Ear Notch Research Articles

Comparison of bovine viral diarrhea virus detection methods: Results of an international proficiency trial

Bovine viral diarrhea virus (BVDV), one of the most important infectious cattle diseases globally, is being combated in multiple countries. The main source for virus transmission within herds and especially to unaffected cattle farms are life-long persistently infected (PI), immunotolerant animals. Therefore, the early identification of PI calves is a major pillar of disease control programs. In addition, rapid and reliable virus identification is necessary to confirm the causative agent in acute clinical cases. Here, we initiated an international interlaboratory proficiency trial in order to evaluate BVDV detection methods.Four ear notch samples and four sera were provided to the participating veterinary diagnostic laboratories (n = 40). Two of the ear notches and two sera contained BVDV and two ear notches and one serum were negative for pestiviruses. The remaining serum was positive for the ovine border disease virus (BDV). The sample panel was analyzed by an ERNS-based ELISA for antigen detection, diverse real-time RT-PCR (RT-qPCR) assays and/or virus isolation. Occasionally, additional typing of the virus strains was performed by sequencing or specific antibody staining of the obtained cell culture isolates. While the antigen ELISA allowed reliable BVDV diagnostics, infectious virus could be isolated only in just under half of the attempts (43.33%). RT-qPCR enabled the sensitive detection of pestiviruses, though an impact of the extraction method on the resulting quantification cycle values was observed. In general, subsequent typing of the detected virus strains is required to differentiate BVDV from BDV infections.In conclusion, for BVDV identification in clinical cases or in the context of disease control, RT-qPCR methods or ERNS antigen ELISAs should be preferentially used.

Práticas de biosseguridade associadas com infecção do vírus da diarreia viral bovina em rebanhos leiteiros no Brasil

ABSTRACT: This study determined the association between biosecurity practices and the status of bovine viral diarrhea virus (BVDV) in dairy production systems. Approximately 280 herds were screened for BVDV virus detection. Following the screening, 68 herds were selected to identify individual BVDV PI animals using an ear notch biopsy and ELISA-antigen. All offspring of the last generation were tested, and the maternal lineage of positive cases was examined. A questionnaire on BVDV biological risk assessment was completed. A multiple correspondence analysis (MCA) was conducted to determine the association between herds with or without BVDV circulation and biosecurity practices. The MCA revealed that farms with virus circulation lacked knowledge about the disease and wrongly perceived their herds as protected, while farms without virus circulation were aware of the disease but considered their herds unprotected. Vaccination practices differed between positive and negative herds, with positive herds using vaccines only for reproductive diseases and negative herds vaccinating for respiratory and reproductive issues. Biosecurity practices such as frequent visitation, contact between animals of different ages, and annual introduction of new animals were linked to viral circulation, while virus-free herds implemented measures like controlled visitation, no contact between different age groups, and quarantine. Lastly, herds with virus circulation acquired pregnant females without prior testing. This study emphasized the crucial role of biosecurity practices in controlling BVDV in dairy herds. It highlighted the sharper risk perception and better application of biosecurity practices in negative herds compared to BVDV-positive herds.

397 Field Demonstration Analyzing the Implementation of Individual Animal Electronic Identification and Genetic Testing in Western Range Sheep Flocks

Abstract The lack of individual performance records derived from an extensively driven production system coupled with impracticality to utilize reproductive technologies makes commercial genetic improvement challenging in small ruminants. We collaborated with five producers throughout California to assess how Electronic ID (EID) and a genotyping panel can augment the productivity of a flock. We sought to study how the data collected from these technologies can hasten genetic improvement towards desirable operational objectives. Familiarity and use of EID tags on ranches at the start of the research varied; thus, allowing for a qualitative reflection on the perceptions of the participants of the usefulness of this technology. We partnered with Superior Farms, a slaughterhouse, to evaluate their targeted genotyping panel, Flock54. Flock54 is a single nucleotide polymorphism (SNP) panel containing 956 markers for assigning pedigree and includes markers linked to quantitative trait loci and/or known single gene causative markers to aid in disease management and fecundity scoring. Ear notch samples were collected from 2,963 animals for analysis. This included 305 total sires as well as their progeny, 989 female lambs and 1,669 male lambs, which were marketed. Nine lambs were positive at a fecundity marker. No other notable results were found from the Flock54 panel. Genomic data was used to ascertain prolific sires on every ranch; however, ram to ewe ratio altered the influence that a prolific sire had on a lamb crop. The lowest ram to ewe ratio was 1:10. Parentage results from this ranch reports the following progeny metrics: Average: 9.9 males (±12.5); Median: 4 males; Max: 61 males, minimum 1 male. In contrast, the greatest ram to ewe ratio was 1:50. Parentage results from this ranch reports the following progeny metrics: Average: 51.9 (± 41.7); Median: 43.5; Max: 135; Minimum: 4,524 carcass samples from terminal lambs at two ranches were processed at Superior Farms` plant and assessed with a USDA inspected camera grading system. Progeny contemporary group deviations were calculated for hot carcass weight, yield grade, ovine cutability calculation (OCC), and OCC yield. After coupling carcass metrics with USDA reported prices, we identified prolific sires within the flock that garnered significantly higher and lower dollar merit at slaughter. A two-tailed Student`s T-test was used on the dollar deviation of edible product sold to identify if there was a statistically significant difference (P-value < 0.05) between a given ram`s progeny and the rest of the progeny slaughtered. We found that twenty-three out of the forty-three rams with progeny from one ranch had a statistically significant dollar deviation difference with a T-value greater than 2.0167. For the other ranch, we found that nineteen out of the thirty-five rams with progeny had a statistically significant difference with a T-value greater than 2.0301.

Biodiversity of pigs of various breeds based on the analysis of mtDNA D-loop

Mitochondrial DNA polymorphism, which is one of the most common genetic markers, is actively used in the study of various animal species. Since the mitochondrial genome has evolved over many years within a species, this has influenced the fixation of mutations and the formation of mitochondrial lineages that share a common origin and form mitochondrial genomes, called mtDNA haplogroups. The objectives of the work were to evaluate the genetic diversity of pigs of two maternal breeds of domestic reproduction based on the analysis of mtDNA D-loop polymorphism and to compare the results obtained with identical sequences from the NCBI database by breed and their geographic distribution. 39 Landrace pigs and 49 Large White pigs were used for the study. MtDNA was isolated from tissue samples (ear notch). The number of haplotypes, haplotypic and nucleotide diversity, and the average number of nucleotide substitutions per site were determined to assess the genetic diversity. 23 polymorphic sites were found: 21 in Landrace pigs, 17 in Large White pigs. A total of ten haplotypes were identified in the study population. The NCBI database found 75 identical sequences for pigs of different breeds bred around the world. After determining the nucleotide sequences of the D-loop fragment of the mtDNA of Landrace and Large White pigs of domestic reproduction and comparing them with the sequences from the NCBI database, the presence of identical sequences in the animals we studied and in the representatives of European and Asian breeds, including commercial and local was established. The materials obtained show that the assessment of mtDNA polymorphism contributes to the identification of phylogenetic relationships between populations, tracing of pedigree processes, and can be considered as an additional criterion of selection and breeding work.

Plasma and Skin Per- and Polyfluoroalkyl Substance (PFAS) Levels in Dairy Cattle with Lifetime Exposures to PFAS-Contaminated Drinking Water and Feed.

Plasma and ear notch samples were removed from 164 Holstein cows and heifers, which had lifetime exposures to per- and polyfluoroalkyl substances (PFAS) through consumption of contaminated feed and water sources. A suite of nine PFAS including five perfluoroalkyl carboxylic acids (PFCA) and four perfluoroalkyl sulfonic acids (PFSA) was quantified in plasma and ear notch samples by liquid chromatography-mass spectrometry. Bioaccumulation of four- to nine-carbon PFCAs did not occur in plasma or skin, but PFSAs longer than four carbons accumulated in both plasma and skin. Exposure periods of at least 1 year were necessary for PFSAs to reach steady-state concentrations in plasma. Neither parity (P = 0.76) nor lactation status (P = 0.30) affected total PFSA concentrations in mature cow plasma. In contrast, lactation status greatly affected (P < 0.0001) total PFSA concentrations in ear notch samples. Skin samples could be used for biomonitoring purposes in instances when on-farm blood collection and plasma preparation are not practical.

PSIII-2 Late-Breaking: Collaboration to Demonstrate the Potential Use and Value of Electronic Identification and DNA Testing in the Sheep Industry

Abstract The dearth of individual performance records derived from an extensively driven production system coupled with the impracticality of utilizing reproductive technologies makes commercial genetic improvement challenging in small ruminants. We collaborated with five producers throughout California to assess how electronic ID (EID) and genetic testing could inform ram selection. Our objective was to determine how the data collected from these technologies could augment genetic improvement on commercial ranches. Familiarity and use of EID tags on ranches at the start of the research varied; thus, allowing for a qualitative reflection on the participant's perceptions of the usefulness of this technology. Ear notch samples were collected and EID ear tags were placed in 2,963 sheep; 305 rams, and 2,658 of their potential progeny. We partnered with Superior Farms (Dixon, CA) to genotype the animals using their Flock54 single nucleotide polymorphism panel which contains 956 markers for assigning parentage, and includes markers linked to quantitative trait loci and/or known single gene causative markers to aid in disease management. SNP data was used to assign paternity and determine sire prolificacy. A total of 524 carcass samples from ram lambs from two of the ranches were processed at Superior Farms' plant and assessed with a USDA-approved camera grading system (e+v, Oranienburg, Germany) that matches EID’s to carcass data. Progeny contemporary group deviations were calculated for hot carcass weight, yield grade, and ovine cutability calculation (OCC) which predicts the yield of edible product on the carcass. The average number of lambs per sire born at each ranch and the mean OCC of the progeny for each sire were compared using a one-way analysis of variance. There was a significant ranch effect on the average progeny number per ram (P &amp;lt; 0.05). The ranch with the lowest ram to ewe ratio (1:10) also has the lowest average progeny per ram and the ranch with the greatest ratio (1:50) has the most progeny per ram. There was a large range in the number of lambs per sire (0-135) within each ranch. There was a significant sire effect (P &amp;lt; 0.05) on average OCC within each progeny contemporary group, meaning some sires were producing offspring with significantly more marketable meat. The use of EIDs in combination with genetic testing, allowed for the collection of individual progeny performance data. This provided insight into sire performance and identified prolific sires that were also producing lambs with superior carcass merit. Producers could use this information to select for high-performing rams; however, there is an unavoidable delay between ram turnout and the collection of progeny carcass data. This limits the practical utility of these data and reinforces the value of having estimated breeding values available at ram purchase to select genetically superior rams.

An unusual complication of ear punching in a laboratory mouse

AbstractEar punching and notching are common methods of identification of laboratory rodents. Concerns have been raised regarding pain and discomfort, infection, thermoregulation and tearing. However, the evidence regarding these potential adverse events is currently limited. In this case report, we describe an unusual complication of ear punching in a laboratory mouse. The mouse had its left ear punched with a single hole at 3 weeks of age. At 11 weeks of age, the mouse caught its left foreleg through its left ear punch up to the level of the shoulder. The mouse's leg was released and the pinna cut to form a notch rather than a hole. To avoid this situation, we suggest that an ear notch be considered rather than an ear punch. This case also highlights the value of daily checks to ensure that the risk of any adverse effects from such unpredictable events is minimised.

Output-based assessment of herd-level freedom from infection in endemic situations: Application of a Bayesian Hidden Markov model

Countries have implemented control programmes (CPs) for cattle diseases such as bovine viral diarrhoea virus (BVDV) that are tailored to each country-specific situation. Practical methods are needed to assess the output of these CPs in terms of the confidence of freedom from infection that is achieved. As part of the STOC free project, a Bayesian Hidden Markov model was developed, called STOC free model, to estimate the probability of infection at herd-level. In the current study, the STOC free model was applied to BVDV field data in four study regions, from CPs based on ear notch samples. The aim of this study was to estimate the probability of herd-level freedom from BVDV in regions that are not (yet) free. We additionally evaluated the sensitivity of the parameter estimates and predicted probabilities of freedom to the prior distributions for the different model parameters. First, default priors were used in the model to enable comparison of model outputs between study regions. Thereafter, country-specific priors based on expert opinion or historical data were used in the model, to study the influence of the priors on the results and to obtain country-specific estimates.The STOC free model calculates a posterior value for the model parameters (e.g. herd-level test sensitivity and specificity, probability of introduction of infection) and a predicted probability of infection. The probability of freedom from infection was computed as one minus the probability of infection. For dairy herds that were considered free from infection within their own CP, the predicted probabilities of freedom were very high for all study regions ranging from 0.98 to 1.00, regardless of the use of default or country-specific priors. The priors did have more influence on two of the model parameters, herd-level sensitivity and the probability of remaining infected, due to the low prevalence and incidence of BVDV in the study regions. The advantage of STOC free model compared to scenario tree modelling, the reference method, is that actual data from the CP can be used and estimates are easily updated when new data becomes available.

Detection of Clinical and Subclinical Lumpy Skin Disease Using Ear Notch Testing and Skin Biopsies.

Lumpy skin disease (LSD) diagnosis is primarily based on clinical surveillance complemented by PCR of lesion crusts or nodule biopsies. Since LSD can be subclinical, the sensitivity of clinical surveillance could be lower than expected. Furthermore, real-time PCR for the detection of LSD viral DNA in blood samples from subclinical animals is only intermittently positive. Therefore, this study aimed to investigate an acceptable, easily applicable and more sensitive testing method for the detection of clinical and subclinical LSD. An animal experiment was conducted to investigate ear notches and biopsies from unaffected skin taken from the neck and dorsal back as alternatives to blood samples. It was concluded that for early LSD confirmation, normal skin biopsies and ear notches are less fit for purpose, as LSDV DNA is only detectable in these samples several days after it is detectable in blood samples. On the other hand, blood samples are less advisable for the detection of subclinical animals, while ear notches and biopsies were positive for LSD viral DNA in all subclinically infected animals by 16 days post infection. In conclusion, ear notches could be used for surveillance to detect subclinical animals after removing the clinical animals from a herd, to regain trade by substantiating the freedom of disease or to support research on LSDV transmission from subclinical animals.

Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples

BackgroundAs a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5’ untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation.ResultsTo enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19–32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage.ConclusionsFurther phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

The Irish Programme to Eradicate Bovine Viral Diarrhoea Virus-Organization, Challenges, and Progress.

A mandatory national Irish bovine viral diarrhoea (BVD) eradication programme, coordinated by Animal Health Ireland, commenced in 2013. Key decisions and programme review are undertaken by a cross-industry Implementation Group (BVDIG) supported by a Technical Working Group. Ear notch tissue is collected from all new-born calves using modified official identity tags, supplemented by additional blood sampling, including for confirmatory testing of calves with initial positive results and testing of their dams. Testing is delivered by private laboratories in conjunction with the National Reference Laboratory, with all results reported to a central database. This database manages key elements of the programme, issuing results to herdowners by short message service messaging supplemented by letters; assigning and exchanging animal-level statuses with government databases of the Department of Agriculture, Food and the Marine to enable legislated restrictions on animal movements; assigning negative herd status based on test results; generating regular reports for programme management and evaluation and providing herd-specific dashboards for a range of users. Legislation supporting the programme has been in place throughout but has not thus far mandated the slaughter of persistently infected (PI) calves. A key challenge in the early years, highlighted by modeling, was the retention of PI animals by some herd owners. This has largely been resolved by measures including graduated financial supports to encourage their early removal, herd-level movement restrictions, ongoing programme communications and the input of private veterinary practitioners (PVPs). A framework for funded investigations by PVPs in positive herds was developed to identify plausible sources of infection, to resolve the status of all animals in the herd and to agree up to three measures to prevent re-introduction of the virus. The prevalence of PI calves in 2013 was 0.66%, within 11.3% of herds, reducing in each subsequent year, to 0.03 and 0.55%, respectively, at the end of 2020. Recent regulatory changes within the European Union for the first time make provision for official approval of national eradication programmes, or recognition of BVD freedom, and planning is underway to seek approval and, in due course, recognition of freedom within this framework by 2023.

A SEARCH FOR GENOMIC VARIANTS ASSOCIATED WITH BODY WEIGHT IN SHEEP BASED ON HIGH DENSITY SNP GENOTYPES ANALYSIS

Body weight is one of the most important economically useful traits, which is characterized by complex inheritance. Therefore, a search for genetic mechanisms affecting its formation is of increased scientific interest. This work presents for the first time the results of the genome-wide association studies in the resource population of sheep (Ovis aries) from backcross (Romanov × Katah-din) × Romanov family, whose body weight was recorded in age dynamics, and SNP profiles were obtained using a high-density DNA chip. We identified 38 SNPs significantly associated with body weight (p < 0.00001) and functional candidate genes which affect skeletal muscle growth, bone scaffold formation, lipid, and carbohydrate metabolism. In addition, age-related changes in the set of significantly significant SNPs are shown. Our aim was to search for genomic variants that affect the body weight of (Romanov × Katahdin) × Romanov backcrosses from the resource population at different age periods. The study was performed on 95 sheep from backcross (Romanov × Katahdin) × Romanov family (Ernst Federal Research Center for Animal Husbandry, 2018-2021). Ear notch samples were taken from backcrosses for extraction of genomic DNA, which isolated using DNA-Extran-2 kits (Syntol LLC, Russia). Animals were genotyped using a high-density DNA chip Ovine Infinium® HD SNP BeadChip (Illumina, Inc., USA) containing ~600 thousand SNP markers. The body weight was recorded at the ages of 6(BW6), 42(BW42), 90(BW90), 180(BW180) and 270 days (BW270). To study genome-wide associations with body weight, we used regression analysis implemented in PLINK 1.90 (--assoc --adjust --qt-means). To confirm the significant effect of SNP and to identify significant regions in the genome of the studied sheep, a test was performed to check null hypotheses according to Bonferroni at a threshold value of p < 1.09×10-7, 0.05/459868. The search for candidate genes located in the region of identified SNPs was performed using the VEP (Ensembl Variant Effect Predictor) tool of Ensembl genome browser 103 (https://www.ensembl.org/index.html). After quality control, 459,868 SNPs were used to perform genome-wide association studies (GWAS). Average body weights in the studied sample were 3.28±0.07, 8.03±0.21, 13.74±0.39, 20.19±0.51, and 22.51±0.50 kg at the age of 6, 42, 90, 180, and 270 days, respectively. We found that in different age periods the set of SNPs associated with the integral indicator of the growth rate, the animal body weight, was different. Thus, out of 38 identified SNPs, 18 SNPs located on OAR2, OAR4, OAR9, and OAR15 were reliably associated with BW6 (p < 0.00001); 3 SNPs located on OAR6 and OAR11 with BW42 (p < 0.00001); 2 SNPs located on OAR10 and OAR19 with BW90 (p < 0.00001); 6 SNPs (p < 0.00001) located on OAR1 and OAR13 with BW180 (p < 0.00001) and 6 SNPs located on OAR1 with BW270 (p < 0.00001). Blocks of 3-5 SNPs were found on OAR1, OAR2, OAR4, and OAR5. Significance levels for six SNPs, including oar3_OAR4_87887519 (p < 7.13×10-8), oar3_OAR4_87889243(p < 1.51×10-7), oar3_OAR9_89145258 (p < 4.95×10-7), oar3_OAR1_192662599 (p < 4.79×10-7), OAR1_208070059.1 (p < 4.79×10-7) and oar3_OAR13_31446454 (p < 6.84×10-7) exceeded the threshold for GWAS (p < 1.09×10-7). Along with known candidate genes associated with body weight in sheep, we found new candidate genes whose effects on this trait have not been previously reported. The functional annotation of the identified candidates showed the presence of genes affecting skeletal muscle growth, bone frame formation, lipid, and carbohydrate metabolism. The obtained data will be useful for the development of marker and genomic selection programs in sheep breeding.

ПОИСК ГЕНОМНЫХ ВАРИАНТОВ, АССОЦИИРОВАННЫХ С ЖИВОЙ МАССОЙ У ОВЕЦ, НА ОСНОВЕ АНАЛИЗА ВЫСОКОПЛОТНЫХ SNP ГЕНОТИПОВ

Body weight is one of the most important economically useful traits, which is characterized by complex inheritance. Therefore, a search for genetic mechanisms affecting its formation is of increased scientific interest. This work presents for the first time the results of the genome-wide association studies in the resource population of sheep (Ovis aries) from backcross (Romanov × Katah-din) × Romanov family, whose body weight was recorded in age dynamics, and SNP profiles were obtained using a high-density DNA chip. We identified 38 SNPs significantly associated with body weight (p < 0.00001) and functional candidate genes which affect skeletal muscle growth, bone scaffold formation, lipid, and carbohydrate metabolism. In addition, age-related changes in the set of significantly significant SNPs are shown. Our aim was to search for genomic variants that affect the body weight of (Romanov × Katahdin) × Romanov backcrosses from the resource population at different age periods. The study was performed on 95 sheep from backcross (Romanov × Katahdin) × Romanov family (Ernst Federal Research Center for Animal Husbandry, 2018-2021). Ear notch samples were taken from backcrosses for extraction of genomic DNA, which isolated using DNA-Extran-2 kits (Syntol LLC, Russia). Animals were genotyped using a high-density DNA chip Ovine Infinium® HD SNP BeadChip (Illumina, Inc., USA) containing ~600 thousand SNP markers. The body weight was recorded at the ages of 6(BW6), 42(BW42), 90(BW90), 180(BW180) and 270 days (BW270). To study genome-wide associations with body weight, we used regression analysis implemented in PLINK 1.90 (--assoc --adjust --qt-means). To confirm the significant effect of SNP and to identify significant regions in the genome of the studied sheep, a test was performed to check null hypotheses according to Bonferroni at a threshold value of p < 1.09×10-7, 0.05/459868. The search for candidate genes located in the region of identified SNPs was performed using the VEP (Ensembl Variant Effect Predictor) tool of Ensembl genome browser 103 (https://www.ensembl.org/index.html). After quality control, 459,868 SNPs were used to perform genome-wide association studies (GWAS). Average body weights in the studied sample were 3.28±0.07, 8.03±0.21, 13.74±0.39, 20.19±0.51, and 22.51±0.50 kg at the age of 6, 42, 90, 180, and 270 days, respectively. We found that in different age periods the set of SNPs associated with the integral indicator of the growth rate, the animal body weight, was different. Thus, out of 38 identified SNPs, 18 SNPs located on OAR2, OAR4, OAR9, and OAR15 were reliably associated with BW6 (p < 0.00001); 3 SNPs located on OAR6 and OAR11 with BW42 (p < 0.00001); 2 SNPs located on OAR10 and OAR19 with BW90 (p < 0.00001); 6 SNPs (p < 0.00001) located on OAR1 and OAR13 with BW180 (p < 0.00001) and 6 SNPs located on OAR1 with BW270 (p < 0.00001). Blocks of 3-5 SNPs were found on OAR1, OAR2, OAR4, and OAR5. Significance levels for six SNPs, including oar3_OAR4_87887519 (p < 7.13×10-8), oar3_OAR4_87889243(p < 1.51×10-7), oar3_OAR9_89145258 (p < 4.95×10-7), oar3_OAR1_192662599 (p < 4.79×10-7), OAR1_208070059.1 (p < 4.79×10-7) and oar3_OAR13_31446454 (p < 6.84×10-7) exceeded the threshold for GWAS (p < 1.09×10-7). Along with known candidate genes associated with body weight in sheep, we found new candidate genes whose effects on this trait have not been previously reported. The functional annotation of the identified candidates showed the presence of genes affecting skeletal muscle growth, bone frame formation, lipid, and carbohydrate metabolism. The obtained data will be useful for the development of marker and genomic selection programs in sheep breeding.

Effect of calf age on bovine viral diarrhea virus tests.

Bovine viral diarrhea virus (BVDV) causes significant economic loss in cattle. Detection of persistently infected (PI) animals is an important control measure, but persistence of maternal antibodies may result in false-negative test results. We assessed the sensitivity and specificity of 2 antigen ELISAs (Idexx BVDV Ag/Serum Plus and BVDV PI X2) and a reverse-transcription real-time PCR (RT-rtPCR; Idexx RealPCR BVDV) assay for detecting PI calves. Ear notch samples were collected from 1,030 calves ~3, 10, 24, and 38 d old (days 3, 10, 24, and 38). All day 38 samples were tested using 2 antigen ELISAs and RT-rtPCR, and any calf that tested positive by any of these tests was blood sampled at ~100 d old (day 100) for antigen and antibody testing by ELISA; samples collected on days 3, 10, and 24 were tested using the antigen ELISAs and PCR. Calves were defined as PI if they were test-positive on day 38 by either ELISA or PCR and were antigen-positive on day 100. Twenty-six calves were PCR BVDV test-positive and one was BVDV PI X2 ELISA-positive at day 38. Five calves were defined as PI, and all tested positive by ELISAs and RT-PCR assay on days 3, 10, and 24. The sensitivity and specificity were 100% for both antigen ELISAs and 96.7% and 100%, respectively, by RT-rtPCR. Test results were not affected by calf age, suggesting that testing for PI calves can be undertaken at any age.

An Assessment of Secondary Clinical Disease, Milk Production and Quality, and the Impact on Reproduction in Holstein Heifers and Cows from a Single Large Commercial Herd Persistently Infected with Bovine Viral Diarrhea Virus Type 2.

The aim of this study was to evaluate secondary clinical disease, milk production efficiency and reproductive performance of heifers and cows persistently infected (PI) with bovine viral diarrhea virus type 2 (BVDV type 2). PI animals (n = 25) were identified using an antigen capture ELISA of ear notch samples. They were distributed into three age groups: ≤ 12 (n = 8), 13 to 24 (n = 6) and 25 to 34 (n = 11) months old. A control group of BVDV antigen ELISA negative female cattle that were age matched to the PI animals was utilized from the same herd. The PI group had a 1.29 higher odds ratio for diarrhea than controls (p = 0.001, IC95% = 1.032–1.623) and 1.615 greater chance of developing bovine respiratory disease (BRD) (p = 0.012, IC95% = 1.155–2.259). The age at first insemination (p = 0.012) and number of insemination attempts required to establish the first pregnancy (p = 0.016) were both higher for PI than controls. Milk production was higher for control cows than PI cows during most of the sampling periods. Somatic cell counts (SCC) were higher in PI cows than the controls at all sampling points across lactation (p ≤ 0.042). PI cattle had a higher incidence of disease, produced less milk, a higher SCC, and poorer reproductive performance than control cattle in this study.

Prevalence and genetic diversity of bovine viral diarrhea virus in dairy herds of China

To determine the nationwide prevalence and genetic diversity of bovine viral diarrhea virus (BVDV) in China, 92 dairy farms with more than 500 animals in 19 provinces of China were surveyed in 2017. At each farm, ear notch samples from calves less than six months old and bulk tank milk (BTM) samples were collected. A total of 901 ear notch samples and 329 BTM samples from 183 tanks were sampled. A total of 20 (20/901, 2.22 %) ear notch samples from 10 (10/92, 10.86 %) farms tested positive for BVDV by IDEXX Antigen Point-of-Care (POC) Test kit and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, 80 of 183 (80/183, 43.7 %) BTM samples from 43 (43/92, 46.7 %) farms were identified as positive by qRT-PCR. The RNA of positive and suspect samples identified by qRT-PCR was subjected to 5′- untranslated region (UTR) amplification by nested RT-PCR and then sequenced. A total of 119 sequences were obtained and phylogenetic analysis of these 5′-UTR sequences revealed the presence of eight different subgenotypes of BVDV-1 including 1a (n = 37, 31.09 %), 1b (n = 5, 4.20 %), 1c (n = 34, 28.57 %), 1d (n = 2, 1.68 %), 1m (n = 25, 21.01 %), 1q (n = 6, 5.04 %), and two unknown subgenotypes which were tentatively typed as “BVDV-1v” (n = 8, 6.72 %) and “BVDV-1w” (n = 2, 1.68 %), respectively. BVDV-1a, 1c, and 1m were the dominant strains, collectively accounting for 80.67 % (96/119) of all sequences. Phylogenetic analysis based on selected N-terminal autoprotease (Npro) sequences confirmed the classification of the 5′-UTR sequences. In conclusion, the prevalence of BVDV persistent infection in dairy cattle was high and genetic diversity was high and increasing, revealing a serious threat to the health of cattle in China and highlighting the need for BVDV control.

Diagnostics in the context of an eradication program: Results of the German bovine viral diarrhea proficiency trial

Bovine viral diarrhea (BVD), one of the most important infectious diseases in cattle, causes major economic losses and significant impact on animal welfare worldwide. The major source for virus spread is persistently infected, immunotolerant calves and, therefore, their early identification is of utmost importance for disease prevention. Here, a ring trial was initiated to control the performance of diagnostic tests used in German regional laboratories in charge of the diagnostics within the country’s BVD control program. A panel of five ear notch and five serum samples was provided for virological analysis. By an antigen ELISA, which was applied 26 times, the status of every sample was correctly identified in any case. In addition, a total of 54 real-time RT-PCR result sets was generated and also in most cases correctly classified. In addition to the virological test panel, a set of six sera and four milk samples was sent to the participating laboratories to be analyzed by serological methods. With serum neutralization tests, an excellent diagnostic sensitivity was achieved. However, one serum and both milk samples - positive for BVDV antibodies - repeatedly tested false negative by some of the used ELISA kits. All negative serum and milk samples were correctly identified by every commercial antibody ELISA. In conclusion, the BVDV proficiency test demonstrated that the used antigen/genome test systems allowed predominantly reliable diagnostics, while for four of the applied nine antibody ELISA kits adjustments are recommended.

Factors associated with the bovine viral diarrhoea (BVD) status in cattle herds in Northwest Germany

In Germany, all calves are tested for the presence of bovine viral diarrhoea/mucosal disease virus (BVDV) virus since January 1, 2011. The basis for this compulsory investigation is the BVDV Federal Regulation (BVDVV), which demands testing of calves before the age of six months and according to the new regulation of June 2016 within four weeks or before entering another stock. In 2012, a questionnaire was sent to 7250 Lower Saxony cattle farmers to identify potential factors associated with the presence of BVDV. Completed questionnaires were received from 2542 farms for further analysis. For BVD status determination of these farms, the diagnostic results of 425,911 ear notch samples of calves as part of the BVD eradication period from June 2010 to December 2013 were used.For the analysis of the completed questionnaires, a univariable analysis was performed by the chi-square or Wilcoxon test for each variable studied. In addition, a multivariable logistic model was performed. Four potential risk factors remained after a backward selection in the final logistic regression model: the dairy production compared to the suckling and other types of production, the herd size, the purchase of animals and the location in western region in comparison with the central and eastern regions. In summary, according to the results of this study, the farm with the highest probability of a BVDV infection in Lower Saxony is a large dairy farm that purchases cattle and is located in a cattle-dense region. When the complete eradication of the virus will be achieved, the results of the present study may help to conduct a risk-oriented monitoring programme.

Comparison of humoral and T-cell-mediated immune responses to a single dose of Bovela® live double deleted BVDV vaccine or to a field BVDV strain

The objective of this study was to determine and compare the humoral and cellular immune responses of calves exposed to a single dose of Bovela® bovine viral diarrhea virus (BVDV) live double deleted vaccine or a field strain virus (FSV) of BVDV type 2 (strain 890). Thirty seronegative, colostrum-deprived 5 month-old Holstein steer calves that tested negative for persistent BVDV by ear notch immunohistochemistry and seronegative to BVDV types 1 and 2 were used. Calves were screened by multi-parameter flow cytometry (MP-FCM) 1 week before vaccination to ensure that they were negative for T cell responses to the BVDV types 1 and 2 viruses in the Bovela® vaccine. Calves were assigned to 3 treatment groups: control (PBS), FSV inoculated, and Bovela® vaccinated. The humoral response was tested by standard serum virus neutralization (SVN) test to BVDV types 1 (Singer strain) and 2 (strain 125). The response by CD4, CD8, and gamma delta (γδ TCR) T cells was evaluated by MP-FCM using individual BVDV types 1 and 2 from Bovela® vaccine as recall antigens at 5, 6, and 7 weeks after vaccination. Activation markers used were upregulation of surface CD25 (IL-2R), intracellular interferon gamma (IFNγ) and intracellular interleukin 4 (IL-4). Each T cell subset was evaluated for increased expression of each activation marker compared to non-antigen stimulated cells of the same animal. All Bovela® vaccinated and FSV inoculated calves produced SVN antibodies to both BVDV types 1 and 2 while control animals remained seronegative throughout the study. The mean (weeks 5, 6, and 7) T cell recall responses to Bovela® BVDV type 1 and type 2 recall antigens were numerically higher in all three T cell subsets (CD4, CD8, and γδ TCR) for all three activation markers (CD25, IFNγ, and IL-4) when compared to either the control animals or to the FSV inoculated animals. These differences were often, but not always, statistically significant (P<0.05)

First Results in the Use of Bovine Ear Notch Tag for Bovine Viral Diarrhoea Virus Detection and Genetic Analysis.

BackgroundInfection due to bovine viral diarrhoea virus (BVDV) is endemic in most cattle-producing countries throughout the world. The key elements of a BVDV control programme are biosecurity, elimination of persistently infected animals and surveillance. Bovine viral diarrhoea (BVD) is a notifiable disease in Belgium and an official eradication programme started from January 2015, based on testing ear notches sampled during the official identification and registration of calves at birth. An antigen-capture ELISA test based on the detection of BVDV Erns protein is used. Ear notch sample may also be used to characterize the genotype of the calf when appropriate elution/dilution buffer is added. Both BVDV antigen-ELISA analysis and animal traceability could be performed.MethodologyWith regards to the reference protocol used in the preparation of ear notch samples, alternative procedures were tested in terms of BVDV analytic sensitivity, diagnostic sensitivity and specificity, as well as quality and purity of animal DNA.Principal Findings/SignificanceThe Allflex DNA Buffer D showed promising results in BVDV diagnosis and genome analyses, opening new perspectives for the livestock industry by the exploitation of the animal genome. Due to the high number of cattle involved in the Belgian official BVDV eradication programme based on ear notch tags sample, a large database on both BVDV status of newborn calves and cattle genome could be created for subsequent different uses (e.g. traceability, determination of parentage, genetic signatures throughout the genome associated with particular traits) evolving through a more integrated animal health.