Disseminated intravascular coagulation in a novel porcine model of severe Staphylococcus aureus sepsis fulfills human clinical criteria.

Sepsis is a common and often fatal complication in human patients in intensive care units. Relevant and well characterized animal models of sepsis may provide valuable information on pathophysiological mechanisms and be a mean of testing new therapeutic strategies. Large animal models of Staphylococcus aureus sepsis are rare, even though S. aureus increasingly affects human patients. Sepsis changes the haemostatic balance and leads to endothelial cell (EC) activation, coagulopathy and, in severe cases, disseminated intravascular coagulation (DIC). The aim of this study was to characterize the haemostatic and vascular alterations in a novel porcine model of severe S. aureus sepsis, investigating whether the changes fulfill the human clinical criteria for DIC. Five pigs were inoculated intravenously with S. aureus and two control animals were sham-inoculated. Blood samples were collected for thromboelastography (TEG) and assessment of plasma-based haemostatic parameters. Tissue was collected for histopathology and reverse transcriptase quantitative real-time polymerase chain reaction for measurement of mRNA encoding EC markers. All infected animals developed DIC; including procoagulant activation represented by hypercoagulable TEG profiles and prolonged clotting time. Histologically, numerous pulmonary thrombi were present in one pig. Inhibitor consumption was represented by decreasing antithrombin levels in infected pigs. Hyaline globules were found in three infected pigs, confirming fibrinolytic activation. EC activation was identified by expression of von Willebrand factor in small vessels together with elevated mRNA encoding activated EC markers. Severe haemostatic and vascular changes fulfilling the human criteria for DIC were therefore seen in all infected pigs. A tendency towards uncompensated DIC was seen in two animals.

The impact of Staphylococcus aureus concentration on the development of pulmonary lesions and cytokine expression after intravenous inoculation of pigs.

Acute respiratory distress syndrome is a common complication in severe sepsis. In pigs, the lungs play an important role in clearing systemic bacterial infections due to pulmonary intravascular macrophages found specifically in pigs. However, this increases the exposure of the porcine lungs to pathogens and potential injury. The authors propose that increasing the concentration of the inoculum without changing the bacterial dose will lead to severe sepsis with pronounced pulmonary lesions. This could potentially create a risk of cytokine spillover to the circulation, leading to an increased systemic response. Eight Danish Landrace pigs, approximately 10 weeks old, were inoculated twice with a low or once with a high concentration of Staphylococcus aureus. Three pigs were sham-inoculated. The animals were grouped based on macro- and microscopic lung lesions. The mRNA expression of local pulmonary inflammatory markers was compared to protein levels of systemic inflammatory markers. The most severe pulmonary lesions were observed in animals receiving the high S. aureus concentration, indicating that severity of lesions is dependent on inoculum concentration rather than total numbers of bacteria. Furthermore, local mRNA expression of inflammatory cytokines appeared to be dependent on the magnitude and severity of tissue destruction, including the ability to confine the lesions. Increasing mRNA levels of serum amyloid A could be a confident marker of severity of pulmonary lesions. Since no correlation was observed between local and systemic levels of inflammatory cytokines, this finding could indicate an ability of the porcine lung to compartmentalize the local inflammatory response and thus restrict systemic contribution.

Quantitative trait loci for clinical mastitis on chromosomes 2, 6, 14 and 20 in Norwegian Red cattle.

Mastitis is the most frequent and costly disease in dairy production and solutions leading to a reduction in the incidence of mastitis are highly demanded. Here a genome-wide association study was performed to identify polymorphisms affecting susceptibility to mastitis. Genotypes for 17 349 SNPs distributed across the 29 bovine autosomal chromosomes from a total of 2589 sires with 1 389 776 daughters with records on clinical mastitis were included in the analysis. Records of occurrence of clinical mastitis were divided into seven time periods in the first three lactations in order to identify quantitative trait loci affecting mastitis susceptibility in particular phases of lactation. The most convincing results from the association mapping were followed up and validated by a combined linkage disequilibrium and linkage analysis. The study revealed quantitative trait loci affecting occurrence of clinical mastitis in the periparturient period on chromosomes 2, 6 and 20 and a quantitative trait locus affecting occurrence of clinical mastitis in late lactation on chromosome 14. None of the quantitative trait loci for clinical mastitis detected in the study seemed to affect lactation average of somatic cell score. The SNPs highly associated with clinical mastitis lie near both the gene encoding interleukin 8 on chromosome 6 and the genes encoding the two interleukin 8 receptors on chromosome 2.

Genome-wide association mapping in Norwegian Red cattle identifies quantitative trait loci for fertility and milk production on BTA12.

Reproductive performance is a critical trait in dairy cattle. Poor reproductive performance leads to prolonged calving intervals, higher culling rates and extra expenses related to multiple inseminations, veterinary treatments and replacements. Genetic gain for improved reproduction through traditional selection is often slow because of low heritability and negative correlations with production traits. Detection of DNA markers associated with improved reproductive performance through genome-wide association studies could lead to genetic gain that is more balanced between fertility and production. Norwegian Red cattle are well suited for such studies, as very large numbers of detailed reproduction records are available. We conducted a genome-wide association study for non-return rate, fertility treatments and retained placenta using almost 1 million records on these traits and 17 343 genome-wide single-nucleotide polymorphisms. Genotyping costs were minimized by genotyping the sires of the cows recorded and by using daughter averages as phenotypes. The genotyped sires were assigned to either a discovery or a validation population. Associations were only considered to be validated if they were significant in both groups. Strong associations were found and validated on chromosomes 1, 5, 8, 9, 11 and 12. Several of these were highly supported by findings in other studies. The most important result was an association for non-return rate in heifers in a region of BTA12 where several associations for milk production traits have previously been found. Subsequent fine-mapping verified the presence of a quantitative trait loci (QTL) having opposing effects on non-return rate and milk production at 18 Mb. The other reproduction QTL did not have pleiotropic effects on milk production, and these are therefore of considerable interest for use in marker-assisted selection.

A genome wide association study for QTL affecting direct and maternal effects of stillbirth and dystocia in cattle.

Dystocia and stillbirth are significant causes of female and neonatal death in many species and there is evidence for a genetic component to both traits. Identifying causal mutations affecting these traits through genome wide association studies could reveal the genetic pathways involved and will be a step towards targeted interventions. Norwegian Red cattle are an ideal model breed for such studies as very large numbers of records are available. We conducted a genome wide association study for direct and maternal effects of dystocia and stillbirth using almost 1 million records of these traits. Genotyping costs were minimized by genotyping the sires of the recorded cows, and using daughter averages as phenotypes. A dense marker map containing 17,343 single nucleotide polymorphisms covering all autosomal chromosomes was utilized. The genotyped sires were assigned to one of two groups in an attempt to ensure independence between the groups. Associations were only considered validated if they occurred in both groups. Strong associations were found and validated on chromosomes 4, 5, 6, 9, 12, 20, 22 and 28. The QTL region on chromosome 6 was refined using LDLA analysis. The results showed that this chromosome most probably contains two QTL for direct effect on dystocia and one for direct effect on stillbirth. Several candidate genes may be identified close to these QTL. Of these, a cluster of genes expected to affect bone and cartilage formation (i.e. SPP1, IBSP and MEPE) are of particular interest and we suggest that these genes are screened in candidate gene studies for dystocia and stillbirth in cattle as well as other species.

Characterization of a QTL region affecting clinical mastitis and protein yield on BTA6.

Quantitative trait loci affecting clinical mastitis were detected and fine mapped to a narrow region on bovine chromosome 6 in the Norwegian Red cattle population. The region includes the casein gene cluster and several candidate genes thought to influence clinical mastitis. The most significant results were found for SNPs within the Mucin 7 gene. This gene encodes an antimicrobial peptide and constitutes part of the first line of defence for the mucosal immune system. Detection of long haplotypes extending several Mb may indicate that artificial selection has influenced the haplotype structures in the region. A search for selection sweeps supports this observation and coincides with association results found both by single SNP and haplotype analyses. Our analyses identified haplotypes carrying quantitative trait loci alleles associated with high protein yield and simultaneously fewer incidences of clinical mastitis. The fact that such haplotypes are found in relative high frequencies in Norwegian Red may reflect the combined breeding goal that is characterized by selection for both milk production and disease resistance. The identification of these haplotypes raises the possibility of overcoming the unfavourable genetic correlation between these traits through haplotype-assisted selection.

Fine mapping of quantitative trait Loci on bovine chromosome 6 affecting calving difficulty.

Calving difficulty is an economically and ethically important trait for dairy cattle breeding. The aim of the present paper was to refine the position of a previously detected quantitative trait locus (QTL) affecting calving difficulty (direct effect) in Norwegian Red dairy cows. A granddaughter design consisting of 18 elite sire families and a total of 713 sons was genotyped for 154 markers spanning the QTL region, and the trait data were analyzed by using a combined linkage and linkage disequilibrium approach. A highly significant QTL was detected in a 150-kb interval between the markers LAP3_281 and BTA-114677. Additionally, there were some indications of a second QTL between the markers BTA-75776 and BTA-75780 located less than 500 kb apart. Several candidate genes may be identified close to these QTL. Of these, a cluster of genes expected to affect bone and cartilage formation may be of particular interest for follow-up studies.

Association analysis of the constructed linkage maps covering TLR2 and TLR4 with clinical mastitis in Norwegian Red cattle.

Toll-like receptors (TLR) are important cell-surface molecules mediating immune responses. Previous studies have identified TLR2 and TLR4 as potential candidate genes for disease resistance. In this study, dense linkage maps comprising single nucleotide polymorphisms (SNPs) have been constructed for the chromosomal regions harbouring TLR2 and TLR4 on bovine chromosome 17 and 8. The most likely marker orders for both regions were compared with the corresponding human map positions and used to reorder bovine scaffolds available from the bovine genome sequence assembly (Btau_3.1). A combined linkage and linkage disequilibrium method was used to investigate possible associations between the TLR genes and mastitis susceptibility recorded in the Norwegian Red cattle population. The analysis did not detect any significant association between the chromosomal regions surrounding TLR2 and TLR4 and mastitis in Norwegian Red cattle.

The origin of selection signatures on bovine chromosome 6.

The extent and pattern of linkage disequilibrium (LD) between closely spaced markers contain information about population history, including past population size and selection history. Selection signatures can be identified by comparing the LD surrounding a putative selected allele at a locus to the putative non-selected allele. In livestock populations, locations of selection signatures identified in this way should be correlated with QTL affecting production traits, as the populations have been under strong artificial selection for these traits. We used a dense SNP map of bovine chromosome 6 to characterize the pattern of LD on this chromosome in Norwegian Red cattle, a breed which has been strongly selected for milk production. The pattern of LD was generally consistent with strong selection in regions containing QTL affecting milk production traits, including a strong selection signature in a region containing a mutation known to affect milk production. The results demonstrate that in livestock populations, the origin of selection signatures will often be QTL for livestock production traits, and illustrate the value of selection signatures in uncovering new mutations with potential effects on quantitative traits.

A genome scan for quantitative trait locus by environment interactions for production traits.

Genotype by environment interactions between milk production traits and production level have often been observed. To increase the power of quantitative trait loci (QTL) detection, QTL by environment interaction was included in QTL analyses for the milk, protein, and fat yields. The aim of the study was to detect QTL with interaction effects with the production environment. The QTL effects were modeled through random regression models for within-herd production level. All autosomes except Bos taurus autosome 6 were included in the analysis. A more detailed study of chromosome 6 is planned. For milk yield, 5 QTL were observed, 2 of which had interaction effects with production level (suggestive linkage). For protein yield, 5 QTL were observed, 3 of which had interaction effects (suggestive linkage). For fat yield, 3 QTL were observed, none of which had interaction effects with the environment (suggestive linkage). Thus, some QTL with interaction effects seemingly exist for milk yield and protein yield. For such QTL, estimated correlations between slope and intercept of the effect (close to 1 or -1) indicated that only 2 alleles were segregating. The study indicates that QTL by environment interactions exist, and that random regression models that describe the environment as herd production level can detect this interaction.

Fine mapping of milk production QTL on BTA6 by combined linkage and linkage disequilibrium analysis.

Combined linkage and linkage disequilibrium analysis were used to refine the position of a previously detected QTL affecting milk production traits on bovine chromosome 6. Through a series of single- and multitrait and single- and multipoint QTL analyses, the QTL could be positioned to a 7.5-cM interval surrounded by the markers BMS2508 and FBN12. The most significant results were found for fat percentage and protein percentage. This effect seemed to be caused by a QTL allele embedded in one specific marker haplotype that caused a reduction in fat and protein yields and a concomitant increase of milk yield, thus resulting in a marked reduction of fat and protein percentages.

A genome scan for quantitative trait loci affecting milk production in Norwegian dairy cattle.

An autosomal genome scan for quantitative trait loci (QTL) affecting milk production traits was carried out on the Norwegian Dairy Cattle population. Six half-sibling families with a total of 285 sons organized according to a granddaughter design were analyzed by a multiple marker regression method. Suggestive QTL for one or several of the five milk traits (milk yield, protein percentage, protein yield, fat percentage and fat yield) were detected on chromosomes 3, 5, 6, 11, 13, 18 and 20. Among these results, the findings on chromosomes 3, 6, and 20 are highly supported by literature. The most convincing result was found close to marker FBN9 on chromosome 6, where a QTL was detected with alleles that cause a marked reduction in both protein and fat percentages and an increase in milk yield. The results for fat and protein percentage were highly significant even after accounting for multiple testing across the genome. Using bootstrapping, a 95% confidence interval for the position of the QTL for the percentage traits on chromosome 6 was estimated to 16 cM.

Quantitative trait loci affecting clinical mastitis and somatic cell count in dairy cattle.

Norway has a field recording system for dairy cattle that includes recording of all veterinary treatments on an individual animal basis from 1978 onwards. Application of these data in a genome search for quantitative trait loci (QTL) verified genome-wise significant QTL affecting clinical mastitis on Chromosome (Chr) 6. Additional putative QTL for clinical mastitis were localized to Chrs. 3, 4, 14, and 27. The comprehensive field recording system includes information on somatic cell count as well. This trait is often used in selection against mastitis when direct information on clinical mastitis is not available. The absence of common QTL positions for the two traits in our study indicates that the use of somatic cell count data in QTL studies aimed for reducing the incidence of mastitis should be carefully evaluated.