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Phage display was used to identify peptide mimics of an immunologically protective nematode glycan (CarLA) by screening a constrained C7C peptide library for ligands that bound to an anti-CarLA mAb (PAB1). Characterisation of these peptide mimotopes revealed functional similarities with an epitope that is defined by PAB1. Mimotope vaccinations of mice with three selected individual phage clones facilitated the induction of antibody responses that recognised the purified, native CarLA molecule which was obtained from Trichostrongylus colubriformis. Furthermore, these mimotopes are specifically recognised by antibodies in the saliva of animals that were immune to natural polygeneric nematode challenge. This shows that antibodies to the PAB1 epitope form part of the mucosal polyclonal anti-CarLA antibody response of nematode immune host animals. This demonstrates that the selected peptide mimotopes are of biological relevance. These peptides are the first to mimic the PAB1 epitope of CarLA, a defined larval glycan epitope which is conserved between many nematode species., Competing Interests: The authors wish to declare no competing interests. This study was funded by the New Zealand Ministry for Business, Innovation and Employment. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The mechanisms underlying resistance to challenge by gastrointestinal nematode parasites in sheep are complex. Using DIGE, we profiled ovine lymph proteins in lambs with host resistance (R), resilience (Ri) or susceptibility (S) to a daily trickle challenge with the nematode Trichostrongylus colubriformis. Efferent intestinal lymph was collected prior to infection (day 1) and on days 5 and 10 post-infection. Eight proteins identified by LC-MS/MS, showed differences relating to host genotype. Of these, Serpin A3-3 and Serpin A3-7 have not been reported previously in the lymph proteome. Three acute phase proteins showed significant differences relating to interactions between breeding line and parasite challenge, including complement C3β, C3α and haptoglobin (Hp) β. In the R lambs C3α was significantly up regulated (P<0.05) on day 10, while in the Ri lambs Hp β was significantly down regulated (P<0.05). In the S lambs, levels of C3β were up regulated and levels of Hp β down regulated (both P<0.05) on day 10. Hence we demonstrate that acute phase inflammation proteins contribute to differences in the innate immune response of sheep to challenge by T. colubriformis. The findings may lead to the development of new approaches to combat nematode infestations in sheep production systems., Biological Significance: Breeding lines of sheep with resistance (R), resilience (Ri) or susceptibility (S) to nematode infections provide an experimental model to examine the biological mechanisms underlying the ability of some sheep to expel worms and remain healthy without the use of an anthelmintic. Using proteomics we identified differences in the expression of acute phase lymph proteins in the R, Ri and S lambs. The results will assist the development of alternative control strategies to manage nematode infections in livestock., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Increased partitioning of amino acids (AA) from skeletal muscle to the intestine and immune system during parasitic infection may be the cause of poor growth in parasitised animals. The effect of an established Trichostrongylus colubriformis infection (6000 L3 T. colubriformis larvae for 6 d (n 5) or kept as parasite-free controls (n 6)) on AA fluxes across the mesenteric-drained viscera, portal-drained viscera (PDV), liver, total splanchnic tissues (TSP) and hindquarters were determined in lambs fed fresh Sulla (Hedysarum coronarium; 800 g DM/d) 48 d post-infection. The lambs were infused with rho-aminohippuric acid (PAH; 723 mg/h) into the mesenteric vein for 8 h to measure TSP plasma flow. Concurrently, indocyanine green (ICG; 14.6 mg/h) was infused into the abdominal aorta to measure plasma flow across the hindquarters. Blood was continuously collected from the mesenteric, portal and hepatic veins, vena cava and the mesenteric artery and plasma harvested. PAH, ICG, AA, metabolite and insulin concentrations were measured. Intestinal worm burdens on day 48 post-infection were higher in the infected lambs (P 0.10). There was a 28 % reduction in the release of AA from the PDV of infected lambs (P < 0.05). The uptakes of most AA were similar in the liver; however, there was increased uptake (P < 0.10) of AA by the TSP of infected lambs. Despite this reduction in AA availability at the liver, there was no effect of parasitic infection on AA uptake across the hindquarters (P < 0.05).

Five groups of ten 7-month-old Romney lambs were challenged twice-weekly (2 x 3,000) with either Ostertagia circumcincta (Groups 1-3) or Trichostrongylus colubrifornzis (Groups 4 and 5) infective-stage larvae (L3) following administration of controlled release capsules (CRC) containing either albendazole (ABZ-CRC) (Groups 2 and 5) or ivermectin (IVM-CRC) (Group 3). Larval challenge comprised a mixture of equal numbers of drug-susceptible and -resistant L3 (OR/TR:OS/TS) during the 14-week period of drug release. Positive faecal egg counts (FEC) were recorded in each group during this period. Eggs recovered from faeces were used in an egg hatch assay (EHA) utilizing different concentrations of ABZ. O. circumcincta eggs from Groups 2 and 3 had significantly higher LC50 values than Group 1; eggs of T. colubriformis from the ABZ-CRC treated Group 5 had a higher LC50 than control Group 4. Following the exhaustion of drug release, larval challenge was changed to consist of only susceptible parasites and EHA performed wveekly to determine whether LC50 declined, which would indicate dilution or replacement of drench survivors. In those animals challenged with O. circumcincta, LC50 values were still significantly higher in Groups 2 and 3 at week 19 than in the previously untreated Group 1. For T. colubriformis, LC50 values declined steadily following exhaustion of drug release and were not significantly different between Groups 4 and 5 by week 20. This decline in LC50, and corresponding rise in FEC, was largely the result of 3 animals which dominated with high FEC. The remaining 6 animals showed little decline in LC50 or rise in FEC. The results demonstrate the ability of persistent drugs to screen for resistant parasites and establish that the period of selective advantage for drug-resistant parasites of both species is significantly longer than the period of drug release from CRCs.

The ability of lactating Romney ewes to resist establishment of ingested infective-stage larvae (L3) of Ostertagia circumcincta and Trichostrongylus colubriformis was measured in the field. Three groups of seven single-lamb-bearing ewes were selected on the basis of uniformity of lambing date from a large flock held on pasture. Either 2, 4 or 6 weeks after parturition, groups of ewes were dosed with 24000 L3 of known oxfendazole-resistant parasite strains; 12000 of each species. Ten to 14 days later the ewes, along with their lambs, were transferred from the field to indoor pens. Twenty-five days after the challenge dose the ewes were drenched with oxfendazole to remove any field-derived infection and 3 days later slaughtered for worm counts. Mean establishment of the resistant parasites was low at all times, with the highest rate recorded being 6.1% for O. circumcincta 2 weeks after parturition. Establishment of O. circumcincta 4 and 6 weeks after parturition, and of T. colubriformis at all times, never exceeded 2%. By comparison, mean establishment in lambs held indoors and parasite free for 13 weeks prior to infection, was 24.9% and 47.1% for O. circumcincta and T. colubriformis, respectively. These results indicate that the lactating ewes were exhibiting a substantial ability to prevent establishment of ingested larvae. The results of this and other similar studies suggest that the dynamics of parasitism in lactating Coopworth and Romney ewes in New Zealand is substantially different to that in Merino ewes in Australia, and that these differences influence optimal strategies for the management of anthelmintic resistance in the two countries.

The effect of albendazole (ABZ)-capsule (CRC) administration on parasite establishment and immunity to ABZ-resistant (RES) and -susceptible (SUS) T. colubriformis was measured in Romney lambs. During 12 weeks of twice-weekly dosing with 3000 parasite larvae (L3), eggs were observed in faeces from CRC-treated and untreated lambs given RES L3, but not CRC-treated lambs given SUS L3. Following the period of trickle challenge all lambs were drenched and, 1 week later, dosed with 20000 SUS L3. Resulting worm burdens were higher in control lambs than in those previously treated with CRCs and challenged with SUS, which in turn were higher than those in the CRC-treated or -untreated lambs previously challenged with RES L3. During the period of trickle challenge, the number of peripheral eosinophils and titres of anti-L3 and anti-adult antibody were raised only in those groups given RES L3. There was no effect of CRC administration. Following drench and challenge, antibody titres and eosinophil numbers increased in the control animals but not in those groups which had received previous trickle infection. The results demonstrate that the larval challenge alone resulted in incomplete though substantial protection against subsequent parasite challenge. The use of CRCs may potentially impact on subsequent animal performance and selection for anthelmintic resistance through a reduced level of immunity.

Groups of lambs either with or without controlled-release albendazole (ABZ) capsules (CRCs) were challenged twice weekly for 6 weeks with either drug-susceptible or drug-resistant Ostertagia circumcincta and Trichostrongylus colubriformis. Groups with and without CRCs remained unchallenged as controls. There was minimal establishment of drug-susceptible parasites of either species in those lambs with CRCs. However, drug-resistant parasites of both species established adult worm burdens in the presence of the capsules. The humoral immune response, as measured by the serum antilarval (L3) antibody (Ab) titre, was pooled for weeks 4-6 and compared for each group. With the exception of anti-T. colubriformis Ab in group 2, anti-L3 Ab titres were significantly higher in all the parasite-challenged groups as compared to the control animals. Also, with the exception of anti-resistant O. circumcincta Ab levels in the CRC-treated animals (group 5), no significant difference was observed in Ab titres between the four groups challenged with either resistant or susceptible larvae. The results demonstrate the inability of CRCs to prevent establishment of drug-resistant parasites and that immune stimulation is not inhibited by the capsules.

The amounts of acetylcholinesterase (AChE) in homogenates of infective-stage larvae of strains, from different countries, of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis resistant or susceptible to benzimidazole (BZ) anthelmintics, were compared using a rapid colorimetric assay. The levels of AChE were much greater in those strains which were BZ-resistant than in BZ-susceptible populations. The larvae of susceptible and resistant H. contortus contained less AChE than did the larvae of the other two species. One-way analysis of variance showed that the observed differences between susceptible and resistant strains were significant in each of the species studied. It is suggested that the extra enzyme may act as an anthelmintic-detoxifying enzyme, may inactivate the active site of the anthelmintic or may prevent binding of the anthelmintic to its receptor.

Infective-stage larvae of trichostrongyle nematodes, either resistant or susceptible to thiabendazole (TBZ), were incubated in eserine, an acetylcholinesterase inhibitor. Paralysis occurred in larvae treated with eserine but significant differences were observed in the percentage of larva immobilized between TBZ-resistant and TBZ-susceptible strains of the nematodes. These differences are probably related to the presence of higher levels of acetylcholinesterase in the TBZ-resistant strains than in the susceptible strains. This could be used as a rapid and inexpensive method of detecting resistance to TBZ in trichostrongyles.