A Nonsense Variant in CCDC65 Gene Causes Respiratory Failure Associated with Increased Lamb Mortality in French Lacaune Dairy Sheep.

We recently demonstrated that the Lacaune deficient homozygous haplotype 6 (LDHH6) potentially hosts a recessive perinatal lethal mutation in Lacaune dairy sheep mapped on OAR3. In the present study, we have analyzed the whole-genome sequences of two Lacaune ram heterozygous carriers of LDHH6. After variant calling and filtering against the variants of 86 non-carrier rams, we have identified a single nucleotide variant (SNV) in the two LDHH6 carriers whose variant allele induced a premature stop codon (p.Glu111*) in the Coiled-Coil Domain Containing 65 (CCDC65) gene. CCDC65 is involved in the assembly of the nexin-dynein regulatory complex for the formation of microtubules in ciliated cells. In order to identify the phenotype in homozygous sheep, we generated at-risk matings (n = 17) between rams and ewes heterozygous for the candidate variant in CCDC65. A total of 16 lambs were born alive with five genotyped as homozygous carriers. The homozygous lambs suffered from respiratory problems, and four of them died within the first month of life. At necropsy, we observed a broad hepatization of lung lobes possibly induced by infectious pneumonia. The management of this lethal recessive allele (frequency of 0.06) through reasoned mating in the Lacaune sheep selection schemes could reduce lamb mortality by 2%.

A validation study of loci associated with mastitis resistance in two French dairy sheep breeds.

BACKGROUND: The identification of loci associated with resistance to mastitis or of the causative mutations may be helpful in breeding programs for dairy sheep as it is for cattle worldwide. Seven genomic regions that control milk somatic cell counts, an indirect indicator of udder infection, have already been identified in sheep (Spanish Churra, French Lacaune and Italian Sardinian-Lacaune backcross populations). In this study, we used a 960 custom-designed ovine single nucleotide polymorphism (SNP) chip in Lacaune and Manech Tête Rousse dairy sheep to validate these seven genomic regions associated with mastitis. RESULTS: The most significant SNP (rs868996547) on Ovis aries chromosome (OAR) 3 was a previously described mutation in the suppressor of cytokine signalling 2 (SOCS2) gene. An antagonist effect of this causal candidate between health and growth in Lacaune sheep was confirmed. Effects of the mutation on the infectious status of the udder, i.e. increases in milk somatic cell counts and bacteria shedding, were also identified. This SNP was not present in the data available on Manech Tête Rousse. Three other regions associated with mastitis were also confirmed on OAR16 (Manech Tête Rousse), 19 (Lacaune) and 2 (both breeds). For the OAR2 region, we validated previously detected SNPs in several other breeds (Sarda, Churra, and Chios). For significant SNPs in the four mastitis regions, the effect varied from 0.24 to 0.67 phenotypic standard deviation of the traits. Two of the mastitis quantitative trait loci (QTL) regions (OAR2 and 16) that we validated here were also associated in opposite ways with milk production traits in both populations. CONCLUSIONS: These results indicate, at least in part, a genomic basis for the trade-off between milk production and mastitis resistance. Four of the seven mastitis QTL regions that were previously identified in independent populations, were confirmed in this study, which demonstrates partial sharing of mastitis-related genetic mechanisms between different distant dairy sheep populations.

Whole blood transcriptome analysis reveals potential competition in metabolic pathways between negative energy balance and response to inflammatory challenge.

Negative Energy Balance (NEB) is considered to increase susceptibility to mastitis. The objective of this study was to improve our understanding of the underlying mechanisms by comparing transcriptomic profiles following NEB and a concomitant mammary inflammation. Accordingly, we performed RNA-seq analysis of blood cells in energy-restricted ewes and control-diet ewes at four different time points before and after intra mammary challenge with phlogogenic ligands. Blood leucocytes responded to NEB by shutting down lipid-generating processes, including cholesterol and fatty acid synthesis, probably under transcriptional control of SREBF 1. Furthermore, fatty acid oxidation was activated and glucose oxidation and transport inhibited in response to energy restriction. Among the differentially expressed genes (DEGs) in response to energy restriction, 64 genes were also differential in response to the inflammatory challenge. Opposite response included the activation of cholesterol and fatty acid synthesis during the inflammatory challenge. Moreover, activation of glucose oxidation and transport coupled with the increase of plasma glucose concentration in response to the inflammatory stimuli suggested a preferential utilization of glucose as the energy source during this stress. Leucocyte metabolism therefore undergoes strong metabolic changes during an inflammatory challenge, which could be in competition with those induced by energy restriction.

A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2) Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model.

Mastitis is an infectious disease mainly caused by bacteria invading the mammary gland. Genetic control of susceptibility to mastitis has been widely evidenced in dairy ruminants, but the genetic basis and underlying mechanisms are still largely unknown. We describe the discovery, fine mapping and functional characterization of a genetic variant associated with elevated milk leukocytes count, or SCC, as a proxy for mastitis. After implementing genome-wide association studies, we identified a major QTL associated with SCC on ovine chromosome 3. Fine mapping of the region, using full sequencing with 12X coverage in three animals, provided one strong candidate SNP that mapped to the coding sequence of a highly conserved gene, suppressor of cytokine signalling 2 (Socs2). The frequency of the SNP associated with increased SCC was 21.7% and the Socs2 genotype explained 12% of the variance of the trait. The point mutation induces the p.R96C substitution in the SH2 functional domain of SOCS2 i.e. the binding site of the protein to various ligands, as well-established for the growth hormone receptor GHR. Using surface plasmon resonance we showed that the p.R96C point mutation completely abrogates SOCS2 binding affinity for the phosphopeptide of GHR. Additionally, the size, weight and milk production in p.R96C homozygote sheep, were significantly increased by 24%, 18%, and 4.4%, respectively, when compared to wild type sheep, supporting the view that the point mutation causes a loss of SOCS2 functional activity. Altogether these results provide strong evidence for a causal mutation controlling SCC in sheep and highlight the major role of SOCS2 as a tradeoff between the host's inflammatory response to mammary infections, and body growth and milk production, which are all mediated by the JAK/STAT signaling pathway.