Local cytokine transcription in naïve and previously infected sheep and lambs following challenge with Teladorsagia circumcincta.

BACKGROUND: The abomasal helminth Teladorsagia circumcincta is one of the most economically important parasites affecting sheep in temperate regions. Infection is particularly detrimental to lambs, in which it can cause pronounced morbidity and severe production losses. Due to the spreading resistance of this parasite to all classes of anthelmintic drugs, teladorsagiosis is having an increasingly severe impact on the sheep industry with significant implications for sheep welfare. Protective immunity develops slowly, wanes rapidly and does not appear to be as effective in young lambs. To investigate the development of immunity to T. circumcincta in sheep and lambs, we used cytokine transcript profiling to examine differences in the abomasal mucosa and gastric lymph node of naïve and previously infected sheep and lambs following challenge. RESULTS: The results of these experiments demonstrated that the abomasal mucosa is a major source of cytokines during abomasal helminth infection. A local Th2-type cytokine response was observed in the abomasal mucosa and gastric lymph node of the previously infected sheep and lambs when compared with those of the naïve during the early stages of infection. In contrast, a pro-inflammatory component more was evident in the abomasal mucosa and gastric lymph node of the naïve sheep when compared with those of the previously infected, which was not observed in the lambs. CONCLUSIONS: The greater levels of Th2-type cytokine transcripts in both the abomasum and gastric lymph node of the previously infected compared with naïve sheep and lambs emphasises the importance of these mechanisms in the immune response to T. circumcincta infection. Younger lambs appear to be able to generate similar Th2-type responses in the abomasum suggesting that the increased morbidity and apparent lack of resistance in younger lambs following continuous or repeated exposure to T. circumcincta is unlikely to be due to a lack of appropriate Th2-type cytokine production.

Macrophage migration is controlled by Tribbles 1 through the interaction between C/EBPß and TNF-α.

In mammals, three Tribbles gene family members have been identified, Tribbles 1, 2 and 3 (Trib1, Trib2 and Trib3). All family members are considered to be pseudokinases in that they contain domains homologous to serine/threonine kinase catalytic cores, but they lack several conserved residues in the ATP-binding pocket. Trib1 is implicated in the inflammatory response pathway through its ability to regulate mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NF-κB) and CCAAT Enhancer Binding Protein (C/EBP). However, its role in macrophages function is unknown. Here, we investigated the functional role of Trib1 in Toll-like receptor-mediated inflammatory responses to IFN-γ in RAW264.7 cells. In gene knock-down experiments in macrophages using small interfering RNAs targeted to Trib1, it was observed that TNF-α production was increased following treatment with IFN-γ and/or TLR2 ligands. Finally, Trib1-silenced macrophages failed to show MCP-1 induced chemokinesis and indicating involvement of Trib1 in controlling of macrophage migration. This work demonstrates that Trib1 contributes to the pro-inflammatory response caused by TLR2 ligands and controls macrophage migration as well as being a biomarker in macrophage-related diseases in both human and veterinary medicine.

Novel gene expression responses in the ovine abomasal mucosa to infection with the gastric nematode Teladorsagia circumcincta.

Infection of sheep with the gastric nematode Teladorsagia circumcincta results in distinct Th2-type changes in the mucosa, including mucous neck cell and mast cell hyperplasia, eosinophilia, recruitment of IgA/IgE producing cells and neutrophils, altered T-cell subsets and mucosal hypertrophy. To address the protective mechanisms generated in animals on previous exposure to this parasite, gene expression profiling was carried out using samples of abomasal mucosa collected pre- and post- challenge from animals of differing immune status, using an experimental model of T. circumcincta infection. Recently developed ovine cDNA arrays were used to compare the abomasal responses of sheep immunised by trickle infection with worm-naïve sheep, following a single oral challenge of 50 000 T. circumcincta L3. Key changes were validated using qRT-PCR techniques. Immune animals demonstrated highly significant increases in levels of transcripts normally associated with cytotoxicity such as granulysin and granzymes A, B and H, as well as mucous-cell derived transcripts, predominantly calcium-activated chloride channel 1 (CLCA1). Challenge infection also induced up-regulation of transcripts potentially involved in initiating or modulating the immune response, such as heat shock proteins, complement factors and the chemokine CCL2. In contrast, there was marked infection-associated down-regulation of gene expression of members of the gastric lysozyme family. The changes in gene expression levels described here may reflect roles in direct anti-parasitic effects, immuno-modulation or tissue repair.

NKp46 defines ovine cells that have characteristics corresponding to NK cells.

Natural killer (NK) cells are well recognized as playing a key role in innate immune defence through cytokine production and cytotoxic activity; additionally recent studies have identified several novel NK cell functions. The ability to study NK cells in the sheep has been restricted due to a lack of specific reagents. We report the generation of a monoclonal antibody specific for ovine NKp46, a receptor which in a number of mammals is expressed exclusively in NK cells. Ovine NKp46+ cells represent a population that is distinct from CD4+ and γδ+ T-cells, B-cells and cells of the monocytic lineage. The NKp46+ cells are heterogenous with respect to expression of CD2 and CD8 and most, but not all, express CD16--characteristics consistent with NK cell populations in other species. We demonstrate that in addition to populations in peripheral blood and secondary lymphoid organs, ovine NKp46+ populations are also situated at the mucosal surfaces of the lung, gastro-intestinal tract and non-gravid uterus. Furthermore, we show that purified ovine NKp46+ populations cultured in IL-2 and IL-15 have cytotoxic activity that could be enhanced by ligation of NKp46 in re-directed lysis assays. Therefore we conclude that ovine NKp46+ cells represent a population that by phenotype, tissue distribution and function correspond to NK cells and that NKp46 is an activating receptor in sheep as in other species.

Foot-and-mouth disease virus can induce a specific and rapid CD4+ T-cell-independent neutralizing and isotype class-switched antibody response in naïve cattle.

The role of T-lymphocyte subsets in recovery from foot-and-mouth disease virus (FMDV) infection in calves was investigated by administering subset-specific monoclonal antibodies. The depletion of circulating CD4(+) or WC1(+) gammadelta T cells was achieved for a period extending from before challenge to after resolution of viremia and peak clinical signs, whereas CD8(+) cell depletion was only partial. The depletion of CD4(+) cells was also confirmed by analysis of lymph node biopsy specimens 5 days postchallenge. Depletion with anti-WC1 and anti-CD8 antibodies had no effect on the kinetics of infection, clinical signs, and immune responses following FMDV infection. Three of the four CD4(+) T-cell-depleted calves failed to generate an antibody response to the nonstructural polyprotein 3ABC but generated a neutralizing antibody response similar to that in the controls, including rapid isotype switching to immunoglobulin G antibody. We conclude that antibody responses to sites on the surface of the virus capsid are T cell independent, whereas those directed against the nonstructural proteins are T cell dependent. CD4 depletion was found to substantially inhibit antibody responses to the G-H peptide loop VP1(135-156) on the viral capsid, indicating that responses to this particular site, which has a more mobile structure than other neutralizing sites on the virus capsid, are T cell dependent. The depletion of CD4(+) T cells had no adverse effect on the magnitude or duration of clinical signs or clearance of virus from the circulation. Overall, we conclude that CD4(+) T-cell-independent antibody responses play a major role in the resolution of foot-and-mouth disease in cattle.

Foot-and-mouth disease virus persists in the light zone of germinal centres.

Foot-and-mouth disease virus (FMDV) is one of the most contagious viruses of animals and is recognised as the most important constraint to international trade in animals and animal products. Two fundamental problems remain to be understood before more effective control measures can be put in place. These problems are the FMDV "carrier state" and the short duration of immunity after vaccination which contrasts with prolonged immunity after natural infection. Here we show by laser capture microdissection in combination with quantitative real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and corroborate by in situ hybridization that FMDV locates rapidly to, and is maintained in, the light zone of germinal centres following primary infection of naïve cattle. We propose that maintenance of non-replicating FMDV in these sites represents a source of persisting infectious virus and also contributes to the generation of long-lasting antibody responses against neutralising epitopes of the virus.