Comparative assessment of two in-house-built isothermal assays for visual detection of African swine fever virus.

Owing to the lack of effective vaccines, current control measures and eradication strategies for the African swine fever virus (ASFV) rely on early detection and stringent stamping-out procedures. In the present study, we developed two independent isothermal amplification assays, namely, loop-mediated isothermal amplification (LAMP) and polymerase spiral reaction (PSR), for quick visualization of the ASFV genome in clinical samples. Additionally, a quantitative real-time PCR (qRT-PCR)-based hydrolysis probe assay was developed for comparative assessment of sensitivity with the developed isothermal assays. The analytical sensitivity of the LAMP, PSR, and qRT-PCR was found to be 2.64 ×105 copies/µL, 2.64 ×102 copies/µL, and 2.64 ×101 copies/µL, respectively. A total of 165 clinical samples was tested using the developed visual assays. The relative accuracy, relative specificity, and relative diagnostic sensitivity for LAMP vs PSR were found to be 95.37% vs 102.48%, 97.46% vs 101.36%, and 73.33% vs 113.33%, respectively.

Characterization of an African swine fever virus outbreak in India and comparative analysis of immune genes in infected and surviving crossbreed vs. indigenous Doom pigs.

Since 2020, African swine fever (ASF) has affected all pig breeds in Northeast India except Doom pigs, a unique indigenous breed from Assam and the closest relatives of Indian wild pigs. ASF outbreaks result in significant economic losses for pig farmers in the region. Based on sequencing and phylogenetic analysis of the B646L (p72) gene, it has been determined that ASFV genotype II is responsible for outbreaks in this region. Recent studies have shown that MYD88, LDHB, and IFIT1, which are important genes of the immune system, are involved in the pathogenesis of ASFV. The differential expression patterns of these genes in surviving ASFV-infected and healthy Doom breed pigs were compared to healthy controls at different stages of infection. The ability of Doom pigs to withstand common pig diseases, along with their genetic resemblance to wild pigs, make them ideal candidates for studying tolerance to ASFV infection. In the present study, we investigated the natural resistance to ASF in Doom pigs from an endemic area in Northeast India. The results of this study provide important molecular insights into the regulation of ASFV tolerance genes.

Incidences of Helicobacter infection in pigs and tracing occupational hazard in pig farmers.

Helicobacter species (spp.) is a gram-negative spiral-shaped motile bacterium that causes gastritis in pigs and also colonizes in the human stomach. The present study assessed the prevalence of Helicobacter spp. in pig gastric mucosa and the stool of pig farmers in Assam, India. A total of 403 stomach samples from pig slaughter points, 74 necropsy samples of pigs from pig farms, and 97 stool samples from pig farmers were collected. Among the pig stomach samples, 43 (20.09%) of those with gastritis showed the presence of Gram-negative, spiral-shaped organisms, while only 3.04% of stomach samples without lesions had these organisms. Scanning Electron Microscopy (SEM) of urease-positive stomach samples revealed tightly coiled Helicobacter bacteria in the mucus lining. Histopathological examination showed chronic gastritis with hemorrhagic necrosis, leucocytic infiltration, and lymphoid aggregates. PCR confirmed the presence of Helicobacter suis in 19.63% of pig stomach samples and 2.08% of pig farmer stool samples. Additionally, 3.12% of the stool samples from pig farmers were positive for Helicobacter pylori. Phylogenetic analysis revealed distinct clusters of Helicobacter suis with other Helicobacter spp. These findings highlight the prevalence of Helicobacter in both pig gastric mucosa and pig farmer stool. The findings highlight the need for improved sanitation and hygiene practices among pig farmers to minimize the risk of Helicobacter infection in humans.

Transcriptome signatures of host tissue infected with African swine fever virus reveal differential expression of associated oncogenes.

African swine fever (ASF) has emerged as a threat to swine production worldwide. Evasion of host immunity by ASF virus (ASFV) is well understood. However, the role of ASFV in triggering oncogenesis is still unclear. In the present study, ASFV-infected kidney tissue samples were subjected to Illumina-based transcriptome analysis. A total of 2463 upregulated and 825 downregulated genes were differentially expressed (p < 0.05). A literature review revealed that the majority of the differentially expressed host genes were key molecules in signaling pathways involved in oncogenesis. Bioinformatic analysis indicated the activation of certain oncogenic KEGG pathways, including basal cell carcinoma, breast cancer, transcriptional deregulation in cancer, and hepatocellular carcinoma. Analysis of host-virus interactions revealed that the upregulated oncogenic RELA (p65 transcription factor) protein of Sus scrofa can interact with the A238L (hypothetical protein of unknown function) of ASFV. Differential expression of oncogenes was confirmed by qRT-PCR, using the H3 histone family 3A gene (H3F3A) as an internal control to confirm the RNA-Seq data. The levels of gene expression indicated by qRT-PCR matched closely to those determined through RNA-Seq. These findings open up new possibilities for investigation of the mechanisms underlying ASFV infection and offer insights into the dynamic interaction between viral infection and oncogenic processes. However, as these investigations were conducted on pigs that died from natural ASFV infection, the role of ASFV in oncogenesis still needs to be investigated in controlled experimental studies.

Clinicopathological and ultrastructural study of African swine fever outbreak in North-East India.

The present investigation focuses on examining the clinical, histopathological, and ultrastructural changes that occurred in pig, during an outbreak of African swine fever (ASF) in 2022 in Assam, India. The disease initially manifested as a per-acute case with high mortality but without any evident clinical signs. Subsequently, some animals exhibited an acute form of the disease characterized by high fever (104-106 °F), anorexia, vomiting, respiratory distress, and bleeding from the anal and nasal orifices. During acute African swine fever virus (ASFV) infections, elevated levels of pro-inflammatory IL-1α, IL-1ß, IL-6, TNF, CCL2, CCL5, and CXCL10 were detected in the palatine tonsil, lymph nodes, spleen, and kidney using qPCR assay. These molecular changes were associated with haemorrhages, edemas, and lymphoid depletion. Postmortem examinations revealed prominent features such as splenomegaly with haemorrhages, haemorrhagic lymphadenitis, severe petechial haemorrhage in the kidney, pneumonia in the lungs, and necrotic palatine tonsil. Histopathological analysis demonstrated lymphocyte depletion in lymphoid organs, multi-organ haemorrhages, and interstitial pneumonia in the lungs. Scanning electron microscopy (SEM) further confirmed lymphocyte depletion in lymphoid organs through lymphocyte apoptosis and kidney damage with distorted tubules due to red blood cell destruction. Transmission electron microscopy reaffirmed lymphocyte apoptosis by observing chromatin condensation and nucleus margination in lymphocytes of lymphoid organs. These findings provide comprehensive insights into the clinical, histopathological, and ultrastructural aspects of ASF outbreak in pigs. Understanding the pathological changes associated with ASF can contribute to improved diagnosis, prevention, and control measures for this highly contagious and economically devastating viral disease.

Meta-Analysis of the Prevalence of Porcine Zoonotic Bacterial Pathogens in India: A 13-Year (2010-2023) Study.

The presence of bacterial pathogens such as Brucella spp., Clostridium spp., E. coli, Listeria monocytogenes, Salmonella spp., Staphylococcus spp., and Streptococcus suis not only hampers pig production but also carries significant zoonotic implications. The present study aims to conduct a comprehensive meta-analysis spanning over 13 years (2010-2023) to ascertain the prevalence of these zoonotic bacterial pathogens in Indian pig populations. The study seeks to synthesize data from diverse geographic regions within India and underscores the relevance of the One Health framework. A systematic search of electronic databases was meticulously performed. Inclusion criteria encompassed studies detailing zoonotic bacterial pathogen prevalence in pigs within India during the specified timeframe. Pertinent information including authors, publication year, geographical location, sampling techniques, sample sizes, and pathogen-positive case counts were meticulously extracted. The meta-analysis of zoonotic bacterial pathogens in Indian pig populations (2010-2023) unveiled varying prevalence rates: 9% Brucella spp., 22% Clostridium spp., 19% E. coli, 12% Listeria monocytogenes, 10% Salmonella spp. and Streptococcus suis, and 24% Staphylococcus spp. The application of random effects further revealed additional variability: 6% Brucella spp., 23% Clostridium spp., 24% E. coli, 14% Listeria monocytogenes, 10% Salmonella spp. and Streptococcus suis, and 35% Staphylococcus spp. Notably, the observed heterogeneity (I2) varied significantly from 87% to 99%. The meta-analysis findings underscore the pervasive nature of these diseases throughout India's pig populations, accentuating the substantial impact of these pathogens on pig health and the potential for zoonotic transmission. The present study reinforces the importance of the adoption of a comprehensive One Health approach that acknowledges the intricate interplay between animal, human and environmental health.

Whole genome sequencing-based cataloguing of antibiotic resistant genes in piggery waste borne samples.

The growing use of antibiotics in livestock is one of the main causes of the rapid global spread of antimicrobial resistance (AMR). However, extensive research on AMR in animals is currently absent. In this article, we provide the bacterial antibiotic resistance genes (ARGs) from piggery waste samples in West Bengal, India, based on whole genome sequencing (WGS). According to the study, there are alarmingly high levels of Enterobacteriaceae in piggery waste, especially slaughterhouse waste, that are resistant to beta-lactam, aminoglycoside, sulphonamide, and tetracycline. We found several plasmids carrying multidrug-resistant Enterobacteriaceae including resistant to last-resort medications like colistin and carbapenems. Our findings will serve as a guide for developing AMR management policies for livestock in India and aid in understanding the current AMR profiles of pigs. To grasp the actual situation with AMR in the pig sector, large scale sample screening must be done.

Genome-wide identification and annotation of SNPs for economically important traits in Frieswal™, newly evolved crossbred cattle of India.

The Frieswal™ is a crossbred cattle evolved by ICAR-Central Institute for Research on Cattle utilizing more than 15,000 cattle maintained at more than 37 military farms spread all over the agro-climatic regions of the country. The ddRAD sequencing method was used to identify and annotate the SNPs and INDELs. The results of variant calling revealed 1,487,851 SNPs and 128,175 INDELs at a read depth of 10. A total of 3,775,079 effects were identified, and majority (66.41%) of the effects were in the intron region of the genome followed by intergenic (21.87%). Majority (99.18%) of the variants had the modifier effect. The results revealed a higher magnitude of transitions as compared to the transversion. The classification of SNPs by functional class revealed a majority of missense (43%) and silent (56%) effects. Out of 26,278 genes identified, 1841 SNPs were annotated in 207 candidate genes responsible for various milk production and reproduction traits. The observed heterozygosity was 0.2804 against the expected heterozygosity value of 0.2978. The overall average inbreeding coefficient (FIS) was 0.0604. The pathway analysis revealed that the prolactin signaling pathway (GO:0038161) was significant biological process complete for both milk production and reproduction traits. The SNP variations can be effectively used as markers for early and accurate identification of the QTLs and for formulating an efficient and effective breed improvement program in Frieswal™ cattle. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03701-0.

Characterization of piggery farm waste-borne bacterial transposable elements associated with antimicrobial resistance phenotypes.

Even though there is a link between antibiotic resistance and the presence of transposable elements few research has looked at the prevalence and distribution of transposable elements/ integrons in piggery farm samples. Present study identified the presence of six transposable elements namely Tn6763 (Accession number: OQ565300), Tn6764, (Accession number: OQ565299), Tn6765 (Accession number: OQ409902), Tn2003 (Accession number: OQ503494), Tn6072 (Accession number: OQ565298) and Tn6020 (Accession number: OQ503493) in piggery farm waste from India which are belongs to Enterobacteriaceae family. In a conjugative experiment, Klebsiella isolates carrying Tn6020 having the resistant phenotypes for nalidixic acid was used as donor cells while Escherichia coli DH5α Cells carrying chloramphenicol resistant plasmid was employed as recipient cells. Transconjugant bacterial colonies were shown to carry the Tn6020 transposable elements with both nalidixic acid (donor cell origin) and chloramphenicol (recipient cell origin) resistant antibiotic phenotypes. Given the presence of transposable elements in 21.4% of resistant Enterobacteriaceae strains, preventative measures are vital for avoiding the spread of mobile genetic resistance determinants in the piggery sector and to monitor their emergence.

Genome wide mining of SNPs and INDELs through ddRAD sequencing in Sahiwal cattle.

The study was conducted in Sahiwal cattle for genome wide identification and annotation of single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs) in Sahiwal cattle. The double digest restriction-site associated DNA (ddRAD) sequencing, a reduced representation method was used for the identification of variants at nucleotide level. A total of 1,615,211 variants were identified at RD10 and Q30 consisting of 1,480,930 SNPs and 134,281 INDELs with respect to the Bos taurus reference genome. The SNPs were annotated for their location, impact and functional class. The SNPs identified in Sahiwal cattle were found to be associated with a total of 26,229 genes. A total of 1819 SNPs were annotated for 209 candidate genes associated with different production and reproduction traits. The variants identified in the present study may be useful to strengthen the existing bovine SNP chips for reducing the biasness over the taurine cattle breeds. The diversity analysis provides the insight of the genetic architecture of the Sahiwal population Studied. The large genetic variations identified at the nucleotide level provide ample scope for implementing an effective and efficient breed improvement programme for increasing the productivity of Sahiwal cattle.

Japanese Encephalitis Virus Genotype III Strains Detection and Genome Sequencing from Indian Pig and Mosquito Vector.

Japanese encephalitis viruses (JEVs) are globally prevalent as deadly pathogens in humans and animals, including pig, horse and cattle. Japanese encephalitis (JE) still remains an important cause of epidemic encephalitis worldwide and exists in a zoonotic transmission cycle. Assam is one of the highly endemic states for JE in India. In the present study, to understand the epidemiological status of JE circulating in pigs and mosquito, particularly in Assam, India, molecular detection of JEV and the genome sequencing of JEV isolates from pigs and mosquitoes was conducted. The genome analysis of two JEV isolates from pigs and mosquitoes revealed 7 and 20 numbers of unique points of polymorphism of nucleotide during alignment of the sequences with other available sequences, respectively. Phylogenetic analysis revealed that the isolates of the present investigation belong to genotype III and are closely related with the strains of neighboring country China. This study highlights the transboundary nature of the JEV genotype III circulation, which maintained the same genotype through mosquito-swine transmission cycles.

Differentially expressed microRNAs in biochemically characterized FrieswalTM crossbred bull semen.

In addition to the transmission of paternal genome, spermatozoa also carry coding as well as noncoding microRNAs (miRNAs) into the female oocyte during the process of biological fertilization. Based on RNA deep sequencing, a total 28 number of differentially expressed miRNAs were cataloged in categorized FrieswalTM crossbred (Holstein Friesian X Sahiwal) bull semen on the basis of conception rate (CR) in field progeny testing program. Validation of selected miRNAs viz. bta-mir-182, bta-let-7b, bta-mir-34c and bta-mir-20a revealed that, superior bull semen having comparatively (p < .05) lower level of all the miRNAs in contrast to inferior bull semen. Additionally, it was illustrated that, bta-mir-20a and bta-mir-34c miRNAs are negatively (p < .01) correlated with seminal plasma catalase (CAT) activity and glutathione peroxidase (GPx) level. Interactome studies identified that bta-mir-140, bta-mir-342, bta-mir-1306 and bta-mir-217 can target few of the important solute carrier (SLC) proteins viz. SLC30A3, SLC39A9, SLC31A1 and SLC38A2, respectively. Interestingly, it was noticed that all the SLCs were significantly (p < .05) expressed at higher level in superior quality bull semen and they are negatively correlated (p < .01) with their corresponding miRNAs as mentioned. This study may reflect the role of miRNAs in regulating few of the candidate genes and thus may influence the bull semen quality traits.

Development of multiplex PCR assay for simultaneous detection of African swine fever, porcine circo and porcine parvo viral infection from clinical samples.

A diagnostic method for simultaneously detecting and distinguishing African Swine Fever (ASF), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV) in clinical specimens is critical for differential diagnosis, monitoring, and control in the field. Three primer pairs were designed and used to create a multiplex PCR assay. In addition, 356 porcine post mortem tissue samples from various parts of India's North Eastern region were tested by the developed multiplex PCR assay to demonstrate its accuracy. Using the designed primers, each of the ASF, PCV2 and PPV target genes was amplified, but no other porcine virus genes were detected. The assay's limit of detection was 102 copies/µl of PCV2, PPV, or ASFV. The detection of PCV2, PPV, and ASF in postmortem tissue samples revealed that they are co-circulating in India's North-Eastern region. The percentage positivity (PP) for PCV2, PPV and ASF single infection were 7.02% (25/356), 3.93% (14/356), and 3.37% (12/356), respectively, while the PP for PCV2& PPV co-infection was 2.80% (10/356), ASF & PCV2 co infection was 1.4% (5/356) and the ASF, PPV& PCV2 co-infection was1.40% (5/356). The results also indicate that the ASF can infect pigs alongside PCV and PPV.

Development of CD163 receptor-based enzyme-linked immunosorbent assay for diagnosis of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is an important economical disease in the global swine industry. The accurate detection of the PRRS virus (PRRSV) antigen is essential for the disease control and prevention programme. In this study, an indirect enzyme-linked immunosorbent test (PRRSVCD163-iELISA) was developed for the detection of the PRRSV antigen in samples of post-mortem swine tissue using the recombinant pig CD163 receptor protein as the capture ligand. The test was found to be specific for PRRSV, with no cross-reactions with other prevalent pig viral pathogens. The assay was validated by testing 217 post-mortem porcine tissue samples and the results were found to be satisfactory with a relative accuracy of 88.88%. Our assay is also quite precise, with intra- and inter-assay CVs of 6% and 10%, respectively. These findings imply that the PRRSVCD163-iELISA developed is capable of detecting the PRRSV antigen in swine post-mortem tissue samples. This research showed that porcine CD163, the PRRSV cellular receptor, can be exploited to build a diagnostic technique for the detection of PRRSV antigen. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03376-z.

Profiling of key heat shock proteins and their relationship with male sexual behavior and seminal characteristics in Kankrej (Bos indicus) breeding bulls during different seasons.

The goal of this study is to use indirect ELISA to determine the concentration of major heat shock proteins (Hsps) in Kankrej (Bos indicus) breeding bulls and their relationship with certain male phenotypic traits including sexual behavior, sperm quality, and bull fertility in different seasons. The seasonal fluctuation in the concentration of three major Hsps (60, 70, and 90) was determined using an indirect enzyme-linked immunosorbent assay (ELISA). According to the findings, Hsps levels are significantly higher during the summer season and are associated with both fresh and post-thawed semen quality traits in Kankrej breeding bulls. The better sexual behavior of bulls and seminal parameters of fresh or thawed semen was observed in the winter season together with the lower concentrations of HSPs. These could suggest negative association between HSPs with bull sexual behavior and seminal parameters. As a result, the concentration of Hsps in breeding bulls may be a useful indicator for determining fertility traits.

Porcine Circovirus type 2 infected myocardial tissue transcriptome signature.

The goal of this study was to compare the global gene expression profile in cardiac tissues of pig infected with porcine circovirus 2 (PCV2) to that of healthy cells. Since PCV2 infection causes severe cardiovascular lesions, the myocardial tissue model was chosen for this study. In High-throughput transcriptome analysis, DESeq2 and CLC genomics workbench analyses revealed a total of 196 significantly differentially expressed genes (DEGs) (p-value < 0.05). Furthermore, 194 transcripts were upregulated, while only two were downregulated (HSPA6 and DNAJA1), with fold changes ranging from 16.293 to -10.002. Among the KEGG canonical pathways targeted by the DEGs in the functional analysis, adrenergic signalling in cardiomyocytes, Cardiac Muscle Contraction, Hypertrophic Cardiomyopathy (HCM), and Dilated Cardiomyopathy (DCM) tends to be enriched. The differentially expressed highly connected (DEHC) biomarker genes in pathogenicity of PCV2 infection, such as LDB3, MYOZ2, CASQ2, TNNT2, MLC2V, MYBPC3, ACTC1, TCAP, TNNI3, TRDN, CSRP3, MYL3, RYR2, LMOD2, MYH7, etc., were identified using protein-protein interaction (PPI) network analysis. The study might provide detailed information on the dysregulated genes and biological pathways in infected myocardial tissues that may be essential for PCV2-related heart pathology.

Comparative CpG methylation kinetic patterns of cis-regulatory regions of heat stress-related genes in Sahiwal and Frieswal cattle upon persistent heat stress.

The kinetic patterns of CpG methylation of the cis-regulatory region of heat stress-related genes on exposed to heat stress (at 42 °C) between the Sahiwal and Frieswal cattle was compared in the present study. Using an in vitro whole blood culture model, cells were continuously exposed to heat stress (at 42 °C) for 6 h. Methylation levels of five genes, viz., GPX1, HSP70, HSP90, c-FOS, and JUN were estimated by SyberGreen-based quantitative methylation-specific PCR (qMSP) assay. CpG methylation kinetics at different time points of heat stress (0.5, 1, 2, 4, 6 h) were analyzed using mixed ANOVA. The initial methylation level, estimated at 37 °C, of HSP70 was significantly high in the Sahiwal breed. A significant (p<0.001) time-dependent hypomethylation of an antioxidant gene (GPX1) CpG islands was detected at the acute phase of the stress. Heat shock protein gene (HSP70) showed a similar CpG methylation kinetics where the hypomethylation was prominent from 1 h and persisted up to 4 h. The heat stress responses of both Sahiwal and Frieswal cattle were identical as there was no distinctiveness in the methylation kinetics of CpG islands of studied genes. The acclimatization of Frieswal cattle-a breed developed in India over the years to the tropical climatic conditions, maybe one of the reasons for this similarity. Thus, the present study results could pave a path to understand the molecular mechanism of heat stress and adaptation of indigenous and crossbred cattle populations to the changing scenario in tropical climate conditions.

Development of a RAPD marker-based classification criterion for quality semen production in Holstein crossbred bulls.

In cattle production systems, an intense selection pressure for production traits has resulted in the decline of fertility traits. To optimize an efficient reproduction system, the inclusion of both male and female fertility traits in the selection process is very much essential. RAPD (Random Amplified Polymorphic DNA) was developed as a molecular biology tool and has been extensively used, to study intra- and interspecific genetic diversity. The present study was undertaken to utilize RAPD primers to investigate the association between DNA markers and semen quality traits viz. Sperm concentration, total sperm count ejaculate and initial sperm motility and thereby to identify good/poor semen producers. DNA isolated from the blood samples of healthy bulls was subjected to RAPD-PCR. The multiple regression analysis followed by independent t test was carried out to identify suitable markers. Based on the results, only 12 bands were identified as marker suitable for any of the quality trait. This includes, OPA2 ~ 760, OPA2 ~ 700, OPA6 ~ 1,200, OPA9 ~ 400, OPA9 ~ 380, OPA12 ~ 970, OPA14 ~ 715, OPA14 ~ 605, OPA16 ~ 485, OPA17 ~ 860 and OPA18 ~ 480. Multiple regression analysis selected, OPA2 ~ 760 and OPA2 ~ 1,750 for sperm concentration and OPA2 ~ 760, OPA2 ~ 700, OPA9 ~ 620, OPA4 ~ 670 and OPA18 ~ 1,015 for total sperm count/ejaculate. But the t test revealed a significant association between OPA2 ~ 760 and total sperm count. Further, discriminant function analysis also identified this marker in the first step itself. The results of the present study can be exploited as a low-cost alternative strategy for identification of good /poor semen producers in crossbred bulls at an early age.

Comparative miRNA signatures among Sahiwal and Frieswal cattle breeds during summer stress.

MicroRNAs (miRNAs) are known to take part in different biological mechanisms, including biotic as well as abiotic cellular stresses. The present investigation was aimed to identify comparative expression profile of differentially expressed miRNAs among Sahiwal (Bos indicus) and Frieswal (Bos indicus × Bos taurus) cattle breeds during summer stress. Stress responses in animals were characterized by recording various physiological parameters, biochemical assays and expression profiling of heat shock protein 70 (Hsp70) during elevated environmental temperature. Ion Torrent-based deep sequencing as well as CLC-genomic analysis identified 322 and 420 Bos taurus annotated miRNAs among Sahiwal and Frieswal, respectively. A total 69 common miRNAs were identified to be differentially expressed during summer among the breeds. Out of the 69, a total 14 differentially expressed miRNAs viz. bta-mir 6536-2, bta-mir-2898, bta-mir-let-7b, bta-mir-425, bta-mir-2332, bta-mir-2478, bta-mir-150, bta-mir142, bta-mir-16a, bta-mir-2311, bta-mir-1839, bta-mir-1248-1, bta-mir-103-2 and bta-mir-181b were randomly selected for qRT-PCR-based validation. bta-mir-2898, bta-mir-6536-1, bta-mir-let-7b, bta-mir-2478, bta-mir-150, bta-mir-16a, bta-mir-2311, bta-mir-1032-b and bta-mir-181-b were significantly (p < 0.01) upregulated during summer among Frieswal in comparison to Sahiwal while, bta-mir 6536-2, bta-mir-2332, bta-mir142, bta-mir-1839 and bta-mir-1248-1 was significantly (p < 0.01) expressed at higher level in Sahiwal in contrast to Frieswal correlation coefficient analysis revealed that bta-mir(s)-150, 16a and 181b are negatively correlated (p < 0.05) with Hsp70 expression. Thus, this study identified that miRNA expression during summer stress can vary between the breeds which may reflect their differential post-transcriptional regulation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02608-4.

Expression pattern of bta-mir-2898 miRNA and their correlation with heat shock proteins during summer heat stress among native vs crossbred cattle.

Earlier studies identified the role of bta-mir-2898 in bovine. Our earlier study identified that, bta-mir-2898 can be over expressed in crossbred cattle during heat stress. Nevertheless the differential expression of bta-mir-2898 among native vs crossbred cattle during summer stress along with it's correlation with different heat shock proteins (HSPs) is not yet studied. In the present context, we studied the differential expression of bta-mir-2898 among Frieswal (Bos indicus x Bos taurus) and Sahiwal (Bos indicus) breeds of cattle during a range of environmental air temperatures and further investigated the correlation of bta-mir-2898 with different HSPs (HSP70, HSP90, HSP60. HSF, HSPB8 and HSP27). It was observed that, at peak air temperature the relative miRNA expression level (p < 0.05) of bta-mir-2898 was 3.4 ± 0.41 and 0.79 ± 0.22 among Frieswal and Sahiwal, respectively. We also observed significant levels (p < 0.05) of mRNA abundance of HSP70, HSP90, HSPB8 and HSP27 among the breeds. In all the cases Sahiwal found to exhibited higher level of HSPs in comparison to Frieswal. Studies revealed that the expression profile of bta-mir-2898 was negatively correlated with the expression of all the HSPs during thermal stress in post anti-mir2898 treated PBMC invitro cultured model originated from both Frieswal and Sahiwal cattle breeds. However, significantly (p < 0.05) higher negative correlations were observed between bta-mir-2898 and HSP70, HSP60 and HSPB8. Present findings highlighted the preliminary role of overexpressed bta-mir-2898 in cattle during thermal stress and its impact on different heat shock proteins.