Cattle DNA Research Articles

Elucidation of fecal inputs into the River Tagus catchment (Portugal) using source-specific mitochondrial DNA, HAdV, and phage markers

Determining the source of fecal contamination in a water body is important for the application of appropriate remediation measures. However, it has been suggested in the extant literature that this can best be achieved using a ‘toolbox’ of molecular- and culture-based methods. In response, this study deployed three indicators (Escherichia coli (EC), intestinal enterococci (IE) and somatic coliphages (SC)), one culture-dependent human marker (Bacteroides (GB-124) bacteriophage) and five culture-independent markers (human adenovirus (HAdV), human (HMMit), cattle (CWMit), pig (PGMit) and poultry (PLMit) mitochondrial DNA markers (mtDNA)) within the River Tagus catchment (n = 105). Water samples were collected monthly over a 13-month sampling campaign at four sites (impacted by significant specific human and non-human inputs and influenced by differing degrees of marine and freshwater mixing) to determine the dominant fecal inputs and assess geographical, temporal, and meteorological (precipitation, UV, temperature) fluctuations. Our results revealed that all sampling sites were not only highly impacted by fecal contamination but that this contamination originated from human and from a range of agricultural animal sources. HMMit was present in a higher percentage (83%) and concentration (4.20 log GC/100 mL) than HAdV (32%, 2.23 log GC/100 mL) and GB-124 bacteriophage with the latter being detected once. Animal mtDNA markers were detected, with CWMit found in 73% of samples with mean concentration of 3.74 log GC/100 mL. Correlation was found between concentrations of fecal indicators (EC, IE and SC), CWMit and season. Levels of CWMit were found to be related to physico-chemical parameters, such as temperature and UV radiation, possibly as a result of the increasing presence of livestock outside in warmer months. This study provides the first evaluation of such a source-associated ‘toolbox’ for monitoring surface water in Portugal, and the conclusions may inform future implementation of surveillance and remediation strategies for improving water quality.

Detection of species adulteration in meat products and Mozzarella-type cheeses using duplex PCR of mitochondrial cyt b gene: A food safety concern in Bangladesh

Food origin authentication is imperative for consumer protection. This study explored meat and milk species identification as a pioneering country-specific report on mislabeling prevalence among processed meat and milk products in Bangladesh. Meat products (64; sausages, burger patty, meatball, kabab) labeled as chicken or beef and Mozzarella-type cheeses (25), made in Bangladesh and of overseas origins, were analyzed for species detection. Two duplex PCR (cattle-buffalo and chicken-pig) were applied with species-specific mitochondrial Cyt b gene primers and determine to be accurate for species identification in meat and milk. Bangladeshi origin beef-labeled products were found to be mixed with buffalo (n = 2) and chicken (n = 5) suggesting up to one third of products might be mislabeled. Such mislabeling would allow these ‘beef’ products to charge a higher price compared with chicken products that command a lower price. Imported meat products were also found mislabeled with buffalo and chicken. Cheese samples, declared as bovine, were found to contain buffalo DNA, and no cattle or buffalo DNA was found in six imported cheese samples. All the meat and cheese products were Halal, as none contained pig DNA. This pilot study shows the majority of products were labelled correctly, but large proportions were not and strict monitoring is recommended to ensure food safety and address consumer preferences, especially religious concerns.

Use of DNA Barcoding Combined with PCR-SFLP to Authenticate Species in Bison Meat Products.

American bison (Bison bison) meat is susceptible to species mislabeling due to its high value and similar appearance to meat from domestic cattle (Bos taurus). DNA barcoding is commonly used to identify animal species. However, as a result of the historical hybridization of American bison and domestic cattle, additional genetic testing is required for species confirmation. The objective of this study was to perform a market survey of bison meat products and verify the species using DNA barcoding combined with polymerase chain reaction-satellite fragment length polymorphism (PCR-SFLP). Bison products (n = 45) were purchased from a variety of retailers. Samples that were positive for domestic cattle with DNA barcoding were further analyzed with PCR-SFLP. DNA barcoding identified bison in 41 products, red deer (Cervus elaphus) in one product, and domestic cattle in three products. PCR-SFLP confirmed the identification of domestic cattle in two samples, while the third sample was identified as bison with ancestral cattle DNA. Overall, mislabeling was detected in 3 of the 45 samples (6.7%). This study revealed that additional DNA testing of species that have undergone historical hybridization provides improved identification results compared to DNA barcoding alone.

Development of loop-mediated isothermal method and comparison with conventional PCR assay for rapid on spot identification of tissue of cattle origin.

Loop-mediated isothermal amplification (LAMP) is a diagnostic method for meat speciation with rapid and minimal equipment requirements. In this study, we developed cattle-specific tube-based LAMP assays targeting mitochondrial Cyt b gene sequence, compared with conventional PCR assay for specificity, sensitivity, and validation of the assay was made. The LAMP reaction was carried at 64°C for 45min, and results were confirmed by SYBR Green I dye and agarose gel-electrophoresis. The specificity of the assays was cross-tested with DNA of buffalo, goat, sheep, and pork. The amplification was observed with samples from cattle only without cross-reactivity with other meat species. The analytical sensitivity of LAMP and PCR method for cattle DNA detection was 0.0001ng and 1ng, respectively. Repeatability of the assay was achieved on samples from known/blind and admixture meat with other than cattle at the relative percentage of 20%, 10%, 5%, and 1%. The study concluded that the developed assay can be easily employed for the rapid identification of tissue of cattle origin in meat and meat products in low resource areas.

Serological and molecular surveys of Anaplasma spp. in Egyptian cattle reveal high A. marginale infection prevalence.

Bovine anaplasmosis is an infectious disease with worldwide distribution. It spreads by various routes mainly through tick bites. This study aimed to investigate bovine related Anaplasma spp. in cattle from three northern governorates of Egypt by serological and molecular assays, to evaluate the associated risk factors and to analyze the phylogeny of revealed A. marginale isolates. During 2020, a total of 650 blood samples were collected from asymptomatic cattle in the governorates of Kafr El-Sheikh (n=240), Menofia (n=230), and Al-Gharbia (n=180). Sera samples were examined using the Anaplasma antibody test kit, cELISA v2. Blood genomic DNA of seropositive cattle was then examined by PCRs specific to A. marginale, A. centrale, and A. bovis. Selected positive samples were subjected to nucleotide sequencing. Risk factors (i.e. geographical area, breed, type of production, sex, age, herd size, season, husbandry system, tick infestation, and application of acaricides) were evaluated by logistic regression approach. In total, 130 cattle (20%, 95% CI: 17.1-23.3) were recorded seropositive for Anaplasma species. Major risk factors associated with seropositivity were being crossbred, dairy cattle, aged more than 5 years, summer season, herd size of below 300, pasture grazing, tick infestation, and not being subjected to regular treatment with acaricides. By using species-specific PCR, only A. marginale was detected. Nucleotide sequencing showed the occurrence of two different msp4 genotypes. This study shows the high prevalence of A. marginale in cattle of Kafr El-Sheikh, Al-Gharbia, and Menofia. However, the connection between Anaplasma species and their tick vectors remains unknown in Egypt and merits further investigations. Since these infections primarily spread through ixodid tick bites, effective ectoparasite control strategies, regular examination of cattle and successful chemoprophylaxis are recommended.

Effect of DNA isolation method and host species on the metagenomic DNA recovery and microbial community coverage

The introduction of next generation sequencing to characterize rumen microbial diversity has expanded the scope of animal nutrition to understand ruminal fermentation in greater details. Although diis technique is highly accurate, it suffers from biases introduced at different stages of sample processing, sequencing and bioinformatics analysis. It is well established that the outcome of metagenomics (using DNA) is affected by the source and method of DNA isolation. The present study aimed to evaluate two popular rumen metagenomic DNA isolation methods, Repeated Bead Beating plus Column (RBBC) and ZR Fecal DNA Microprep Kit, for their effect on the DNA quality and quantity, and microbial community assessment in four ruminant species, namely cattle, buffalo, goat and sheep. Metagenomic DNA was isolated from total rumen contents using these methods, and the quality and quantity of the purified DNA were determined. The coverage of microbial diversity was assessed by the Terminal Restriction Fragment Length Polymorphism (TRFLP) technique using two restriction enzymes (Mspl and BsuRI). The number and peak area of terminal restriction fragments (TRFs) were used to compare the efficiency of the DNA isolation methods across the species. The study established that although the metagenomic DNA recovery was greater using the RBBC method, the quality of the purified DNA was superior with die ZR Fecal DNA kit. Significantly greater microbial diversity (P<0.05) was also noticed in cattle, goat and sheep rumen metagenomic DNA isolated using the ZR Fecal DNA kit. Further, the overall comparison between the methods, irrespective of the species, also revealed significantly (P< 0.001) greater microbial diversity coverage in the ZR Fecal DNA isolation kit than the RBBC method. In conclusion, the current study revealed that die ZR Fecal DNA Kit was comparatively better than the RBBC method for assessing microbial diversity in me rumen metagenomic samples of cattle, buffalo, goat and sheep.

Production of embryos and a live offspring using post mortem reproductive material from bison (Bison bison bison) originating in Yellowstone National Park, USA

Bison from Yellowstone National Park (YNP) have an important genetic history. As one of the few wild herds of bison with no evidence of cattle DNA introgression and a large enough population to maintain genetic diversity, they are considered a conservation priority for the species. Unfortunately, there is a high prevalence of the zoonotic disease brucellosis in the herd. Part of the management strategy for controlling the disease and herd size in YNP is to remove bison from the population during the winter migration out of the park. This interagency management cull provides an opportunity to collect a large number of oocytes from a wild bison population for genetic banking and research purposes. During the winters of 2014–2018, which is the nonbreeding season for bison, oocytes were collected post mortem and used to determine the effects of donor reproductive maturity and pregnancy status on oocyte quality and in vitro fertilization (IVF) outcomes, and to demonstrate the feasibility of producing healthy offspring. Cumulus oocyte complexes (COCs) were placed into an in vitro embryo production (IVP) system, and on days 7, 7.5, and 8 of in vitro culture (Day 0 = day of in vitro fertilization) embryos were assessed for developmental stage and quality prior to vitrification. Embryos were then stored in liquid nitrogen until the breeding season when a subset were warmed, cultured for 6 h, evaluated for survival, and transferred to healthy bison recipients. There were no significant differences in the ability of recovered COCs to support blastocyst development based on female reproductive maturity or pregnancy status (juvenile 79/959 (8.2%) vs sexually mature 547/6544 (8.4%); non-pregnant 188/2302 (8.2%) vs pregnant 556/6122 (9.1%)). Following the transfer of 15 embryos to 10 recipients, one healthy female calf was born. This work demonstrates that live offspring can be generated from COCs collected from YNP bison post mortem in the non-breeding season, and that gamete recovery can be a valuable tool for conservation of valuable genetics for this species while mitigating diseases like brucellosis.

Molecular evidence confirms occurrence of Rhipicephalus microplus Clade A in Kenya and sub-Saharan Africa

BackgroundThe tick vector Rhipicephalus microplus which transmits Babesia spp. and rickettsial pathogens has not been reported in Kenya since 1998. More recently, the pathogenic Babesia bovis has been detected in cattle blood DNA. The status of R. microplus in Kenya remains unknown. This study employed morphological and molecular tools to characterize R. microplus originating from Kenya and assess the genetic relationships between Kenyan and other African R. microplus genotypes.MethodsTicks were collected in south-eastern Kenya (Kwale County) from cattle and characterized to investigate the existence of R. microplus. Genetic and phylogenetic relationships between the Kenyan and other annotated R. microplus reference sequences was investigated by analysis of the cytochrome c oxidase subunit 1 (cox1) gene. To further characterize Kenyan ticks, we generated low coverage whole genome sequences of two R. microplus, one R. decoloratus and R. appendiculatus. A B. bovis specific TaqMan probe qPCR assay was used to detect B. bovis in gDNA from R. microplus ticks.ResultsOccurrence of R. microplus was confirmed in Kwale County, Kenya. The Kenyan R. microplus cox1 sequences showed very high pairwise identities (> 99%) and clustered very closely with reference African R. microplus sequences. We found a low genetic variation and lack of geographical sub-structuring among the African cox1 sequences of R. microplus. Four complete mitochondrial (mt) genomes for two R. microplus, one R. decoloratus and one R. appendiculatus were assembled from next generation sequence data. The mitochondrial genome sequences of the two Kenyan R. microplus ticks clustered closely with reference genome sequences from Brazil, USA, Cambodia and India forming R. microplus Clade A. No B. bovis was detected in the Kwale R. microplus DNA.ConclusionsThese findings confirm the presence of R. microplus in Kenya and suggest that R. microplus Clade A is prevalent in cattle in sub-Saharan Africa. These and other recent findings of widespread occurrence of R. microplus in Africa provide a strong justification for urgent surveillance to determine and monitor the spread of R. microplus and vector competence of Boophilus ticks for B. bovis in Africa, with the ultimate goal of strategic control.

Comparative whole genome DNA methylation profiling across cattle tissues reveals global and tissue-specific methylation patterns

BackgroundEfforts to improve animal health, and understand genetic bases for production, may benefit from a comprehensive analysis of animal genomes and epigenomes. Although DNA methylation has been well studied in humans and other model species, its distribution patterns and regulatory impacts in cattle are still largely unknown. Here, we present the largest collection of cattle DNA methylation epigenomic data to date.ResultsUsing Holstein cattle, we generated 29 whole genome bisulfite sequencing (WGBS) datasets for 16 tissues, 47 corresponding RNA-seq datasets, and 2 whole genome sequencing datasets. We did read mapping and DNA methylation calling based on two different cattle assemblies, demonstrating the high quality of the long-read-based assembly markedly improved DNA methylation results. We observed large differences across cattle tissues in the methylation patterns of global CpG sites, partially methylated domains (PMDs), hypomethylated regions (HMRs), CG islands (CGIs), and common repeats. We detected that each tissue had a distinct set of PMDs, which showed tissue-specific patterns. Similar to human PMD, cattle PMDs were often linked to a general decrease of gene expression and a decrease in active histone marks and related to long-range chromatin organizations, like topologically associated domains (TADs). We tested a classification of the HMRs based on their distributions relative to transcription start sites (TSSs) and detected tissue-specific TSS-HMRs and genes that showed strong tissue effects. When performing cross-species comparisons of paired genes (two opposite strand genes with their TSS located in the same HMR), we found out they were more consistently co-expressed among human, mouse, sheep, goat, yak, pig, and chicken, but showed lower consistent ratios in more divergent species. We further used these WGBS data to detect 50,023 experimentally supported CGIs across bovine tissues and found that they might function as a guard against C-to-T mutations for TSS-HMRs. Although common repeats were often heavily methylated, some young Bov-A2 repeats were hypomethylated in sperm and could affect the promoter structures by exposing potential transcription factor binding sites.ConclusionsThis study provides a comprehensive resource for bovine epigenomic research and enables new discoveries about DNA methylation and its role in complex traits.

Investigating new SNPs of CAST, CAPN and SCD genes in 5’UTR of Bali cattle

Abstract. Jakaria, Ulum MF, Lestari D, Akwila S, Sihite DEWT, Priyanto R, Muladno, Sumantri C. 2020. Investigating new SNPs of CAST, CAPN and SCD genes in 5’UTR of Bali cattle. Biodiversitas 21: 2971-2976. The present investigation aims to explore specific SNPs of Bali cattle, focusing mainly on CAST, CAPN, and SCD genes in 5'UTR. DNA of Bali cattle (25 individuals) from Breeding Centre of Bali cattle in Bali Province, Indonesia, were extracted according to the protocol of GenAID. Amplification of the CAST, CAPN, and SCD genes were designed using the Primer3 program based on reference sequences (access numbers AH014526.2, AH009246.3, and AY241932). Furthermore, the PCR product was sequenced through forward primer sequencing. The data sequence results were analyzed using the Bioedit, MEGA7, Popgen 3.2, and DnaSP6 V6 programs. The results showed that SNPs were detected at g.921C&gt;T and g.922T&gt;G in 5'UTR for the CAST gene between the reference of GenBank AH014526.2 and Bali cattle, but it was monomorphic within the population. The high polymorphism of SNPs was found in g.232G&gt;T for the CAPN gene and g.134A&gt;del for the SCD gene. However, the SNP g.246C&gt;del SCD gene was detected as monomorphic in this region. Moreover, this result shows that low nucleotide diversities (Pi) for CAST, CAPN, and SCD genes in 5'UTR of Bali cattle were 0.00422, 0.00632, and 0.00449, respectively. This finding was important for developing marker-assisted selection (MAS) to improve the meat quality of Bali cattle.

Bartonella bovis and Bartonella chomelii infection in dairy cattle and their ectoparasites in Algeria

Bartonella are blood-borne and vector-transmitted bacteria, some of which are zoonotic. B. bovis and B. chomelii have been reported in cattle. However, no information has yet been provided on Bartonella infection in cattle in Algeria. Therefore, 313 cattle from 45 dairy farms were surveyed in Kabylia, Algeria, in order to identify Bartonella species infecting cattle using serological and molecular tests. In addition, 277 ticks and 33 Hippoboscidae flies were collected. Bartonella bovis and B. chomelii were identified as the two species infecting cattle. Bartonella DNA was also amplified from 6.8 % (n = 19) of ticks and 78.8 % (n = 26) of flies. Prevalence of B. bovis DNA in dairy cattle was associated both with age and altitude. This study is the first one to report of bovine bartonellosis in Algeria, both in dairy cattle and in potential Bartonella vectors, with the detection of B. bovis DNA in tick samples and B. chomelii in fly samples.

DGAT1 K232A polymorphism is associated with milk production traits in Chinese cattle

The production traits of cattle, especially milk trait, are of great significance to human life. A quantitative trait loci (QTL) associated with milk fat content was detected in the centromeric region of cattle chromosome 14. This QTL harbors a strong candidate gene called DGAT1 responsible for the milk quality. A non-conservative substitution of lysine by alanine (K232A) was found in DGAT1 gene producing a strong effect on milk composition and yield. The lysine (K allele) is associated with increased milk fat content, while the decreased milk fat content is linked to the alanine (A allele) amino acid. To estimate the frequencies of the DGAT1 K232A polymorphism in Chinese cattle breeds, PCR and DNA sequencing methods were used to investigate the polymorphism of DGAT1 K232A in a total of 682 individuals, including 655 Chinese cattle and 27 Holstein cattle. The results demonstrated that the frequency of K allele gradually elevated from the northern group to the southern group of native Chinese cattle, whereas the frequency of A allele showed a contrary pattern, displaying a significant geographical difference across native Chinese cattle breeds. Our results confirm that the southern cattle group has higher milk fat content than that of the northern group.

On-Site Detection of Tissues of Buffalo Origin by Loop-Mediated Isothermal Amplification (LAMP) Assay Targeting Mitochondrial Gene Sequences

Meat fraud and adulteration is a growing concern in the marketplace today. Species identification in food product is among primary duties of food office control. In this study, a one-step, species-specific, sensitive, and rapid loop-mediated isothermal amplification (LAMP) assay was developed for identification of tissues of buffalo origin. A set of six LAMP primers were designed by targeting mitochondrial cyt b gene. The reaction temperature of LAMP and component of reaction mixture were optimized as 64 °C amplification temperature, 6 U/μL concentration of Bst DNA polymerase, 3 mM concentration of MgSO4, 0.4 mM concentration of dNTP, 0.6 M concentration of betaine, and 1:6 ratio of outer and inner primer pair. Successful amplifications were confirmed using SYBR Green I dye and agarose gel electrophoresis. The specificity of assay was tested with DNA of cattle, goat, sheep, and pig where amplification was observed exclusively in buffalo. The analytical sensitivity of the LAMP assay for buffalo DNA detection was 0.1 pg. The applicability check of developed assay carried out on known/coded samples and binary meat admixture substantiated the accuracy of assay. Supernatant of tissue exposed to Phire Animal Tissue Direct PCR master mix treatment was observed rich enough with DNA of target species and successfully amplified under optimized LAMP reaction. The comparison of results on the samples undergone pre-standardized species-specific PCR taken as gold standard shown complete match with developed species-specific LAMP assay. Hence, the developed species-specific LAMP assays are effective in detection of tissue of buffalo origin.

Resultados negativos para Borrelia burgdorferiem vacas e carrapatos de região fronteiriça entre Brasil e Paraguai: relato de caso

Lyme borreliosis is an infectious disease caused by bacteria of the genus Borrelia. In ruminants, most infections are asymptomatic, but the animals can present myalgia, lameness, laminitis, arthritis, synovitis, neurological symptoms, and also decreased production and abortion. The objective was to investigate Borrelia burgdorferi DNA in cows and cattle ticks on a small dairy farm in a border region. Blood samples and ticks were collected from Holstein cows with a history of decreased milk production and abortions. Borrelia burgdorferi DNA was extracted from blood samples using a commercial extraction kit, and from ticks using an alkaline hydrolysis solution for subsequent nested-PCR. Serum and tick samples did not present Borrelia burgdorferi DNA, and 100% of the ticks were identified as Rhipicephalus (Boophilus) microplus. Although this study shows negative results it contributes to understanding the epidemiology of this etiological agent in Parana and in Brazil, since there are few studies on bovine species. The negative results of this work demonstrate that the animals and ticks researched were not exposed to Borrelia burgdorferi, however, as it is a property located in a border region, the sanitary monitoring of the herd must be performed constantly since this is a region. vulnerable to the entry of potential threats to human, animal and environmental health from vectors and pathogenic microorganisms, given the large extension of the land border with the neighboring country and which also has different health status.

74 Analysis of chromosomal abnormality of bovine IVF embryos based on next-generation sequencing

It has been suggested that bovine IVF embryos have a higher frequency of occurrence of chromosomal abnormalities than invivo-fertilised embryos, which may explain low pregnancy success, but the details have not been clarified (Yao et al. 2018 Sci. Rep. 8, 7460). In this study, chromosomal aneuploidy in blastocysts of bovine IVF and invivo-fertilised was analysed by copy number variations (CNVs) based on next-generation sequencing. The IVF bovine embryos were cultured in well of-the-well culture dishes (LinKID micro25: Dai Nippon Printing) containing 125µL of CR1aa supplemented with 5% calf serum at 38.5°C in 5% O2 and 5% CO2 for 8 days after insemination. Invitro development of embryos was monitored using time-lapse cinematography (Sugimura et al. 2010 Biol. Reprod. 83, 970-78). Invivo embryos were produced by collection of a superstimulated Japanese Black cow. Embryos that reached the blastocyst stage were divided into inner cell mass (ICM) and trophectoderm (TE) fractions by a micromanipulator with a blade. The TE and ICM samples were biopsied individually from 10 IVF and 4 invivo-derived embryos, and extracted DNA was amplified using the SurePlex DNA amplification System (Illumina). The whole-genome amplified DNA libraries were sequenced using MiSeq (Illumina). The sequencing reads were mapped onto the Bos taurus reference genome ARS-UCD1.2, obtained from the National Center for Biotechnology Information. In all 29 autosomal chromosomes and the X chromosome, CNV analysis was performed by CNV-seq (Xie and Tammi 2009 BMC Bioinformatics 10, 80). Male or female Japanese Black cattle DNA sequence was used for the reference genome. The parameter of CNV-seq was run with P-value=0.001, log2=0.6, and window size=1M. Four IVF embryos showed chromosomal duplications or deletions in either ICM- or TE-cell samples (4/10, 40%). The CNV loci between ICM and TE cells were relatively similar in each embryo. One of them was a code 1-expanded blastocyst with normal cleavage. Interestingly, CNV was not identified in another code 1-expanded blastocyst that underwent direct cleavage from 1 cell to 3 or more cells. In invivo embryos, only one embryo had a CNV (1/4, 25%). Observed CNVs in both IVF and invivo embryos were segmental duplication or deletion in each chromosome. Hence, to improve pregnancy success in bovine IVF embryos, cytogenetic evaluation may be useful for quality evaluation of embryos that are prone to chromosomal abnormalities, as well as morphological scoring.

Single Nucleotide Polymorphism on Exon 2 Leptin Gene of Pasundan Cattle

Single Nucleotide Polymorphism on exon 2 of leptin gene which changes encoding from Arginine to Cysteine could change the function of Leptin as an obese gene which regulated feed intake and energy expenditure. The aims to analyze point mutation based on SNP exon 2 of Leptin gene of Pasundan cattle. This research used 49 DNA of Pasundan cattle from BPPT SP Cijeungjing. Multiplication of DNA was done by Polymerase Chain Reaction (PCR). Analyze of SNP on exon 2 Leptin gene based on nucleotide sequence from direct sequencing of PCR product which was alligned with leptin gene sequence from gene bank using Bioedit and MEGA 5.2 program. The results showed nucleotide sequence of exon 2 Leptin gene in Pasundan cattle is different from Leptin gene sequence of Bos indicus and Bos taurus. It was found one synonymous SNP that did not change amino acids Serine encoding on S17S, and two non-synonymous SNP which altered amino acids encoding, i.e R25C and R25H. In Pasundan cattle, the frequency of C allele (42.86%) was higher than A allele (31.64%) and T allele (25.50%). Six genotypes were identified i.e. CC (24.49%), CT (32.65%), CA (4.08%), TT (8.16%) and TA (2.05 %), AA (28.57%).

Detection of bovine herpesvirus 4 DNA in cattle by realtime PCR.

BoHV-4 is poorly understood. Data on the circulation of the virus among animals and its role in infectious diseases insufficient. Aimes and goals. Development of real-time PCR for detecting the BoHV-4 and studying the frequency of its presence in samples from sick animals. The nucleotide sequences of the glycoprotein L gene served as a target for amplification. The sequences of reference strains published in GenBank were used to analyze and design the primers. Studies were conducted in 3 regions of Western Siberia on 5 large dairy farms. 27.7% of samples contained the virus. The virus was present as a monoagent in nasal cavity of calves (80.0%), lungs (46.2%) and bronchial lymph nodes (38.5%) in pneumonia. In the cases of diarrhea the virus was detected in 20%, and in cows with gynecological pathology in 10.0%. In respiratory diseases of calves the virus was detected in association with BoHV-1 (21.6%) and BoCV (20.3%), and in gynecological pathology of cows with BVDV1 (6%). According to the phylogenetic analysis of 5 identified virus isolates, four belonged to the American branch and one to the European branch. The circulation of American strains occurred in the territory of the Republic of Kazakhstan (1), Tyumen (1) and Novosibirsk (2) regions, and the European - in the Novosibirsk region. The search for viruses involved to the infectious pathology, as well as studying the genetic diversity of viruses circulating on a particular farm including imported from other countries, is relevant.

Ancient cattle DNA reveals a bullish tale

Ancient cattle DNA reveals a bullish tale

The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.

Multiplex PCR assay for identification and quantification of bovine and equine in minced meats using novel specific nuclear DNA sequences

The 2013 horsemeat scandal demonstrated the importance and significance of developing analytical methods to accurately identify and quantify bovine and equine components in meats. In this study, we successfully screened the bovine- and equine-specific nuclear DNA sequences for detection of bovine and equine fractions in minced meats, respectively. Primers and probes designed from bovine- and equine-specific nuclear DNA sequences were critically analysed against 53 species, and then their specificity was evaluated against 23 species through PCR. The conservation of primers and probes binding regions was tested in 44 different individuals of bovine or equine. The limit of detection (LOD) is 0.05 ng in both conventional and real-time PCR, and the limit of quantification (LOQ) is down to 5%. Real-time PCR confirmed that bovine- and equine-specific nuclear DNA sequences are present as a fixed copy number in the genome. A multiplex conventional PCR method was established to effectively discriminate bovine (223 bp), equine (197 bp) and reference (129 bp) in one reaction. A multiplex real-time PCR was developed and confirmed in five binary mixtures of cattle and horse DNA, exhibiting adequate parameters of trueness (R.E. ≤ 9.35%) and precision (R.S.D. ≤ 8.88%). Furthermore, we confirmed that our method held good potential in the detection of meat products in real life. The method and strategy developed in this study will play an important role in combating adulteration and maintaining a fair market.