In Vitro Antibacterial Evaluation of Terminalia chebula as an Alternative of Antibiotics against Bovine Subclinical Mastitis

The extent of subclinical mastitis in three breeds of cattle, Kankrej, Gir, and Crossbred, was performed at cattle farms in Anand town of Gujarat State, India. The prevalence of subclinical mastitis in crossbred cattle was higher compared to local breed of cattle. Causative agents identified using 16S rDNA polymerase chain reaction (PCR)-based molecular method were Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus megaterium. In vitro antibacterial activity of ethyl acetate extract of plant Terminalia chebula (Combretaceae) was checked by agar well diffusion method against four isolated and molecularly identified microorganisms. Ethyl acetate extract shows antimicrobial activity with varying magnitudes against all identified isolates. Among the three different concentrations, 500?µg/mL conc. of extract is as effective as that of standard amoxicillin. In vitro results support the use of plant extract from T. chebula as an alternative to antibiotics therapy against bovine subclinical mastitis.     

Comparative gene frequencies of nucleoside phosphorylase from cattle of Bos taurus and Bos indicus derivation in Brazil

1. Two nucleoside phosphorylase (NP) phenotypes were detected in 844 animals from four distinct genetic groups of Bos taurus and Bos indicus derivation. 2. Bos indicus breeds like Guzerat (Kankrej), Gir, Nellore (Ongole) and Indubrazil presented an NP-H frequency of 1.00, 0.928, 0.776 and 0.754 respectively, while the Canchim breed, a Bos taurus-Bos indicus crossbred cattle (5/8 Charolais-3/8 Zebu) presented a frequency of 0.372. 3. The high frequency detected from the NP-H allele in the Bos indicus breeds strongly suggests that this enzyme is a genetic marker for cattle and that it probably has a very high frequency in all Indian breeds.     

Evidence for pleiotropism and recent selection in the PLAG1 region in Australian Beef cattle

A putative functional mutation (rs109231213) near PLAG1 (BTA14) associated with stature was studied in beef cattle. Data from 8199 Bos taurus, Bos indicus and Tropical Composite cattle were used to test the associations between rs109231213 and various phenotypes. Further, 23 496 SNPs located on BTA14 were tested for association with these phenotypes, both independently and fitted together with rs109231213. The C allele of rs109231213 significantly increased hip height, weight, net food intake, age at puberty in males and females and decreased IGF‐I concentration in blood and fat depth. When rs109231213 was fitted as a fixed effect in the model, there was an overall reduction in associations between other SNPs and these traits but some SNPs remained associated (P < 10^?4). Frequency of the mutant C allele of rs109231213 differed among B. indicus (0.52), B. taurus (0.96) and Tropical Composite (0.68). Most chromosomes carrying the C allele had the same surrounding 10 SNP haplotype, probably because the C allele was introgressed into Brahman from B. taurus cattle. A region of reduced heterozygosity surrounds the C allele; this is small in B. taurus but 20 Mb long in Brahmans, indicating recent and strong selection for the mutant allele. Thus, the C allele appears to mark a mutation that has been selected almost to fixation in the B. taurus breeds studied here and introduced into Brahman cattle during grading up and selected to a frequency of 0.52 despite its negative effects on fertility.     

A comparison of Ixodes rubicundus (Acari: Ixodidae) infestations on Friesian and Bonsmara cattle in South Africa

This study was conducted to compare the relative resistance of crossbred Bos indicus X B. taurus Bonsmara and B. taurus Friesian cattle to Ixodes rubicundus (Karoo paralysis tick) infestations. During periods of peak abundance of the ticks, Friesian oxen harboured almost twice or more than twice as many ticks as either Bonsmara oxen or cows. During periods of low tick abundance tick burdens on both cattle breeds were closely similar. It is envisaged that cattle can play an important role in an integrated control strategy against the Karro paralysis tick.     

Large-scale in vitro embryo production and pregnancy rates from Bos taurus, Bos indicus, and indicus-taurus dairy cows using sexed sperm

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir (Bos indicus, n = 617), Holstein (Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO₂ in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10⁶ sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos.     

Distribution of <Emphasis Type="Italic">BoLA-DRB3</Emphasis> allelic frequencies and identification of a new allele in the Iranian cattle breed Sistani (<Emphasis Type="Italic">Bos indicus</Emphasis>)

The distribution of the frequencies of BoLA-DRB3 gene alleles in the Iranian cattle breed Sistani was studied by the PCR-RFLP (“hemi-nested”) assay using restriction endonucleases RsaI, HaeIII and BstYI. In the examined cattle breed (65 animals) 32 alleles have been identified one of which being described for the first time (6.15% frequency). The nucleotide sequence of the polymorphic region of exon 2 of this allele has been determined and submitted in the GenBank database under accession number DQ486519. The submitted sequence has maximum homology (92%) with the previously described sequence DRB3-mRNA from Bos indicus (AccN X79346) and differs from it by 24 nucleotide substitutions which result in 16 amino acid substitutions. The peptide (on the basis of the reconstructed amino acid sequence) has 89% identity to the sequence encoded by the BIDRBF 188 locus (Bos indicus). The results obtained permit the sequence described by us to be considered as a new allele of the BoLA-DRB3 gene (DRB3.2 ^* X). The total frequency of the main six alleles (DRB3.2*8, *10, *11, *20, *34 and *X) occurring with a frequency of over 5% is about 60% in Iranian Sistani cattle. Fifteen alleles have <1% frequency. The highest frequency was observed for DRB3.2*8 allele (21.54%) like in other previously described breeds of Bos indicus (up to 23.07%). The Iranian breed Sistani has a high level of similarity by the spectrum of BoLA-DRB3 alleles and their frequencies to other Bos indicus breeds and significantly differs by these criteria from the Bos Taurus breeds. The Iranian Sistani herd under study includes alleles associated with to resistance to leukemia (DRB3.2*11 and *23) and to different forms of mastitis (DRB3.2* 2, *7, *11, *23 and *24) although their frequencies are low (from 0.77 to 5.37%). On the whole, a high level of diversity of BoLA-DRB3 gene alleles and the availability of alleles associated with resistance to different diseases makes this breed of interest for breeding practice. The article is published in the original.     

Microsatellite analysis of genetic population structure of zebu cattle (Bos indicus) breeds from north-western region of India

The present study aims to understand the existing genetic diversity and structure of six native cattle breeds (Rathi, Tharparkar, Nagori, Mewati, Gir, and Kankrej) adapted to the north-western arid and semi-arid region of India based on microsatellite loci. Various diversity estimates, mean number of alleles (12.84); effective number of alleles (5.02); gene diversity (0.769), and observed heterozygosity (0.667) reflected the existence of substantial within-breed diversity in all the investigated cattle breeds. Mean estimates of F-statistics: F(IT) = 0.144 ± 0.023, F(IS) = 0.071 ± 0.021, and F(ST) = 0.078 ± 0.014 were significantly different from zero (P < 0.05). The interbreed relationships indicated moderate level of breed differentiation between the six cattle breeds with least differentiation between Kankrej-Mewati pair. The phylogeny structuring further supported close grouping of Kankrej and Mewati breeds. Correspondence analysis plotted Rathi, Tharparkar, and Gir individuals into three separate areas of multivariate space; whereas, Kankrej, Mewati, and Nagori cattle showed low breed specific clustering. This reflected the existence of discrete genetic structure for Tharparkar, Rathi, and Gir, the prominent dairy breeds of the region; whereas, admixture was observed for Kankrej, Mewati, and Nagori individuals.     

Comprehensive analysis of the mitochondrial DNA diversity in Chinese cattle

Complete mitochondrial DNA D‐loop sequences of 1105 individuals were used to assess the diversity of maternal lineages of cattle populations in China. In total, 250 taurine and 88 zebu haplotypes were identified. Five main haplogroups—T1a, T2, T3, T4 and T5—were identified in Bos taurus, whereas Bos indicus harbored two haplogroups—I1 and I2. Our results suggest that the distribution of T1a in Asia was concentrated mainly in the northeast region (northeast China, Korea and Japan); haplogroups T2, T3 and T4 were predominant in Chinese cattle; and T5 was sporadically detected in Mongolian and Pingwu cattle. In contrast to the widespread presence of I1, I2 was distributed only in southwestern China (Yunnan‐Guizhou Plateau and the Tibet Autonomous Region) and Xinjiang Uygur Autonomous Region. This is the first time that all five taurine haplogroups and two zebu haplogroups have been found in Mongolian cattle. In addition, eight individuals in Tibetan cattle carried the Bos grunniens mtDNA type. The high mtDNA diversity (H = 0.904 ± 0.008) and the weak genetic structure among the 57 Chinese cattle breeds/populations are consistent with their complex historical background, migration route and ecological environment.     

Evaluation of genetic diversity and population structure of west‐central Indian cattle breeds

Evaluations of genetic diversity in domestic livestock populations are necessary to implement region‐specific conservation measures. We determined the genetic diversity and evolutionary relationships among eight geographically and phenotypically diverse cattle breeds indigenous to west‐central India by genotyping these animals for 22 microsatellite loci. A total of 326 alleles were detected, and the expected heterozygosity ranged from 0.614 (Kenkatha) to 0.701 (Dangi). The mean number of alleles among the cattle breeds ranged from 7.182 (Khillar) to 9.409 (Gaolao). There were abundant genetic variations displayed within breeds, and the genetic differentiation was also high between the Indian cattle breeds, which displayed 15.9% of the total genetic differentiation among the different breeds. The genetic differentiation (pairwise F_ST) among the eight Indian breeds varied from 0.0126 for the Kankrej–Malvi pair to 0.2667 for Khillar–Kenkatha pair. The phylogeny, principal components analysis, and structure analysis further supported close grouping of Kankrej, Malvi, Nimari and Gir; Gaolao and Kenkatha, whereas Dangi and Khillar remained at distance from other breeds.     

Complete mitogenome reveals genetic divergence and phylogenetic relationships among Indian cattle (Bos indicus) breeds

Indigenous cattle of India belong to the species, Bos indicus and they possess various adaptability and production traits. However, little is known about the genetic diversity and origin of these breeds. To investigate the status, we sequenced and analyzed the whole mitochondrial DNA (mtDNA) of seven Indian cattle breeds. In total, 49 single-nucleotide variants (SNVs) were identified among the seven breeds analyzed. We observed a common synonymous SNV in the COII gene (m.7583G?>?A) of all the breeds studied. The phylogenetic analysis and genetic distance estimation showed the close genetic relationship among the Indian cattle breeds, whereas distinct genetic differences were observed between Bos indicus and Bos taurus cattle. Our results indicate a common ancestor for European Zwergzebu breed and South Indian cattle. The estimated divergence time demonstrated that the Bos indicus and Bos taurus cattle lineages diverged 0.92 million years ago. Our study also demonstrates that ancestors of present zebu breeds originated in South and North India separately ～30,000 to 20,000 years ago. In conclusion, the identified genetic variants and results of the phylogenetic analysis may provide baseline information to develop appropriate strategies for management and conservation of Indian cattle breeds.     

Microsatellite Analysis of Genetic Population Structure of Zebu Cattle (Bos indicus) Breeds from North-Western Region of India

The present study aims to understand the existing genetic diversity and structure of six native cattle breeds (Rathi, Tharparkar, Nagori, Mewati, Gir, and Kankrej) adapted to the north-western arid and semi-arid region of India based on microsatellite loci. Various diversity estimates, mean number of alleles (12.84); effective number of alleles (5.02); gene diversity (0.769), and observed heterozygosity (0.667) reflected the existence of substantial within-breed diversity in all the investigated cattle breeds. Mean estimates of F-statistics: F_IT = 0.144 ± 0.023, F_IS = 0.071 ± 0.021, and F_ST = 0.078 ± 0.014 were significantly different from zero (P < 0.05). The interbreed relationships indicated moderate level of breed differentiation between the six cattle breeds with least differentiation between Kankrej-Mewati pair. The phylogeny structuring further supported close grouping of Kankrej and Mewati breeds. Correspondence analysis plotted Rathi, Tharparkar, and Gir individuals into three separate areas of multivariate space; whereas, Kankrej, Mewati, and Nagori cattle showed low breed specific clustering. This reflected the existence of discrete genetic structure for Tharparkar, Rathi, and Gir, the prominent dairy breeds of the region; whereas, admixture was observed for Kankrej, Mewati, and Nagori individuals.     

The relative influence of temperature and humidity on cutaneous function in Bos indicus and Bos taurus females

The relative importance of dry- and wet-bulb temperatures on cutaneous function inBos indicus andBos taurus females under humid tropical climatic conditions was evaluated. The parameters investigated were sweating rate and skin temperature, while the species utilised were zebu White Fulani (Bos indicus) and German Brown and German Black and White (Bos taurus).The sweating rate, irrespective of breed, differed with the site of sampling and was more influenced by dry-bulb (59%) than by wet-bulb temperature (41%). Skin temperature responded more to wet-bulb temperature in White Fulani and German Black and White cattle, but not in German Brown cattle.It is concluded that the response of the animals, with respect to sweating, was similar but that the efficiency of sweating, judged by the lowering of skin temperature, was higher inBos indicus than inBos taurus. This, in part, may explain the higher degree of comfort demonstrated byBos indicus under tropical conditions.     

Virulence determinants and antimicrobial resistance of E. coli isolated from bovine clinical mastitis in some selected dairy farms of Bangladesh

E. coli is one of the major significant pathogens causing mastitis, the most complex and costly diseases in the dairy industry worldwide. Present study was undertaken to isolate, detect the virulence factors, phylogroup, antimicrobial susceptibility and antimicrobial resistance genes in E. coli from cows with clinical mastitis. A total of 68 milk samples comprising 53 from clinical mastitis and 15 from apparently healthy cattle were collected from four different established dairy farms in Bangladesh. E. coli was isolated from the milk samples and identified by PCR targeting malB gene and sequencing of 16S rRNA gene. E. coli isolates were screened by PCR for the detection of major virulence genes (stx, eae and cdt) of diarrheagenic E. coli followed by phylogenetic grouping. Antimicrobial susceptibility of the E. coli isolates was determined by disk diffusion test and E. coli showing resistance was further screened for the presence of antimicrobial resistance genes. E. coli was isolated from 35.8% of the mastitis milk samples but none from the apparently healthy cattle milk. All the E. coli isolates were negative for stx, eae and cdt genes and belonged to the phylogenetic groups A and B1 which comprising of commensal E. coli. Antibiotic sensitivity testing revealed 84.2% (16/19) of the isolates as multidrug resistant. Highest resistance was observed against amoxicillin (94.5%) followed by ampicillin (89.5%) and tetracycline (89.5%). E. coli were found resistant against all the classes of antimicrobials used at the farm level. Tetracycline resistance gene (tetA) was detected in 100% of the tetracycline resistant E. coli and blaTEM-1 was present in 38.9% of the E. coli isolates. Findings of this study indicate a potential threat of developing antimicrobial resistance in commensal E. coli and their association with clinical mastitis. Occurrence of multidrug resistant E. coli might be responsible for the failure of antibiotic therapies in clinical mastitis as well as pose potential threat of transmitting and development of antibiotic resistance in human.     

<Emphasis Type="Italic">IGF-1</Emphasis> and <Emphasis Type="Italic">IGF-1R</Emphasis> gene polymorphisms in East Anatolian Red and South Anatolian Red cattle breeds

The aim of this study was to determine the allele and genotype frequencies of the Insulin like growth factor 1 gene (IGF-1) and Insulin like growth factor 1 receptor gene (IGF-1R) polymorphisms in East Anatolian (EAR) and South Anatolian Red cattle (SAR). Polymorphisms in both genes are claimed to affect economic parameters like body weight and subcutaneous back fat. For the study, blood samples were collected from 50 SAR and 50 EAR cattle. In both breeds, high frequencies of allele B of IGF-1, which was supposed to have positive effect on carcass traits and allele A of IGF-1R, related to milk traits, were observed. Therefore, no relationship of the polymorphisms studied with economic traits was observed, as both breeds have respectively low carcass and milk parameters. However, distribution of allele frequencies of IGF-1 and IGF-1R in SAR and EAR cattle was similar with Zebu cattle that support Bos indicus introgression to Anatolian breeds.     

Genomic clues of the evolutionary history of Bos indicus cattle

Together with their sister subspecies Bos taurus, zebu cattle (Bos indicus) have contributed to important socioeconomic changes that have shaped modern civilizations. Zebu cattle were domesticated in the Indus Valley 8000 years before present (YBP). From the domestication site, they expanded to Africa, East Asia, southwestern Asia and Europe between 4000 and 1300 YBP, intercrossing with B. taurus to form clinal variations of zebu ancestry across the landmass of Afro‐Eurasia. In the past 150 years, zebu cattle reached the Americas and Oceania, where they have contributed to the prosperity of emerging economies. The zebu genome is characterized by two mitochondrial haplogroups (I1 and I2), one Y chromosome haplogroup (Y3) and three major autosomal ancestral groups (Indian‐Pakistani, African and Chinese). Phenotypically, zebu animals are recognized by their hump, large ears and excess skin. They are rustic, resilient to parasites and capable of bearing the hot and humid climates of the tropics. Many resources are available to study the zebu genome, including commercial arrays of SNP, reference assemblies and publicly available genotypes and whole‐genome sequences. Nevertheless, many of these resources were initially developed to support research and subsidize industrial applications in B. taurus, and therefore they can produce bias in data analysis. The combination of genomics with precision agriculture holds great promise for the identification of genetic variants affecting economically important traits such as tick resistance and heat tolerance, which were naturally selected for millennia and played a major role in the evolution of B. indicus cattle.     

Engineering Disease Resistant Cattle

Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk. Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis causing organisms. Care will be taken to avoid altering milk’s nutritional and manufacturing properties. Multi-cistronic constructs may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Influence of Diet Composition on Cattle Rumen Methanogenesis: A Comparative Metagenomic Analysis in Indian and Exotic Cattle

Comparative metagenomics approach has been used in this study to discriminate colonization of methanogenic population in different breeds of cattle. We compared two Indian cattle breeds (Gir and Kankrej) and two exotic cattle (Holstein and Jersey) breeds. Using a defined dietary plan for selected Indian varieties, the diet dependent shifts in microbial community and abundance of the enzymes associated with methanogenesis were studied. This data has been compared with the available rumen metagenome data from Holstein and Jersey dairy cattle. The abundance of genes for methanogenesis in Holstein and Jersey cattle came from Methanobacteriales order whereas, majority of the enzymes for methanogenesis in Gir and Kankrej cattle came from Methanomicrobiales order. The study suggested that by using slow/less digestible feed, the propionate levels could be controlled in rumen; and in turn, this would also help in further reducing the hydrogenotrophic production of methane. The study proposes that with the designed diet plan the overall methanogenic microbial pool or the individual methanogens could be targeted for development of methane mitigation strategies.     

Multiplex PCR assay for species identification of bovine mastitis pathogens

Aim: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. Methods and Results: A two‐tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10³ CFU ml^?1. A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. Conclusion: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. Significance and Impact of the Study: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.     

Polymorphism of mitochondrial DNA (mtDNA) in cattle and buffaloes

Mitochondrial DNA (mtDNA) from two breeds of cattle, viz., [Hariana (Bos indicus), Holstein (Bos taurus)] and Indian water buffalo (Bubalis bubalus), was analyzed using 13 restriction endonucleases which recognized an average of about 40 six-base sites. Polymorphism among cattle was detected with six of these enzymes. The two Holstein differed at six sites, whereas the Hariana breed (Bos indicus) did not show any site polymorphism. Surprisingly, the Hariana type differed by only one site from one of the Holstein types. The total size of buffalo mtDNA was estimated to be 16.4 kb. Polymorphism within the Murrah buffalo breed was observed with respect to aBglI site. Scarcely any of the restriction fragments of buffalo mtDNA matched those of cattle mtDNA.