Nematodirus Battus Research Articles

Nematodirus battus 50 years on--a realistic vaccine candidate?

It has been 50 years since the parasitic nematode of lambs, Nematodirus battus, was first described. This parasite has several interesting features; in particular, it induces a rapid, protective immune response in infected young lambs (< 3 months of age), which is not observed if lambs are infected with other trichostrongyle nematodes. Indeed, protection against most gastrointestinal nematodes only develops once lambs are over five to six months old. In this article I suggest that N. battus offers an opportunity to improve our understanding of protective immune responses in young lambs, and could therefore hold a key to rational anti-nematode vaccine developments, based on natural antigens.

Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies

In vitro and in vivo studies were conducted to determine possible direct anthelmintic effects of condensed tannins towards different ovine gastrointestinal nematodes. A larval development/viability assay was used to investigate the effect of a condensed tannin extract (Quebracho) towards larvae of Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus vitrinus. The development to infective larvae and their viability was assessed in all three species and LD 50 values were calculated. The presence of Quebracho extract in the cultures decreased the viability of L 3 in all species; the LD 50 were not significantly different for the different species. Forty-eight sheep were allocated to one of eight groups and were infected with a single dose of either 4000 L 3 H. contortus (groups 1 and 2) or 5000 L 3 T. colubriformis and 5000 L 3 Nematodirus battus simultaneously (groups 3–6) or 10,000 L 3 of T. circumcincta (groups 7 and 8). From day 28 until day 31 of the experiment, sheep infected with the intestinal species were drenched with Quebracho extract at 4, 8 or 16% w/w of food intake, or remained as undrenched controls; sheep infected with the abomasal species were either drenched with Quebracho extract at 8% w/w of food intake or remained as undrenched controls. All sheep were slaughtered 4 days after the end of the drenching period. Sheep infected with the intestinal species and drenched with 16% w/w Quebracho had lower FEC compared to sheep drenched with 8% w/w ( P<0.05), which in turn were lower than in sheep either drenched with 4% Quebracho or which remained undrenched ( P<0.05). The lowest intestinal worm burden was recovered from sheep drenched with 8% w/w Quebracho extract ( P<0.05). The administration of Quebracho extract at 8% of food intake for 3 days did not affect FEC or worm burdens in sheep infected with the abomasal species compared to controls.

Efficacy of doramectin injectable against Oestrus ovis and gastrointestinal nematodes in sheep in the southwestern region of France

A study was conducted to evaluate the therapeutic efficacy of doramectin administered intramuscularly at a dose rate of 200 μg/kg to sheep harbouring naturally acquired infections of gastrointestinal nematodes and Oestrus ovis in the southwestern region of France. On day 0, 24 sheep were selected on the basis of positive faecal egg counts (>100 EPG) and positive assessment of O. ovis infection (including positive O. ovis antibody level and positive clinical score). The sheep were randomly allocated to a non-medicated control group (T1) or a doramectin-treated group (T2) of 12 animals each. On day 0, sheep in group T2 received a single intramuscular injection of doramectin (200 μg/kg), whereas those in group T1 received an intramuscular injection of saline solution (sodium chloride, 0.02 ml/kg). Individual faecal egg counts were performed on days 0, 1, 2, 3, 4, 5, 6, 7, and 14. Between days 14 and 16, all sheep were slaughtered, and worm and O. ovis burdens were determined. In doramectin-treated sheep, faecal egg counts had decreased to zero by day 4 for all recovered types of nematode eggs: strongyles, Nematodirus sp., Trichuris sp., and Rhabditidae sp. For strongyles, Nematodirus sp., and Rhabditidae, the percentage reductions in faecal egg counts (geometric means) of doramectin-treated sheep, compared to the non-medicated control sheep were 100% from days 4–7. For Trichuris sp., they were 100, 99.7, 99.9, and 100% on days 4, 5, 6, and 7, respectively. On day 14, percentage reductions were 100% for Nematodirus sp. and Rhabditidae, and 99.8 and 99.1% for strongyles and Trichuris sp., respectively. At necropsy, only adult nematodes and mainly first-stage O. ovis larvae were recovered. Doramectin was highly efficacious against the adult stages of Teladorsagia circumcincta (100%), Nematodirus battus (100%), Nematodirus filicollis (99.9%), Oesophagostomum venulosum (99.8%), and Trichuris sp. (99.3%). It was also 100% efficacious against first-stage larvae of O. ovis. No abnormal clinical signs or adverse reactions in any of the sheep treated with doramectin were observed.

The Dynamics, Prevalence and Impact of Nematode Infections in Organically Raised Sheep in Sweden

A three-year survey (1997–99) was carried out on organically reared sheep flocks throughout Sweden. The aim was to determine the prevalence and intensity of nematode infections and to establish relationships between sheep management practices and parasite infections. Faecal samples from ewes and lambs were collected from 152 organic flocks around lambing-time and during the grazing-period for analysis. Results were compared with the different management practices that farmers use to prevent parasitism in their flocks. A high proportion of the flocks was infected with nematodes. The most prevalent species were Haemonchus contortus, Teladorsagia circumeincta, Trichostrongylus axei, T. colubriformis and Chabertia ovina and infections progressively increased during summer in lambs grazing on permanent pastures. Severity of parasitic infection in lambs was highly dependent on egg output from the ewes. H. contortus was found in 37% of the flocks, even at latitudes approximating the Polar Circle. Nematodirus battus was recorded for the first time in Sweden during the course of this study. Lambs turned out onto permanent pasture showed higher nematode faecal egg counts (epg) than lambs that had grazed on pastures, which had not carried sheep the previous year. This beneficial effect of lambs grazing non-infected pastures persisted if the ewes were treated with an anthelmintic before turn-out and if the lambs were kept on pastures of low infectivity after weaning. In lambs, the prevalence and the magnitude of their egg counts were higher during autumn in flocks where lambs were slaughtered after 8 months of age, compared with flocks where all lambs were slaughtered before this age. These results will be used in providing advice to farmers of ways to modify their flock management in order to minimise the use of anthelmintics, but at the same time efficiently produce prime lambs.

Vaccination of young lambs against infection with Nematodirus battus using gamma irradiated larvae

Helminthologically naÏve 6-week-old Suffolk lambs were given 1–3 doses of 20 000 γ-irradiated infective larvae (L3) of the nematode Nematodirus battus at weekly intervals. Following an anthelmintic drench they were challenged with 50 000 viable L3 at 10 weeks of age. Nematode worm burdens 14 days post-challenge showed a significant (P<0.01) 66% reduction in the single vaccine dose group. The two and three dose groups had mean worm burdens which were 30 and 42% lower than controls, respectively, although these were not statistically significant. There was little measurable stimulation of the immune system in the vaccinated lambs, suggesting that the repeatedly dosed animals may have developed immunological unresponsiveness to the parasite.

Relationships of Nematodirus species and Nematodirus battus isolates (Nematoda: Trichostrongyloidea) based on nuclear ribosomal DNA sequences.

Nuclear ribosomal sequence data from the internal transcribed spacers (ITS-1 and ITS-2), 5.8S subunit, and regions of the 18S and 28S genes were used to investigate sequence diversity among geographic samples of Nematodirus battus, and to infer phylogenetic relationships among Nematodirus species. Phylogenetic analysis of these data yielded strong support for relationships among species, depicting Nematodirus helvetianus and Nematodirus spathiger as sister-taxa and a clade of these 2 species and Nematodirus filicollis. This tree is consistent with caprine bovids as ancestral hosts, with a subsequent host shift to Bovinae in N. helvetianus. Eleven of 14 N. battus sequences were unique, with 19 variable sites among sequences representing 5 geographic samples. The lowest number of variable nucleotide sites was observed in samples representing apparently recent introductions to the United States and Canada, which is consistent with a population bottleneck concomitant with translocation. Comparison of directly sequenced polymerase chain reaction products and clones revealed evidence for intraindividual variation at some of the sequence sites, and this pattern of variation and that within geographic samples indicates incomplete rDNA repeat homogenization within species. This pattern of variation is not conducive for inferring phylogenetic relationships among sequences representing N. battus or addressing the putative history of introduction.

Efficacy of an ivermectin controlled-release capsule against nematode and arthropod endoparasites in sheep

Five controlled trials were conducted in Germany or in the United Kingdom, using 74 female sheep of merino or Dorset horn breeds, to evaluate the efficacy of an ivermectin controlled-release...

Differences in the second internal transcribed spacer of four species of Nematodirus (Nematoda: Molineidae)

Genetic differences among Nematodirus spathiger, Nematodirus filicollis, Nematodirus helvetianus and Nematodirus battus in the nucleotide sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA ranged from 3.9 to 24.7%. Pairwise comparisons of their ITS-2 sequences indicated that the most genetically similar species were N. spathiger and N. helvetianus. N. battus was the most genetically distinct species, with differences ranging from 22.8 to 24.7% with respect to the other three species. Some of the nucleotide differences among species provided different endonuclease restriction sites that could be used in restriction fragment length polymorphism studies. The ITS-2 sequence data may prove useful in studies of the systematics of molineid nematodes.

Isoelectric Focusing of Soluble Proteins in the Characterization of Species and Isolates of Nematodirus (Nematoda: Trichostrongyloidea)

Isoelectric focusing was performed on extracts from Nematodirus spathiger, Nematodirus filicollis, Nematodirus helvetianus, and 3 geographic isolates of Nematodirus battus. Gender-specific differences were noted within species; however, the overall protein profile of each species and isolate was distinct and reproducible and allowed unequivocal differentiation. A coefficient of similarity (Sm) for males of each species and isolate was calculated, and a dendrogram, based on evaluation of Sm by the unweighted pair-group method with arithmetic means, was produced. Although cluster analysis of the 3 isolates of N. battus indicates the North American and Weybridge isolates are similar, interpretation of the relationships and thus the history of introduction based on these data is equivocal. Isoelectric focusing is a robust method for establishing identity and has great utility in diagnostics. However, in the absence of selective histochemical staining, interpretation of identity and homology for specific bands and banding patterns is problematic, thus limiting the utility of this method for phylogenetic inference.

Persistence of immunity to Nematodirus battus infection in lambs

Fifty-four Greyface Suffolk lambs aged 3 months were allocated to six groups of seven and one group of 12. Three groups were infected continuously with Nematodirus battus larvae (L 3) over a 7-week period and three groups remained worm-free. One week after the last larval dose all six groups were treated with anthelmintic and challenged with a single dose of 30 000 N. battus L 3 either 1, 6 or 12 weeks post-treatment (PT) and killed 10 days later. A seventh continuously infected and treated group ( n = 12) was segregated into four sub-groups of three lambs which were used as tissue cell count controls and provided data on local cellular responses prior to challenge. Lambs in the first sub-group were killed immediately after anthelmintic treatment and those in the other sub-groups were killed on the same day that the lambs in the other main groups were challenged. Overall post-challenge worm burdens did not differ significantly between previously infected and challenge control groups although they were significantly reduced in both treatment groups by Week 12 PT. The principal manifestation of acquired immunity that was maintained throughout 12 weeks without further infection was retardation in larval development There was also evidence of preferential rejection of male worms from immune lambs. Local mast cell, but not eosinophil, responses were significantly enhanced by previous infection and persisted up to Week 12 PT. The numbers of bone marrow eosinophils were significantly increased as a result of previous infection and this response persisted up to Week 12 PT. During primary infection anti-L 4 and anti-adult worm IgG responses were significantly increased in the previously infected lambs by Day 42 post-infection. Eosinophil responses during this period did not differ between groups. The inflammatory cell responses, coupled with the parasitological observations, suggest that immunity to previous infection is maintained for up to 12 weeks PT without further antigenic stimulation. This ‘immunological memory’ may have waned partially after 6 weeks PT although the superimposition of age resistance may have masked the effect.

The effect of dexamethasone on resistance of older lambs to infection with Nematodirus battus

Old lambs (8 months of age) infected with 50,000 L3 of Nematodirus battus had larvae developing normally in their tissues on day 4 post-infection (P.I.), but by day 8 P.I. there were only 4105 +/- 1044 worms left in the alimentary tract. Some of these worms contained crystals in their intestine. Eight-month-old lambs treated with dexamethasone and infected with 50,000 L3 of N. battus contained a mean worm burden of 7878 +/- 1262 on 18 days P.I. Untreated 8-month-old lambs similarly infected were virtually worm free by day 18 P.I. Peripheral eosinophilia became elevated in the untreated lambs over the course of infection and, at post-mortem, the tissue mast cell and eosinophil counts were much higher than in the dexamethasone treated group. Although the phenomenon of age resistance is thought to have a strong immunological component, there may also be other physiological factors, resulting in fewer nematodes and lower fecundity of the worms.

Prevalence and seasonal changes in the gastro-intestinal helminths of Nigerian goats.

A total of 120 gastro-intestinal tracts and 960 faecal samples were examined to assess the prevalence and seasonal changes in the gastro-intestinal helminth parasites of Red Sokoto (maradi) goats slaughtered at Ibadan between May 1991 and April 1992. Egg types of strongyles, Strongyloides, Trichuris, Skrjabinema, Dicrocoelium and Moniezia were encountered in 93%, 83%, 44%, 0.9%, 2.3% and 31% of the faecal samples respectively. However, only strongyle, Strongyloides and Trichuris eggs occurred in large numbers and were more common during the rainy season than in the dry season. The parasites recorded and their prevalences were Haemonchus contortus (90.0%), H. ovis (5.0%), Strongyloides papillosus (80.8%), Trichostrongylus colubriformis (78.3%), T. axei (69.2%), Trichuris ovis (72.5%), T. globulosa (38.3%), Oesophagostomum columbianum (67.5%), Cooperia curticei (58.3%) Gaigeria pachyscelis (40.8%), Skrjabinema ovis (5.0%), Nematodirus battus (5.8%), Moniezia expansa (29.2%), M. benedeni (10.0%), Paramphistomum spp. (5.0%) and Cysticercus tenuicollis (33.3%). Haemonchus ovis is reported for the first time in Nigeria. Mixed infections were most prevalent. Young goats were more commonly infected and had higher worm counts than adult goats. Only Haemonchus, Trichostrongylus, Strongyloides and Cooperia spp. occurred in large numbers. Irrespective of the age of the goats, higher worm counts were generally encountered during the rainy season than in the dry season. The results are discussed in relation to the control of helminthiasis in grazing animals in Nigeria.

The serum immune response of young lambs to a primary infection with Nematodirus battus.

Young lambs (6-8 weeks of age) were infected with 50000 infective stage larvae of the trichostrongyle nematode Nematodirus battus. Initial mean adult worm burdens of 23884 +/- 1678 (S.E.M.) at day 14 post-infection (p.i.) were reduced significantly (P < 0.01) to a mean burden of 5383 +/- 1397 by day 21 p.i. Blood samples were taken and serum analysed using ELISA and Western blotting techniques for IgG antibodies specific for N. battus. Pre-infection serum from young naive animals showed IgG recognition of adult and larval N. battus whole worm homogenate. Recognition of adult antigens became reduced during the first 14 days after infection, followed by a subsequent increase beginning between days 14 and 18 p.i. and peaking at day 28 p.i. IgM recognition of N. battus antigens showed a single response peak, starting between days 14 and 18 p.i., without the pre-infection elevated titre as observed with IgG. These results show that young lambs can mount a significant serum response to a primary infection with N. battus during rejection of adult worms and that lambs receive from their dams IgG antibodies that are capable of recognizing adult and larval N. battus during the first few weeks of life.

Vasoactive intestinal polypeptide-like and peptide histidine isoleucine-like proteins excreted/secreted by Nippostrongylus brasiliensis, Nematodirus battus and Ascaridia galli.

Vasoactive intestinal polypeptide (VIP)-like protein was detected by dot blot analysis in the excretions/secretions (E/S) of Nematodirus battus and Ascaridia galli and was confirmed in the E/S of Nippostrongylus brasiliensis. ELISA analysis showed that N. brasiliensis E/S contained the highest proportion of VIP-like protein (28.04 pmoles/mg of total E/S protein) and A. galli E/S contained the lowest (10.89 pmoles/mg of total E/S protein). Peptide histidine isoleucine (PHI)-like protein was detected by dot blot analysis in the E/S products of N. brasiliensis, N. battus and A. galli. ELISA analysis suggested that A. galli E/S contained the highest proportion of PHI (20.77 nmoles/mg of total E/S protein) and N. battus E/S contained the lowest (0.67 nmoles/mg of total E/S protein). The possible significance of VIP-like and PHI-like substances in the E/S of gastrointestinal nematodes is discussed.

Effect of dietary protein on the regulation of populations of Nematodirus battus by lambs

Two groups of seven and two groups of six lambs were offered either a complete basal ruminant diet (13·2 per cent crude protein [ CP]) (groups 2 and 4) or the same diet supplemented with fish meal (18·3 per cent CP) (groups 1 and 3). Groups 1 and 2 were infected daily for seven weeks with Nematodirus battus larvae (L 3) and groups 3 and 4 served as uninfected challenge controls. All the groups were treated with anthelmintic in the eighth week, challenged with a single dose of 30,000 N battus L 3 one week later and killed nine days after the challenge. Although protein supplementation tended to enhance the regulation of the population of N battus in the lambs which had been infected continuously, the effect was not statistically significant. The worm burdens in both the groups of previously infected lambs were significantly reduced both in number and size (P<0·001) and they had a lower proportion of male worms than the uninfected challenge controls. The lambs could be segregated into high or low responders on the basis of their worm burdens, and there was a significant reduction in worm burdens (P<0·001) and size (P<0·01) in parallel with the lambs' responsiveness. The identification of high and low responders shows that when the dietary protein supply is adequate the predominant effect of the host on the pathogenicity of its parasites is the host's genetically-determined susceptibility.

Dietary protein influences upon immunity to Nematodirus battus infection in lambs

Several indices of the immune response to Nematodirus battus infection in lambs offered differing levels of dietary protein were quantified. Lambs were offered either a complete basal ruminant diet (13.2% crude protein (CP)) or the same diet supplemented with fish meal as a source of rumen bypass protein (18.3% CP). Lambs from each dietary treatment group were given either a 7-week continuous trickle infection with N. battus L 3 or remained uninfected. All lambs were drenched with anthelmintic at week 8 post-infection (PI), challenged with a single dose of 30 000 N. battus L 3 1 week later, and killed 9 days post-challenge (PC). Previous infection induced a significant reduction in worm burdens ( p < 0.001) and enhancement of immune responses when compared to challenge controls. Among previously infected lambs, protein supplementation did not reduce worm burdens significantly, although there was a trend for fewer worms in the supplemented lambs. However, a significant increase in mucosal globule leucocyte ( p < 0.05) and eosinophil ( p < 0.05) numbers was evident. Supplementation ( p < 0.05) and previous infection ( p < 0.001) both enhanced serum anti-worm IgG titres over time. Peripheral blood eosinophil counts were not affected by supplementation but were significantly elevated over time as a result of previous infection ( p < 0.001). Since there were no significant differences in worm burdens of supplemented and unsupplemented previously infected lambs, it was of interest to determine whether lambs possessed an innate ability to regulate their parasite burden. Hence they were re-grouped based on an arbitrary cut-off burden of 1000 worms. Hihh responders (HR) had burdens below 1000 worms, while low responders (LR) had burdens above this value and challenge controls were pooled. The data were re-analyzed based on these groupings and showed significant reduction in worm burdens between all three groups ( p < 0.001). Globule leucocytes were the only cell type that appeared to be significantly more abundant in the intestinal mucosa of HR ( p < 0.001). Serum antibody responses ( p < 0.05) and peripheral blood eosinophil counts ( p < 0.01) were significantly elevated over time in accord with the degree of responsiveness. The results of this study suggest that supplementation of protein upon an adequate basal diet of lambs previously exposed to N. battus does not significantly enhance worm regulation despite significant increases in cellular and antibody responses. The immunity acquired is characterized by reduction in worm burdens, elevated anti-worm antibodies and a cellular inflammatory response. The identification of HR and LR essentially shows that when the protein supply is adequate, the predominant host effect influencing the pathogenicity of the parasites is the level of genetically determined susceptibility of the host.

Comparison of two enzyme immunoassays for the detection of Haemonchus contortus infections in sheep

Two enzyme-linked immunosorbent assays (ELISA) using either excretory/secretory (ES) products or crude somatic antigens (CSA) of adult Haemonchus contortus were compared for their ability to detect antibodies against H. contortus in sheep. Serum samples obtained from a group of 32 H. contortus mono-infected sheep were tested in the two ELISAs and the obtained data were compared with the results of the faecal examinations of these sheep. The first sheep became patent 3 weeks post infection (p.i.) and all sheep had positive egg counts at week 5 p.i. The first antibodies against H. contortus were detected 1 week p.i. and all sheep were found positive by both ELISAs at week 4 p.i. Using sera from a large number of H. contortus-infected sheep a difference in sensitivity between the ES ELISA (97.7%) and the CSA ELISA (89.2%) was found. The specificity of each assay was determined by testing sera obtained from sheep with mono-infections of H. contortus, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Ostertagia circumcincta, Nematodirus battus, Cooperia curticei, Fasciola hepatica, Taenia ovis or Eimeria spp. The specificity of the ES ELISA was 87.2%, whereas the specificity of the CSA ELISA was 82.7%.

Cross antigenicity among ovine trichostrongyloidea. Preliminary report

A preliminary trial on the extent of cross-antigenicity among the sheep strongylids Haemonchus contortus, Trichostrongylus colubriformis, Teladorsagia circumcincta and Nematodirus battus in 2.5- to 4-month-old lambs has been carried out using ELISA and Western blotting (WB). Cross antigenicity was tested using soluble extracts from adult and third stage larvae (L3) of H. contortus as antigenic source probed with sera from lambs with monospecific heterologous infections. There was cross-antigenicity between L3 of H. contortus and Trichostrongylus colubriformis in ELISA and WB. Immunodetection results with adult H. contortus antigen showed a closer relationship to Teladorsagia circumcincta. Certain heterologous sera reacted with H. contortus antigens more strongly than the homologous one, but sera from the H. contortus-infected animals had reactivity around the 25 kDa region from adult antigens which could have potential diagnostic use.

Cloning and Sequence Analysis of the Small Subunit Ribosomal RNA Gene from Nematodirus battus

The small subunit ribosomal RNA gene (SSU rRNA) from the nematode parasite, Nematodirus battus, was cloned from amplified genomic DNA using polymerase chain reaction primers complementary to the 5' and 3' ends of Haemonchus contortus small subunit ribosomal DNA (SSU rDNA). The 1,758 base pair cloned fragment was sequenced in both directions using internally generated primers and aligned with those from the genus Haemonchus. Results indicated a 1.9% sequence difference between the genera with a 14.2% difference between N. battus and Caenorhabditis elegans. Data presented here in conjunction with previously published work comparing rDNA sequences within the genus Haemonchus demonstrate limited divergence in this group of genes between these subfamilies and suggest further that this molecule alone may be inappropriate for assessing phylogenetic relatedness within the superfamily Trichostrongyloidea.

Activity of morantel citrate against strains of benzimidazole-resistant nematodes of sheep in the United Kingdom

The activity of morantel citrate (5.94 mg/kg base) was determined in laboratory tests against field isolates of benzimidazole-resistant nematodes. Its efficacies against adult and seven-day-old worms were 100 per cent and 100 per cent for Cooperia curticei, 95.1 per cent and 69.8 per cent for Haemonchus contortus and 100 per cent and 82 per cent for Ostertagia circumcincta. Morantel citrate was 100 per cent effective against benzimidazole-susceptible Nematodirus battus and Trichostrongylus vitrinus, and it reduced faecal egg counts by 97.9 per cent in sheep infected naturally with benzimidazole-resistant H contortus and O circumcincta.