ 1380 G/A), TLR4 (+ 10 C/T) and TLR4 (+ 399 C/T) loci were significantly ( P ≤ 0.05) associated with bovine brucellosis. The odds ratios (OR) of CG and CC genotypes versus GG genotype were 0.31 (0.12-0.82; 95 % CI) and 0.18 (0.03-1.06; 95 % CI) at SLC11A1 (+ 1066 C/G) locus in cases of brucellosis affected cattle. For TLR1 (+ 1380 G/A) locus, the OR for AG and AA genotypes versus GG genotypes were 0.15 (0.05-0.44; 95 % CI) and 0.26 (0.04-1.47; 95 % CI) which indicated that proportion of GG homozygote was significantly higher in brucellosis affected animals as compared to control. At TLR1 (+ 1446 C/A) locus the OR of AC genotype versus CC genotype was 0.24 (0.08-0.68; 95 % CI) which revealed that relative proportion CC genotypes was significantly higher in case population. The TLR4 (+ 10 C/T) locus had three genotypes (TT, CT and CC) where OR of CT and CC genotypes versus TT genotype were near to zero. The OR of CT genotypes versus CC genotypes was 8.25 (0.94-71.92; 95 % CI) at TLR4 (+399 C/T) locus and indicated that CT genotype had higher odds of bovine brucellosis than control animals. [ABSTRACT FROM AUTHOR]

Bovine herpesvirus 1 (BoHV-1) is the most common viral pathogen found in bovine semen, causing numerous reproductive disorders leading to economic losses to the cattle industry. For rapid detection of BoHV-1 in bovine semen, in this study, we applied a loop-mediated isothermal amplification (LAMP) assay. The assay could be completed within 90 min, including total DNA isolation, target amplification, and visual interpretation of positive or negative results with the naked eye. The assay detected as little as 10 fg of BoHV-1 DNA per reaction. The analytical sensitivity of the assay was 0.2 TCID BoHV-1 per reaction, which was 100 times more sensitive than conventional PCR and comparable to TaqMan real-time PCR. The applicability of the assay was assessed by analysing 118 semen samples collected from breeding bulls. On comparison with TaqMan real-time PCR, the LAMP assay had a diagnostic sensitivity of 97 %, specificity of 100 %, and accuracy of 99.2 % for detection of BoHV-1 in bovine semen. The LAMP assay developed in this study is a rapid, sensitive, and cost-effective alternative for detection of BoHV-1 in bovine semen. [ABSTRACT FROM AUTHOR]

The antiviral potential of small interfering RNAs (siRNAs) targeting rabies virus (RV) polymerase (L) and nucleoprotein (N) genes delivered through lentiviral vector was investigated. For in vitro evaluation, siRNAs expressing BHK-21 cell lines (BHK-L and BHK-N) were developed using transduction with Lenti-L and Lenti-N lentiviruses encoding siRNAs against RV-L and N genes, respectively. When these cell lines were challenged in vitro with RV Pasteur virus-11 (PV-11) strain, there was reduction in number of RV-specific foci and target gene transcripts indicating inhibitory effect on RV multiplication. For in vivo evaluation, mice were treated intracerebrally with lentiviruses and challenged with 20 LD of RV challenge virus standard-11 (CVS-11) strain by intramuscular route in masseter muscle. Five out of eight mice treated with Lenti-N survived indicating 62.5 % protection. The control and Lenti-L-treated mice died within 7-10 days indicating lethal nature of challenge virus and no protection. These results demonstrated that siRNA targeting RV-N could not only inhibit RV multiplication, but also conferred protection in mice against lethal RV challenge. These findings have implication on therapeutic use of siRNA targeting RV-N against RV infection. [ABSTRACT FROM AUTHOR]

Small interfering RNAs (siRNAs) targeting rabies virus (RV) glycoprotein (G) and nucleoprotein (N) genes were evaluated as antiviral agents against rabies virus in vitro in BHK-21 cells. To select effective siRNAs targeting RV-G, a plasmid-based transient co-transfection approach was used. In this, siRNAs were expressed as short hairpin RNAs (shRNAs), and their ability to inhibit RV-G gene expression was evaluated in cells transfected with a plasmid expressing RV-G. The nine different siRNAs designed to target RV-G exhibited varying degrees of knockdown of RV-G gene expression. One siRNA (si-G7) with considerable effect in knockdown of RV-G expression also demonstrated significant inhibition of RV multiplication in BHK-21 cells after in vitro challenge with the RV Pasteur virus-11 (PV-11) strain. A decrease in the number of fluorescent foci in siRNA-treated cells and a reduction (86.8 %) in the release of RV into infected cell culture supernatant indicated the anti-rabies potential of siRNA. Similarly, treatment with one siRNA targeting RV-N resulted in a decrease in the number of fluorescent foci and a reduction (85.9 %) in the release of RV. As a dual gene silencing approach where siRNAs targeting RV-G and RV-N genes were expressed from single construct, the anti-rabies-virus effect was observed as an 87.4 % reduction in the release of RV. These results demonstrate that siRNAs targeting RV-G and N, both in single and dual form, have potential as antiviral agent against rabies. [ABSTRACT FROM AUTHOR]

BMPs are multifunctional growth factors implicated in regulating the ovarian function as key intra-ovarian factors. Biological effects of BMPs are mediated through binding with membrane bound receptors like BMPR-IB and initiating downstream Smad signaling pathway. FecB mutation, regarded as a loss of function mutation in the BMPR-IB gene was identified in certain sheep breeds having high fecundity. Similar type of fecundity genes in goats have not been discovered so far. Hence, the current study was designed to investigate the effects of BMPR-IB gene modulation on granulosa cell function in goats. The BMPR-IB gene was knocked out using CRISPR-Cas technology in granulosa cells and cultured in vitro with BMP-4 stimulation for three different durations In addition, the FecB mutation was introduced in the BMPR-IB gene applying Easi-CRISPR followed by BMP-4/7 stimulation for 72 h. Steroidogenesis and cell viability were studied to explore the granulosa cell function on BMPR-IB gene modulation. BMPRs were found to be expressed stage specifically in granulosa cells of goats. Higher transcriptional abundance of R-Smads, LHR and FSHR indicating sensitisation of Smad signaling and increased gonadotropin sensitivity along with a significant reduction in the cell proliferation and viability was observed in granulosa cells upon BMPR-IB modulation. The inhibitory action of BMP-4/7 on P4 secretion was abolished in both KO and KI cells. Altogether, the study has revealed an altered Smad signaling, steroidogenesis and cell viability upon modulation of BMPR-IB gene in granulosa cells similar to that are documented in sheep breeds carrying the FecB mutation. [ABSTRACT FROM AUTHOR]

A total of 9 SNPs located in TFRC and ACK1 genes of SSC13q41 genomic region were examined for their association with the adhesion pattern of native Indian pigs using local isolate of diarrhoeagenic E. coli. Phenotypic evaluation of adhesion pattern of 150 pigs revealed 116 animals positive for adhesion, whereas 34 animals had non-adhesive phenotype. Among the adhesive animals, 6, 87 and 23 pigs were strongly adhesive, weakly adhesive and adhesive, respectively. PCR–RFLP study revealed 8 polymorphic SNPs with low to moderate PIC ranging from 7.39 to 37.25% and low to high heterozygosities (8–70%). The loci g.291 C > T, rs81218930 C > T, rs318751568 C > T of TFRC and g.93222 C > A g.94600 C > T of ACK1 showed significant departure from HWE. The genotypic frequencies of the SNPs as well as the haplotypes did not differ significantly (P > 0.05) across the adhesion patterns except one SNP (ACK1-g.107371 A > C). Among the g.107371 A > C genotypes observed, CA was associated with non-adhesive phenotype. Furthermore, TFRC mRNA expression levels were found to be significantly (P < 0.05) different among various adhesive phenotypes, whereas that of ACK1 was significantly (P < 0.05) different between non-adhesive and adhesive groups. The significant association of SNP (ACK1-g.107371 A > C), which was also previously reported to influence ETECF4 mediated diarrhoea susceptibility, implicates its wider application in genetic control of piglet diarrhoea. Furthermore, the up-regulation of TFRC gene expression in adhesive group supports its proposed role in activation of immune cells against E. coli and intracellular iron transport. [ABSTRACT FROM AUTHOR]