Differentially expressed genes associated with Staphylococcus aureus mastitis in dairy goats.

To study gene expression within the mammary glands of dairy goats with mastitis, mRNA was collected from milk somatic cells (MSCs) of left udder halves challenged with Staphylococcus aureus and right udder halves infused with PBS, as control, at different time points (0, 12, 24 and 48h post-infection). Transcriptional profiles were investigated using bovine cDNA microarrays; of the total 288 differentially expressed genes identified with ANOVA analysis (False Discovery Rate=0.05, 1.5-fold change), 26, 36 and 16 genes were down-regulated at 12, 24 and 48h post-infection, respectively, while 60, 141 and 9 genes were up-regulated at the same corresponding time points. The expression profiles clearly changed at 24h post-infection with 177 genes significantly altered, corresponding to a 10-fold increase of S. aureus bacterial count in milk from infected udders. Differential expression of selected genes (CD2BP2, BCAP31, MHCII, FOSL2, MAPK13, ILT5 and JUNB) was also confirmed by real-time PCR at the different time points considered, showing high correlation with the microarray measurements and high reliability of the microarray analyses. The most readily inducible classes of genes in caprine MSCs infected with S. aureus were pro-inflammatory cytokines, chemokines and their receptors; IL-1alpha, lymphotoxin alpha, granulocyte chemotactic protein (CXCL6), and IL-2 receptor gamma were all up-regulated in infected udders versus healthy controls. This study identified a number of differentially expressed genes induced by S. aureus intramammary infection and demonstrates the intricacy of the patterns of gene expression that influence host response to a complex pathogen of significant relevance to both human and veterinary medicine.

An assessment of opportunities to dissect host genetic variation in resistance to infectious diseases in livestock.

This paper reviews the evidence for host genetic variation in resistance to infectious diseases for a wide variety of diseases of economic importance in poultry, cattle, pig, sheep and Atlantic salmon. Further, it develops a method of ranking each disease in terms of its overall impact, and combines this ranking with published evidence for host genetic variation and information on the current state of genomic tools in each host species. The outcome is an overall ranking of the amenability of each disease to genomic studies that dissect host genetic variation in resistance. Six disease-based assessment criteria were defined: industry concern, economic impact, public concern, threat to food safety or zoonotic potential, impact on animal welfare and threat to international trade barriers. For each category, a subjective score was assigned to each disease according to the relative strength of evidence, impact, concern or threat posed by that particular disease, and the scores were summed across categories. Evidence for host genetic variation in resistance was determined from available published data, including breed comparison, heritability studies, quantitative trait loci (QTL) studies, evidence of candidate genes with significant effects, data on pathogen sequence and on host gene expression analyses. In total, 16 poultry diseases, 13 cattle diseases, nine pig diseases, 11 sheep diseases and three Atlantic salmon diseases were assessed. The top-ranking diseases or pathogens, i.e. those most amenable to studies dissecting host genetic variation, were Salmonella in poultry, bovine mastitis, Marek's disease and coccidiosis, both in poultry. The top-ranking diseases or pathogens in pigs, sheep and Atlantic salmon were Escherichia coli, mastitis and infectious pancreatic necrosis, respectively. These rankings summarise the current state of knowledge for each disease and broadly, although not entirely, reflect current international research efforts. They will alter as more information becomes available and as genome tools become more sophisticated for each species. It is suggested that this approach could be used to rank diseases from other perspectives as well, e.g. in terms of disease control strategies.

Diagnostic markers for diseases: SELDI-TOF profiling of pig sera for PRRS.

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious viral disease causing severe losses to the pig industry. Most weaning piglets are likely to be exposed to the infection and show at least asymptomatic PRRS viremia strongly related to productive performance. The aims of this study were to set up experimental conditions for pig sera proteomic profiling and to identify biomarkers that differentiate weaning asymptomatic piglets positive to PRRS viremia from negative controls (PCR tested) with potential predictive value for the subsequent occurrence of clinical PRRS. Protein profiles were generated by SELDI-TOF MS using the Bio-Rad Chips WCX, IMAC30 and H50. The discovery phase revealed that a consistent number of highly significant protein peaks can be detected by the WCX and IMAC30 surfaces; however none of these peaks were statistically confirmed by the subsequent validation phase, highlighting that serum concentration of the contaminant and most abundant proteins is a crucial parameterfor SELDI-TOF MS studies. Current protocols are being furtheroptimized and adapted to pig sera to reduce the unfavourable effects of the most abundant proteins and to increase the number of potential detectable biomarkers. Furthermore, proteomic fingerprint profiling has been shown to be a promising diagnostic tool that, in the future, may be useful to provide also insights into the mechanisms of early viral infection in vivo.

Analysis and mapping of CACNB4, CHRNA1, KCNJ3, SCN2A and SPG4, physiological candidate genes for porcine congenital progressive ataxia and spastic paresis.

The cause of porcine congenital progressive ataxia and spastic paresis (CPA) is unknown. This severe neuropathy manifests shortly after birth and is lethal. The disease is inherited as a single autosomal recessive allele, designated cpa. In a previous study, we demonstrated close linkage of cpa to microsatellite SW902 on porcine chromosome 3 (SSC3), which corresponds syntenically to human chromosome 2. This latter chromosome contains ion channel genes (Ca(2+), K(+) and Na(+)), a cholinergic receptor gene and the spastin (SPG4) gene, which cause human epilepsy and ataxia when mutated. We mapped porcine CACNB4, KCNJ3, SCN2A and CHRNA1 to SSC15 and SPG4 to SSC3 with the INRA-Minnesota porcine radiation hybrid panel (IMpRH) and we sequenced the entire open reading frames of CACNB4 and SPG4 without finding any differences between healthy and affected piglets. An anti-epileptic drug treatment with ethosuximide did not change the severity of the disease, and pigs with CPA did not exhibit the corticospinal tract axonal degeneration found in humans suffering from hereditary spastic paraplegia, which is associated with mutations in SPG4. For all these reasons, the hypothesis that CACNB4, CHRNA1, KCNJ3, SCN2A or SPG4 are identical with the CPA gene was rejected.

Radiation hybrid mapping of 18 positional and physiological candidate genes for arthrogryposis multiplex congenita on porcine chromosome 5.

We report the chromosomal assignment of 18 porcine genes to human homologues using the INRA-Minnesota swine radiation hybrid panel (IMpRH). These genes (CACNA1C, COL2A1, CPNE8, C3F, C12ORF4, DDX11, GDF11, HOXC8, KCNA1, MDS028, TMEM106C, NR4A1, PHB2, PRICKLE1, Q6ZUQ4, SCN8A, TUBA8 and USP18) are located on porcine chromosome 5 (SSC5) and represent positional and functional candidates for arthrogryposis multiplex congenita (AMC), which maps to SSC5. CPNE8, PRICKLE1, Q6ZUQ4 and TUBA8 were mapped to the interval for pig AMC between microsatellites SW152 and SW904. Three SNPs in TUBA8 co-segregated with the AMC phenotype in 230 pigs of our research population without recombination and could be used as a genetic marker test for AMC. In addition, we provide evidence that a small chromosomal region of HSA22q11.2 evolutionarily corresponds to SSC5q12-q22 (and contains the human homologues of porcine SW152, Q6ZUQ4, TUBA8 and USP18), while the regions flanking HSA22q11.2 on SSC5 correspond to HSA12p13 and HSA12q12. We identified seven distinct chromosomal blocks, further supporting extensive rearrangements between genes on HSA12 and HSA22 in the AMC region on SSC5.

Application of bovine microsatellite markers for genetic diversity analysis of Swiss yak (Poephagus grunniens).

In order to assess the applicability of bovine microsatellite markers for population genetic studies in Swiss yak, 131 bovine microsatellite markers were tested on a panel of 10 animals. Efficient amplification was observed for 124 markers (94.6%) with a total of 476 alleles, of which 117 markers (94.3%) were polymorphic. The number of alleles per locus among the polymorphic markers ranged from two to nine. Seven loci (ILSTS005, BMS424B, BMS1825, BMS672, BM1314, ETH123 and BM6017) failed to amplify yak genomic DNA. Two cattle Y-chromosome specific microsatellite markers (INRA126 and BM861) amplified genomic DNA from both male and female yaks. However, two additional markers on cattle Y-chromosome (INRA124 and INRA189) amplified DNA from only males. Of the polymorphic markers, 24 microsatellites proposed by CaDBase for within- and cross-species comparisons and two additional highly polymorphic markers (MHCII and TGLA73) were used to investigate the genetic variability and the population structure of a Swiss yak herd that included 51 additional animals. The polymorphic information content ranged from 0.355 to 0.752, while observed heterozygosity (HO) ranged from 0.348 to 0.823. Furthermore, a set of 13 markers, organized into three multiplex polymerase chain reactions, was evaluated for routine parentage testing. This set provided an exclusion probability in a family of four yaks (both parents and two offspring) of 0.995. These microsatellites serve as useful tools for genetic characterization of the yak, which continues to be an important domestic livestock species.

Application of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis).

The aim of the present study was to assess the applicability of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis). A total of 127 microsatellite markers were tested on a male and a young female Saola. An efficient amplification was observed for 123 markers (96.8%), 73 markers (59.3%) were polymorphic. Four loci (BM2304, BMS1928, BMS779 and ILSTS006) on cattle chromosomes 1, 4, 7 and 8, respectively, failed to amplify in Saola. Two cattle Y-chromosome-specific microsatellite markers (INRA126 and BM861) were successfully amplified from both sexes in Saola. However, two additional markers (INRA124 and INRA189) on Y-chromosome failed to amplify in the female animal. These results show that most of the bovine microsatellite markers are applicable in Saola and therefore they can be used to study the phylogenetic relationships and the genetic diversity of the Saola population.