Quantification of cytokine mRNA levels in sheep infected with Teladorsagia (Ostertagia) circumcincta

Abstract

The infection of grazing sheep with Teladorsagia (Ostertagia) circumcincta, one of the major gastrointestinal (GI) nematode parasites, is almost unavoidable. Even though studies have reported that nutritional supplementation can improve the host immune response, the mechanism has not been documented yet under in vivo conditions. Evidences show that cytokines play a vital role in host immune response against GI nematode parasite infection. Therefore, the current study examined the host-parasite interaction and the influence of some important factors (nutrition and immunosuppression) on the host immune response using cytokines (mRNA levels) and faecal egg count (FEC: eggs per gram of fresh faeces (e.p.g)). Basic assumptions about Thl/Th2 subsets in cytokine pattern were also examined in this current study.

A Real-Time PCR assay was developed to 'quantify six important cytokine mRNA levels from sheep abomasal biopsy samples. The uniform spread of four of them (interferon gamma - IFNγ, interleukin (IL) 2, IL4 and IL10) within an abomasum was confirmed in a subsequent uniformity study.

The influence of dietary protein supplementation on the immune response was investigated in a subsequent trial in which twenty, two-tooth pregnant cannulated ewes were used. They were assigned to two groups, high (HP, n=10) and low protein (LP, n=10) and were trickle infected with T. circumcincta and T. colubriformis from six weeks prior to parturition until six weeks after parturition FEC and cytokine mRNA levels (from abomasal biopsy tissues) were examined during this period. Even though protein supplementation did not influence the FEC (statistically), a 50% reduction in mean FEC was observed in the HP group. The expression of IFNy (p<0.05), IL2 (p<0.05) and IL10 (p<0.001) were higher in the LP group than in the LP group. Likewise, IFNγ and IL2 mRNA levels were higher (p<0.05) during the period before than after parturition.

In another experiment, the effect of immunosuppression on the immune response was examined in twelve, cannulated sheep, which were assigned either to a control (n=6) or an immunosuppressed (n=6) group. For convenience, four periods: 1 - immune establishment; 2 - challenge (with ex sheathed larvae); 3 - immunosuppression (in one group of sheep) and 4 -rechallenge of immune (control) and immunosuppressed groups were established. During period 1, all sheep were infected with T. circumcincta for five weeks and resistance was confirmed by low FEC (<300 epg). All sheep were challenged with 200,000 ex sheathed L3 of T. circumcinta in period 2 and biopsy samples were taken at each of 24 h and 10 min before challenge and at 10, 30, 120 min and 24 and 48 h after challenge for cytokine mRNA analysis. During period 3 of four weeks, one group was immunosuppressed while the other group was maintained as control but all animals were infected with T. circumcincta and FEC monitored. Period 4 was carried out exactly as described for the period 2. Data from six animals (n=3) were analyzed as six animals were excluded either due to failure of the cannula or death of animals before the end of the trial. The FEC of both control and immunosuppressed groups were less than 500 until two weeks after first immunosuppression, but the immunosuppressive group subsequently had a FEC of 2000 epg 22 days, and counts remained almost the same (1900 FEC) for 25 days, after first immunosuppressive injection. All cytokine mRNA levels except IL2 (P=0.062) were higher (p<0.05) in the immunosuppressed group than in the control group.

High frequencies of samples with undetectable mRNA levels were observed both in protein and immunosuppression experiments and thus the sampling technique was shifted to peripheral blood mononuclear cells (PBMC). A house keeping gene (β-actin) was used to screen good quality RNA for all samples before being used for mRNA test and this modified sampling technique was then applied in the final experiment.

To examine the development of immune responses from naivety to resistance against T. circumcincta, 15 naive lambs were assigned to either an infected (inf-n=8) or control (con-n=7) group. In addition to FEC and cytokine mRNA levels, feed intake (FI) and body weight (BW) were also measured throughout the trial. The FEC of inf lambs started from 0 and peaked at 425 e.p.g (six weeks after first dosing (AFD)) before a typical decline to 0 e.p.g by day 63 as immunity developed. Lower BW (mean loss of 1.3kg) was observed from two weeks AFD till the end of experiment but was not statistically different. A small reduction (non significant) in PI of the inf group was observed compared to con group from five to eight weeks AFD. All cytokine mRNA levels of inf lambs expressed a clear difference from that of con and were significantly (p<0.05) reduced three days AFD (first sampling AFD). The expression of IFNγ, IL2 and IL10 showed four distinct stages whereas IL4 mRNA copies expressed two distinct stages compared to con. Despite findings of positive significant (p<0.05) correlation between the pairs IL10-IL2 and IL10-IFNγ in all control animals, this pattern tended to reduce in infected animals, where significant correlations were observed in 75% and 62.5% of animals, respectively; Interestingly, ILl0 mRNA showed no significant correlation with IL4 in 62.5 and 75% of con and inf group animals. This lack of association between IL10 and IL4 mRNA levels may also provide evidence for the down-regulatory activity of IL10 secreted by T regulatory cells. Reduction in the selected cytokine mRNA profile was observed during the first blood samplings in both inf and con lambs suggesting a stress effect.

This reliable quantification assay developed for cytokine mRNA under in vivo conditions could be used as a tool for immunological experiments in future and the cytokine mRNA patterns observed in the study provide a basis for future in vivo studies.