Comparative Transcriptome Analysis of Milk Somatic Cells During Lactation Between Two Intensively Reared Dairy Sheep Breeds

In dairy sheep industry, milk production dictates the value of a ewe. Milk production is directly related to the morphology and physiology of the mammary gland; both being designated targets of breeding strategies. Although within a flock breeding parameters are mutual, large differences in milk production among individual ewes are usually observed. In this work, we tested two of the most productive dairy sheep breeds reared intensively in Greece, one local the Chios breed and one foreign the Lacaune breed. We used transcriptome sequencing to reveal molecular mechanisms that render the mammary gland highly productive or not. While highly expressed genes (caseins and major whey protein genes) were common among breeds, differences were observed in differentially expressed genes. ENSOARG00000008077, as a member of ribosomal protein 14 family, together with LPCAT2, CCR3, GPSM2, ZNF131, and ASIP were among the genes significantly differentiating mammary gland’s productivity in high yielding ewes. Gene ontology terms were mainly linked to the inherent transcriptional activity of the mammary gland (GO:0005524, GO:0030552, GO:0016740, GO:0004842), lipid transfer activity (GO:0005319) and innate immunity (GO:0002376, GO:0075528, GO:0002520). In addition, clusters of genes affecting zinc and iron trafficking into mitochondria were highlighted for high yielding ewes (GO:0071294, GO:0010043). Our analyses provide insights into the molecular pathways involved in lactation between ewes of different performances. Results revealed management issues that should be addressed by breeders in order to move toward increased milk yields through selection of the desired phenotypes. Our results will also contribute toward the selection of the most resilient and productive ewes, thus, will strengthen the existing breeding systems against a spectrum of environmental threats

Repeatability of Health and Welfare Traits and Correlation with Performance Traits in Dairy Goats Reared under Low-input Farming Systems

The objectives of the study were to estimate the repeatability of health and welfare traits and investigate their association with performance in three breeds of dairy goats reared under low-input farming systems in Greece. A total of 1210 goats of Eghoria (n = 418), Skopelos (n = 429), and Damascus (n = 363) breeds were assessed. Udder health, parasitic resistance, welfare, milk yield and quality, and body condition score were recorded monthly for two milking periods. Udder health records included somatic cell count (SCC) and total viable count (TVC). Based on combinations of SCC and TVC and thresholds set at >10(6) cells/mL and >2 × 10(4) cfu/mL, respectively, additional udder health phenotypes were defined. Parasitism included myiasis, tick infestation, gastrointestinal nematode (GIN) and cestode faecal egg count (FEC), and lungworm faecal larval count (FLC). Infection with each of the endoparasites was defined based on FEC/FLC. Welfare assessment parameters included the presence of ear and horn injuries, ocular and nasal discharge, body and udder abscesses, injury and lesions on the skin of different regions, diarrhoea, hernias, overgrown hooves, arthritis, lameness, and udder asymmetry. Trait repeatability and animal correlations were estimated. Significant (p < 0.05) repeatability was reported for all udder health and most welfare traits in all breeds, GIN and cestode FEC, and GIN and lungworm infection in Eghoria, and myiasis in Skopelos. Correlations of health and most of welfare traits with performance were non-significant or favourable. Overall, results demonstrate potential to improve health and welfare of the studied breeds without compromising performance

Genetic profile of scrapie codons 146, 211 and 222 in the PRNP gene locus in three breeds of dairy goats

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds

A genome-wide association study reveals novel SNP markers associated with resilience traits in two Mediterranean dairy sheep breeds

Genetic selection for higher productivity increased dairy sheep susceptibility to diseases and environmental stressors, challenging their health and welfare status and production efficiency. Improving resilience to such stressors can enhance their ability to face these challenges without compromising productivity. Our objective was to estimate genomic heritability and perform genome-wide association studies (GWAS) to detect SNPs and candidate genes associated with three proxy traits for resilience (milk somatic cell count—SCC, lactation persistency—LP, body condition score—BCS) of Chios and Frizarta dairy ewes. We used genome-wide genotypes of 317 Chios and 346 Frizarta ewes. Individual records of milk yield and BCS, and milk samples were collected monthly for two consecutive milking periods; samples were analyzed to determine SCC. The LP was calculated as the regression coefficient of daily milk yield on days from lambing. Within breed, variance components analyses and GWAS were performed using genomic relatedness matrices in single-trait animal linear mixed models. Genomic-based heritability estimates were relatively high (BCS: h2 = 0.54 and 0.55, SCC: h2 = 0.25 and 0.38, LP: h2 = 0.43 and 0.45, for Chios and Frizarta ewes, respectively), compared to previous pedigree-based studies. The GWAS revealed 7 novel SNPs associated with the studied traits; one genome-wide and two suggestive significant SNPs for SCC (Frizarta: rs403061409, rs424064526 and rs428540973, on chromosomes 9, 1 and 12, respectively), one suggestive significant SNP for BCS (Chios: rs424834097 on chromosome 4) and three suggestive significant SNPs for LP (Frizarta: rs193632931 and rs412648955 on chromosomes 1 and 6, Chios: rs428128299 on chromosome 3). Nineteen candidate genes were detected: two for BCS (Chios: POT1, TMEM229A), thirteen for SCC (Frizarta: NTAQ1, ZHX1, ZHX2, LOC101109545, HAS2, DERL1, FAM83A, ATAD2, RBP7, FSTL1, CD80, HCLS1, GSK3B) and four for LP (Frizarta: GRID2, FAIM, CEP70—Chios: GRIP1). Present results show that resilience in the studied dairy sheep breeds is heritable and advance existing knowledge on the genomic background of SCC, LP, and BCS. Future research will quantify effects of different alleles of significant SNPs on the studied traits and search for possible correlations among traits to facilitate their effective incorporation in breeding programs aiming to improve resilience.</p

Genetic profile of scrapie codons 146, 211 and 222 in the PRNP gene locus in three breeds of dairy goats

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds

A genome-wide association study reveals novel SNP markers associated with resilience traits in two Mediterranean dairy sheep breeds

Genetic selection for higher productivity increased dairy sheep susceptibility to diseases and environmental stressors, challenging their health and welfare status and production efficiency. Improving resilience to such stressors can enhance their ability to face these challenges without compromising productivity. Our objective was to estimate genomic heritability and perform genome-wide association studies (GWAS) to detect SNPs and candidate genes associated with three proxy traits for resilience (milk somatic cell count—SCC, lactation persistency—LP, body condition score—BCS) of Chios and Frizarta dairy ewes. We used genome-wide genotypes of 317 Chios and 346 Frizarta ewes. Individual records of milk yield and BCS, and milk samples were collected monthly for two consecutive milking periods; samples were analyzed to determine SCC. The LP was calculated as the regression coefficient of daily milk yield on days from lambing. Within breed, variance components analyses and GWAS were performed using genomic relatedness matrices in single-trait animal linear mixed models. Genomic-based heritability estimates were relatively high (BCS: h2 = 0.54 and 0.55, SCC: h2 = 0.25 and 0.38, LP: h2 = 0.43 and 0.45, for Chios and Frizarta ewes, respectively), compared to previous pedigree-based studies. The GWAS revealed 7 novel SNPs associated with the studied traits; one genome-wide and two suggestive significant SNPs for SCC (Frizarta: rs403061409, rs424064526 and rs428540973, on chromosomes 9, 1 and 12, respectively), one suggestive significant SNP for BCS (Chios: rs424834097 on chromosome 4) and three suggestive significant SNPs for LP (Frizarta: rs193632931 and rs412648955 on chromosomes 1 and 6, Chios: rs428128299 on chromosome 3). Nineteen candidate genes were detected: two for BCS (Chios: POT1, TMEM229A), thirteen for SCC (Frizarta: NTAQ1, ZHX1, ZHX2, LOC101109545, HAS2, DERL1, FAM83A, ATAD2, RBP7, FSTL1, CD80, HCLS1, GSK3B) and four for LP (Frizarta: GRID2, FAIM, CEP70—Chios: GRIP1). Present results show that resilience in the studied dairy sheep breeds is heritable and advance existing knowledge on the genomic background of SCC, LP, and BCS. Future research will quantify effects of different alleles of significant SNPs on the studied traits and search for possible correlations among traits to facilitate their effective incorporation in breeding programs aiming to improve resilience

Investigation of the relationship between lameness, milk production and rearing methods of Chios dairy sheep

The overall objective of the present study was to investigate the incidence of lameness in Chios dairy sheep flocks. Two further objectives were to assess the effect of rearing methods on incidence of lameness and the effect of lameness on milk yield of ewes. Finally, the genetic profile of the breed, concerning resistance to footrot, was assessed. The research was carried out in three consecutive stages. The first stage included the epidemiological investigation of lameness and the assessment of the relationship between lameness and rearing methods. Data concerning lameness and rearing methods were collected from 66 Chios sheep flocks using a questionnaire. Thereafter, 9 flocks were selected and were subject of investigation on regular visits every 14 days over one milking period, to assess the locomotion score of each individual ewe. The importance of factors affecting incidence, severity and duration of lameness was assessed using general linear models. The incidence of lameness was 6.8%. The commonest causes of lameness were footrot (66.4%), white line abscesses (16.4%) and white line disease (11.8%). The incidence of lameness was significantly lower in large size flocks (P<0.05) and in flocks that had access to grazing. Moreover, the stocking density in sheep sheds had a significant negative effect (P<0.001) on the incidence of lameness. Also, the severity of lameness was higher during winter months (P<0.05) and its duration was significantly lower in ewes treated with injectable antibiotics (P<0.05). At the second stage, the effect of lameness on milk yield was investigated. A total of 283 Chios ewes, from two flocks were used. The flocks were visited on a weekly basis. Data concerning daily milk yield of each individual ewe were collected. Comparisons of data between lame ewes and the other ewes were performed using general linear models and milk curves were calculated. Lame ewes had significantly lower (about 270 gr/ewe/day) daily milk yield (D.M.Y.) when compared to the rest of the ewes. Also, the reduction on D.M.Y. of lame ewes started before the onset of lameness and lasted several weeks causing a significant reduction on their total milk yield per lactation (almost 20%) when compared to the average flock. In the third stage, the polymorphisms of the DQA2 gene in Chios sheep were studied. A total of 385 sheep were randomly selected from 30 flocks; 18 sheep were lame due to footrot. Blood samples were collected from the 385 sheep in order to isolate DNA for further analysis. The DNA samples were analyzed and the DQA2 gene was typed using PCR-single strand conformational polymorphism. Genotypic and allelic frequencies were calculated with counting and the effect of the DQA2 gene on footrot was assessed using a general linear model. A total of 20 DQA2 genes and 78 genotypes were detected in the sampled population. Allele K and genotype KK were the most common (with prevalence being at 31.7% and 12.0%, respectively). The overall effect of genotype on the prevalence of footrot was statistically significant (P<0.05), whereas allele Ε was associated with higher susceptibility to footrot.Η παρούσα ερευνητική εργασία είχε ως στόχο τη διερεύνηση της συχνότητας εμφάνισης χωλοτήτων σε ποίμνια προβάτων της γαλακτοπαραγωγού φυλής Χίου. Ταυτόχρονα, μελετήθηκαν οι επιπτώσεις των μεθόδων εκτροφής στη συχνότητα εμφάνισης χωλοτήτων, καθώς και η επίδραση των χωλοτήτων στην γαλακτοπαραγωγή των προβατίνων. Τέλος, διερευνήθηκε το γενετικό υπόβαθρο της φυλής σε ό,τι αφορά την ανθεκτικότητά της στην ποδοδερματίτιδα. Η έρευνα πραγματοποιήθηκε σε τρεις αυτοτελείς φάσεις. Η πρώτη φάση αφορούσε στην επιδημιολογική διερεύνηση των χωλοτήτων και την εκτίμηση της σχέσης μεταξύ χωλότητας και μεθόδων εκτροφής. Συλλέχθηκαν στοιχεία που αφορούσαν στις χωλότητες και στις μεθόδους εκτροφής από 66 ποίμνια της φυλής Χίου με τη συμπλήρωση ερωτηματολογίου. Στη συνέχεια, επιλέχτηκαν 9 ποίμνια όπου γίνονταν επισκέψεις κάθε 14 ημέρες, κατά τη διάρκεια μιας περιόδου αρμέγματος, για την εκτίμηση του Δ.Κ. σε όλες τις προβατίνες. Η εκτίμηση των παραγόντων που επηρεάζουν τη συχνότητα εμφάνισης, τη σοβαρότητα και τη διάρκεια των χωλοτήτων έγινε με γραμμικά στατιστικά πρότυπα. Η συχνότητα εμφάνισης χωλοτήτων ήταν 6,8%. Τα συχνότερα αίτια ήταν η ποδοδερματίτιδα (66,4%), τα αποστήματα (16,4%) και η νόσος της λευκής γραμμής (11,8%). Η συχνότητα εμφάνισης χωλοτήτων ήταν σημαντικά μικρότερη στα μεγαλύτερα ποίμνια (P<0.05) και στα ποίμνια που έβγαζαν τα ζώα για βόσκηση (P<0.05), ενώ η διαθέσιμη επιφάνεια δαπέδου ανά προβατίνα στο προβατοστάσιο σχετιζόταν αρνητικά (P<0.001) με την εμφάνιση χωλοτήτων. Επίσης, η χωλότητα ήταν περισσότερο σοβαρή κατά τους χειμερινούς μήνες (P<0.05) και η διάρκειά της ήταν σημαντικά μικρότερη στα ζώα που χορηγήθηκαν αντιβιοτικά (P<0.05). Στη δεύτερη φάση, υπολογίστηκε η επίδραση των χωλοτήτων στη γαλακτοπαραγωγή. Επιλέχτηκαν συνολικά 283 προβατίνες της φυλής Χίου από δύο ποίμνια. Οι επισκέψεις στα ποίμνια γίνονταν σε εβδομαδιαία βάση. Συλλέγονταν δεδομένα ημερήσιων ατομικών γαλακτομετρήσεων για το σύνολο των ζώων. Έγιναν συγκρίσεις των ζώων με χωλότητα και των υπόλοιπων ζώων με τη χρήση γενικών γραμμικών μοντέλων και χαράχτηκαν οι καμπύλες γαλακτοπαραγωγής. Διαπιστώθηκε ότι στις προβατίνες που παρουσίαζαν χωλότητα, η μέση ημερήσια γαλακτοπαραγωγή (Μ.Η.Γ.) ήταν σημαντικά μειωμένη (περίπου κατά 270 γρ./προβατίνα/ημέρα) σε σύγκριση με τα υπόλοιπα ζώα. Επίσης, η μείωση στη Μ.Η.Γ. των ζώων με χωλότητα ξεκινούσε πριν την εκδήλωση της χωλότητας και διαρκούσε αρκετές εβδομάδες μετά την αποδρομή της, προκαλώντας σημαντική μείωση και στη συνολική γαλακτοπαραγωγή ανά γαλακτική περίοδο (μείωση περίπου 20%) σε σύγκριση με τα υπόλοιπα ζώα. Η τρίτη φάση αφορούσε στη διερεύνηση των πολυμορφισμών του γονιδίου DQA2 σε πρόβατα της φυλής Χίου. Από 30 εκτροφές επιλέχτηκαν τυχαία 385 πρόβατα, σε 18 από τα οποία είχε διαγνωστεί ποδοδερματίτιδα. Από τα 385 πρόβατα λήφθηκαν δείγματα αίματος και έγινε απομόνωση του DNA. Στη συνέχεια, η παρουσία πολυμορφισμών στο γονίδιο DQA2 διερευνήθηκε με την τεχνική PCR-single strand conformational polymorphism (SSCP). Υπολογίστηκε η συχνότητα εμφάνισης των γενοτύπων και των αλληλομόρφων, καθώς και η επίδραση του γονιδίου DQA2 στην εμφάνιση ποδοδερματίτιδας με τη βοήθεια γραμμικού προτύπου. Βρέθηκαν συνολικά 20 αλληλόμορφα του γονιδίου DQA2 (το K αλληλόμορφο ήταν το σημαντικότερο με συχνότητα εμφάνισης περίπου 31,7%) και 78 γενότυποι, από τους οποίους ο KK ήταν ο συχνότερος (12,0%). Επίσης, βρέθηκε ότι η συνολική επίδραση του γενοτύπου στην εμφάνιση ποδοδερματίτιδας ήταν σημαντική (P<0.05), ενώ το αλληλόμορφο Ε σχετιζόταν με αυξημένη ευαισθησία στη νόσο (P<0.05)

Point-of-Care Diagnostics for Farm Animal Diseases: From Biosensors to Integrated Lab-on-Chip Devices

Zoonoses and animal diseases threaten human health and livestock biosecurity and productivity. Currently, laboratory confirmation of animal disease outbreaks requires centralized laboratories and trained personnel; it is expensive and time-consuming, and it often does not coincide with the onset or progress of diseases. Point-of-care (POC) diagnostics are rapid, simple, and cost-effective devices and tests, that can be directly applied on field for the detection of animal pathogens. The development of POC diagnostics for use in human medicine has displayed remarkable progress. Nevertheless, animal POC testing has not yet unfolded its full potential. POC devices and tests for animal diseases face many challenges, such as insufficient validation, simplicity, and portability. Emerging technologies and advanced materials are expected to overcome some of these challenges and could popularize animal POC testing. This review aims to: (i) present the main concepts and formats of POC devices and tests, such as lateral flow assays and lab-on-chip devices; (ii) summarize the mode of operation and recent advances in biosensor and POC devices for the detection of farm animal diseases; (iii) present some of the regulatory aspects of POC commercialization in the EU, USA, and Japan; and (iv) summarize the challenges and future perspectives of animal POC testing

The ‘French heartworm’ in Greece: A countrywide serological survey of Angiostrongylus vasorum infection by combined detection of circulating antigens and specific antibodies

Angiostrongylus vasorum is a nematode of the superfamily Metastongyloidea and was first described in the south of France in the nineteenth century where its biology was extensively studied in the 1960ies, leading to the common use of the byname "the French heartworm". The life cycle of the parasite is indirect including slugs and snails as intermediate hosts, while the adults live in the right heart and pulmonary arteries. Since then, many studies have been conducted in different European countries. However, there is scant knowledge regarding the prevalence and distribution of this parasite in Greece. Therefore, the objective of the present study was to investigate the current seroprevalence of A. vasorum in dogs in Greece. Overall 1000 apparently healthy and randomly selected dogs, presented in veterinary clinics, were involved at national level (n = 66 locations). Individual serum samples were obtained and tested using two in-house ELISAs by combining the detection of specific antibodies against the parasite and of circulating antigens of A. vasorum. Overall, 3.0% (n = 30, CI 95% 2.1 to 4.3%) of the sampled dogs were seropositive to at least one of the two ELISAs: 0.4% (n = 4, CI 95% 0.2 to 1.0%) were positive by both ELISAs, while 1.0% (n = 10, CI 95% 0.5 to 1.8%) were seropositive only for specific antibodies and 1.6% (n = 16, CI 95% 1.0 to 2.6%) were antigen-positive only. This is the first multicentric seroepidemiological study about A. vasorum in dogs from Greece. We showed that this parasite circulates in the local canine population and further studies should be carried out to elucidate its significance in the country

Aetiology, Risk Factors, Diagnosis and Control of Foot-Related Lameness in Dairy Sheep

During the last twenty years, considerable research efforts have recognized the consequences of foot-related lameness primarily in cattle, and meat and wool sheep. Despite the lack of extensive epidemiological studies, field observations and isolated research reports in dairy sheep have suggested that the problem might be more severe in semi-intensive and intensive farming systems. Footrot, contagious ovine digital dermatitis, ovine interdigital dermatitis, white line disease, and pedal joint abscess are the most common causes of foot-related lameness. Dichelobacter nodosus, Fusobacterium necrophorum, Treponema spp., and Actinomyces pyogenes are the most significant foot-related lameness-associated pathogens. Despite a documented hereditary predisposition, environmental factors are the most important in determining the occurrence of foot-related lameness. Moist and warm environment, increased parity and milk yield, inappropriate housing conditions and infrastructures, inadequate hygiene status, imbalanced nutrition, and insufficient foot care are the most critical risk factors. Furthermore, a foot-lameness control plan should include targeted implementation of claw trimming and footbathing, evidence-based planning of hygiene measures in preventive veterinary practices (i.e., antibiotic administration, vaccinations against footrot), selective breeding to footrot resistance, and, most importantly, the continuous training of farming personnel. Controlling foot-lameness in dairy sheep is critical in determining the well-being of animals, and strongly affects the farm&rsquo;s profitability and sustainability