Monoclonal Antibodies Specific to Water Buffalo (Bubalus bubalis) Myxovirus Resistance Protein_1

Recombinant bubaline (Bubalus bubalis) myxovirus resistance protein 1 (bbMx1) was successfully expressed by an Escherichia coli expression system. After immunization and cell fusion, a set of ten mouse hybridomas producing mAbs to bbMx1 was established. The ten corresponding mAbs were further characterized using indirect ELISA, western blot analysis and immunocytofluorescent staining. Eight mAbs, designated 11C7, RD5, NF3, 9D1, FD4, PE6, 11A7 and 10F5, displayed binding abilities and specificity in the three formats. Moreover, combining NF3 (for capture) with RD5 (for detection), 9D1 with RD5, FD4 with 11C7 or PE6 with 11C7 generated a strong signal in a prototype sandwich-ELISA. The results suggest that the mAbs developed and characterized here provide an excellent starting point for developing diagnostic tools aimed at detecting viral infections in the water buffalo, whatever using immunoblotting, immunocytostaining or sandwich-ELISA

Genome-wide association for milk production and lactation curve parameters in Holstein dairy cows

The aim of this study was to identify genomic regions associated with 305-day milk yield and lactation curve parameters on primiparous (n = 9,910) and multiparous (n = 11,158) Holstein cows. The SNP solutions were estimated using a weighted single-step genomic BLUP approach and imputed high-density panel (777k) genotypes. The proportion of genetic variance explained by windows of 50 consecutive SNP (with an average of 165 Kb) was calculated, and regions that accounted for more than 0.50% of the variance were used to search for candidate genes. Estimated heritabilities were 0.37, 0.34, 0.17, 0.12, 0.30 and 0.19, respectively, for 305-day milk yield, peak yield, peak time, ramp, scale and decay for primiparous cows. Genetic correlations of 305-day milk yield with peak yield, peak time, ramp, scale and decay in primiparous cows were 0.99, 0.63, 0.20, 0.97 and -0.52, respectively. The results identified three windows on BTA14 associated with 305-day milk yield and the parameters of lactation curve in primi- and multiparous cows. Previously proposed candidate genes for milk yield supported by this work include GRINA, CYHR1, FOXH1, TONSL, PPP1R16A, ARHGAP39, MAF1, OPLAH and MROH1, whereas newly identified candidate genes are MIR2308, ZNF7, ZNF34, SLURP1, MAFA and KIFC2 (BTA14). The protein lipidation biological process term, which plays a key role in controlling protein localization and function, was identified as the most important term enriched by the identified genes

Expert-based development of a generic HACCP-based risk management system to prevent critical negative energy balance in dairy herds

The objective of this study was to develop a generic risk management system based on the Hazard Analysis and Critical Control Point (HACCP) principles for the prevention of critical negative energy balance (NEB) in dairy herds using an expert panel approach. In addition, we discuss the advantages and limitations of the system in terms of implementation in the individual dairy herd. For the expert panel, we invited 30 researchers and advisors with expertise in the field of dairy cow feeding and/or health management from eight European regions. They were invited to a Delphi-based set-up that included three inter-correlated questionnaires in which they were asked to suggest risk factors for critical NEB and to score these based on 'effect' and 'probability'. Finally, the experts were asked to suggest critical control points (CCPs) specified by alarm values, monitoring frequency and corrective actions related to the most relevant risk factors in an operational farm setting. A total of 12 experts (40 %) completed all three questionnaires. Of these 12 experts, seven were researchers and five were advisors and in total they represented seven out of the eight European regions addressed in the questionnaire study. When asking for suggestions on risk factors and CCPs, these were formulated as 'open questions', and the experts' suggestions were numerous and overlapping. The suggestions were merged via a process of linguistic editing in order to eliminate doublets. The editing process revealed that the experts provided a total of 34 CCPs for the 11 risk factors they scored as most important. The consensus among experts was relatively high when scoring the most important risk factors, while there were more diverse suggestions of CCPs with specification of alarm values and corrective actions. We therefore concluded that the expert panel approach only partly succeeded in developing a generic HACCP for critical NEB in dairy cows. We recommend that the output of this paper is used to inform key areas for implementation on the individual dairy farm by local farm teams including farmers and their advisors, who together can conduct herd-specific risk factor profiling, organise the ongoing monitoring of herd-specific CCPs, as well as implement corrective actions when CCP alarm values are exceeded

From QTL to QTN

Most traits of economical importance in animal production are quantitative i.e. they are characterized by a continuous variation of phenotypic values. Examples for such traits are carcass weight, milk production and lean meat content. The phenotype of an animal for a quantitative trait depends on its genotype at several loci (called quantitative trait loci, QTL) as well as on environmental factors. Up to date, a large number of QTLs have been identified in farm animals by segregation analysis either within commercial populations or in crossbreed populations. Animal geneticists face now the challenge to identify the causative mutations lying behind these QTLs. In this thesis, we report the identification of the causative mutation for a major QTL influencing muscle development, fat deposition and heart size in pig. Previous studies have mapped this locus to the distal end of pig chromosome 2p. Furthermore, they have hypothesized that the causative mutation(s) may lie in an element regulating the expression of insulin-like growth factor 2 (IGF2). Firstly, we sequenced the IGF2 region in the pig and made comparative sequence analysis with available human and mouse sequences. We then used an identity-by-descent approach and managed to pinpoint the causative mutation to a G‡A transition located in an evolutionary conserved CpG island in IGF2 intron 3 (IGF2-intron3-G3070A). Subsequently, we used electrophoretic mobility shift assay and transient transfection experiments and showed that the QTN (quantitative trait nucleotide) abrogates the binding of a putative repressor. We completed our study by determining the core binding site of this transacting factor and by performing DNase I footprinting of the CpG island containing the QTN. In addition, we identified an IGF2 antisense transcript (IGF2-AS) and showed that its expression was also influenced by the QTN. The discovery of mutations causing QTLs in farm animals opens great future prospects. Besides evident practical breeding interests there are also major scientific interests, as understanding the mechanism causing the QTL effects will broaden our general knowledge on how the genome operates

From QTL to QTN: Identification of a Quantitative Trait Nucleotide Influencing Muscle Development and Fat Deposition in Pig

Most traits of economical importance in animal production are quantitative i.e. they are characterized by a continuous variation of phenotypic values. Examples for such traits are carcass weight, milk production and lean meat content. The phenotype of an animal for a quantitative trait depends on its genotype at several loci (called quantitative trait loci, QTL) as well as on environmental factors. Up to date, a large number of QTLs have been identified in farm animals by segregation analysis either within commercial populations or in crossbreed populations. Animal geneticists face now the challenge to identify the causative mutations lying behind these QTLs. In this thesis, we report the identification of the causative mutation for a major QTL influencing muscle development, fat deposition and heart size in pig. Previous studies have mapped this locus to the distal end of pig chromosome 2p. Furthermore, they have hypothesized that the causative mutation(s) may lie in an element regulating the expression of insulin-like growth factor 2 (IGF2). Firstly, we sequenced the IGF2 region in the pig and made comparative sequence analysis with available human and mouse sequences. We then used an identity-by-descent approach and managed to pinpoint the causative mutation to a GA transition located in an evolutionary conserved CpG island in IGF2 intron 3 (IGF2-intron3-G3070A). Subsequently, we used electrophoretic mobility shift assay and transient transfection experiments and showed that the QTN (quantitative trait nucleotide) abrogates the binding of a putative repressor. We completed our study by determining the core binding site of this transacting factor and by performing DNase I footprinting of the CpG island containing the QTN. In addition, we identified an IGF2 antisense transcript (IGF2-AS) and showed that its expression was also influenced by the QTN. The discovery of mutations causing QTLs in farm animals opens great future prospects. Besides evident practical breeding interests there are also major scientific interests, as understanding the mechanism causing the QTL effects will broaden our general knowledge on how the genome operates

Molecular characterization of a region in IGF2 intron 3 harbouring a quantitative trait nucleotide affecting muscle growth in the pig

A paternally expressed quantitative trait locus (QTL) affecting muscle growth, fat deposition and heart size has been identified at the distal tip of pig chromosome 2p. We have recently shown that the QTL is caused by a G→A transition at position IGF2-intron3-3072.This localizes the mutation in an evolutionary conserved CpG-island that is hypomethylated in skeletal muscle. Furthermore, we have demonstrated by electrophoretic mobility shift assay and transient tranfection analyses of murine C2C12 myoblasts that the quantitative trait nucleotide (QTN) prevents the binding of a repressor in vitro. In the present study we report DNase I footprinting of the 300bp surrounding the QTN. The characterization of this CpG-island will help us to further elucidate the mechanisms underlying the QTL effect as well as to increase our general knowledge of the complex regulation of IGF2expression

Characterization of the bovine pseudoautosomal boundary: Documenting the evolutionary history of mammalian sex chromosomes

Here, we report the sequence characterization of the bovine pseudoautosomal boundary (PAB) and its neighborhood. We demonstrate that it maps to the 5′ end of the GPR143 gene, which has concomitantly lost upstream noncoding exons on the Y chromosome. We show that the bovine PAB was created ∼20.7 million years ago by illegitimate intrachromatid recombination between inverted, ruminant-specific Bov-tA repeats. Accordingly, we demonstrate that cattle share their PAB with all other examined ruminants including sheep, but not with cetaceans or more distantly related mammals. We provide evidence that, since its creation, the ancestral ruminant PAB has been displaced by attrition, which occurs at variable rates in different species, and that it is capable of retreat by attrition erasure. We have estimated the ratio of male to female mutation rates in the Bovidae family as ∼1.7, and we provide evidence that the mutation rate is higher in the recombining pseudoautosomal region than in the adjacent, nonrecombining gonosome-specific sequences

Detection and eradication of Helicobacter spp. infection in laboratory mouse colony

Endemic infection with Helicobacter spp. is frequently detected in research mice colonies. So far, several species have been isolated including H. hepaticus, H. bilis, H. muridarum, H. typhlonicus and H. rodentium as the most prevalent ones. The transmission of this pathogen is through fecal-oral route, followed by persistent long-term shedding in infected individuals. In immunocompetent mice the infection is subclinical, however, emerging evidence show that it can have significant health implications leading to typhlocolitis, intestinal and hepatic tumors and mammary gland neoplasia. Moreover, the infection alters reproduction successes with increase in fetal resorptions, decline in neonatal weight and decreased number of weaned pups detected in both experimentally and naturally infected mice. In addition to the impact on animal health and welfare, the infection might have confounding effects on obtained research results; therefore, regular screening and eradication are key for obtaining a pathogen free colony. In order to eliminate a naturally acquired Helicobacter infection in our animal facility, we have attempted an eight-week long treatment protocol with amoxicillin, clarithromycin, metronidazole and bismuth, administered in drinking water. This treatment route is more economical, time-efficient and noninvasive compared to other alternatives for eradication such as treatment with medicated diet, oral gavage or cross fostering. To determine the results of the administrated therapy we have performed qPCR of DNA extracted from fecal pellets