Eosinophil and IgA responses in sheep infected with Teladorsagia circumcincta

Infections of sheep with the gastrointestinal nematode Teladorsagia circumcincta are characterised by increased concentrations of IgA, eosinophilia and mastocytosis but the interactions between these immune responses are unclear. We investigated the kinetics of the parasite-specific IgA and eosinophil responses in controlled infections in lambs to determine if there were any associations with subsequent worm growth and survival. IgA and eosinophil responses had very similar kinetics and variation in both responses accounted for more of the variation in adult worm length than either trait alone suggesting that IgA and eosinophils interact in regulating the growth of T. circumcincta. Curiously, those animals with higher peak eosinophil responses had longer worms at slaughter emphasising the intriguing complexity of the immune responses to these parasitic infections.     

Resistance of Teladorsagia circumcincta in goats to ivermectin in the Netherlands

Resistance of Teladorsagia circumcincta in goats to ivermectin In 2005, suspected ivermectin resistance of gastrointestinal nematodes was investigated in 17 goats on a farm in the Netherlands, using a faecal egg count reduction test combined with faecal larval cultures. Eight goats were treated orally with 0.2 mg/ kg ivermectin on 1 August 2005 and the other nine goats served as untreated controls. Faeces were collected from all goats on 1 August and on 12 August. Faecal egg counts were reduced by 80.6% in goats receiving ivermectin. On the basis of faecal larval cultures and the egg counts, ivermectin was found to be 100% effective against Haemonchus contortus, 15.0% effective against Teladorsagia circumcincta, and 93.3% effective against Trichostrongylus spp. The effect of levamisole and oxfendazole on gastrointestinal nematodes was also evaluated. Nine goats were treated subcutaneously with 5 mg/kg levamisole and seven goats orally with 4.5 mg/kg oxfendazole on 12 September On the basis of faecal egg counts performed on 12 and 22 September, levamisole reduced the total egg count by 95.4% and oxfendazole reduced the egg count by 85.8%. Differentiation of pooled faecal larval cultures revealed that levamisole was 99.9% effective against H. contortus, 81.9% effective against T. circumcincta, and 99.5% against Trichostrongylus spp. Oxfendazole was 75.5% effective against H. contortus, 33.2% effective against T. circumcincta, and 100% effective against Trichostrongylus spp. On 28 October the nine goats that were still present on the farm were orally treated with 0.4 mg/kg ivermectin. Faecal egg counts and differentiation of pooled larval cultures of faecal samples collected on 28 October and 9 November, respectively, revealed that total egg counts were reduced by 93.1%. Ivermectin was 49.1% effective against T. circumcincta but 100% effective against H. contortus and Trichostrongylus spp. Thus H. contortus appears to be resistant to oxfendazole and T. circumcinta appears to be resistant to oxfendazole and ivermectin. This is the first documented case of ivermectin resistance of a gastrointestinal nematode of small ruminants in the Netherlands.     

Resistance to prophylactic treatment with macrocyclic lactone anthelmintics in Teladorsagia circumcincta

Sixty-four Romney ewe lambs were allocated to 12 groups on the basis of liveweight. Four groups (n=5) were administered oral ivermectin (IVM), 4 (n=6) oral moxidectin (MOX) and the remaining 4 (n=5) controlled-release capsules containing IVM (IVM-CRCs). Nine and 10 days later, the groups within each treatment type were challenged with infective-stage larvae (L3) of 1 of 4 different isolates of Teladorsagia circumcincta (two doses each of 5000 L3). The first of these (S) was known to be anthelmintic-susceptible; the second (OR) was a multiple anthelmintic-resistant strain recovered from the field following therapeutic failure of both ivermectin and moxidectin and subsequently maintained in the laboratory without further anthelmintic selection; the third (R) was derived from OR but had been passaged for five generations indoors with each generation being screened with all three broad-spectrum anthelmintic classes; and the fourth (RxS) was an F1 cross between the R and S isolates. As anticipated, because of its limited residual activity, IVM had no significant effect on the establishment, 9 and 10 days post-treatment, of any of the parasite isolates. In contrast MOX, which has greater residual activity, was highly effective at preventing the establishment of the S isolate but showed no significant effect against the OR, R or RxS isolates. The IVM-CRC was also highly effective at preventing the establishment of the S isolate and furthermore it significantly reduced establishment of both the OR and RxS isolates, although it had no significant effect against the R isolate. The results suggest that with respect to the establishment of T. circumcincta L3s following anthelmintic treatment, macrocyclic lactone (ML) resistance is effectively a dominant trait in the presence of MOX, while it behaves as a partially dominant/recessive trait under treatment with IVM-CRCs. The potential implications of this finding in relation to selection for ML resistance in T. circumcincta are discussed.     

Pyrosequencing analysis of the beta-tubulin gene in Spanish Teladorsagia circumcincta field isolates

Benzimidazole (BZ) resistance in gastrointestinal nematodes has been associated with single nucleotide polymorphisms (SNPs) at codons 200, 167 and 198 in the beta-tubulin isotype 1 gene and, recently, these SNPs have also been found in macrocyclic lactone (ML) resistant strains of Haemonchus contortus. On this basis, we have studied the same putative SNPs in Spanish Teladorsagia circumcincta field isolates by pyrosequencing. Single L3 (infective 3rd stage larvae) from five sheep flocks were tested after confirming their BZ susceptibility and degree of ivermectin (IVM) resistance. According to the Faecal Egg Count Reduction Test (FECRT) one flock was classified as IVM susceptible, another one was resistant, and the rest had a suspicion of resistance to IVM. DNA extraction was carried out on 598 single L3 and 56% of these were identified as T. circumcincta after the amplification of a species-specific ITS2 fragment. The number of L3 analyzed for the SNPs 198/200 was 255 and for the SNP 167 was 187. Results clearly indicate no resistance-associated SNPs were present at any codon, before or after treatment. Therefore, all T. cicumcincta L3 were designated as susceptible homozygous genotypes for all SNPs. The absence of the mutations in these populations would argue against resistance haplotypes being present in the parasite population prior to drug treatment, at least in Spanish T. circumcincta.     

Teladorsagia circumcincta antibodies in serum and milk samples in experimentally infected lactating ewes

The aim of this study was to evaluate the IgG activity in serum and milk samples of experimentally infected lactating ewes with the nematode Teladorsagia circumcincta as well as its relationship to the degree of infection. In a previous study 28 pregnant ewes were divided into two homogeneous groups, with high (H) and low (L) level of parasitism, respectively. Blood and milk samples were taken weekly after lambing until the end of the lactation, 126 days post-partum (pp). IgG against T. circumcincta were measured by means of an indirect ELISA. The kinetic of the immunoglubulins in serum samples showed a very low activity at the beginning but gradually increased throughout the lactation; H group showed higher values most of the sampled days than L group. Contrary, IgG in milk samples remained high during the first month pp, then decreased around 38% and by the end of the study rose again. Antibodies in both samples were correlated and especially during the second month of lactation (r=0.3; p<0.001). With the aim to correlate the immune response and the degree of infection we found an inverse relationship (r=-0.2; p<0.05) during the second month of lactation between eggs and IgG in serum. However the correlation with immunoglobulins in milk was positive, mainly, on the last third of lactation (r=0.2; p<0.01). As a conclusion, the individual detection of total IgG antibodies against T. circumcincta in lactating ewes is highly dependent on the stage of lactation. Therefore, these associations should be confirmed under field conditions.     

Cross-reactive antigens of Haemonchus contortus adult worms in Teladorsagia circumcincta infected goats.

The adult Haemonchus contortus somatic antigens responsible for cross-reactivity have been analysed using serum samples from goat kids infected and reinfected with Teladorsagia circumcincta. Goat kids infected with T. circumcincta had similar serum ELISA values against somatic antigens of H. contortus as goats infected with H. contortus itself. Immunoblotting confirmed this extensive cross-reactivity particularly in the molecular weight range 105-29 kDa. However, peptides with high (195, 152 and 119 kDa) or low (23 kDa) molecular weight were only faintly recognized by heterologous sera.     

A key mechanism of pathogenesis in sheep infected with the nematode Teladorsagia circumcincta

Infection of sheep with the abomasal nematode Teladorsagia circumcincta can cause a relative protein deficiency and reduce growth rate in growing lambs. A key event appears to be the destruction of junctions between epithelial cells. If the infection is heavy or prolonged, this leads to increased mucus production, hyperplasia, decreased acid production, gastrinemia, inappetance and pepsinogenemia. The severity of the infection depends upon the extent of concurrent infection, the nutritional status of the host and genetically controlled variation in the ability to mount protective immune responses.     

Teladorsagia circumcincta egg output at the onset of natural and induced lactation in ewes

Nineteen ewes maintained under 'worm-free' conditions received anthelmintic and 1 week later were dosed on 5 days each week with 500 Teladorsagia circumcincta larvae for between 10 and 12.5 weeks. Faecal egg counts were determined twice weekly. There were three groups: pregnant (P) ewes (n = 6) which each reared twin lambs, hormone-treated (H) ewes (n = 7) which were not pregnant and were given exogenous hormones (dexamethasone, oestradiol-17 beta, progesterone) for 37 days to induce udder development and milk production, and untreated barren (B) ewes (n = 6). The plasma concentrations of oestradiol-17 beta, prolactin and progesterone were measured. Starting 10-15 days before birth in Group P, there was a biphasic periparturient rise in egg count and the onset of the first peak in egg count preceded the parturient peak in plasma prolactin concentration by several days. Most Group H (5/7) and all Group B ewes remained highly resistant to Teladorsagia infection throughout, despite an induced rise in prolactin concentrations in Group H ewes. It was concluded that factors other than prolactin initiate the periparturient rise but that prolactin is one factor which maintains the response once lactation commences.     

Green tea flavan-3-ols and oligomeric proanthocyanidins inhibit the motility of infective larvae of Teladorsagia circumcincta and Trichostrongylus colubriformis in vitro

The effects of a hot water infusion and an aqueous acetone extract of green tea (Camellia sinensis) on the motility of infective larvae of the sheep nematodes Teladorsagia circumcincta and Trichostrongylus colubriformis were investigated under in vitro conditions. The infusion and extract dose-dependently inactivated the infective larvae as assessed by the larval migration inhibition (LMI) assay. To determine the components responsible for the inhibitory activity, the hot water infusion and aqueous acetone extract of green tea were fractionated on Sephadex LH-20 and the green tea extract fractions (GTE-I-VIII) characterised by mass spectrometry. The larvae were exposed to increasing concentrations of these GTE fractions. Fractions containing epigallocatechin gallate (EGCG) and proanthocyanidin oligomers were most effective. GTE fractions were more effective against T. circumcincta than T. colubriformis larvae as assessed by the LMI assay.     

Proteomic approach to identify candidate effector molecules during the in vitro immune exclusion of infective Teladorsagia circumcincta in the abomasum of sheep

In the present study we have employed an in vitro organ challenge model to study the post-challenge responses in parasite na?ve and immune gastric tissue of sheep, in an attempt to identify the host derived factors involved in immune exclusion of Teladorsagia circumcincta larvae. Proteins present in the epithelial cells and mucus from ovine abomasa following parasite challenge in previously na?ve and immune animals were analysed through Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-Tof)-MS and shotgun proteomics. MALDI-ToF analysis of epithelial cell lysates revealed that a number of proteins identified were differentially expressed in na?ve and immune cells. These included intelectin and lysozymes, which were present at higher levels in epithelial cell lysates derived from immune samples. A large number of proteins were identified in the mucosal wash from immune tissue which were not present in the mucosal wash of the na?ve tissue. Some of these proteins were present in washes of immune tissue prior to the parasite challenge including immunoglobulin A, galectin 14 and 15 and sheep mast cell protease 1. However, other proteins, such as calcium activated chloride channel and intelectin were only detected in the washings from the challenged tissue. The latter may be related to an enhanced mucus release, which may result in entrapment of infective larvae and thus reduced establishment in tissue that has been previously challenged with the parasite. In conclusion, several proteins have been identified which may be involved, either directly or indirectly, in the exclusion and immune elimination of incoming infective larvae. In the present study, the usefulness of the in vitro model has been confirmed, and the global proteomic approach has identified proteins that had not previously been associated with parasite exclusion from abomasal mucosa, such as the calcium activated chloride channel.     

Haemonchus contortus and Ostertagia circumcincta: fenbendazole treatment and abomasal permeability changes in sheep

Effects of fenbendazole (7.5 mg/kg of body weight infused during 90 minutes) on abomasal transmural potential difference (PD), pH, and ionic concentrations were determined in 8 lambs fitted with permanent abomasal cannulae. Four sheep were treated before inoculation and on day 28 after inoculation, with 200,000 3rd-stage Ostertagia circumcincta larvae and 4 others were similarly inoculated with 15,000 3rd-stage Haemonchus contortus larvae. In worm-free lambs, the unique effect was an increase in Na+ concentration of the abomasal contents. The O circumcincta or H contortus-inoculated lambs had an increase in PD, pH, and Na+ concentrations. Treatment further increased these concentrations, but the effects on PD occurred and ceased earlier in sheep inoculated with O circumcincta than in those inoculated with H contortus. The results indicated that part of the changes were linked to substances produced by the parasites or by the host. The increase in PD and pH was associated with a decrease in K+ concentration and indicated that these substances stimulated HCO3- transfer.     

Effect of infection with Teladorsagia circumcincta on milk production and composition in Assaf dairy sheep

Infection by gastrointestinal nematodes (GIN) in grazing sheep can negatively influence the growth of lambs and milk yield. In the current study we evaluated the effect of high and low levels of infection with Teladorsagia circumcincta on production parameters of lactating ewes. Two groups of ewes were infected with 50,000 T. circumcincta third stage larvae (L3) 30 days before lambing. To obtain a high level of infection, Group HI was challenged with 30,000 L3 30 days post-partum (pp) and again 90 days pp with the same dose. The low infection group (LI) was treated with an intraruminal controlled-release albendazole bolus one-month pp. We determined the effect of the differing parasite loads on milk yield, milk composition, weight gain, and body condition (BC). Throughout the trial the milk yield was higher in the LI group and, consequently, at the end of the lactation, this group produced 11.1% more milk than the HI group. In the final third of lactation there was an overall increase in milk fat, protein, and lactose although the factor most affected by infection level was the protein percentage, with an increment of 11.9% in the HI group. The somatic cell counts remained low during the experiment indicating the absence of udder infection. Regarding weight and BC, significant differences were found only in BC although LI ewes weighed 2% more than the HI group. In conclusion, a low level of GIN infection in sheep leads to a better BC and a higher milk production with high protein percentage.     

Duration of activity of topical eprinomectin against experimental infections with Teladorsagia circumcincta and Trichostrongylus colubriformis in goats

The immediate as well as the persistent anthelmintic efficacies of topically applied eprinomectin were evaluated in goats against induced infections with Teladorsagia circumcincta (2800 L3) and Trichostrongylus colubriformis (6000 L3). Twenty-three culled dairy goats were allocated to the following groups: control animals (group 1), animals treated 21 days prior to nematode infection (group 2), animals treated 7 days prior to nematode infection (group 3) and animals treated 21 days after nematode infection (group 4). Eprinomectin was applied at twice the cattle dose rate (1.0 mg/kg BW). According to the groups, necropsies were undertaken 28 days after nematode infection (groups 1-3) or 14 days after the anthelmintic treatment (group 4). Worm counts were determined for abomasum and small intestine. The curative anthelmintic efficacy of eprinomectin at 1.0 mg/kg BW on existing worm burdens was 100% against T. circumcincta and T. colubriformis. Quite similar worm burdens reductions were observed when eprinomectin was administered 7 days before infection whereas they were only 52.4 and 17.8% for T. circumcincta and T. colubriformis, respectively, for an administration of the drug 21 days prior to the nematode infection.     

Some observations on immunologically mediated inhibited Teladorsagia circumcincta and their subsequent resumption of development in sheep

Two similar experiments were conducted with groups of yearling sheep which had been trickle infected with Teladorsagia circumcincta for 6 weeks, treated with anthelmintic then challenged with a single dose of 50,000 larvae. Ten days after challenge these sheep contained significantly fewer worms and a significantly higher proportion of arrested early fourth stage larvae than controls which had not received the trickle infection. However, by 19 or 23 days after challenge most of the arrested worms had resumed development, a process which was accelerated in a group treated with corticosteroids. It was concluded that under these conditions arrested development of Teladorsagia was a short-lived, immunologically mediated phenomenon which could be reversed by immuno-suppressing the sheep.     

Effects of protein supply and reproductive status on local and systemic immune responses to Teladorsagia circumcincta in sheep

The effects of protein supply and reproductive status on circulating antibody responses and local inflammatory cell counts were investigated in parasitized sheep, with local immune responses assessed through a recently refined abomasal cannulation methodology. We hypothesized that if breakdown of immunity has a nutritional basis, then protein scarcity would result in a breakdown of immunity to Teladorsagia circumcincta in both periparturient and non-reproducing (barren) ewes. Twin-bearing and barren, abomasally cannulated ewes were fed at either 0.8 or 1.3 times protein requirements from 3 weeks before until 6 weeks after parturition (n = 6). All sheep were trickle infected at a rate of 10,000 infective larvae (L3) per day, for 3 days per week throughout the experiment. Faecal egg counts remained virtually zero in all barren ewes, whilst protein supplementation reduced faecal egg counts in the periparturient ewes during most of the periparturient period. Final worm burdens, taken at 6 weeks into lactation, were lower for the barren ewes than for the lactating ewes, whilst protein supplementation reduced worm burdens in the latter. Protein supply did not affect mucosal mast cell counts, which were consistently higher for the barren ewes than the periparturient ewes, but were temporarily decreased around parturition. Barren ewes and protein supplemented lactating ewes had higher globule leukocyte counts than the unsupplemented lactating ewes. Protein supplementation increased eosinophil counts in the lactating ewes though only during the later part of the lactation period. Plasma IgA anti-L3 antibody was similar for all ewes, but IgE anti-L3 antibody was higher for the protein supplemented periparturient ewes compared to the unsupplemented periparturient ewes and all barren ewes. It is likely that the combination of low protein requirements and large body protein reserves did not result in breakdown of immunity to T. circumcincta for the barren ewes. These results suggest that changes in mucosal mast cell and eosinophil counts are not necessarily associated with changes in host resistance to T. circumcincta. However, the data support the view that increased globule leukocyte counts and plasma IgE anti-L3 antibody may be associated with nutritionally improved expression of immunity in periparturient ewes.