Carbon dioxide is an absolute requirement for exsheathment of some, but not all, abomasal nematode species.

The first step in the infection process of grazing ruminants by gastrointestinal nematodes is the exsheathment of the third-stage larva (L3). Exsheathment of various species can be achieved in vitro using carbon dioxide (CO2) under the appropriate temperature and pH conditions. However, it remains unclear whether elevated CO2 levels are an absolute requirement for exsheathment. Exsheathment of four abomasal species was investigated in both the presence and absence of CO2, in either rumen fluid (cow or sheep) or buffer (standard or enriched). Exsheathment of Ostertagia ostertagi, Teladorsagia circumcincta and Ostertagia leptospicularis was observed in CO2-depleted rumen fluid and enriched buffer (respectively 46%, 22% and 15% in rumen fluid and 28% 18% and 26% in enriched buffer after 24 h). The level of this response was dependent on the species as well as the medium, and exsheathment was significantly higher in the presence of CO2. For Haemonchus contortus, exsheathment could only be achieved under CO2-saturated conditions. In conclusion, even though these parasite species exsheath in the same environment, there were significant differences in the minimal requirements to trigger their exsheathment. Some abomasal species were capable of exsheathment in the absence of CO2, which is likely facilitated by cofactors present in the rumen fluid and/or enriched buffer.

Anthelmintic resistance in goat herds-In vivo versus in vitro detection methods.

Anthelmintic resistance (AR) is a serious threat to animal health and has a major economic impact worldwide due to production and financial losses. The aim of this study was to determine the occurrence of AR on 30 goat farms in Slovakia during the pasturing seasons and to compare three widely used in vitro and in vivo methods for detecting AR in field conditions. A three-year survey was conducted during the pasturing seasons of 2014-2016. Goats on each farm were split into treated and control groups and were treated by recommended (5 mg/kg body weight) and double doses (10 mg/kg b.w.) of albendazole. Comparisons between percent reduction in a faecal egg count reduction test (FECRT) and an egg hatch test (EHT) and the presence of L3 larvae in a larval development test (LDT) using resistant concentrations of benzimidazole (BZ) were monitored after treatment. The FECRT indicated percent reductions of 69.2-86.2% for the single dose and of 36.3-45.4% for the double dose. The EHT indicated that all farms had BZ-resistant nematodes. Low (<15% hatching) and high (>15% hatching) levels of resistance were detected on 13 and 17 farms, respectively. The LDT failed to detect resistant larvae on seven farms but detected low and high levels of resistance on seven and 14 farms, respectively. The data indicate a moderate correlation between in vitro and in vivo tests for detecting BZ resistance among the 30 goat farms. The hatching detected by the EHT and the presence of L3 larvae by the LDT at resistant BZ concentrations provided reasonable identification of low levels of resistance in the parasite populations, but the use of a double dose for a treatment may underestimate the real occurrence of low levels of resistant parasites on goat farms.

A stochastic model to investigate the effects of control strategies on calves exposed to Ostertagia ostertagi.

Predicting the effectiveness of parasite control strategies requires accounting for the responses of individual hosts and the epidemiology of parasite supra- and infra-populations. The first objective was to develop a stochastic model that predicted the parasitological interactions within a group of first season grazing calves challenged by Ostertagia ostertagi, by considering phenotypic variation amongst the calves and variation in parasite infra-population. Model behaviour was assessed using variations in parasite supra-population and calf stocking rate. The model showed the initial pasture infection level to have little impact on parasitological output traits, such as worm burdens and FEC, or overall performance of calves, whereas increasing stocking rate had a disproportionately large effect on both parasitological and performance traits. Model predictions were compared with published data taken from experiments on common control strategies, such as reducing stocking rates, the 'dose and move' strategy and strategic treatment with anthelmintic at specific times. Model predictions showed in most cases reasonable agreement with observations, supporting model robustness. The stochastic model developed is flexible, with the potential to predict the consequences of other nematode control strategies, such as targeted selective treatments on groups of grazing calves.

Anthelmintic effects of forage chicory (Cichorium intybus) against gastrointestinal nematode parasites in experimentally infected cattle.

Two experiments studied the effects of dietary chicory against gastrointestinal nematodes in cattle. In Experiment (Exp.) 1, stabled calves were fed chicory silage (CHI1; n = 9) or ryegrass/clover hay (CTL1; n = 6) with balanced protein/energy intakes between groups. After 16 days, all calves received 10 000 Ostertagia ostertagi and 66 000 Cooperia oncophora third-stage larvae (L3) [day (D) 0 post-infection (p.i.)]. In Exp. 2, calves were assigned to pure chicory (CHI2; n=10) or ryegrass/clover (CTL2; n = 10) pastures. After 7 days, animals received 20 000 O. ostertagi L3/calf (D0 p.i.) and were moved regularly preventing pasture-borne infections. Due to poor regrowth of the chicory pasture, CHI2 was supplemented with chicory silage. At D40 p.i. (Exp. 1) and D35 p.i. (Exp. 2) calves were slaughtered for worm recovery. In Exp.1, fecal egg counts (FEC) were similar between groups. However, O. ostertagi counts were significantly reduced in CHI1 by 60% (geometric mean; P < 0·01), whereas C. oncophora burdens were unaffected (P = 0·12). In Exp. 2, FEC were markedly lowered in CHI2 from D22 p.i onwards (P < 0·01). Ostertagia ostertagi adult burdens were significantly reduced in CHI2 by 66% (P < 0·001). Sesquiterpene lactones were identified only in chicory (fresh/silage). Chicory shows promise as an anti-Ostertagia feed for cattle and further studies should investigate its on-farm use.

The parasitic phase of Ostertagia ostertagi: quantification of the main life history traits through systematic review and meta-analysis.

Predictive models of parasite life cycles increase our understanding of how parasite epidemiology is influenced by global changes and can be used to support decisions for more targeted worm control. Estimates of parasite population dynamics are needed to parameterize such models. The aim of this study was to quantify the main life history traits of Ostertagia ostertagi, economically the most important nematode of cattle in temperate regions. The main parameters determining parasite density during the parasitic phase of O. ostertagi are (i) the larval establishment rate, (ii) hypobiosis rate, (iii) adult mortality and (iv) female fecundity (number of eggs laid per day per female). A systematic review was performed covering studies from 1962 to 2007, in which helminth-naïve calves were artificially infected with O. ostertagi. The database was further extended with results of unpublished trials conducted at the Laboratory for Parasitology of Ghent University, Belgium. Overall inverse variance weighted estimates were computed for each of the traits through random effects models. An average establishment rate (±S.E.) of 0.269±0.022 was calculated based on data of 27 studies (46 experiments). The establishment rate declined when infection dose increased and was lower in younger animals. An average proportion of larvae entering hypobiosis (±S.E.) of 0.041 (±0.009) was calculated based on 27 studies (54 experiments). The proportion of ingested larvae that went into hypobiosis was higher in animals that received concomitant infections with nematode species other than O. ostertagi (mixed infections). An average daily adult mortality (±S.E.) of 0.028 (±0.002) was computed based on data from 28 studies (70 experiments). Adult mortality was positively correlated with infection dose. A daily fecundity (±S.E.) of 284 (±45) eggs per female was found based on nine studies (10 experiments). The average female sex ratio of O. ostertagi based on individual animal data (n=75) from six different studies was estimated to be 0.55. We believe that this systematic review is the first to summarise the available data on the main life history traits of the parasitic phase of O. ostertagi. In conclusion, this meta-analysis provides novel estimates for the parameterization of life cycle-based transmission models, explicitly reports measures of variance around these estimates, gives evidence for density dependence of larval establishment and adult mortality, shows that host age affects larval establishment and, to our knowledge, provides the first evidence for O. ostertagi of a female-biased sex ratio.

Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection.

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm's Excretory/secretory antigens.

Transcriptome analyses reveal protein and domain families that delineate stage-related development in the economically important parasitic nematodes, Ostertagia ostertagi and Cooperia oncophora.

BACKGROUND: Cooperia oncophora and Ostertagia ostertagi are among the most important gastrointestinal nematodes of cattle worldwide. The economic losses caused by these parasites are on the order of hundreds of millions of dollars per year. Conventional treatment of these parasites is through anthelmintic drugs; however, as resistance to anthelmintics increases, overall effectiveness has begun decreasing. New methods of control and alternative drug targets are necessary. In-depth analysis of transcriptomic data can help provide these targets. RESULTS: The assembly of 8.7 million and 11 million sequences from C. oncophora and O. ostertagi, respectively, resulted in 29,900 and 34,792 transcripts. Among these, 69% and 73% of the predicted peptides encoded by C. oncophora and O. ostertagi had homologues in other nematodes. Approximately 21% and 24% were constitutively expressed in both species, respectively; however, the numbers of transcripts that were stage specific were much smaller (~1% of the transcripts expressed in a stage). Approximately 21% of the transcripts in C. oncophora and 22% in O. ostertagi were up-regulated in a particular stage. Functional molecular signatures were detected for 46% and 35% of the transcripts in C. oncophora and O. ostertagi, respectively. More in-depth examinations of the most prevalent domains led to knowledge of gene expression changes between the free-living (egg, L1, L2 and L3 sheathed) and parasitic (L3 exsheathed, L4, and adult) stages. Domains previously implicated in growth and development such as chromo domains and the MADF domain tended to dominate in the free-living stages. In contrast, domains potentially involved in feeding such as the zinc finger and CAP domains dominated in the parasitic stages. Pathway analyses showed significant associations between life-cycle stages and peptides involved in energy metabolism in O. ostertagi whereas metabolism of cofactors and vitamins were specifically up-regulated in the parasitic stages of C. oncophora. Substantial differences were observed also between Gene Ontology terms associated with free-living and parasitic stages. CONCLUSIONS: This study characterized transcriptomes from multiple life stages from both C. oncophora and O. ostertagi. These data represent an important resource for studying these parasites. The results of this study show distinct differences in the genes involved in the free-living and parasitic life cycle stages. The data produced will enable better annotation of the upcoming genome sequences and will allow future comparative analyses of the biology, evolution and adaptation to parasitism in nematodes.

Relationship between susceptibility of Blackface sheep to Teladorsagia circumcincta infection and an inflammatory mucosal T cell response.

Teladorsagia circumcincta is the most economically important gastrointestinal (abomasal) nematode parasite of sheep in cool temperate regions, to which sheep show genetically-varying resistance to infection. Lambs, from parents with genetic variation for resistance, were trickle infected with L3 larvae over 12 weeks. 45 lambs were identified with a range of susceptibilities as assessed by: adult worm count at post mortem, faecal egg count (FEC) and IgA antibody levels. This project investigated the correlation of T cell cytokine expression and resistance to infection at the mature stage of response, when the resistant lambs had excluded all parasites.Histopathology showed only minor changes in resistant animals with a low level lymphocyte infiltration; but in susceptible lambs, major pathological changes were associated with extensive infiltration of lymphocytes, eosinophils and neutrophils.Absolute quantitative RT-qPCR assays on the abomasal lymph node (ALN) revealed a significant positive correlation between IL6, IL21 and IL23A transcript levels with adult worm count and FEC. IL23A was also negatively correlated with IgA antibody levels. Significantly positive correlation of TGFB1 levels with adult worm count and FEC were also seen in the abomasal mucosa. These data are consistent with the hypothesis that the inability to control L3 larval colonization, adult worm infection and egg production is due to the activation of the inflammatory Th17 T cell subset.

The comparative morphology of Marshallagia marshalli and Ostertagia occidentalis (Nematoda: Strongylida, Trichostrongylidae) by scanning electron microscopy.

The systematics of the Ostertagiinae is unsettled with no agreement on how many genera and species are present in cattle and sheep. Ten species of Ostertagiinae are commonly parasitic in cattle and sheep. In the global fauna, six of 13 ostertagiine genera are endemic to Iran. The life cycle of Ostertaginae is direct and ingested third-stage larvae after exsheatment in the rumen, penetrate the gastric glands in the abomasal mucosa where two parasitic moults occur before the L5 emerges from the gland. In the present work, Marshallagia marshalli and Ostertagia occidentalis, collected from the abomasums of sheep from Mashhad, Iran, is described. The association of light and scanning electron microscopy (SEM) allowed a detailed analysis of the morphology and ultrastructure of these nematodes. The male body length of M. marshalli and O. occidentalis were 9.3-10.20 and 9.60-10.50 mm, respectively. The female body length of M. marshalli and O. occidentalis were 10.10-15.30 and 10.4-15.70 mm, respectively. One of cervical papillae is seen 333 and 250 µm from the anterior end of male and female body surface in O. occidentalis and 287.5 and 200 µm from the anterior end of male and female body surface in M. marshalli, respectively. The size of cervical papillae is 13.3 µm in male and 10 µm in female in O. occidentalis and 9.33 µm in male and 8.57 µm in female in M. marshalli. Some other taxonomic features of M. marshalli and O. occidentalis, such as details of cephalic region, the system of longitudinal and surface cuticular ridges (synlophe), the orientation of rays of the copulatory bursa, localization of vulva, morphology of vulvar flap, and posterior end of females are also documented by SEM.

A calcium-activated nucleotidase secreted from Ostertagia ostertagi 4th-stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods.

Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

Anthelmintic resistance in cattle nematodes on 13 south-west Victorian properties.

OBJECTIVE: To report the level of anthelmintic resistance on 13 commercial cattle properties in south-west Victoria, Australia. PROCEDURE: Between 2006 and 2009 worm egg count reduction tests were conducted on calves on the 13 properties. Samples were collected 10-14 days post anthelmintic treatment and worm egg counts and larval differentiation tests were conducted. Resistance was defined if there was less than 95% reduction (lower confidence limit <90%) in the faecal worm egg count for the particular genus. RESULTS: The percentage of properties with anthelmintic resistance in at least one species was 54% for benzimidazole (BZ), 100% for levamisole (LEV) and for ivermectin (IVM) it was 100% for the half-dose (0.1 mg/kg) and 62% for the full dose (0.2 mg/kg). A substantial frequency of resistance was detected in Ostertagia ostertagi to BZ (5/11), LEV (3/3) and IVM (5/11), in Trichostrongylus spp. to BZ (4/7) and in Cooperia spp. to IVM (6/11). No resistance to LEV was detected in Trichostrongylus or Cooperia spp. Suspected IVM-resistant Trichostrongylus spp. and BZ-resistant Cooperia spp. were only detected on one property each. CONCLUSION: This is the first Australian report of macrocyclic lactone-resistant O. ostertagi in the refereed literature. The frequency of resistance in O. ostertagi to BZ, LEV and IVM and in Trichostrongylus spp. to BZ in the present study appears higher than levels detected in the 2004-05 New Zealand survey, whereas the resistance frequency in Cooperia spp. to IVM and BZ was less.

Analysis of the translationally controlled tumour protein in the nematodes Ostertagia ostertagi and Caenorhabditis elegans suggests a pivotal role in egg production.

The translationally controlled tumour protein (TCTP) is a conserved protein which has been described for a wide range of eukaryotic organisms including protozoa, yeasts, plants, nematodes and mammals. Several parasitic organisms have been shown to actively secrete TCTP during host infection as part of their immuno-evasive strategy. In this study, we have studied TCTP in Ostertagia ostertagi, a parasitic nematode of cattle, and in the free-living nematode Caenorhabditis elegans. An analysis of the transcription and expression patterns showed that TCTP was present in the eggs of both species. This localisation is consistent for some other Strongylida such as Teladorsagia circumcincta, Cooperia oncophora and Haemonchus contortus. TCTP was also detected at low levels in excretory-secretory material from adult O. ostertagi worms. The role of TCTP in nematode biology was also investigated by RNA interference in C. elegans. Knock-down of C. elegans tctp (tct-1) transcription reduced the numbers of eggs laid by the hermaphrodite in the F(0) and F(1) generations by 90% and 72%, respectively, indicating a pivotal role of TCTP in reproduction.

Identification and characterization of a novel specific secreted protein family for selected members of the subfamily Ostertagiinae (Nematoda).

It has been shown that the bovine abomasal parasite, Ostertagia ostertagi, drastically modulates its microenvironment, causing epithelial cell damage, accumulation of inflammatory cells and pH changes in the stomach. The mechanisms used by the parasite to change the abomasal environment are largely unknown, but an important role has been attributed to excretory-secretory (ES) products from the parasite. In this study we have identified proteins representing a novel ES protein family, characterized by the SCP/Tpx-1/Ag5/PR-1/Sc7 protein motif. These proteins were named Oo-AL1 and Oo-AL2 (O. ostertagi ASP-like protein). Both proteins contain a signal peptide and 1 predicted N-glycosylation site. The transcript for Oo-AL1 was present from the L4 stage onwards in both male and female adult worms, whereas the Oo-AL2 transcript was hardly detectable. Western blots of somatic extracts and ES products from different developmental stages of O. ostertagi, probed with anti-Oo-AL1 antibodies, revealed Oo-AL proteins in the ES products of adult worms. An analysis of the nematode genome and EST databases indicated that these novel ES proteins are unique to O. ostertagi and its relative, Teladorsagia circumcincta, suggesting a key function in these abomasal parasites.

Associations between anti-Fasciola hepatica antibody levels in bulk-tank milk samples and production parameters in dairy herds.

Our primary objective was to determine the relationships between Fasciola-specific antibody levels in bulk-tank milk and measures of productivity to estimate economic losses that are associated with Fasciola infections. A bulk-tank milk sample was collected in March 2004 from 1105 dairy herds in Flanders and the antibody levels against Fasciola hepatica (ODRf) and Ostertagia ostertagi (ODRo) were determined. The association of ODRf with four production parameters (milk yield, milk-protein %, milk-fat % and inter-calving interval) was assessed by multivariable linear-regression models. Production data were available for 463 out of the 1105 herds sampled. An increase in ODRf from the 25% quantile (0.428) to the 75% quantile (1.064) was associated with a decrease in the annual average milk yield of 0.7kg/(cowday) (P=0.002), with a decrease in the average milk-fat % of 0.06% (P<0.001) and with an increase of the mean inter-calving interval of 4.7 days (P=0.03). No significant relationship was found with the average milk-protein %. When the relationships of ODRf and ODRo with milk yield were tested simultaneously, we saw an additive rather than synergistic effect of concurrent infections.

Biological control of gastrointestinal parasitic nematodes using Duddingtonia flagrans in sheep under natural conditions in Mexico.

This investigation was aimed to evaluate the use of an oral bio-preparation containing Duddingtonia flagrans chlamydospores for the control of sheep gastrointestinal parasitic nematodes under the Mexican cold high plateau conditions. Two groups of gastrointestinal parasitic nematode naturally infected sheep, were randomly selected and located into two free-gastrointestinal nematode larvae paddocks. Group 1 received once a week a supplement containing D. flagrans chlamydospores mixed with oats and molasses. Group 2 received a similar supplement without any fungal material. After 5 months grazing animals were discarded from the experiment and two groups of free-nematode "tracer" sheep were located into the same paddocks to collect larvae from the contaminated pastures. Animals were slaughtered and necropsied and the nematodes were obtained and counted. A screening of the number of gastrointestinal nematode larvae present on the grass was performed and compared between the two grazing areas. The results showed 56% reduction in the Ostertagia (Teladorsagia) circumcincta and 94% reduction in the Nematodirus sp. population of the "tracer" sheep who grazed on the D. flagrans-treated sheep area, compared to the nematode population in animals grazed on the non-treated area. The results of the number of larvae on the grazing pastures showed a 51.1% reduction for H. contortus, and 100% for Cooperia sp. in the area with fungi. In the case of Trichostrongylus sp. no reduction was observed, when compared to the control group.

Efficacy and specificity of RNA interference in larval life-stages of Ostertagia ostertagi.

RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, beta-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.

Population dynamics of nematode parasites of reindeer in the sub-arctic.

Nematode parasite infections of semi-domestic reindeer grazing in their natural habitat in northern Finland were monitored for approximately 2 years. This was achieved by monthly faecal egg counts of male and female calves and adult females from an experimental reindeer herd, in addition to estimating the acquisition of nematode infection from pasture using tracer reindeer calves. The most abundant parasite was Ostertagia gruehneri in the worm counts of tracer animals and in faecal egg counts of adult female reindeer. Capillaria sp. eggs were detected in calves and adults, but Nematodirinae eggs were only recovered from calves. Faecal egg counts showed variations between months for each nematode species, with male and female calves shedding similar numbers of eggs. During each year, calves shed more Capillaria sp. eggs than adult female reindeer, but similar numbers of O. gruehneri eggs. Egg counts of O. gruehneri were more abundant in late summer-autumn (July-September), whereas Capillaria sp. and the Nematodirinae dominated the winter months (November-February). The seasonal trends of adult worm burdens of O. gruehneri in the tracers paralleled the egg count patterns. Capillaria sp. was not detected in tracer worm counts. Tracer worm burdens showed that the proportion of inhibited larvae of O. gruehneri and Nematodirinae steadily increased from spring to early winter, followed by a decline and a commensurate increase in the number of adult parasites in the second summer. This investigation showed that parasite transmission occurs continuously throughout the year for nematode parasites of reindeer in northern Finland.

In vivo anthelmintic activity of Dorycnium rectum and grape seed extract against Ostertagia (Teladorsagia) circumcincta and Trichostrongylus colubriformis in sheep.

AIM: To assess the in vivo anthelmintic activity of condensed tannins (CT) in the forage species Dorycnium rectum and Medicago sativa, and in an extract from grape (Vitus vinifera) seeds (GSE), against two species of parasite, Teladorsagia (Ostertagia) circumcincta and Trichostrongylus colubriformis, at different stages of their life cycle, in sheep that were parasite-naïve or previously exposed to nematodes. METHODS: In Trial 1, a factorial treatment structure was used to compare faecal nematode egg counts (FEC) and worm burdens in 40 weaned Romney lambs fed either the CT-containing forage D. rectum (12% dry matter; DM) or M. sativa (lucerne; 0.2% DM). Twenty naïve and 20 previously-exposed lambs were drenched free of parasites then reinfected with known species and numbers of parasites, and housed in pens indoors on a diet of lucerne pellets and chaffed hay. Groups of lambs (n=5 lambs per group) were fed one of the forages over one of two time periods within the parasite's life cycle. Six to nine days after the last feeding of fresh forages, faecal samples were collected for FEC, and all lambs were slaughtered and worm counts conducted. In Trial 2, 12 Suffolk x Romney lambs were surgically implanted with an abomasal cannula and then housed indoors in metabolism crates. After infection with parasites, six lambs were infused continuously over a 14-day period with a commercially available CT GSE (96% DM, made up to 34 g/L in water); the remaining lambs were infused with water. During infusion, samples were collected for egg hatch and larval development assays. After infusion, samples were collected for FEC, and all lambs were slaughtered and worm counts conducted. RESULTS: In Trial 1, there was a significant (p<0.001) difference in burdens of O. circumcincta between naïve lambs and those previously exposed to parasites, but no other differences were recorded. In Trial 2, lambs infused with GSE had significantly (p<0.05) fewer T. colubriformis at slaughter and significantly (p<0.001) fewer eggs hatched in the egg hatch assay (EHA) than for lambs infused with water. Overall, the differences attributable to GSE were small in magnitude, being an 11% drop in egg hatch, and an 18% drop in numbers of adult T. colubriformis after 14 days of continuous infusion. No other differences were recorded. CONCLUSION: The results indicate that the in vivo anthelmintic activity of these CT sources is, at best, modest and is unlikely to be of any practical value. Further, these data emphasise that in vitro activity is an unreliable indicator of in vivo efficacy for CT-containing forages and extracts.

Confirmation of resistance to ivermectin by Cooperia curticei in sheep.

AIM: To investigate the occurrence of resistance to a full dose of oral ivermectin by Cooperia curticei in sheep. METHODS: Twelve lambs on a sheep and cattle property in the North Island of New Zealand were randomly allocated to one of two equal-sized groups. One group was treated orally with a single dose of ivermectin at the manufacturer's recommended dose rate of 0.2 mg/kg, while the other remained as an untreated control. Worm counts were carried out post mortem on the abomasa and small intestines of all animals in both groups 7 days after treatment. RESULTS: While treatment with ivermectin reduced the numbers of all other worm genera to almost zero, those of Ostertagia(= Telodorsagia) circumcincta and C. curticei were reduced by only 37% and 19%, respectively. CONCLUSIONS: These results provide clear evidence of resistance to ivermectin by O. circumcincta and C. curticei. They also appear to represent the first record of macrocylic lactone (ML) resistance in C. curticei in sheep in New Zealand or elsewhere.

Evaluation of the stability of Ostertagia ostertagi ELISA microtitre plates over time using cow milk samples.

The objective of this study was to assess the stability of ELISA plates prepared with one of three blocking agents and used with one of two conjugates at various time intervals after preparation of the plates. Two of the blocking agents used were commercially available: one termed stabilgaurd (stab) and one manufactured by SVANOVA Biotek AB Inc. (svan). The third blocking agent used was bovine serum albumin (bsa). A polyclonal rabbit anti-bovine IgG (poly) and an anti-bovine IgG monoclonal (mono) conjugate were used. Eighteen composite individual cow milk samples collected late in lactation (200-400 days in milk) were used in this study. An indirect microtitre plate ELISA that used the Ostertagia ostertagi antigen was used to quantify antibodies against the parasite, present in the milk samples. Each of six blocking agent/conjugate combinations (called systems) were used to test 18 milk sub-samples at 1, 4 and 24 weeks after blocking the plates. Plates blocked with stab and svan were kept at room temperature and an additional set were incubated at 37 degrees C so as to mimic long term storage (about 1 year) and tested only once at 4 weeks. Those blocked with bsa were frozen at -20 degrees C. Concordance correlation coefficients (CCC) and reproducibility were used to assess the agreement between test results conducted on the same milk sample at the various test-times using a particular system. Generally, there was good agreement between tests conducted at different times for all systems. However, the svan-mono and bsa-poly systems had the best agreement with overall CCC values of 96% and 93%, respectively. The svan-poly system had the lowest CCC of 75%. The CCC and reproducibility ranked the systems in a similar way. The high CCC between tests done using plates kept at room temperature and ones incubated at 37 degrees C, suggested that plates would be stable up to a year after blocking. The storage of plates blocked with svan and stab agents under room temperature, makes them more convenient to use and transport relative to bsa-blocked plates that have to be frozen.