Influence of pathogenic bacteria species present in the postpartum bovine uterus on proteome profiles.

In the first 2-3 weeks after parturition >90% of dairy cows will have some form of uterine infection. Uterine contamination with pathogens, such as Trueperella (formerly Arcanobacterium) pyogenes increases the risk of developing more severe endometritis, which can reduce conception rates. In this study, we compared the uterine proteome of cows infected with Trueperella pyogenes with that of uninfected cows, using 2D gel electrophoresis, and identified annexins A1 and A2 (ANXA1 and ANXA2), apolipoprotein A-1, calprotectin (S100A9), cathelicidin, enolase 1 (ENO1), peptidoglycan recognition protein 1 (PGLYRP1), phosphoglycerate mutase 1 (PGAM1), serine dehydratase (SDS) and serine protease inhibitors (SERPIN) B1, B3 and B4 proteins as differing in abundance in endometritis. Subsequently, levels of ten of these proteins were monitored in uterine samples collected from a herd of lactating, dairy cows at 15 and 42 days post-partum (DPP). The levels were compared with the cytology scores of the samples and the bacterial species isolated from the uterus. Cathelicidin, PGLYRP1, SERPINB1 and S100A9 levels at 15DPP showed strong positive correlations (r=0.78, 0.80, 0.79, and 0.68 respectively; P<0.001) with % of polymorphonuclear neutrophils (PMN). When compared with other bacterial pathogens identified, Streptococcus agalactiae and Truperella pyogenes induced increased expression of the indicator proteins, suggesting that these organisms may adversely affect the subsequent ability of the cow to conceive. Interestingly, there was no difference in the proportion of cows pregnant at 6 and 17 weeks after start of mating between the cows with high or low %PMN.

Effect of asynchronous transfer on bovine embryonic development and relationship with early cycle uterine proteome profiles.

The uterus provides the nurturing environment that supports the growth of the early preimplantation bovine conceptus. To determine critical time points of uterine influence, in vitro-produced Day 7 blastocysts were transferred into synchronous (Day 7) uteri and asynchronous uteri (Days 5 or 9). Embryo growth was evaluated 7 and 15 days after transfer and compared with that of embryos generated by AI. Conceptuses recovered from asynchronous Day 9 transfers were fourfold larger than synchronous transfer or gestational Day 14 AI conceptuses; by 15 days after transfer, differences were less marked. Two-dimensional gel electrophoresis was used to compare the histotroph protein composition of uterine luminal flushings (ULF) on Days 5 and 9 after oestrous to determine any protein differences that would promote embryo growth. The ULF were collected by serially flushing the uteri of the same heifers and mature cows at different times of the cycle. Ten proteins that differed in abundance between Day 5 and 9 were identified by mass spectrometry. Three, namely phosphoserine aminotransferase 1, purine nucleoside phosphorylase and aldose reductase, were verified by western blot analysis as more abundant on Day 9 (P<0.002). Myostatin was present in only in Day 9 ULF, whereas tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and legumain were only detected in Day 14 ULF. Although mature cows had lower progesterone concentrations on Days 5 and 14 (P<0.05) and tended to have less TIMP2 than heifer groups, no other protein differences were detected. Thus, the embryo growth-enhancing environment on Day 9 was associated with temporal changes in the expression of several proteins of the histotroph.

Host-defence-related proteins in cows' milk.

Milk is a source of bioactive molecules with wide-ranging functions. Among these, the immune properties have been the best characterised. In recent years, it has become apparent that besides the immunoglobulins, milk also contains a range of minor immune-related proteins that collectively form a significant first line of defence against pathogens, acting both within the mammary gland itself as well as in the digestive tract of the suckling neonate. We have used proteomics technologies to characterise the repertoire of host-defence-related milk proteins in detail, revealing more than 100 distinct gene products in milk, of which at least 15 are known host-defence-related proteins. Those having intrinsic antimicrobial activity likely function as effector proteins of the local mucosal immune defence (e.g. defensins, cathelicidins and the calgranulins). Here, we focus on the activities and biological roles of the cathelicidins and mammary serum amyloid A. The function of the immune-related milk proteins that do not have intrinsic antimicrobial activity is also discussed, notably lipopolysaccharide-binding protein, RNase4, RNase5/angiogenin and cartilage-glycoprotein 39 kDa. Evidence is shown that at least some of these facilitate recognition of microbes, resulting in the activation of innate immune signalling pathways in cells associated with the mammary and/or gut mucosal surface. Finally, the contribution of the bacteria in milk to its functionality is discussed. These investigations are elucidating how an effective first line of defence is achieved in the bovine mammary gland and how milk contributes to optimal digestive function in the suckling calf. This study will contribute to a better understanding of the health benefits of milk, as well as to the development of high-value ingredients from milk.

The abundance of milk cathelicidin proteins during bovine mastitis.

Current on-farm methods for detecting mastitis in dairy cows have limitations with their specificity and sensitivity, particularly at an early stage of infection. There is therefore a need to explore new approaches for detecting early and subclinical mastitis. This study examined the expression of a group of neutrophil-specific proteins, the cathelicidins, in milk samples from naturally occurring as well as experimentally induced mastitis infections. Immunoblot analysis indicated that cathelicidin proteins are only observed in infected quarters and demonstrate a high correlation with somatic cell count (SCC) during the onset of infection. In most of the infections examined, cathelicidin was detected prior to the observation of clinical symptoms and at SCC counts as low as 6.2 × 10(3)cells/mL. In naturally occurring mastitis the correlation between cathelicidin and infection status is not as strong, with 25% of pathogen-positive milk samples containing no detectable cathelicidin. This may reflect the varying levels of neutrophil concentration and activity at different stages or severities of infection. Our results indicate that milk cathelicidin levels increase following intramammary infection and cathelicidin-based biomarkers may assist in the detection of preclinical mastitis or determining the stage of infection.

The constituents of Microctonus sp. parasitoid venoms.

Purified RNA transcripts from venom glands dissected from the parasitoid wasp Microctonus hyperodae were copied, cloned and sequenced using traditional dideoxy sequencing methods. Using mass spectrometry analysis of the trypsinised PAGE gel protein bands we identified the RNA transcripts for the 3 most abundant proteins found in the venom and hence obtained their full protein sequence. Other abundant transcripts were also further sequenced. To reduce the effort required to obtain transcript information we dissected venom glands from a second parasitoid, Microctonus aethiopoides (Morocco biotype). The RNA transcripts were purified and reverse transcribed but instead of cloning the cDNA it was directly sequenced using Roche GS20 pyrosequencing. Results from a single GS20 sequencing run provided data similar to that obtained by the traditional methods used in analysing transcripts from M. hyperodae in a fraction of the time and cost. Comparing the transcripts between the two species showed that a similar range of genes are expressed with the putative orthologs of seven of the eight full length genes characterised from M. hyperodae being found in M. aethiopoides. Pyrosequencing should provide a valuable new method for rapidly sampling transcripts from a wide range of specialised insect tissues.

The bovine salivary proteins BSP30a and BSP30b are independently expressed BPI-like proteins with anti-Pseudomonas activity.

The family of BPI-like proteins are thought to play a role in innate immunity of the airways and oral cavity. They have similarities with bactericidal/permeability-increasing protein (BPI), an important host defence molecule in mammals, in the nucleotide sequence of their mRNAs, organisation of exons and their predicted protein structure. We compared the expression and function of 2 of the 13 known bovine BPI-like proteins, BSP30a and b, which together constitute 30% of the total protein in bovine saliva. Despite their recent divergence, we found that the two proteins have unique expression patterns, considerable inter- and intra-animal variation in abundance and differ in their glycosylation status. Recombinant and native BSP30a and b exhibited growth suppression activity against Pseudomonas aeruginosa and Streptococcus pneumoniae. Native BSP30a and b had no significant lipopolysaccharide-binding activity. These data provide functional evidence supporting a role for the BPI-like proteins in host defence and suggest that BSP30a and b contribute to the growth suppression function of bovine saliva through a mechanism independent of LPS opsonisation.

Analysis of secreted aspartic proteinases from Candida albicans: purification and characterization of individual Sap1, Sap2 and Sap3 isoenzymes.

The recently discovered secreted aspartic proteinase multi-gene (SAP) family in Candida albicans has complicated assessment of proteolytic activity as a factor in the onset and development of Candida infections. Differential expression of the SAP genes under various conditions, as well as possible variation in the properties of the individual isoenzymes, have consequences for immunological detection, for targeted drug design and possibly for pathogenicity. It is therefore important to be able to monitor Sap isoenzyme profiles in different strains of C. albicans cultures, and to know the biochemical properties of each isoenzyme. We have employed a simple purification protocol based on strong anion exchange chromatography for the direct analysis of C. albicans Sap isoenzymes from culture filtrates, as well as recovery of individual Sap1, Sap2 and Sap3 products. In the case of Sap1, this involved development of an overexpression system using the pEMBLyex4 vector transformed into Saccharomyces cerevisiae. The C. albicans strains ATCC 10231 and 10261 were shown to produce different ratios of Sap2 and Sap3 under the same conditions. Analysis of all three purified proteins by gel electrophoresis, immunoblotting and proteinase assays which were designed to evaluate pH dependence, thermal stability and substrate specificity revealed similar but distinct properties for each isoenzyme. Although Sap3 was shown to be antigenically more similar to Sap2 than was Sap1, it was less similar in terms of thermal stability and activity at low pH, being more stable and more active.