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.     

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.     

The immunology and genetics of resistance of sheep to Teladorsagia circumcincta

Teladorsagia circumcincta is one of the most economically important gastrointestinal nematode parasites of sheep in cool temperate regions, to which sheep show genetically-varying resistance to infection. This is a very common parasite and viable sheep production requires the extensive use of anthelmintic drugs. However, the emergence of drug-resistant parasites has stimulated the search for alternative control strategies to curb production losses. Lambs become infected soon after weaning and begin to control parasite burden within 8–10 weeks of continual infection. This control is an acquired characteristic mediated by the development of parasite-specific antibodies. This paper describes the immunology associated with resistance and susceptibility, focussing on differential T cell activation that regulates the production of specific effector mechanisms. It continues by summarizing the methods used to identify genes that could be exploited as molecular markers of selection for resistance. In particular it focusses on the link between understanding the molecular immunology of infection and the identification of candidate genes for selection.     

Cloning and structural analysis of partial acetylcholine receptor subunit genes from the parasitic nematode Teladorsagia circumcincta

Nematode nicotinic acetylcholine receptors (nAChRs) are the sites of action for the anthelmintic drug levamisole. Recent findings indicate that the molecular mechanism of levamisole resistance may involve changes in the number and/or functions of target nAChRs. Accordingly, we have used an RT-PCR approach to isolate and characterise partial cDNA clones (tca-1 and tca-2) encoding putative nAChR subunits from the economically important trichostrongyloid, Teladorsagia circumcincta. The predicted tca-1 gene product is a 248 aa fragment (TCA-1) which contains structural motifs typical of ligand-binding (alpha-) subunits, and which shows very high sequence similarities (98.8 and 97.2% amino acid identities) to the alpha-subunits encoded by tar-1 and hca-1 from Trichostrongylus colubriformis and Haemonchus contortus, respectively. Sequence analyses of partial tca-1 cDNAs from one levamisole-resistant and two susceptible populations of T. circumcincta revealed polymorphism at the predicted amino acid level, but there was no apparent association of any particular tca-1 allele with resistance. tca-2 encodes a 67 aa fragment (TCA-2) containing the TM4 transmembrane domain and carboxyl terminus of a putative nAChR structural (non-alpha) subunit. The deduced amino acid sequence of TCA-2 shows highest similarity (75% amino acid identity) to ACR-2, a structural subunit involved in forming levamisole-gated ion channels in Caenorhabditis elegans, but low similarity (43% identity) to the corresponding regions of TAR-1 and HCA-1. tca-2 is the first nAChR subunit gene of this type to be isolated from parasitic nematodes, and it provides a basis for further characterisation of structural subunits in trichostrongyloids.     

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.     

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.     

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.     

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.     

Characterisation of two triple resistant field isolates of Teladorsagia from Scottish lowland sheep farms

The anthelmintic resistance status of two field isolates derived from farms (farm A and B) located near Edinburgh were examined using both controlled efficacy tests (CET) and faecal egg count reduction tests (FECRT). Efficacies against fenbendazole (FBZ), levamisole (LEV) and ivermectin (IVM) and, for one isolate, against combinations of these anthelmintics and moxidectin were determined in na?ve lambs, artificially infected with the isolates and treated with the compounds at the manufacturers recommended dose rates. (FBZ, 5mg/kg bodyweight (BW); LEV, 7.5mg/kg BW; IVM, 0.2mg/kg BW; Moxidectin (MOX) 0.2mg/kg BW). In both field isolates, the predominant species found pre-treatment and the only species found post-treatment was Teladorsagia circumcincta. Resistance to FBZ, LEV and IVM was confirmed in CET and FECRT on farm A and to the latter two compounds on farm B, which had a history of benzimidazole resistance and where TBZ resistance was also demonstrated using an egg hatch assay (EHA). For the farm A isolate CET efficacies against FBZ; IVM; LEV; FBZ + IVM; FBZ + LEV; FBZ, LEV + IVM and MOX were 59, 60, 88, 94,93, 92 and 98%, respectively. The CET efficacies for the farm B isolate were 51% and 72% for LEV and IVM, respectively.     

Studies using Teladorsagia circumcincta in an in vitro direct challenge method using abomasal tissue explants

An in vitro direct challenge (IVDC) method in which abomasal tissue explants maintained at 37 degrees C in Hanks/Hepes solution (pH 7.6) in a high oxygen concentration (incubator gassed with pure oxygen and sealed prior to use) were-challenged with exsheathed third stage larvae of Teladorsagia circumcincta has been used to investigate the tissue association phase of larval establishment. Studies using na?ve and recently challenged immune sheep and goat abomasal tissues have shown significant reductions (P < 0.05) in the numbers of tissue associated larvae in material from immune animals. The mechanisms that affect the tissue association process appear to be labile since more larvae were recovered from the tissue digests of previously infected sheep that had not been recently exposed to larval challenge in comparison to those that had been recently challenged (P < 0.05). The method has also been used to demonstrate the influence of protein nutrition on the establishment of larvae in abomasal tissues and region specific differences in the efficacy of exclusion mechanisms. The technique appears to have potential as a means of investigating the crucial first phase of the process of establishment.     

Failure of acid secretion during infection of the ovine abomasum by the nematode parasite Teladorsagia circumcincta

Elevation of the abomasal pH of ruminants is a well known consequence of nematode parasitism. Many have argued that the nematodes seek to increase pH to promote their survival. Indeed, there is limited evidence that substances released by the parasites are capable of directly influencing parietal cell activity. In one experimental series, excretions/secretions (ES) of Teladorsagia circumcincta reduced the apparent activity of rabbit gastric glands. Worm ES were shown to contain ammonia, probably accounting for the observed activity, and the effects of ES/ammonia are almost certainly an in-vitro phenomenon only. An alternative hypothesis contends that the loss of acid secretion arises as a consequence of the profound host response elicited by the presence of the parasites. Invariably, presence of parasites is accompanied by an inflammatory response in which eosinophils are conspicuous. Recent research has shown that exudates from inflamed tissues were also inhibitory to gastric gland activity and that during infection, expression of the pro-inflammatory cytokine interleukin 1-beta, the most potent known inhibitor of acid secretion by parietal cells, was increased. Further, the elevation of abomasal pH may prove to have adverse effects on the ability of the parasites to maintain their position within the abomasum.     

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.     

Thermotolerance of L3 Ostertagia (Teladorsagia) circumcincta and some of its enzymes

Parasitic nematodes of ruminants can be expected to experience temperatures in excess of 40 degrees C in faeces on pasture and, perhaps, in the host. L3 Ostertagia (Teladorsagia) circumcincta survived for at least 90 min at 45 degrees C in vitro in water, but the larvae were inactivated rapidly by only slightly higher temperatures. The glycolytic enzymes hexokinase and pyruvate kinase were inactivated in a similar temperature range, whereas malate dehydrogenase maintained its activity at temperatures in excess of 50 degrees C. These data imply that the loss of glycolytic activity might explain the loss of larval motility at temperatures between 45 degrees C and 50 degrees C.     

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.