Divergent ghrelin expression patterns in sheep genetically resistant or susceptible to gastrointestinal nematodes.

Gastrointestinal nematodes are a major problem for pastoral ruminant production systems. This problem could be reduced by the application of breeding strategies that select for nematode resistant sheep, but no suitable molecular markers are available. Research selection flocks containing lines that are resistant (R) or susceptible (S) to gastrointestinal nematodes provide an excellent resource for discovering selectable markers, and for studying the underlying mechanisms of an effective anti-nematode response. In this study we have used a combination of quantitative real time PCR assays and ELISA to determine if nematode challenge impacts on the expression of the satiety-regulating hormone ghrelin. The expression responses were then compared between the selection flock R and S lines. The results show that the basal levels of ghrelin in plasma were greater than 2-fold higher in nematode naïve S line sheep. Three days after a primary nematode challenge divergent ghrelin expression patterns were observed between the selection lines, with levels increasing in R sheep while decreasing in S sheep. After a secondary challenge this trend was repeated, but following a third challenge ghrelin expression levels rose in both R and S sheep, by which time the S animals had acquired an effective immune response to the nematodes, as measured by a significant reduction in faecal egg output. Importantly, this phenomenon was observed in gene expression studies in gut tissues and also in ELISA measurements of ghrelin peptide levels in plasma. A regression analysis showed that ghrelin transcript expression in the gut accounted for >40% of the variation in faecal egg count measured following Haemonchus or Trichostrongylus infection. We therefore hypothesise that the direction of ghrelin expression (up or down) immediately following nematode exposure may play an important role in regulating the differing anti-nematode responses that occur in the R and S lines. Such differences identify ghrelin as a previously unrecognized factor influencing the acquisition of immunity to nematodes.

Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to Trichostrongylus colubriformis and Haemonchus contortus.

Resistance to an acute gastrointestinal nematode (GIN) infection is dependent on the ability of the host to recognise the parasite and mount a protective Th2 response. It is hypothesised that lambs which are genetically susceptible to GIN will differentially up-regulate Th1-type genes and therefore remain susceptible to chronic parasitism compared with genetically resistant lambs which will differentially up-regulate Th2-type genes and clear the parasite infection. Two selection flocks, in which lines of Merino sheep produced lambs genetically resistant or susceptible to GIN, were acutely challenged once or thrice with either Haemonchus contortus or Trichostrongylus colubriformis. Faecal-egg counts (FECs), and plasma and tissue anti-parasite (H. contortus or T. colubriformis) antibody isotype responses showed that resistant animals challenged three times with T. colubriformis established a protective Th2 response (negligible FEC, IgG1 and IgE) whereas susceptible animals required multiple challenges to establish a significant IgG1 response despite FECs remaining high. Trichostrongylus colubriformis elicited a more pronounced host response than H. contortus. RNA extracted from tissues at the site of each parasite infection and associated lymph nodes were interrogated by microarray and quantitative PCR analyses to correlate host gene expression to FECs and antibody responses. The IFN-gamma inducible gene cxcl10 was up-regulated in the susceptible line of the Trichostrongylus selection flock sheep after a tertiary challenge with the parasites H. contortus and T. colubriformis. However, a uniform pattern of genes was not up-regulated in resistant animals from both selection flocks during both parasite infections, suggesting that the mode of host resistance to these parasites is different, although some similarities in host susceptibility were apparent.

Identification of immune genes and proteins involved in the response of bovine mammary tissue to Staphylococcus aureus infection.

BACKGROUND: Mastitis in dairy cattle results from infection of mammary tissue by a range of micro-organisms but principally coliform bacteria and Gram positive bacteria such as Staphylococcus aureus. The former species are often acquired by environmental contamination while S. aureus is particularly problematic due to its resistance to antibiotic treatments and ability to reside within mammary tissue in a chronic, subclinical state. The transcriptional responses within bovine mammary epithelial tissue subjected to intramammary challenge with S. aureus are poorly characterised, particularly at the earliest stages of infection. Moreover, the effect of infection on the presence of bioactive innate immune proteins in milk is also unclear. The nature of these responses may determine the susceptibility of the tissue and its ability to resolve the infection. RESULTS: Transcriptional profiling was employed to measure changes in gene expression occurring in bovine mammary tissues sampled from three dairy cows after brief and graded intramammary challenges with S. aureus. These limited challenges had no significant effect on the expression pattern of the gene encoding beta-casein but caused coordinated up-regulation of a number of cytokines and chemokines involved in pro-inflammatory responses. In addition, the enhanced expression of two genes, S100 calcium-binding protein A12 (S100A12) and Pentraxin-3 (PTX3) corresponded with significantly increased levels of their proteins in milk from infected udders. Both genes were shown to be expressed by mammary epithelial cells grown in culture after stimulation with lipopolysaccharide. There was also a strong correlation between somatic cell count, a widely used measure of mastitis, and the level of S100A12 in milk from a herd of dairy cows. Recombinant S100A12 inhibited growth of Escherichia coli in vitro and recombinant PTX3 bound to E. coli as well as C1q, a subunit of the first component of the complement cascade. CONCLUSION: The transcriptional responses in infected bovine mammary tissue, even at low doses of bacteria and short periods of infection, probably reflect the combined contributions of gene expression changes resulting from the activation of mammary epithelial cells and infiltrating immune cells. The secretion of a number of proinflammatory cytokines and chemokines from mammary epithelial cells stimulated by the bacteria serves to trigger the recruitment and activation of neutrophils in mammary tissue. The presence of S100A12 and PTX3 in milk from infected udder quarters may increase the anti-bacterial properties of milk thereby helping to resolve the mammary tissue infection as well as potentially contributing to the maturation of the newborn calf epithelium and establishment of the newborn gut microbial population.

Gastrointestinal nematode challenge induces some conserved gene expression changes in the gut mucosa of genetically resistant sheep.

Sheep have a varying ability to resist infection with gastrointestinal nematodes. This ability is due in part to genetic differences that exist between individuals. In order to define these differences we have used real-time PCR to quantify gene expression responses in the gut mucosal surface of genetically resistant and susceptible sheep, following a nematode challenge. Expression profiles were determined in response to two different nematode species, Haemonchus contortus and Trichostrongylus colubriformis, and in divergent sheep originating from two different genetic backgrounds. Results show that the response generated differs between resistant and susceptible animals and is further impacted by the origin of the sheep and nematode species used for challenge. However, some conserved features of a response mounted by a resistant or a susceptible animal were identified. Genes found to be more abundantly expressed in resistant animals include markers of an early inflammatory response, several Toll-like receptors (TLR2, 4, 9) and free radical producing genes (DUOX1 and NOS2A). Conversely, genes differentiating susceptible animals indicate a prolonged response and development of a chronic inflammatory state, characterised by elevated expression of members of the NF-kappabeta signalling pathway (IKBKB and NFKBIA) together with delayed expression of regulatory markers such as IL2RA (CD25), IL10 and TGFbeta2. While multiple nematode response pathways were identified, the identification of conserved aspects of the response which associate with resistance provides evidence that alternative nematode control strategies, such as breeding for resistant animals, may be feasible.

Identification of immuno-reactive proteins from a sheep gastrointestinal nematode, Trichostrongylus colubriformis, using two-dimensional electrophoresis and mass spectrometry.

Gastrointestinal nematode infections of livestock animals are prevalent and costly problems worldwide. Currently, infections are controlled by anthelmintic chemicals but increasing drug resistance has prompted research interest to shift towards alternative methods of control such as vaccine development and selection of worm-resistant animals. The present study analyses proteins from Trichostrongylus colubriformis infective L3s that are recognised by IgG of immune sheep. Following protein separation via two-dimensional electrophoresis and Western blot probing with plasma from sheep resistant to T. colubriformis, mass spectrometry-based proteomic analyses were used to identify immuno-reactive protein spots. We were able to identify 28 immune targets, including aspartyl protease inhibitor, enolase, chaperone proteins, galectin, glycolytic enzymes, kinase, phosphatase and structural muscle proteins such as myosin, paramyosin, calponin and DIM-1. The data suggest that immune responses to T. colubriformis are dispersed over a relatively large number of parasite antigens, including several cytoplasmically expressed proteins. The results have new implications for understanding the molecular mechanisms that underpin host-parasite interaction during gastrointestinal nematode infections.