Phylogenetic genotyping, virulence genes and antimicrobial susceptibility of Escherichia coli isolates from cases of bovine mastitis.

The study described in this research communication used phylogenetic genotyping to identify virulence genes and antimicrobial susceptibility in Escherichia coli recovered from cases of bovine mastitis. From 385 mastitic milk samples, 30 (7.8%) isolates were confirmed as E. coli. Most isolates (80%) belonged to phylo-group A. These 30 E. coli isolates were also screened for 11 different virulence genes. The majority of isolates (63%) harbored no virulence gene. Only 11 (37%) isolates tested positive for two virulence genes, either the iron uptake gene iucD in 3 (10%) isolates or the serum resistance gene traT in 2 (7%) isolates or both traT and iucD in 6 (20%) isolates. The E. coli isolates showed highest susceptibility to gentamicin, meropenem, and pipracillin. Most isolates were resistant to ampicillin, cefotaxime and streptomycin. This study suggests that mastitis causing E. coli might originate from commensal bacteria and that the presence of these virulence genes, common in extra-intestinal pathogenic E. coli (ExPEC) strains could be attributed to high genetic variability of mastitis-causing E. coli.

Folic acid supplementation regulates milk production variables, metabolic associated genes and pathways in perinatal Holsteins.

Perinatal period is the critical time in dairy cattle due to negative energy balance and high milk production stress. Being a key role in biosynthesis and methylation cycle, folic acid is considered essential for lactational and metabolic performance in dairy cattle. Thus, the current study was designed to evaluate the effect of folic acid supplementation on milk production phenotypic traits in periparturient cows. Transcriptomic screening was performed for milk production and metabolism-associated differentially expressed genes. The 123 cows having similar parity, weight and expected date of calving were randomly selected and divided into three groups; A (n = 41, folic acid 240 mg/500 kg cow/day), B (n = 40, FA 120 mg/500 kg cow/day) and C (Control, n = 42). Folic acid was supplemented for 21 days (14 days pre- and seven days post-calving), and three samples of blood lymphocytes were taken on day seven post-calving from each folic acid-treated and control group. In addition, the milk samples for each folic acid-treated group have been collected at 2nd, 3rd and 4th month of lactation. The increase in average milk yield noticed in group B were significantly (p-value < .05) higher than C and A. However, the data showed no noteworthy differences for milk fat and milk protein among the three groups. The transcriptomic analysis revealed that folic acid treatment regulated many key metabolic-related genes (DGAT2, ALOX5, LAP3, GPAT3, GGH, ALDOA, TKT) and pathways (glycolysis, folate biosynthesis, glutathione metabolism, etc.) in periparturient dairy cattle. It was concluded from the above findings that 120 mg/500 kg of folic acid quantity could be considered as a standard during the periparturient period to enhance the milk production performance of dairy cows. The transcriptomic profile revealed several metabolic and milk production-associated genes which could be a useful addition to the marker selection for the enhancement of metabolism and milk production of periparturient dairy cows.

Significant genetic effects of JAK2 and DGAT1 mutations on milk fat content and mastitis resistance in Holsteins.

Improving the production traits and resistance against mastitis in dairy cattle is a challenge for animal scientists across the globe. The present study was designed to investigate the genetic effects of single nucleotide polymorphisms (SNPs) in Janus kinase 2 (JAK2) and diacylglycerol acyltransferase (DGAT1) genes with production and mastitis-related traits. Four SNPs in JAK2 and one in DGAT1 were analyzed through Chinese Cow's SNPs Chip-I (CCSC-I) and genotyped in a population of 312 Chinese Holsteins. Our findings demonstrated that milk fat percentage, somatic cell count (SCC), somatic cell score (SCS), serum cytokines interleukin 6 (IL-6) and interferon gamma (IFN-γ) showed significant associations (P < 0.05) with at least one or more identified SNPs. Consequently, the analysis based on haplotypes amongst the SNPs in JAK2 revealed noteworthy (P < 0.05) association with SCC and IL-6. Collectively, our results verified the pleiotropic ability of detected SNPs in bovine JAK2 and DGAT1 for milk fat percentage as well as mastitis-related traits. The significant SNPs in both the genes could serve as powerful genetic markers to minimize mastitis risk. In addition, besides SCC and SCS, the IFN-γ and IL-6 could also be used as indicators of improved genetic resistance against mastitis.

Unmapped reads from cattle RNAseq data: A source for missing and misassembled sequences in the reference assemblies and for detection of pathogens in the host.

Usually, reads from transcriptome sequencing data unmapped to the target species' reference genome are disregarded. A recent RNAseq project on the new fatal disease Bovine Neonatal Pancytopenia had indicated an unexplained immune response signature to a double-stranded RNA virus. To unravel its background, contigs were de novo assembled from unmapped RNAseq reads and aligned against the bovine genome assemblies and multispecies NCBI databases. Lack of genuine virus sequence contigs rejected the hypothesis of a live virus being causal for the unexplained immune response. Alignment data also demonstrated incomplete bovine reference genome assemblies. In addition, we found that several parasite and virus genome reference assemblies in NCBI were contaminated with bovine DNA and confirmed recombination of bovine DNA into BVD virus strains. Exploring unmapped reads can extract useful biological information regarding the presence of microorganisms and can highlight issues with reference genome assemblies of host and pathogen species.

Polymorphisms in epigenetic and meat quality related genes in fourteen cattle breeds and association with beef quality and carcass traits.

Improvement for carcass traits related to beef quality is the key concern in beef production. Recent reports found that epigenetics mediates the interaction of individuals with environment and nutrition. The present study was designed to analyze the genetic effect of single nucleotide polymorphisms (SNPs) in seven epigenetic-related genes (DNMT1, DNMT3a, DNMT3b, DNMT3L, Ago1, Ago2, and HDAC5) and two meat quality candidate genes (CAPN1 and PRKAG3) on fourteen carcass traits related to beef quality in a Snow Dragon beef population, and also to identify SNPs in a total of fourteen cattle populations. Sixteen SNPs were identified and genotyped in 383 individuals sampled from the 14 cattle breeds, which included 147 samples from the Snow Dragon beef population. Data analysis showed significant association of 8 SNPs within 4 genes related to carcass and/or meat quality traits in the beef populations. SNP1 (13154420A>G) in exon 17 of DNMT1 was significantly associated with rib-eye width and lean meat color score (p<0.05). A novel SNP (SNP4, 76198537A>G) of DNMT3a was significantly associated with six beef quality traits. Those individuals with the wild-type genotype AA of DNMT3a showed an increase in carcass weight, chilled carcass weight, flank thicknesses, chuck short rib thickness, chuck short rib score and in chuck flap weight in contrast to the GG genotype. Five out of six SNPs in DNMT3b gene were significantly associated with three beef quality traits. SNP15 (45219258C>T) in CAPN1 was significantly associated with chuck short rib thickness and lean meat color score (p<0.05). The significant effect of SNP15 on lean meat color score individually and in combination with each of other 14 SNPs qualify this SNP to be used as potential marker for improving the trait. In addition, the frequencies of most wild-type alleles were higher than those of the mutant alleles in the native and foreign cattle breeds. Seven SNPs were identified in the epigenetic-related genes. The SNP15 in CAPN1 could be used as a powerful genetic marker in selection programs for beef quality improvement in the Snow Dragon Beef population.

Genetic effects of single nucleotide polymorphisms in JAK2 and STAT5A genes on susceptibility of Chinese Holsteins to mastitis.

The JAK-STAT pathway plays a key role in host immunity. The present study was designed to evaluate the effects of single nucleotide polymorphisms (SNPs) in STAT5A and JAK2 genes on some serum cytokines, mastitis and milk production traits. Two SNPs (SNP1 43046497A/C and SNP2 43047829G/A) in STAT5A, and four SNPs in JAK2 (SNP3 39652267A/G, SNP4 39630048C/T, SNP5 39631044G/A, and SNP6 39631175T/C) were revealed and genotyped in 268 Chinese Holstein cattle. Fixed model was used to analyze the association of SNPs with phenotypes by general linear model procedure of SAS 9.1. SNP1 and SNP4 were significantly associated with IL-6 and IL-17 (P < 0.05), respectively. In JAK2 gene, SNP3 was highly significant (P < 0.01) and SNP5 was significant (P < 0.05) in association with SCC, whereas, the association of SNP6 was found significant (P < 0.05) with both SCC and SCS. Combination genotype analysis revealed that SNPs in JAK2 gene significantly associated with SCC and SCS were associated significantly with the corresponding phenotypes in combinations as well. The GG genotype of SNP3 individually and in any combination genotypes showed lowest SCC. The dominant effect of SNP1, SNP5 and SNP6 was found highly significant (P < 0.01) on the corresponding phenotypes (IL-6, SCC and SCS). As for haplotype analysis, two haplotypes were revealed between the two SNPs of STAT5A gene and four haplotypes amongst four SNPs in JAK2 gene; strong linkage disequilibrium (D' > 0.9) was observed between all these haplotypes. The results imply that the identified SNPs could be powerful markers to select dairy cattle with improved genetic resistance against mastitis.

Toxoplasma gondii infection in small ruminants from Khyber Pakhtunkhwa, Pakistan: Seroprevalence, spatial distribution and associated risk factors.

The primary aim of this investigation was to assess the occurrence of Toxoplasma gondii infection among sheep and goats in five districts within the southern region of Khyber Pakhtunkhwa province, Pakistan. Additionally, the study aimed to pinpoint potential factors associated with the spread of this infection. Serum samples were gathered from a total of 3505 small ruminants, comprising 1810 sheep and 1695 goats. These samples were then subjected to testing for anti-Toxoplasma antibodies using the Toxo-Latex slide Agglutination test. Concurrently, a predetermined questionnaire was employed to gather data on possible risk factors. The collected data underwent bivariate frequency analysis and multivariable logistic regression analyses. The findings revealed that 20.08% of the examined animals had positive results for anti-Toxoplasma antibodies. Notably, the prevalence of T. gondii was notably higher among sheep, with 21.8% of the sheep testing positive, as opposed to the 18% of tested goats (P = 0.007). The seroprevalence percentages were comparable across the five districts for both types of animals. For goats, the prevalence ranged from 16.8% to 20.1%, while for sheep, it ranged from 20.2% to 24%. Among the younger population (≤1 year), the seroprevalence was statistically lower at 17.7% compared to the older population (>1 year) at 23.1% (P < 0.001). Female animals demonstrated a higher prevalence (21.9%) in contrast to their male counterparts (17.8%) (P = 0.002). Animals reared for breeding and milking purposes displayed a greater seroprevalence (23.9%) compared to those intended for sale (17.7%) (P < 0.001). Furthermore, animals kept under a transhumant production system displayed the lowest prevalence (18.9%), followed by sedentary (21.7%) and nomadic (31.2%) herds (P < 0.001). The outcomes of this study highlight the importance of implementing management interventions to curtail T. gondii infection among animals within the Khyber Pakhtunkhwa province of Pakistan. Such measures hold the potential to reduce associated public health risks and enhance the profitability of farmers.

Alarming multidrug resistance in Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis: Antibiotic resistance patterns and occurrence of selected resistance genes.

Bovine mastitis is a widespread and costly disease that affects dairy farming globally, characterized by mammary gland inflammation. Bovine intramammary gland infection has been associated with more than 135 different pathogens of which Staphylococcus aureus is the main etiology of sub-clinical mastitis (SCM). The current study was designed to investigate the prevalence, antibiotic resistance pattern, and the presence of antibiotic resistance genes (mecA, tetK, aacA-aphD and blaZ) in S. aureus isolated from the raw milk of cows with subclinical mastitis. A total of 543 milk samples were collected from lactating cows such as Holstein Friesian (n = 79), Sahiwal (n = 175), Cholistani (n = 107), and Red Sindhi (n = 182) from different dairy farms in Pakistan. From the milk samples microscopic slides were prepared and the somatic cell count was assessed to find SCM. To isolate and identify S. aureus, milk was streaked on mannitol salt agar (MSA) plates. Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+). All the biochemically confirmed S. aureus isolates were molecularly identified using the thermonuclease (nuc) gene. The antibiotic resistance pattern of all the S. aureus isolates was evaluated through the disc diffusion method. Out of 543 milk samples, 310 (57.09%) were positive for SCM. Among the SCM-positive samples, S. aureus was detected in 30.32% (94/310) samples. Out of 94 isolates, 47 (50%) were determined to be multidrug resistant (MDR). Among these MDR isolates, 11 exhibited resistance to Cefoxitin, and hence were classified as methicillin-resistant Staphylococcus aureus (MRSA). The S. aureus isolates showed the highest resistance to Lincomycin (84.04%) followed by Ampicillin (45.74%), while the least resistance was shown to Sulfamethoxazole/Trimethoprim (3.19%) and Gentamycin (6.38%). Polymerase chain reaction (PCR) analysis revealed that 55.31% of the isolates carried blaZ gene, 46.80% carried tetK gene, 17.02% harbored the mecA gene, whereas, aacA-aphD gene was found in 13.82% samples. Our findings revealed a significant level of contamination of milk with S. aureus and half (50%) of the isolates were MDR. The isolated S. aureus harbored various antibiotic resistance genes responsible for the absorbed phenotypic resistance. The alarmingly high prevalence of MDR S. aureus isolates and MRSA strains in these cases possess a serious risk to public health, emphasizes the urgent need to address this issue to protect both human and animal health in Pakistan.

Diversity, prevalence and risk factors associated to gastrointestinal tract parasites in wild and domestic animals from Pakistan.

The gastrointestinal tract (GIT) of animals contains various helminth parasites, responsible for subclinical and clinical parasitism. The health situation of animals is seriously affected by these parasites and leads massive financial losses for the livestock trade. The present study was conducted to determine the prevalence and associated risk factors of GIT parasites in small and large ruminants of Mardan district and zoo animals of Golden Life Safari Park Khyber Pakhtunkhwa. A total of 419 fecal samples were collected including 117 from goats, 110 from cattle, 80 from sheep, 66 from buffaloes, 40 from birds, four from deer, and two from leopards. The fecal samples were subjected to sedimentation and floatation to identify parasite eggs. Of a total of 419 fecal samples, 215 (51.31%) were found positive for GIT parasites eggs. The prevalence of GIT parasites was high in sheep (65%), followed by goats (62.39%), cattle (42.70%), and buffaloes (37.87%). In birds, GIT parasite eggs were identified in 11 (45.83%) and 7 (43.75%) fecal samples of Pheasant and Peacock, respectively. However, an absence of infection was noted in fecal samples from deer and leopards. Eggs of GIT Parasites belonging to one superfamily and four different genera were recognized in fecal samples of domestic animals and buffaloes, which were Trichostrongyloidea (19.81%), Strongyloides (9.31%), Trichuris (7.77%), Monezia (6.44%), and Fasciola (4.53%). For bird, the coccidia group comprises the highest representation of parasites (2.63%), followed by the Trichuris genus (1.67%). Settlement, management system, anthelminthic medications and body condition score were significantly associated with the occurrence of GIT parasites in feces (P < 0.05). Animals from rural settlements, rearing under poor management systems, with thin (poor) body condition and under warmed were more affected by GIT parasites. The diversity and the high prevalence of GIT parasites reported in this study highlights the urgent need for intervention through treatment and modification of management practices in the investigated areas of Pakistan.

Antimicrobial Usage and Detection of Multidrug-Resistant Staphylococcus aureus: Methicillin- and Tetracycline-Resistant Strains in Raw Milk of Lactating Dairy Cattle.

Staphylococcus aureus is a prominent cause of food-borne diseases worldwide. Enterotoxigenic strains of this bacteria are frequently found in raw milk, and some of these strains are resistant to antimicrobials, posing a risk to consumers. The main objectives of this study were to determine the antimicrobial resistance pattern of S. aureus in raw milk and to detect the presence of mecA and tetK genes in it. A total of 150 milk samples were obtained aseptically from lactating cattle, including Holstein Friesian, Achai, and Jersey breeds, maintained at different dairy farms. The milk samples were checked for the presence of S. aureus, and it was detected in 55 (37%) of them. The presence of S. aureus was verified by culturing on selective media, gram staining, and performing coagulase and catalase tests. Further confirmation was performed through PCR with a species-specific thermonuclease (nuc) gene. Antimicrobial susceptibility testing of the confirmed S. aureus was then determined by using the Kirby-Bauer disc diffusion technique. Out of the 55 confirmed S. aureus isolates, 11 were determined to be multidrug-resistant (MDR). The highest resistance was found to penicillin (100%) and oxacillin (100%), followed by tetracycline (72.72%), amikacin (27.27%), sulfamethoxazole/trimethoprim (18.18%), tobramycin (18.18%), and gentamycin (9.09%). Amoxicillin and ciprofloxacin were found to be susceptible (100%). Out of 11 MDR S. aureus isolates, the methicillin resistance gene (mecA) was detected in 9 isolates, while the tetracycline resistance gene (tetK) was found in 7 isolates. The presence of these methicillin- and tetracycline-resistant strains in raw milk poses a major risk to public health, as they can cause food poisoning outbreaks that can spread rapidly through populations. Our study concludes that out of nine empirically used antibiotics, amoxicillin, ciprofloxacin, and gentamicin were highly effective against S. aureus compared to penicillin, oxacillin, and tetracycline.

Individual and Community-Level Risk Factors for Giardiasis in Children under Five Years of Age in Pakistan: A Prospective Multi-Regional Study.

OBJECTIVES: This study aimed to estimate the prevalence of Giardia lamblia infection and identify associated risk factors at both individual and community levels in a pediatric population in different agroecological zones of Khyber Pakhtunkhwa, Pakistan. METHODS: A community-based cross-sectional study was conducted from March to December 2022. Using stratified sampling, 1026 households were recruited from nine agroecological zones. Stool samples were collected from 1026 children up to the age of five years and processed for detection of Giardia using a commercial ELISA kit. Data on potential risk factors were collected using a pre-structured questionnaire. A multivariable logistic regression model was used to identify risk factors associated with giardiasis. RESULTS: The study found that the prevalence of giardiasis in the study area was 3.31%. Children aged 13-24 months were found to be at higher risk for giardiasis. Illiterate mothers and fathers attending daycare institutions/kindergartens, mothers not practicing hand washing during critical times, households with companion animals, and homes where stray dogs/cats enter were identified as predictors of giardiasis at the individual level. Children living in sub-mountain valleys use un-piped water, inadequate domestic water storage vessels, drink un-boiled or unfiltered water, live near rubbish heaps or un-paved streets/pathways, and have unimproved latrine facilities were identified as risk factors of giardiasis at the community level. CONCLUSIONS: The study highlights the need for integrated intervention approaches at both individual and community levels to reduce the incidence of giardiasis in Khyber Pakhtunkhwa, Pakistan. Interventions aimed at promoting behavioral change and providing safe and adequate water sources, combined with individual-level interventions such as hand washing and awareness of giardiasis prevention methods, would be critical to addressing this health concern. Inter-sectoral collaboration between the health sector and other sectors would also be necessary to achieve meaningful progress in reducing the incidence of giardiasis in resource-limited areas.

Epidemiological analysis of anaplasmosis in cattle from Khyber Pakhtunkhwa, Pakistan.

Background and Aim: Anaplasmosis, a tick-borne disease affecting livestock caused by the bacteria Anaplasma, poses a global concern. This study aimed to estimate the prevalence, spatiotemporal variation, and associated risk factors of anaplasmosis in cattle from the Bannu and Lakki Marwat districts of Khyber Pakhtunkhwa, Pakistan. Materials and Methods: This study used 197 cattle exhibiting clinical symptoms of anaplasmosis in natural settings. Microscopic examination was used to estimate the prevalence. Potential risk factors, such as sampling regions and months, gender, breed, and age were studied. Results: The study revealed an overall anaplasmosis prevalence of 19.79%. Bannu district exhibited a higher occurrence at 22.10%, compared to Lakki Marwat district at 17.64%. Young cattle (<2 years) demonstrated a notably higher incidence of anaplasmosis (26.78%) compared to adults (>5 years), which had a prevalence of 12.35% (p < 0.05). Female cattle (22.36%) were more susceptible than male cattle (11.11%). Prevalence peaked in June (45.71%) and was lowest in February (3.57%). Crossbred cattle had a higher prevalence (23.52%) than purebred cattle (11.47%). Conclusion: Anaplasmosis can be effectively controlled using a comprehensive approach encompassing selective breeding for resilience, targeted care of young calves and females, effective tick control during warmer months, consistent use of insecticides, and proactive risk factor management. Raising awareness among farmers through diverse channels, including media, is pivotal to bolster tick-borne disease management strategies.

Integrated analysis of transcriptome and milk metagenome in subclinical mastitic and healthy cows.

OBJECTIVE: Abnormally increased somatic cell counts in milk is usually a sign of bovine subclinical mastitis. Mutual interaction between the host and its associated microbiota plays an important role in developing such diseases. The main objective of this study was to explore the difference between cows with elevated somatic cell counts and healthy cattle from the perspective of host-microbe interplay. METHODS: A total of 31 milk samples and 23 bovine peripheral blood samples were collected from Holstein dairy cattle to conduct an integrated analysis of transcriptomic and metagenomics. RESULTS: The results showed that Ralstonia and Sphingomonas were enriched in cows with subclinical mastitis. The relative abundance of the two bacteria was positively correlated with the expression level of bovine TCN1 (Transcobalamin 1 encoding gene) and UPP1 (uridine phosphorylase 1 encoding gene). Moreover, functional analysis revealed a distinct alternation in some important microbial biological processes. CONCLUSION: These results reveal the relative abundance of Ralstonia and Sphingomonas other than common mastitis-causing pathogens varied from healthy cows to those with subclinical mastitis and might be associated with elevated SCC. Potential association was observed between bovine milk microbiota composition and the transcriptional pattern of some genes, thus providing new insights to understand homeostasis of bovine udder.

Emerging Roles of Noncoding RNAs in Bovine Mastitis Diseases.

Non-coding RNAs (ncRNAs) are an abundant class of RNA with varying nucleotide lengths. They have been shown to have great potential in eutherians/human disease diagnosis and treatments and are now gaining more importance for the improvement of diseases in livestock. To date, thousands of ncRNAs have been discovered in the bovine genome and the continuous advancement in deep sequencing technologies and various bioinformatics tools has enabled the elucidation of their roles in bovine health. Among farm animals' diseases, mastitis, a common inflammatory disease in cattle, has caused devastating economic losses to dairy farmers over the last few decades. Here, we summarize the biology of bovine mastitis and comprehensively discuss the roles of ncRNAs in different types of mastitis infection. Based on our findings and relevant literature, we highlighted various evidence of ncRNA roles in mastitis. Different approaches (in vivo versus in vitro) for exploring ncRNA roles in mastitis are emphasized. More particularly, the potential applications of emerging genome editing technologies, as well as integrated omics platforms for ncRNA studies and implications for mastitis are presented.

Conductive copper glue constructs a reversible and stable zinc metal anode interface for advanced aqueous zinc ion battery.

Aqueous zinc (Zn) ion battery (AZIB) has become one of the research hotspot in the field of energy storage due to its low cost, green environmental protection, high theoretical capacity, and high safety. However, the unrestrained growth of the dendrites leads to the occurrence of side reactions, such as corrosion of the electrodes and generation of hydrogen, which reduces the coulombic efficiency and performance of the battery. Herein, a simple method reports pasting a conductive copper glue (CCG) coating on the surface of Zn anode to improve the serious dendrite growth. The coating has strong intermolecular interaction and high conductivity, which not only avoids the occurrence of side reactions but also facilitates the uniform deposition of Zn2+ ions, preventing dendrite formation. The symmetrical battery assembled with Zn anode modified by CCG coating delivers longer cycle life (167 h) and lower voltage hysteresis (≈26 mV), which is much better than that of bare Zn symmetrical battery (30 h, ≈67 mV). Furthermore, the full battery assembly with modified Zn anode and stainless steel (SS) supported V2O5 nanospheres (VO-SS) cathode exhibit high capacity and long cycle life (113.5 mAh g-1 after 4000 cycles at 4.8 A g-1).

Identification of Novel mRNA Isoforms Associated with Acute Heat Stress Response Using RNA Sequencing Data in Sprague Dawley Rats.

The molecular mechanisms underlying heat stress tolerance in animals to high temperatures remain unclear. This study identified the differentially expressed mRNA isoforms which narrowed down the most reliable DEG markers and molecular pathways that underlie the mechanisms of thermoregulation. This experiment was performed on Sprague Dawley rats housed at 22 °C (control group; CT), and three acute heat-stressed groups housed at 42 °C for 30 min (H30), 60 min (H60), and 120 min (H120). Earlier, we demonstrated that acute heat stress increased the rectal temperature of rats, caused abnormal changes in the blood biochemical parameters, as well as induced dramatic changes in the expression levels of genes through epigenetics and post-transcriptional regulation. Transcriptomic analysis using RNA-Sequencing (RNA-Seq) data obtained previously from blood (CT and H120), liver (CT, H30, H60, and H120), and adrenal glands (CT, H30, H60, and H120) was performed. The differentially expressed mRNA isoforms (DEIs) were identified and annotated by the CLC Genomics Workbench. Biological process and metabolic pathway analyses were performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A total of 225, 5764, and 4988 DEIs in the blood, liver, and adrenal glands were observed. Furthermore, the number of novel differentially expressed transcript lengths with annotated genes and novel differentially expressed transcript with non-annotated genes were 136 and 8 in blood, 3549 and 120 in the liver, as well as 3078 and 220 in adrenal glands, respectively. About 35 genes were involved in the heat stress response, out of which, Dnaja1, LOC680121, Chordc1, AABR07011951.1, Hsp90aa1, Hspa1b, Cdkn1a, Hmox1, Bag3, and Dnaja4 were commonly identified in the liver and adrenal glands, suggesting that these genes may regulate heat stress response through interactions between the liver and adrenal glands. In conclusion, this study would enhance our understanding of the complex underlying mechanisms of acute heat stress, and the identified mRNA isoforms and genes can be used as potential candidates for thermotolerance selection in mammals.

The prevalence, risk factors analysis and evaluation of two diagnostic techniques for the detection of Cryptosporidium infection in diarrheic sheep from Pakistan.

BACKGROUND: Cryptosporidium spp. is recognized as an opportunistic zoonotic parasite that infects humans as well as wild and domestic animals. This enteric protozoan is a major cause of diarrhea in humans and animals and often result in death due to severe dehydration. The present study was designed to investigate the prevalence, identification of various risk factors and evaluation of sensitivity of the two diagnostic techniques for rapid and correct detection of Cryptosporidium infection in diarrheic sheep in Pakistan. METHODS: A total of 360 fecal samples were collected and processed for detection of Cryptosporidium infection after proper preservation. These samples were properly stained with modified Ziehl-Neelsen acid staining and then examined under simple microscope at 100x magnification for confirmation of Cryptosporidium oocysts. The same samples were again processed through simple PCR for confirmation of the Cryptosporidium spp. RESULTS: The age wise prevalence was detected through simple microscopy and PCR. We found highest prevalence at the age of ≤1 year followed by 1-2 years of age while the lowest prevalence was recorded at the age of ≥ 2-3 years of sheep and found significant difference between different ages (P<0.05). The sex wise prevalence showed the highest prevalence in male (♂) animals detected compared to female (♀). The overall prevalence was detected 27.08% and 18.80% through PCR and simple microscopy, respectively, and significant difference between two diagnostic techniques were observed (P<0.05). Considering the seasonality, the highest prevalence was recorded through simple microscopy in autumn, summer, and spring, while the lowest in winter. These results were confirmed through PCR. CONCLUSION: It was concluded that molecular detection is the most efficient, specific and sensitive technique for detection of Cryptosporidium infection than simple microscopy. Moreover sheep is the major potential source of infection to other wild and domestic animals including humans.

Population diversity of sheep bot fly, Oestrus ovis (Linné, 1758) (Diptera: Oestridae: Oestrinae), using SDS polyacrylamide gel electrophoresis.

The study was planned to evaluate the inter, and intra population genetic variation in general protein banding pattern in Oestrus ovis larvae, by using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The larvae were collected from slaughtered goats head from five different locations (AAS, PN, LA, GM, and BC) of Karachi, Pakistan. The data obtained was subjected to POPGENE (Population Genetic Analysis) software for analysis. The polymorphic loci within populations ranged from 45.45% to 90.91%. Polymorphic loci observed in all populations were 90.91%. The expected heterozygosity observed was 0.182 ± 0.096 in all populations. The chi-square test showed 5 out of 11 loci at H-W equilibrium. The overall fixation index (FST) value was 0.108, showing that the likelihood of subpopulations being differentiated from one another is about 11 percent. The gene flow value (Nm = 2.065) was higher, showing that genes flow occurs between populations. The values of genetic identity were greater, and genetic distance were smaller among all the populations, which means that all the populations were more alike and closer to each other. It was concluded that there was no sympatric and parapatric population differentiation observed among all the population of O. ovis and the populations of the five different locations were not genetically and reproductively isolated from each other.

Association of Aberrant DNA Methylation Level in the CD4 and JAK-STAT-Pathway-Related Genes with Mastitis Indicator Traits in Chinese Holstein Dairy Cattle.

The present study was designed to evaluate the gene expression and DNA methylation level in the promoter region of the CD4 and the JAK-STAT-pathway-related genes. A total of 24 samples were deployed in the gene expression and 118 samples were used in the DNA methylation study. Student's t-tests were used to analyze the gene expression and DNA methylation. The evaluation of DNA methylation in promoter regions of JAK2 and STAT5A revealed hypo-methylation levels of CpG sites and higher gene expression in cows diagnosed with mastitis as compared to the healthy control, and vice versa in those with CD4. DNA methylation was negatively correlated with gene expression in JAK2, STAT5A, and CD4 genes. Six, two, and four active transcription factors were identified on the CpG sites in the promoter regions of JAK2, STAT5A, and CD4 genes, respectively. Regarding correlation analysis, the DNA methylation levels of CD4 showed significantly higher positive correlations with somatic cell counts (p < 0.05). Findings of the current study inferred that aberrant DNA methylation in the CpG sites at the 1 kb promoter region in JAK2, STAT5A, and CD4 genes due to mastitis in cows can be used as potential epigenetic markers to estimate bovine mastitis susceptibility in dairy cattle.

Prevalence and risk factors assessment of mange mites in livestock of Malakand Division, Pakistan.

Mange mites represent a serious problem in livestock farming due to their worse effect on animals health, reducing milk and meat production. The status of mange mites prevalence was unknown from the livestock present in Malakand Division, Pakistan. Hence, the present study was conducted to determine the prevalence and risk factors assessment of mange mites in livestock of Malakand Division, Pakistan. Villages and livestock herds were visited for data collection and sampling for one year from September 2017 to August 2018. The diagnosis of mange infestation was carried out from skin scrapings collected from symptomatic animals. A total of 1437 animals were screened (240 sheep, 658 cows, 340 buffalos, 199 goats) from selected localities of the Malakand Division. The overall prevalence was 2.37%, with the highest prevalence in buffalos (5.0%) and lowest (0.42%) in sheep (p < 0.05). The locality also significantly affect the prevalence of mange mites in livestock, with a higher percentage (8.14%) in the Ouch area. The prevalence was higher in winter (3.78%) than in summer (1.07%). The prevalence was high (p < 0.05) in young animals (≤1 year) (4.7%) as compared to adults (>1 year) (1.15%). Gender was also a significant risk factor for mange infestation. The infestation was higher in females (2.85%) as compared to males (0.64%) (p < 0.05). Herd size of less than ten animals had a significantly high prevalence rate (3.69%) compared to >10 animals (1.89%). Animals having an infection history >15 days infected more than animals having infection history ≤15 days. This study documented for the first-time epidemiology of mange mites in livestock of Malakand Division, Pakistan. Further studies involving molecular characterization of mange mites will increase our understanding on the spread of mange mites in Malakand Division livestock.