Characterization and expression profiling of buffalo IFN-lambda family.

Interferon lambda (IFN-λ) is an important type III interferon triggered mainly by viral infection. IFN-λ binds to their heterodimeric receptors and signals through JAK-STAT pathways similar to type I IFN. In this study, we deduced the buffalo IFN-λ sequences through the polymerase chain reaction, and then studied IFN-λ's expression patterns in different tissues, and post induction with poly I:C and live MRSA using RT-qPCR. The full-length sequences of buffalo IFN-λ3, IFN-λ receptors, and a transcript variant of IFN-λ4 were determined. IFN-λ1 is identified as a pseudogene. Virus response elements and a recombination hotspot factor was observed in the regulatory region of IFN-λ. The IFN-λ3 expressed highest in lungs and monocytes but IFN-λ4 did not. The expression of Interferon Lambda Receptor 1 was tissue specific, while Interleukin 10 Receptor subunit beta was ubiquitous. Following poly I:C induction, IFN-λ3 expression was primarily observed in epithelial cells as opposed to fibroblasts, displaying cell type-dependent expression. The cytosolic RNA sensors were expressed highest in endometrial epithelial cells, whereas the endosomal receptor was higher in fibroblasts. 2',5'-oligoadenylate synthetase expressed higher in fibroblasts, myxoma resistance protein 1 and IFN-stimulated gene 56 in epithelial cells, displaying cell-specific antiviral response of the interferon stimulated genes (ISGs). The endometrial epithelial cells expressed IFN-λ3 after live S. aureus infection indicating its importance in bacterial infection. The induction of IFN-λ3 was S. aureus isolate specific at the same multiplicity of infection (MOI). This study elucidates the IFN-λ sequences, diverse expression patterns revealing tissue specificity, and specificity in response to poly I:C and bacterial stimuli, emphasising its crucial role in innate immune response modulation.

Antimicrobial and antibiofilm effect of cannabinoids from Cannabis sativa against methicillin-resistant Staphylococcus aureus (MRSA) causing bovine mastitis.

Antimicrobial resistance (AMR) poses a serious threat to human, animal, and plant health on a global scale. Search and elimination techniques should be used to effectively counter the spread of methicillin-resistant Staphylococcus aureus (MRSA) infections. With only a few novel drugs in clinical development, the quest for plant-based alternatives to prevent the spread of antibiotic resistance among bacteria has accelerated. Treatment of MRSA infections is challenging owing to rapidly emerging resistance mechanisms coupled with their protective biofilms. In the present research, we examined the antibacterial properties of ten plant-derived ethanolic leaf extracts. The most effective ethanolic leaf extract against MRSA in decreasing order of zone of inhibition, Cannabis sativa L. > Syzygium cumini > Murraya koenigii > Eucalyptus sp. > while Aloe barbadensis, Azadirachta indica, had very little impact. Mangifera indica, Curcuma longa, Tinospora cordifolia, and Carica papaya did not exhibit inhibitory effects against MRSA; hence, Cannabis was selected for further experimental study. The minimal inhibitory concentration (MIC) of Cannabis sativa L. extract was 0.25 mg ml-1 with 86% mortality. At a sub-MIC dosage of 0.125 mg ml-1, the biofilm formation was reduced by 71%. The two major cannabinoids detected were cannabidiol and delta-9-tetrahydrocannabinol (Δ9-THC), which were majorly attributed to substantial inhibitory action against MRSA. The time-kill kinetics demonstrated a bactericidal action at 4 MIC over an 8-20-h time window with a 90% reduction in growth rate. The results from SEM, and light microscopy Giemsa staining revealed a reduction in cells in the treated group with increased AKP activity, indicating bacterial cell membrane breakdown. These findings suggested cannabinoids may be a promising alternative to antibiotic therapy for bovine biofilm-associated MRSA.

Expression and characterization of novel chimeric endolysin CHAPk-SH3bk against biofilm-forming methicillin-resistant Staphylococcus aureus.

The continuous evolution of antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) due to the misuse of antibiotics lays out the need for the development of new antimicrobials with higher activity and lower resistance. In this study, we have expressed novel chimeric endolysin CHAPk-SH3bk derived from LysK to investigate its antibacterial activity against planktonic and biofilm-forming MRSA. The molecular docking and MD simulation results identified critical amino acids (ASP47, ASP56, ARG71, and Gly74) of CHAPk domain responsible for its catalytic activity. Chimeric endolysin CHAPk-SH3bk showed an effective binding to peptidoglycan fragment using 14 hydrogen bonds. The in-vitro antibacterial assays displayed higher activity of CHAPk against planktonic MRSA with 2-log10 reduction in 2 h. Both CHAPk and CHAPk-SH3bk displayed bactericidal activity against MRSA with ∼4log10 and ∼3.5log10 reduction in 24 h. Biofilm reduction activity displayed CHAPk-SH3bk reduced 33 % and 60 % of hospital-associated ATCC®BAA-44™ and bovine origin SA1 respectively. The CHAPk treatment reduced 47 % of the preformed biofilm formed by bovine-origin MRSA SA1. This study indicates an effective reduction of preformed MRSA biofilms of human and animal origin using novel chimeric construct CHAPk-SH3bk. Stating that the combination and shuffling of different domains of phage endolysin potentially increase its bacteriolytic effectiveness against MRSA.

Development of B cell epitopes-based enzyme linked immune sorbent assay for detection of bovine anti-Mullerian hormone.

The present study aimed to generate antibodies against predicted B cell epitopic peptides encoding bAMH for developing different ELISA models. Sandwich ELISA was determined to be an excellent technique for assessing bAMH in bovine plasma based on sensitivity tests. The assay's specificity, sensitivity, inter- and intra-assay CV, recovery %, Lower limit of quantification (LLOQ), and Upper limit of quantification (ULOQ) were determined. The test was selective since it did not bind to AMH-related growth and differentiation factors (LH and FSH) or non-related components (BSA, progesterone). The intra-assay CV was 5.67%, 3.12%, 4.94%, 3.61% and 4.27% for 72.44, 183.11, 368.24, 522.24 and 732.25 pg/ml AMH levels, respectively. At the same time, the inter-assay CV was 8.77%, 7.87%, 4.53%, 5.76% and 6.70% for 79.30, 161.27, 356.30, 569.33 and 798.19 pg/ml AMH levels, respectively. The average (Mean ± SEM) recovery percentages were 88-100%. LLOQ was 5 pg/ml and ULOQ at 50 µg/ml (CV < 20%). In conclusion, we developed a new highly sensitive ELISA against bAMH using epitope specific antibodies.

Adaptive Selection in the Evolution of Aquaglyceroporins in Mammals.

Aquaporins (AQPs) are integral membrane proteins responsible for water transport across cellular membranes in both prokaryotes and eukaryotes. A subfamily of AQPs, known as aquaglyceroporins (AQGPs), facilitate the transport of small solutes such as glycerol, water, and other solutes across cellular membranes. These proteins are involved in a variety of physiological processes, such as organogenesis, wound healing, and hydration. Although AQPs have been studied extensively in different species, their conservation patterns, phylogenetic relationships, and evolution in mammals remain unexplored. In the present study, 119 AQGP coding sequences from 31 mammalian species were analysed to identify conserved residues, gene organisation, and most importantly, the nature of AQGP gene selection. Repertoire analysis revealed the absence of AQP7, 9, and 10 genes in certain species of Primates, Rodentia, and Diprotodontia, although not all three genes were absent in a single species. Two Asparagine-Proline-Alanine (NPA) motifs located at the N- and C-terminal ends, aspartic acid (D) residues, and the ar/R region were conserved in AQP3, 9, and 10. Six exons encoding the functional MIP domain of AQGP genes were found to be conserved across mammalian species. Evolutionary analysis indicated signatures of positive selection in AQP7, 9, and 10 amongst different mammalian lineages. Furthermore, substitutions of certain amino acids located close to critical residues may alter AQGP functionality, which is crucial for substrate selectivity, pore formation, and transport efficiency required for the maintenance of homeostasis in different mammalian species.

Production of MSTN Gene-Edited Embryos of Buffalo Using the CRISPR/Cas9 System and SCNT.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and somatic cell nuclear transfer (SCNT) have been used to produce genome-edited farm animal species for improved production and health traits; however, these tools are rarely used in the buffalo and can play a pivotal role in milk and meat production in tropical and subtropical countries. In this study, we aimed to produce myostatin (MSTN) gene-edited embryos of the Murrah buffalo using the CRISPR/Cas9 system and SCNT. For this, fibroblast cells were electroporated with sgRNAs carrying all-in-one CRISPR/Cas9 plasmids targeting the first exon of the MSTN gene. Following puromycin selection, single-cell clonal populations were established and screened using the TA cloning and Sanger sequencing methods. Of eight single-cell clonal populations, one with a monoallelic and another with a biallelic heterozygous gene editing event were identified. These two gene-edited clonal cell populations were successfully used to produce blastocyst-stage embryos using the handmade cloning method. This work establishes the technical foundation for generation of genome-edited cloned embryos in the buffalo.

Evaluation of Virulence, Antimicrobial Resistance and Biofilm Forming Potential of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Bovine Suspected with Mastitis.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that poses a significant threat in cases of chronic mastitis in dairy animals. The ability of MRSA to persist in the host is attributed to various virulence factors, genes encoding surface adhesins, and determinants of antibiotic resistance, which provide it a survival advantage. This investigation focused to determine the virulence factors, antimicrobial resistance (AMR) profile and biofilm production potential of 46 MRSA isolates from 300 bovine mastitis milk samples. The AMR profile revealed a high level of resistance, with 46 and 42 isolates resistant to cefoxitin and oxacillin, respectively, followed by 24 and 12 isolates resistant to lomefloxacin and erythromycin, respectively. Only 2 isolates resistant to tetracycline and none were resistant to chloramphenicol. The study also evaluated various virulence factors such as coa (n = 46), nuc (n = 35) hlg (n = 36), pvl (n = 14), tsst-1(n = 28) spa (n = 39) and enterotoxin genes sea (n = 12) and seg (n = 28) and identified antibiotic resistance determinants mecA and blaZ in 46 and 27 isolates, respectively. Intercellular adhesion genes icaA and icaD were present in 40 and 43 isolates, respectively and surface adhesion genes ebps, fnbpA, eno, sasG, cna, and bap were found in 43, 40, 38, 26, 21 and 1 isolates, respectively. Microtiter plate (MTP) assay revealed that 29 MRSA isolates were capable of producing biofilms, whereas 17 were not. Biofilms producing MRSA isolates possessed adhesion genes, virulence factors, toxin genes and AMR genes that may act synergistically towards a chronic disease progression, illness and severe damage to the udder, which generally last for several months and very challenging to cure.

Potential status of A1 and A2 variants of bovine beta-casein gene in milk samples of Indian cattle breeds.

This study aimed to determine the polymorphism in 7th exon of beta-casein gene (CSN2) gene in seven domestic (Kosali, Tharparkar, Gangatiri, Sahiwal, Gir, Khariar, Motu) and two exotic cattle breeds (Jersey and Holstein-Friesian). Genomic DNA was extracted from 1000 milk samples, and the C > A polymorphism in CSN2 was determined using the tetra-primer amplification refractory mutation system-polymerase chain reaction method. In all Indigenous cattle breeds, the mean frequency of A1A2 and A2A2 genotypes was 0.19 and 0.80, respectively. The A1A1 genotype was absent in all seven domestic cattle breeds. The frequency of the A2A2 genotype was highest in the Gir breed (0.93). However, the Sahiwal, Tharparkar, and Motu breeds also had a higher frequency of A2A2 genotype compared to other breeds. In contrast, Gangatiri breed of India showed lowest frequency of A2A2 genotype. The mean A1 and A2 allele frequency was 0.09 and 0.91, respectively. In exotic breeds, the mean frequencies of the A1A1, A1A2, and A2A2 genotypes were 0.42, 0.55, and 0.03, respectively. Similarly, the mean A1 and A2 allele frequency was 0.69 and 0.31, respectively. This study suggests the high potential of Gir, Sahiwal, Tharparkar, and Motu cattle for A2 milk production since they carry a favorable A2 genotype.

Development of an on-site lateral flow immune assay based on mango leaf derived colloidal silver nanoparticles for rapid detection of Staphylococcus aureus in milk.

In order to ensure food safety, screening food samples for the presence of pathogens has been categorised as a legal testing item throughout the globe. One of the most prevalent zoonotic bacteria transmitted through dairy milk is Staphylococcus aureus. Given the limitations of the conventional detection methods, in the current study we desigined a competitive lateral flow immune assay (LFIA) using colloidal silver nanoparticles derived from mango leaves for the detection of Staphylococcus aureus in cow milk. SpA, a recombinant protein of Staphylococcus aureus, was used to raised hyperimmune sera used for developing the assay followed by conjugation with the synthesized nanoparticles. To increase the specificity of the assay, the milk samples were prenriched with selective agar exclusively require for Staphyloccocus aureus. The assay was found to be completed within 7-8 h by observing test and control lines in LFIA strips. The developed assay was found to specifically detect the bacteria as low as 1000 cfu/ml of milk samples. With a total 230 number of raw and clinical mastitis milk samples, the assay was validated and achieved relative accuracy, specificity, and sensitivity values of 97.39, 98.03, and 96.1%, respectively. The developed LFIA, which uses economically feasible and stable silver nanoparticles derived from mango leaves, has the potential for routine screening of milk samples for the presence of Staphylococcus aureus, especially in low-resource settings, allowing for early diagnosis, which facilitates effective treatment for the dairy animals and prevents the transmission of the disease in consumers.

CRISPR/cas9 cassette targeting Escherichia coli blaCTX-M specific gene of mastitis cow milk origin can alter the antibiotic resistant phenotype for cefotaxime.

CTX-M beta-lactamases are one of the most important extended spectrum beta-lactamase (ESBL) resistance enzymes found in E. coli. In the present study, 59% of E. coli isolates from mastitis cow milk were reported to be positive for ESBL types. The prevalence of beta-lactam (ß-lactam) antibiotic resistance was reported to be 84%, 72.7%, 52.27%, 50%, and 45.4% for cefotaxime, cefepime, cefuroxime, oxacillin, and cephalexine, respectively. The blaCTX-M gene was found in 65% (n = 17) of the E. coli isolates when they were genotyped. Further, the use of a CRISPR/cas9 cassette to target the E. coli blaCTX-M gene revealed changes in antibiotic phenotypes for cefotaxime.

Novel aadA5 and dfrA17 variants of class 1 integron in multidrug-resistant Escherichia coli causing bovine mastitis.

Mobile genetic elements (MGEs) are associated with the emergence of multidrug resistance in extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. This study explores the role of class 1 integrons and IS26 elements in breaching taxonomic barriers. A total of 110 E. coli bacteria were isolated from 300 clinical mastitis milk samples. The 98% E. coli isolates were extended-spectrum beta-lactamase- producers. About 83% of these isolates carried co-resistance for fluoroquinolones. The co-existence of (extended-spectrum beta-lactamase + quinolone resistance determining region mutations) and (extended-spectrum beta-lactamase + plasmid-mediated quinolone resistance genes) was found in 76% and 44% of isolates, respectively. The MGEs were detected in 88% of isolates with IS26 in 82% and class 1 integrase in 40% of isolates. The types of class 1 integron gene cassettes detected includes dfrA7, (dfrA17 + aadA5), and (dfrA1 + aadA1). We discovered 2 and 4 novel variants of the dfrA17 and aadA5 genes, respectively. We report a variant of aadA5 with mutation E235G in the Indian subcontinent earlier reported only in a human clinical isolate from Belgium. About 19 isolates carried IS26 linked to integrase gene intI1 with an internal deletion of 265 bp in the 5`CS of integrase gene intI1, earlier reported only in E. coli ST131 isolates from human clinical, wastewater samples. This study suggests intercontinental dissemination of antibiotic resistant genes (ARGs) across different microbiomes via mobile genetic elements. KEY POINTS: • The role of mobile genetic elements in the emergence of multidrug-resistant E. coli in bovine mastitis. • Novel variants of the aadA5 (aminoglycoside adenyl transferase) and dfrA17 (dihydrofolate reductase) genes were identified in pathogenic E. coli isolated from bovine mastitis in class 1 integron gene cassette. • Sequence analysis of mobile genetic components revealed the physical connection between IS26 and intI1 genes with an internal deletion in 5'CS of class 1 integrase.

Beta-defensins as marker for male fertility: a comprehensive review†.

Bovine male fertility in animals has a direct impact on the productivity of dairy herds. The epididymal sperm maturations involve extensive sperm surface modifications to gain the fertilizing ability, especially by absorptions of the plethora of biomolecules, including glycoprotein beta-defensins (BDs), enzymes, organic ions, protein, and phospholipids. Defensins are broad-range nonspecific antimicrobial peptides that exhibit strong relations with innate and adaptive immunity, but their roles in male fertility are relatively recently identified. In the course of evolution, BD genes give rise to different clusters with specific functions, especially reproductive functions, by undergoing duplications and nonsynonymous mutations. BD polymorphisms have been reported with milk compositions, disease resistance, and antimicrobial activities. However, in recent decades, the link of BD polymorphisms with fertility has emerged as an appealing improvement of reproductive performance such as sperm motility, membrane integrity, cervical mucus penetration, evading of uterus immunosurveillance, oviduct cell attachment, and egg recognition. The reproductive-specific glycosylated BD class-A BDs (CA-BDs) have shown age- and sex-specific expressions in male reproductive organs, signifying their physiological pleiotropism, especially in the sperm maturation and sperm transport in the female reproductive tract. By considering adult male reproductive organ-specific BD expressions, importance in sperm functionalities, and bioinformatic analysis, we have selected two bovine BBD126 and BBD129 genes as novel potential biomarkers of bovine male fertility. Despite the importance of BDs, however, genomic characterization of most BD genes across most livestock and nonmodel organisms remains predictive/incomplete. The current review discusses our understanding of BD pleiotropic functions, polymorphism, and genomic structural attributes concerning the fertilizability of the male gamete in dairy animals.

Analysis of amplification and association polymorphisms in the bovine beta-defensin 129 (BBD129) gene revealed its function in bull fertility.

ß-defensins are adsorbable on the sperm surface in the male reproductive tract (MRT) and enhance sperm functional characteristics. The beta-defensin 129 (DEFB129) antimicrobial peptide is involved in sperm maturation, motility, and fertilization. However, its role in bovine fertility has not been well investigated. This study examines the relationship between the bovine BBD129 gene and Bos indicus x Bos taurus bull fertility. The complete coding sequence of BBD129 mRNA was identified by RNA Ligase Mediated-Rapid Amplification of cDNA End (RLM-RACE) and Sanger sequencing methodologies. It consisted of 582 nucleotides (nts) including 5' untranslated region (UTR) (46nts) and 3'UTR (23nts). It conserves all beta-defensin-like features. The expression level of BBD129 was checked by RT-qPCR and maximal expression was detected in the corpus-epididymis region compared to other parts of MRT. Polymorphism in BBD129 was also confirmed by Sanger sequencing of 254 clones from 5 high fertile (HF) and 6 low fertile (LF) bulls at two positions, 169 T > G and 329A > G, which change the S57A and N110S in the protein sequence respectively. These two mutations give rise to four types of BBD129 haplotypes. The non-mutated TA-BBD129 (169 T/329A) haplotype was substantially more prevalent among high-fertile bulls (P < 0.005), while the double-site mutated GG-BBD129 (169 T > G/329A > G) haplotype was significantly more prevalent among low-fertile bulls (P < 0.005). The in silico analysis confirmed that the polymorphism in BBD129 results in changes in mRNA secondary structure, protein conformations, protein stability, extracellular-surface availability, post-translational modifications (O-glycosylation and phosphorylation), and affects antibacterial and immunomodulatory capabilities. In conclusion, the mRNA expression of BBD129 in the MRT indicates its region-specific dynamics in sperm maturation. BBD129 polymorphisms were identified as the deciding elements accountable for the changed proteins with impaired functionality, contributing to cross-bred bulls' poor fertility.

Global epidemiology of CTX-M-type ß-lactam resistance in human and animal.

CTX-M ESBL are widely found in animal and human infections. For better understanding of CTX-M variations and epidemiology, a total of 2210 CTX-M sequences were retrieved from NCBI as at 20 December 2020. The maximum incidences of CTX-M were reported in China (n = 508), USA (n = 354) and Japan (n = 180). Single amino acid substitution in the domain region of CTX-M ESBL lead to survival benefits to the bacteria. A total of 31 different variations were found of which D240G was the most common followed by A77V and V103I substitutions. The variations in CTX-M enzymes were explained continent-wise revealing the maximum variation reported in America followed by Asia and Europe of which D240G substitution was the most prevalent. India contained only three variations (E166A, P167S D240G) found in New Delhi, Karnataka, West Bengal and Tamil Nadu. The P167 and D240 were under strong positive selection with dN/dS calculation.

Virulence and enterotoxin gene profile of methicillin-resistant Staphylococcus aureus isolates from bovine mastitis.

Bovine mastitis is a major infectious disease affecting dairy animals resulting in enormous economic losses, prolonged antibiotic treatment, reduced milk yield and death of livestock. Emergence of Methicillin-resistant Staphylococcus aureus (MRSA) among bovine mastitis is matter of concern for animal health and dairy industry. The present study was conducted to detect the distribution of virulence and enterotoxin genes among MRSA isolates from bovine mastitis. Out of 500 milk samples, 126 isolates were identified as Staphylococcus and from these only 56 were S. aureus. S.aureus were resistant to cefoxitin (75%), ceftazidime (75%), amoxicillin (71.4%), cefodaxime (67.8%), cefepime (66.1%), oxacillin (64.3%), norfloxacin (60.7%) and gentamicin (58.9%). Only 42 isolates were identified as MRSA strains among staphylococci isolates. MRSA were harbouring virulence genes; mecA (100%), coa (100%) and nuc (100%). The other virulence factors such as hlg (80.9%, 34/42), pvl (47.6%, 20/42) and spa (92.8%, 39/42) were also reported. Molecular characterisation of enterotoxin genes revealed that out of 42 tested isolates 11 were found negative (26%) for any enterotoxin gene whereas 7 (16.6%), 6 (14.3%), 18 (42.8%), 1 (2.3%), 26 (61.9%),27(64.2%),3 (7.1%) were found positive for sea, seb, sec, sed, seg, sei, and seq enterotoxin respectively.

Structural Elucidation of Inter-CARD Interfaces involved in NOD2 Tandem CARD Association and RIP2 Recognition.

Nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) recognizes the muramyl dipeptide and activates the NF-κB signaling cascade following its interaction with receptor-interacting protein 2 (RIP2) via caspase recruitment domains (CARDs). The NOD2-RIP2 interaction is not understood well due to inadequate structural information. Using comparative modeling and multimicrosecond timescale molecular dynamics simulations, we have demonstrated the association of NOD2-CARDs (CARDa-CARDb) and their interaction with RIP2CARD. Our results suggest that a negatively charged interface of NOD2CARDa and positively charged type-Ia interface of NOD2CARDb are crucial for CARDa-CARDb association and the type-Ia interface of NOD2CARDa and type-Ib interface of RIP2CARD predicted to be involved in 1:1 CARD-CARD interaction. Moreover, the direct interaction of NOD2CARDb with RIP2CARD signifies the importance of both CARDs of NOD2 in RIP2-mediated CARD-CARD interaction. Altogether, the structural results could help in understanding the underlying molecular details of the NOD2-RIP2 association in higher and lower eukaryotes.

PU.1 is involved in the transcriptional up-regulation of RNA and DNA sensing pathway genes in buffalo fibroblasts.

PU.1, CEBPA, and CEBPB are Lineage Determining Transcription Factors (LDTFs) that play roles in biological processes such as cell differentiation and the immune system regulation including the innate immune pathways. The roles of these LDTFs in the innate RNA and DNA sensing pathways have received little attention. We show that in buffalo fibroblasts, PU.1 causes the mRNA up-regulation of the RNA and DNA sensors such as RIG-I (65.1 fold), MDA5 (20.4 fold), IFI16-l (8.0 fold), and cGAS (60.5 fold) while CEBPA does the same but to a lesser extent (RIG-I-26.4 fold, MDA5-10.8 fold, IFI16-l- 3.3 fold and cGAS-8.6 fold). CEBPB does not appear to have a role in the up-regulation of these genes. PU.1 expression also primes the cells to develop a strong immune response against the dsRNA virus mimic polyinosinic:polycytidylic acid (poly I:C) by significantly up-regulating Interferon-ß (14.9 fold change with p-value <0.0001). CEBPA up-regulates Interferon-ß to a lower level than PU.1 (4.7 fold change with p-value 0.0024), whereas CEBPB exhibits non-significant up-regulation (2.1 fold with p-value of 0.1449). As PU.1 robustly up-regulates the nucleic acid sensing pathways, it can prove to be useful in improving the defence against viruses that can cause losses to animal husbandry.

Characterization of Anti-Müllerian Hormone (AMH) Gene in Buffaloes and Goats.

The Anti-Müllerian Hormone (AMH) is a member of the transforming growth factor beta (TGF-ß) superfamily, playing a significant role in cell proliferation, differentiation and apoptosis. In females, AMH is secreted throughout their reproductive life span from ovaries, whereas in males it is secreted by gonadal cells at a very early stage of testicular development. AMH is a promising marker of ovarian reserve in women and can be used to measure the female reproductive lifespan. In the present study, we cloned and sequenced the GC rich AMH gene from Indian riverine buffalo (Bubalus bubalis) and goat (Capra hircus). Obtained sequences were compared to the AMH sequences of other mammals, and corresponding amino acid sequences revealed that the caprine and bovine AMH sequences are more closely related to each other than to those of other mammals. Furthermore, we analyzed the chromosomal localization of AMH genes in mammalian species to understand potential syntenic relationship. The AMH gene is localized between the sequences for the SF3A and JSRP1 genes and maintains this precise location in relation to other nearby genes. The dN/dS ratio of AMH gene did not indicate any pressure for either positive or negative selection; thus, the physiological function of the AMH gene in the reproduction of these two ruminant species remains very vital. Similar to other mammals, the AMH gene may be an important indicator for regulating female reproductive biology function in bovine, cetacean, caprine, and camelidae.

In-silico Analysis of Angiotensin-converting Enzyme 2 (ACE2) of Livestock, Pet and Poultry Animals to Determine its Susceptibility to SARS-CoV- 2 Infection.

BACKGROUND: Novel coronavirus SARS-CoV-2 is responsible for the COVID-19 pandemic. It was first reported in Wuhan, China, in December 2019, and despite the tremendous efforts to control the disease, it has now spread almost all over the world. The interaction of SARSCoV- 2spike protein and its acceptor protein ACE2 is an important issue in determining viral host range and cross-species infection, while the binding capacity of spike protein to ACE2 of different species is unknown. OBJECTIVE: The present study has been conducted to determine the susceptibility of livestock, poultry and pets to SARS-CoV-2. METHODS: We evaluated the receptor-utilizing capability of ACE2s from various species by sequence alignment, phylogenetic clustering and protein-ligand interaction studies with the currently known ACE2s utilized by SARS-CoV-2. RESULT: In-silico study predicted that SARS-CoV-2 tends to utilize ACE2s of various animal species with varied possible interactions. The probability of the receptor utilization will be greater in horse and poor in chicken, followed by ruminants. CONCLUSION: Present study predicted that SARS-CoV-2 tends to utilize ACE2s of various livestock and poultry species with greater probability in equine and poor in chicken. The study may provide important insights into the animal models for SARS-CoV-2 and animal management for COVID- 19 control.

Functional milk proteome analysis of genetically diverse goats from different agro climatic regions.

Goat milk, a choice of substitution to mother's milk for its composition, fulfils nutritional requirement of infants, pregnant mothers and older people. The present study was carried out to unravel the milk proteome profiles from geographically and genetically diverse goat breeds by gel based 2DE and nLC-MS/MS. A total of 1307 functional proteins comprising casein and other low abundance proteins were identified. Gene annotations revealed that the majority of the proteins were involved in binding function, catalytic activity and structural molecules and localised in nucleus and membrane. The distinguished proteins were involved in 144 KEGG pathways in information processing, metabolism, cellular process, organismal systems and diseases. The large number of proteins and peptides including bioactive peptides were reported from goat milk from diverse agro-climatic regions of India indicating their significant potential for human health applications. SIGNIFICANCE: Goat milk in India is used in various Ayurvedic formulations to treat a number of ailments and allergies as well as for nutraceutical formulations. The study identifies milk protein variants both at protein and DNA level and subsequent identification of proteins by 2DE and nLC-MS/MS resulting in a proteome comprising of 1307 proteins. The specific proteins and peptides having significant role in immune regulation, disease pathways, cellular growth and metabolism have been identified. The results contribute to goat milk protein and peptide database which is very limited. We identified proteins for specific functional categories and associated them with different pathways for studying functional diversity of goat milk proteins. The proteins and peptides identified can be used for multiple human health application.