Determination of N-acetylneuraminic and N-glycolylneuraminic acids in unprocessed milk of four cattle breeds.

This research communication reports concentrations of two sialic acids (SA), N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic (Neu5Gc), in fresh milk from different cow breeds throughout lactation. According to published studies, the two SA types found in animal-derived products have diverse and conflicting effects on human health, but SA content is not routinely analysed in individual milk cows samples. We measured the content of Neu5Ac and Neu5Gc in milk from Holstein Friesian (HO), Simmental (SM), Simmental × Holstein crossbred (SM×HO), and Podolica (POD) cows at 60 and 120 d following calving. HO, SM and SM×HO were reared in an intensive production while POD were raised in an extensive system. Results showed that total Neu5Ac was overall thirty times more abundant than Neu5Gc, and their concentrations were higher at 120 d than at 60 d (P < 0.001). Neu5Gc values were greater in HO, SM, and SM × HO than in POD (P < 0.001), while HO had a higher Neu5Ac value than the other three breeds (P < 0.001). These findings shed light on the differences in SA content among cow breeds and lay the groundwork for future research to select animals that produce milk with desirable characteristics for human health.

Global transcriptomic profiles of circulating leucocytes in early lactation cows with clinical or subclinical mastitis.

Bovine mastitis, an inflammatory disease of the mammary gland, is classified as subclinical or clinical. Circulating neutrophils are recruited to the udder to combat infection. We compared the transcriptomic profiles in circulating leukocytes between healthy cows and those with naturally occurring subclinical or clinical mastitis. Holstein Friesian dairy cows from six farms in EU countries were recruited. Based on milk somatic cell count and clinical records, cows were classified as healthy (n = 147), subclinically (n = 45) or clinically mastitic (n = 22). Circulating leukocyte RNA was sequenced with Illumina NextSeq single end reads (30 M). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21, followed by GO enrichment analysis. Both subclinical and clinical mastitis caused significant changes in the leukocyte transcriptome, with more intensive changes attributed to clinical mastitis. We detected 769 DEGs between clinical and healthy groups, 258 DEGs between subclinical and healthy groups and 193 DEGs between clinical and subclinical groups. Most DEGs were associated with cell killing and immune processes. Many upregulated DEGs in clinical mastitis encoded antimicrobial peptides (AZU1, BCL3, CAMP, CATHL1, CATHL2, CATHL4,CATHL5, CATHL6, CCL1, CXCL2, CXCL13, DEFB1, DEFB10, DEFB4A, DEFB7, LCN2, PGLYRP1, PRTN3, PTX3, S100A8, S100A9, S100A12, SLC11A1, TF and LTF) which were not upregulated in subclinical mastitis. The use of transcriptomic profiles has identified a much greater up-regulation of genes encoding antimicrobial peptides in circulating leukocytes of cows with naturally occurring clinical compared with subclinical mastitis. These could play a key role in combatting disease organisms.

Identification of the complete coding cDNAs and expression analysis of B4GALT1, LALBA, ST3GAL5, ST6GAL1 in the colostrum and milk of the Garganica and Maltese goat breeds to reveal possible implications for oligosaccharide biosynthesis.

BACKGROUND: Milk sialylated oligosaccharides (SOS) play crucial roles in many biological processes. The most abundant free SOS in goat's milk are 3'sialyllactose (3'-SL), 6'sialyllactose (6'-SL) and disialyllactose (DSL). The production of these molecules is determined genetically by the expression of glycosyltransferases and by the availability of nucleotide sugar substrates, but the precise mechanisms regulating the differential patterns of milk oligosaccharides are not known. We aimed to identify the complete cDNAs of candidate genes implicated in SOS biosynthesis (B4GALT1, LALBA, ST3GAL5, ST6GAL1) and to analyse their expression during lactation in the Garganica and Maltese goat breeds. Moreover, we analysed the colostrum and milk contents of 3'-SL, 6'-SL and disialyllactose (DSL) and the possible correlations between expressed genes and SOS. RESULTS: We identified the complete coding cDNAs of B4GALT1 (HQ700335.1), ST3GAL5 (KF055858.2), and ST6GAL1 (HQ709167.1), the single nucleotide polymorphism (SNPs) of these genes and 2 splicing variants of the ST6GAL1 cDNA. RT-qPCR analysis showed that LALBA and ST6GAL1 were the genes with the highest and lowest expression in both breeds, respectively. The interaction effects of the breeds and sampling times were associated with higher levels of B4GALT1 and ST3GAL5 gene expression in Garganica than in Maltese goats at kidding. B4GALT1, LALBA, and ST3GAL5 gene expression changed from kidding to 60 and 120 days in Maltese goats, while in Garganica goats, a difference was observed only for the LALBA gene. Breed and lactation effects were also found for SOS contents. Positive correlations of B4GALT1, LALBA, ST3GAL5, and ST6GAL1 with 3'-SL/6'SL and DSL were found. CONCLUSIONS: The genetic effect on the oligosaccharide content of milk was previously highlighted in bovines, and this study is the first to investigate this effect in two goat breeds (Garganica and Maltese) during lactation. The genetic variability of candidate genes involved in SOS biosynthesis highlights their potential role in affecting gene expression and ultimately biological function. The investigation of gene regulatory regions as well as the examination of other sialyltransferase genes will be needed to identify the genetic pattern leading to a higher SOS content in the autochtonous Garganica breed and to protect it using a focused breeding strategy.

Characterization of leukocyte subsets in buffalo (Bubalus bubalis) with cross-reactive monoclonal antibodies specific for bovine MHC class I and class II molecules and leukocyte differentiation molecules.

Although buffaloes (Bubalus bubalis) are a major component of the livestock industry worldwide, limited progress has been made in the study of the mechanisms regulating the immune response to pathogens and parasites affecting their health and productivity. This has been, in part, attributable to the limited availability of reagents to study immune responses in buffalo. As reported here, a set of cross-reactive monoclonal antibodies (mAbs), developed against bovine, ovine and caprine leukocyte differentiation molecules (LDM) and major histocompatibility complex (MHC) molecules, were identified and used to compare expression of LDM in Italian and Egyptian buffalo. The results show most of the epitopes identified with the mAbs are conserved on LDM and MHC I and II molecules in both lineages of buffalo. Comparison of the composition of lymphocyte subsets between buffalo and cattle revealed they are similar except for expression of CD2 and CD8 on workshop cluster one (WC1) positive γδ T cells. In cattle, CD8 is expressed on a subset of CD2+/WC1- γδ T cells that are present in low frequency in blood of young and old animals, whereas, CD8-/CD2-/WC1+ γδ T cells are present in high frequency in young animals, decreasing with age. In the buffalo, CD2 is expressed on a subset of WC1+ γδ T cells and CD8 is expressed on all WC1+ γδ T cells. The availability of this extensive set of mAbs provides opportunities to study the immunopathogenesis of pathogens and parasites affecting the health of buffalo.

Salmonella Typhimurium infection primes a nutriprive mechanism in piglets.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important cause of acute food- borne zoonoses worldwide, typically carried by pigs. It is well known that Salmonella has evolved a wide array of strategies enabling it to invade the host, but little information is available on the specific host responses to Salmonella infections. In the present study, we used an in vivo approach (involving piglets infected with a virulent or an attenuated S. Typhimurium strain) coupled to histological and proteomic analysis of the cecum mucosa, to highlight the host pathways activated during S. Typhimurium infection. We confirm the complex host-pathogen interaction. Our data showed that the metabolic and the cytoskeleton organization functions were the most significantly altered. In particular, the modifications of energy metabolic pathway could suggest a "nutriprive" mechanism, in which the host reduce its metabolic and energetic status to limit Salmonella infection. This study could represent a preliminary approach, providing information useful to better understand the host-Salmonella interaction.

Splicing variants of SERPINA1 gene in ovine milk: characterization of cDNA and identification of polymorphisms.

The serine protease inhibitor, clade A, member 1 (SERPINA1) is the gene for a protein called alpha-1-antitrypsin (AAT), which is a member of the serine protease inhibitor (serpin) superfamily of proteins. By conformational change, serpins control several chemical reactions inhibiting the activity of proteases. AAT is the most abundant endogenous serpin in blood circulation and it is present in relatively high concentration in human milk as well as in bovine and porcine colostrum. Here we report for the first time the molecular characterization and sequence variability of the ovine SERPINA1 cDNA and gene. cDNAs from mammary gland and from milk were PCR amplified, and three different transcripts (1437, 1166 and 521bp) of the SERPINA1 gene were identified. We amplified and sequenced different regions of the gene (5' UTR, from exon 2 to exon 5 and 3' UTR), and we found that the exon-intron structure of the gene is similar to that of human and bovine. We detected a total of 97 SNPs in cDNAs and gene sequences from 10 sheep of three different breeds. In adult sheep tissues a SERPINA1 gene expression analysis indicated a differential expression of the three different transcripts. The finding reported in this paper will aid further studies on possible involvement of the SERPINA1 gene in different physiological states and its possible association with production traits.

Milk fatty acid variability: effect of some candidate genes involved in lipid synthesis.

In this work, the genetic variation of milk FA was investigated in three different bovine breeds, the Jersey, the Piedmontese and the Valdostana, and at different lactation stages. All animals were genotyped for 21 Single Nucleotide Polymorphisms located within nine candidate genes involved in lipid synthesis: diacylglycerol acyltransferase 1 and 2 (DGAT1, 2); stearoyl-CoA desaturase (SCD); growth hormone receptor (GHR); fatty acid synthase (FASN); acyl-CoA dehydrogenase (ACAD); fatty acid binding protein (FABP4); lipoprotein lipase (LPL); and leptin gene (LEP). The highest milk-fat Jersey breed also showed the highest content of saturated FA. Throughout lactation, the breeds showed a similar variation in the FA, with a decrease in the short-chain, this was accompanied by a general increase in the long chain FA at the end of lactation. The increase in long chain saturated FA was particularly evident in the case of the Jersey. The effect of SCD gene on the C14 desaturation index was confirmed; the DGAT1 gene was polymorphic only in the Jersey breed, but its effect was confirmed only on milk fat content; three further potential candidate genes were identified: first, the FABP4 gene, which was found to influence medium and long chain FA in all the breeds, but not the desaturation indices; second, the FASN gene, which was found to influence the amount of PUFA in the Piedmontese and the Valdostana, and third, the LPL gene, which was found to affect fat content in the Piedmontese.

Massive screening of copy number population-scale variation in Bos taurus genome.

BACKGROUND: Copy number variations (CNVs) represent a significant source of genomic structural variation. Their length ranges from approximately one hundred to millions of base pair. Genome-wide screenings have clarified that CNVs are a ubiquitous phenomenon affecting essentially the whole genome. Although Bos taurus is one of the most important domestic animal species worldwide and one of the most studied ruminant models for metabolism, reproduction, and disease, relatively few studies have investigated CNVs in cattle and little is known about how CNVs contribute to normal phenotypic variation and to disease susceptibility in this species, compared to humans and other model organisms. RESULTS: Here we characterize and compare CNV profiles in 2654 animals from five dairy and beef Bos taurus breeds, using the Illumina BovineSNP50 genotyping array (54001 SNP probes). In this study we applied the two most commonly used algorithms for CNV discovery (QuantiSNP and PennCNV) and identified 4830 unique candidate CNVs belonging to 326 regions. These regions overlap with 5789 known genes, 76.7% of which are significantly co-localized with segmental duplications (SD). CONCLUSIONS: This large scale screening significantly contributes to the enrichment of the Bos taurus CNV map, demonstrates the ubiquity, great diversity and complexity of this type of genomic variation and sets the basis for testing the influence of CNVs on Bos taurus complex functional and production traits.

A second generation radiation hybrid map to aid the assembly of the bovine genome sequence.

BACKGROUND: Several approaches can be used to determine the order of loci on chromosomes and hence develop maps of the genome. However, all mapping approaches are prone to errors either arising from technical deficiencies or lack of statistical support to distinguish between alternative orders of loci. The accuracy of the genome maps could be improved, in principle, if information from different sources was combined to produce integrated maps. The publicly available bovine genomic sequence assembly with 6x coverage (Btau_2.0) is based on whole genome shotgun sequence data and limited mapping data however, it is recognised that this assembly is a draft that contains errors. Correcting the sequence assembly requires extensive additional mapping information to improve the reliability of the ordering of sequence scaffolds on chromosomes. The radiation hybrid (RH) map described here has been contributed to the international sequencing project to aid this process. RESULTS: An RH map for the 30 bovine chromosomes is presented. The map was built using the Roslin 3000-rad RH panel (BovGen RH map) and contains 3966 markers including 2473 new loci in addition to 262 amplified fragment-length polymorphisms (AFLP) and 1231 markers previously published with the first generation RH map. Sequences of the mapped loci were aligned with published bovine genome maps to identify inconsistencies. In addition to differences in the order of loci, several cases were observed where the chromosomal assignment of loci differed between maps. All the chromosome maps were aligned with the current 6x bovine assembly (Btau_2.0) and 2898 loci were unambiguously located in the bovine sequence. The order of loci on the RH map for BTA 5, 7, 16, 22, 25 and 29 differed substantially from the assembled bovine sequence. From the 2898 loci unambiguously identified in the bovine sequence assembly, 131 mapped to different chromosomes in the BovGen RH map. CONCLUSION: Alignment of the BovGen RH map with other published RH and genetic maps showed higher consistency in marker order and chromosome assignment than with the current 6x sequence assembly. This suggests that the bovine sequence assembly could be significantly improved by incorporating additional independent mapping information.

Double muscling in Marchigiana beef breed is caused by a stop codon in the third exon of myostatin gene.

Double muscling is a partially recessive trait present in some beef breeds. It shows a high frequency in some breeds, while in others the frequency is low, and double-muscled individuals are rare. The double muscling is caused by an allelic series of mutations that cause a loss of function of the myostatin gene ( GDF8). We describe here a new mutation in the myostatin gene in Marchigiana breed, a typical beef breed of Central Italy, in which rare double-muscling individuals have been described. A PCR product of the third exon was sequenced in subjects phenotypically showing double muscling, and a G > T transversion was discovered that introduces a premature stop codon. The variant found adds to the large series of mutations present in cattle, and particularly to the only two causative of double muscling in the third exon. A PCR-RFLP test is described for the rapid and effective identification of both heterozygous and homozygous subjects. It was applied to a larger survey carried on the same and also in two other beef breeds, Chianina and Romagnola. Further individuals carrying the new variant were found in Marchigiana, but none in the other breeds. The results may be important for a better comprehension of the role of myostatin in muscular development, for commercial use and for the inference of phylogeny of this gene.