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Development of Trichostrongylus colubriformis and Trichostrongylus vitrinus, parasites of ruminants in the rabbit and comparison with Trichostrongylus retortaeformis.

The parasitic phase of development of both Trichostrongylus colubriformis and Trichostrongylus vitrinus, parasites of ruminants, was studied in detail in the rabbit. In T. colubriformis, the third moult appeared by 4 days after infection (DAI) and the last moult occurred between 10 and 11 DAI. In T. vitrinus, the third moult occurred between 8 and 11 DAI and the last one between 12 and 15 DAI. The prepatent period lasted 16-17 days for T. colubriformis and 20 days for T. vitrinus. The chronology of the life cycles and the distribution of the parasites along the small intestine for various Trichostrongylus spp. from lagomorphs and ruminants in the natural host or in the experimental host were compared. All of these biological parameters indicated a lower level of adaptation of T. vitrinus compared to the other species of Trichostrongylus. The results are fully compatible with the evolutionary scheme based on morphological analyses.

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Trichostrongylus angistris n. sp. from the red duiker Cephalophus natalensis A. Smith, 1834 and a redescription of Trichostrongylus minor Mönnig, 1932.

Trichostrongylus angistris n. sp. was found in the abomasa of 13 red duiker Cephalophus natalensis A. Smith, 1834, culled in the Charter's Creek Nature Reserve, Natal. The species is closely related to Trichostrongylus minor Mönnig, 1932 and can be differentiated from it by the shorter dorsal ray and the different shape of the gubernaculum and spicules. The shoes of the spicules of T. minor are set at an angle to the long axis, while those of T. angistris are curved. Upon re-examination, the Trichostrongylus spp., tentatively identified as Trichostrongylus capricola Ransom, 1907 and Trichostrongylus vitrinus Looss, 1905, proved to be T. angistris. In this paper, T. angistris is compared with T. capricola and T. vitrinus and T. minor is redescribed.

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A comparison of the infectivity of cryopreserved versus unfrozen infective larvae of Haemonchus contortus, Trichostrongylus colubriformis and Trichostrongylus axei. Results of the Onderstepoort Veterinary Institute and collaborators from 1977 to the present.

The infectivity for sheep of cryopreserved infective larvae (L3) of various strains of Haemonchus contortus, Trichostrongylus colubriformis and Trichostrongylus axei is compared using previously published results of trials conducted at the Onderstepoort Veterinary Institute laboratories, and of collaborators. The means and ranges of development were similar for both frozen and unfrozen larvae of two of the three worm species reviewed. A mean of 33.4% (range, 12.7-63.0%) of cryopreserved H. contortus L3 developed, compared to a mean of 43.7% (range 2.4-78.7%) of unfrozen L3 of this worm species. The corresponding values for T. colubriformis were 33.0% (range 10.3-62.7%), and 33.5% (range 8.3-52.2%), respectively. In the case of T. axei, the development of the cryopreserved L3 (tested in only three trials) was markedly lower than that of unfrozen L3 in the single trial in which the latter was evaluated. It is concluded that development of cryopreserved L3 is probably similar to that of unfrozen L3 and that, for several reasons, maintaining nematode larvae in the frozen state in liquid nitrogen is a much superior method to that of one which entails cycling worm strains continually in their final hosts.

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A comparison of the first internal transcribed spacer of ribosomal DNA in seven species of Trichostrongylus (Nematoda: Trichostrongylidae).

The first internal transcribed spacer (ITS-1) of the ribosomal DNA of seven species of Trichostrongylus was sequenced. The length of ITS-1 in the different species varied from 387 to 390 bases. The G + C content of the ITS-1 sequences were approximately 42%. Little or no intraspecific variation was detected in the three species. Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus vitrinus, for which multiple isolates from different geographical regions were sequenced. In contrast, the level of ITS-1 sequence differences between species ranged from 1.3% to 5.7%. The greatest sequence differences were detected between T. tenuis, the parasite species which infects birds and the six species found in mammals. Some of the nucleotide differences occurred at sites corresponding to recognition sites for restriction endonucleases. These results are compared with previous data obtained for the second internal transcribed spacer (ITS-2). The ITS-1 data indicate that this region of rDNA may also be useful for systematic studies in trichostrongylid nematodes.

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A middle-repetitive DNA sequence element in the sheep parasitic nematode, Trichostrongylus colubriformis.

A novel repetitive DNA sequence in the sheep parasitic nematode Trichostrongylus colubriformis was cloned and sequenced. A 1.1 kb repetitive sequence (Tc15) which hybridized with DNA from T. colubriformis but not with DNA from two other parasitic nematodes, Haemonchus contortus and Ostertagia circumcincta, or sheep was further characterized. Southern blot analysis showed that the repeat hybridized to a range of fragments in restriction digested T. colubriformis DNA and existed in multiple copy number tandem arrays. However, to define clearly the repetitive monomeric unit further screening of phagemid libraries containing BamH I restriction fragments using a subclone of Tc15 as a probe was carried out. Restriction map and sequence data were compiled for 3 clones containing a 145 bp highly repetitive sequence (designated TcREP) which shared homology with the original pTc15 clone. TcREP hybridized to a tandemly repeating sequence monomer of 145 bp in T. colubriformis DNA which was cloned from various genetic environments in the T. colubriformis genome. TcREP homologous sequences were also found in the genomes of two other species of the same genus (Trichostrongylus axei and Trichostrongylus vitrinus) but not in a fourth species (Trichostrongylus rugatus).

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Regulation of Trichostrongylus vitrinus and T colubriformis populations in naturally infected sheep in the Netherlands.

During a study on the epidemiology of gastrointestinal helminth infections in sheep at the University of Utrecht (1974 to 1977) information was obtained on the population regulation of small intestinal Trichostrongylus species. Comparison of the cumulative burdens in tracer lambs with those in flock lambs showed an accumulation of Trichostrongylus species in the latter until autumn followed by an expulsion of worms. The dominating species, T vitrinus, was expelled earlier than T colubriformis. The proportion of inhibited early third stage Trichostrongylus species larvae in tracer lambs was low, but it tended to increase in autumn. In most flock lambs the levels of inhibition were also low. In non-lactating ewes, populations consisted predominantly of inhibited early third stage larvae but when adults were present they tended to be mainly T colubriformis. Similar populations were seen in some lactating ewes. In other lactating ewes Trichostrongylus species populations with low proportions of inhibited early third stage larvae which were dominated by T vitrinus, indicated a relaxation of resistance around parturition.

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Differences in the second internal transcribed spacer (ribosomal DNA) between five species of Trichostrongylus (Nematoda: Trichostrongylidae).

The second internal transcribed spacer (ITS-2) of the ribosomal DNA of 5 species of Trichostrongylus has been sequenced. The ITS-2 of the 5 species was 237 or 238 bases in length, and had a GC content of approximately 30%. No evidence of intraspecific variation was detected in the ITS-2 sequence of T. colubriformis, T. vitrinus or T. retortaeformis, irrespective of the life cycle stage examined. There was evidence, however, of variation at five positions in the ITS-2 sequence of T. vitrinus samples and at one position in T. axei, indicating intra-individual variation in the sequence of different copies of the ribosomal DNA. Nonetheless, there were consistent sequence differences between the five Trichostrongylus species examined. The level of interspecific differences in nucleotide sequence was low (1.3-7.6%), with the species infecting birds (T. tenuis) being genetically more different to the four species found in mammals. Some of the nucleotide differences between species occurred at the recognition sites of endonucleases, which makes them of important diagnostic value for species identification. Also of significance are the recognition sites for several enzymes located within the regions of sequence homology for the five species of Trichostrongylus. These may prove useful in distinguishing between genera of trichostrongyle nematodes.

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Climate and the distribution of intestinal Trichostrongylus spp. of sheep.

The relative prevalences of Trichostrongylus vitrinus, T. colubriformis and T. rugatus in sheep in south-west Western Australia were examined for evidence of associations with climatic conditions in different localities. The region has a mediterranean-type climate with hot, dry summers and cool, wet winters. The intestinal Trichostrongylus spp. populations in sheep sampled comprised 50% T. vitrinus, 38% T. colubriformis and 12% T. rugatus. The prevalence of T. vitrinus was negatively correlated with mean autumn, winter and spring temperatures of a locality. The prevalence of T. colubriformis was positively correlated with mean autumn, winter and spring temperatures. The prevalence of T. rugatus was not correlated with temperature of any season, but was negatively correlated with mean annual rainfall and length of growing season of a locality. There were suggestions of association of amount of rainfall of a locality and prevalence of T. rugatus was not correlated with temperature of any season, but was negatively correlated with mean annual rainfall and length of growing season of a locality. There were suggestions of association of amount of rainfall of a locality and prevalence of T. colubriformis and of differences in seasonal prevalence of T. vitrinus, but the significance of these was obscured by confounding of some climatic factors. The differences in prevalence of the three Trichostrongylus spp. at different locations was attributed to differences in preferred ambient conditions for development and survival of the free-living stages.

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Distinguishing morphological features of the third larval stage of ovine Trichostrongylus spp.

In this paper we describe a procedure that enables the identification of species of infective third stage (L(3)) Trichostrongylus larvae. Lambs were infected with putatively monospecific infections of three species of Trichostrongylus commonly found in New Zealand (T. axei, T. colubriformis and T. vitrinus) and Teladorsagia (Ostertagia) circumcincta. After recovering L(3) from faecal cultures, the lambs were slaughtered and adult male worms recovered and examined for spicule morphology to verify identification. L(3) were examined for morphological features and measurement of their length. Further L(3) were exsheathed and examined under high power optics to observe posterior morphological features (tubercles). The posterior of T. colubriformis has a three-tubercle structure whereas T. vitrinus has a single tubercle and T. axei none. However, the tails of T. circumcincta also lack tubercles and thus T. axei cannot be readily distinguished from them on this feature. The range of lengths of L(3) of Trichostrongylus spp. (600-858 microm) and T. circumcincta (700-914 microm) were found to overlap considerably. The shape of the anterior end of these two species differs and this in combination with length provides an indication of the proportion of T. axei and T. circumcincta in a culture. A combination of tubercle number, with overall length and anterior morphology of L(3), can be used to differentiate nematode populations of T. axei, T. colubriformis, T. vitrinus and T. circumcincta.

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The natural regulation of Trichostrongylus spp. populations in young grazing sheep.

The dynamics of Trichostrongylus axei and intestinal Trichostrongylus spp. were studied in grazing lambs over a 2-year period. Monthly comparisons were made between worm counts of experimental lambs continuously exposed to infection from 3 to 7 months of age, and tracer lambs which were allowed to graze on the experimental plots for successive 4-week periods. It was found that irrespective of the level of larval intake, experimental lambs were unable to regulate their worm burdens until at least 5 months of age. When larval intake was high, resistance to establishment of larvae apparently occurred at approximately 6 months of age followed by rejection and resistance to reinfection. In conditions of low larval intake, worms accumulated with no apparent control operating in lambs up to 7 months of age. These results suggest that a rapid turnover of worm populations does not occur in Trichostrongylus spp., and that in young lambs, worms accumulate until sufficient exposure to infection stimulates resistance to larval establishment and ultimately rejection of adults.

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Development and survival of Haemonchus contortus and Trichostrongylus sp. on pasture in Cameroon.

The development and survival of Haemonchus contortus and Trichostrongylus sp. on pasture at Bamenda, Cameroon were studied by spreading faeces containing strongyle eggs from mixed infections on 12 experimental grass plots from April 1988 to March 1989. Development of eggs to infective larvae during the rainy season (April to October) took place within one week. The infective larvae of H. contortus survived for 11 +/- 2 weeks (with a range of 7 to 13 weeks) during this period while those of Trichostrongylus sp. survived for 9 +/- 2 weeks (with a range of 6 to 12 weeks). No larvae were recovered from eggs spread on pasture during the dry season, except in December when unseasonal rains fell within 9 days of spread of the faeces. Rainfall was identified as the most important climatic condition affecting the development and survival of infective larvae on pastures at Bamenda.

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The effects of immunization of sheep with Trichostrongylus colubriformis larvae on worm burdens acquired during grazing.

The effects of immunization of sheep with Trichostrongylus colubriformis larvae on worm burdens acquired during grazing. International Journal for Parasitology 19: 177-181. Romney sheep, reared helminth-free in pens to 5 months of age, were immunized against Trichostrongylus colubriformis by giving two doses of 200,000 T. colubriformis infective larvae at 15 day intervals to assess protection from natural challenge during grazing. Five immunized sheep and five unimmunized sheep were grazed on infested pasture for 4 weeks, and were then returned to the pens for 4 weeks before slaughter. Worm burdens, gastrointestinal histology and mucus antiparasite activity were examined at slaughter. Faecal egg counts and haematological examinations were carried out at regular intervals throughout the trial. Significant protection (P less than 0.05) was afforded immunized sheep against adult T. colubriformis (87%), T. axei (67%), Nematodirus spathiger (91%) and Ostertagia spp. (42%). Greater numbers of immature Nematodirus spp. and Ostertagia spp. were present in immunized sheep Overall, a significant (P less than 0.05) 42% reduction in total nematode burdens was afforded by immunization of the sheep with T. colubriformis larvae. Immunized sheep had significantly (P less than 0.05) more globule leukocytes, mast cells and eosinophils in gastrointestinal tissue and significantly (P less than 0.05) higher levels of mucous antiparasite activity than unimmunized sheep. Haematological observations showed some sheep had transient eosinophilia during immunization or grazing. Both immunized and unimmunized sheep showed depressed (P less than 0.05) total leukocyte counts during grazing which returned to pre-grazing levels within 1 week of return of the sheep to the pens. Overall, haematological parameters reflected parasite challenge and were unrelated to worm burdens acquired.

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Lack of intraspecific variation in the second internal transcribed spacer (ITS-2) of Trichostrongylus colubriformis ribosomal DNA.

The nucleotide sequence of the second internal transcribed spacer (ITS-2) was determined for three populations of the parasitic nematode Trichostrongylus colubriformis which differed in their susceptibility to benzimidazole anthelmintics and/or in their geographical origin. No intraspecific variation was found in the ITS-2 sequence, indicating that this region of rDNA is inadequate to discriminate between resistant and susceptible populations of T. colubriformis, but it may prove useful for distinguishing between species of Trichostrongylus.

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Abundant variation in microsatellites of the parasitic nematode Trichostrongylus tenuis and linkage to a tandem repeat.

An understanding of how genes move between and within populations of parasitic nematodes is important in combating the evolution and spread of anthelmintic resistance. Much has been learned by studying mitochondrial DNA markers, but autosomal markers such as microsatellites have been applied to only a few nematode species, despite their many advantages for studying gene flow in eukaryotes. Here, we describe the isolation of 307 microsatellites from Trichostrongylus tenuis, an intestinal nematode of red grouse. High levels of variation were revealed at sixteen microsatellite loci (including three sex-lined loci) in 111 male T. tenuis nematodes collected from four hosts at a single grouse estate in Scotland (average He = 0.708; mean number of alleles = 12.2). A population genetic analysis detected no deviation from panmixia either between (F(ST) = 0.00) or within hosts (F(IS) = 0.015). We discuss the feasibility of developing microsatellites in parasitic nematodes and the problem of null alleles. We also describe a novel 146-bp repeat element, TteREP1, which is linked to two-thirds of the microsatellites sequenced and is associated with marker development failure. The sequence of TteREP1 is related to the TcREP-class of repeats found in several other trichostrongyloid species including Trichostrongylus colubriformis and Haemonchus contortus.

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Observations of the sheath extension of the third stage, infective larvae of Trichostrongylus rugatus.

The length of the sheath extension of third stage (L(3)), infective larvae of Trichostrongylus rugatus is reported. The total length and length of the sheath extension of L(3) recovered from the faeces of sheep originating from two localities in South Australia were measured. The total length of larvae was 615-722 microm and the length of the sheath extension was 46-56 microm (mean 51.5). The larvae of T. rugatus comprised up to 72% of the larvae recovered. Worm free sheep were infected with larvae from the two localities and L(3) were recovered following culture of faeces from these experimentally infected animals. L(3) measured 640-746 microm in total length with a sheath extension of 46-56 microm (mean 51.4). Adult nematodes recovered from gastro-intestinal tracts were identified as T. rugatus. The length of the sheath extension used in conjunction with the total length is a characteristic that enables confident differentiation of infective larvae of T. rugatus from other Trichostrongylus species infecting sheep in southern Australia and Haemonchus contortus.

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Attempts to generate immunity against Trichostrongylus colubriformis and Haemonchus contortus in young lambs by vaccination with viable parasites.

The ability of young Merino lambs to achieve protective immunity following vaccination via viable nematode infections was assessed. Lambs were infected from 1 month of age by repeated continuous low dose (trickle) administration of Trichostrongylus colubriformis or Haemonchus contortus infective larvae (L3), or by truncated infections with high doses of viable T. colubriformis L3. After 7 weeks all groups were drenched with anthelmintic and at 3 months of age they were re-infected with the homologous species. Protection was assessed by faecal egg counts at 3, 4, 5, 6 and 7 weeks after challenge, and worm count at 7 weeks after challenge. Young lambs were partially protected by 3 months of age against Trichostrongylus by trickle infection. This protection correlated with local mast cell and T-cell priming, increased numbers of local antigen-presenting cells and T-cells and increased worm-specific antibody titres in the intestine. However, there was no evidence that young lambs were capable of immunologically recognising H. contortus antigens following trickle infection, nor did trickle infection significantly protect young lambs against Haemonchus challenge.

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Induction of protective immunity to Trichostrongylus colubriformis in neonatal merino lambs.

The premise that any bias of immune reactivity in neonatal lambs towards T-helper (TH)2 responses could benefit the induction of protection against gastrointestinal nematodes was investigated. In two trials, lambs were either trickle-immunised with 2000 infective larvae of Trichostrongylus colubriformis (TcL3), 3 times weekly from the day of birth for 6 weeks or inoculated with a recombinant T. colubriformis 17 kDa antigen in incomplete Freund's adjuvant (IFA). In trial 1, trickle immunised and control neonates challenged at 7 weeks of age had similar worm counts 10 days after challenge, but from 25 days, significant reductions (P<0.01) in mean faecal egg count and worm count in excess of 75% were displayed by the immunised lambs. The results of a second, similar trial, gave 85-91% reductions in parasitism in trickle immunised neonates (P<0.001) and around 50% protection in neonates vaccinated with recombinant 17 kDa antigen. Parasitism in immunised neonates in Trial 2 was significantly reduced (P<0.001) compared to that in 4-month-old animals. Antibody responses in trickle-immunised (protected) and challenge control (infected) neonates were almost exclusively of the IgG1 isotype compared to vaccinated animals which exhibited increased levels of anti-17kD IgG2. Trichostrongylus colubriformis infection, but not specific vaccination, induced interleukin-5 production by mesenteric lymph node cells. The results offer the tantalising prospect of generating protective immunity to gastrointestinal parasites prior to weaning in sheep; this was most effectively generated by viable parasites in this investigation.

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