Time of onset and target of immune reactions in sheep with acquired immunity against Haemonchus contortus

Adams D. B. 1982. Time of onset and target of immune reactions in sheep with acquired immunity against Haemonchus contortus. International Journal for Parasitology12: 439–443. Nonimmune sheep and sheep rendered immune by infection with the abomasal nematode, Haemonchus contortus, were infected with the parasite and treated at various times with the glucocorticosteroid, dexamethasone. The results show that this immunosuppressant drug abolished acquired but not innate immunity to H. contortus and that acquired responses were not important in restraining the fecundity of adult worms during primary infection. By treating immune sheep with dexamethasone during reinfection, it was shown that the responses acting against the establishment of infection commence later than the fourth day after larval administration and are complete by the seventh day. H. contortus more advanced in development than the fourth-larval stage were relatively insusceptible to this manifestation of acquired immunity.     

Changes in blood leukocytes,bone marrow and lymphoid organs in sheep infected with Haemonchus contortus

Haemonchus contortus infection, which causes a blood loss anaemia in sheep, depleted leukocytes and produced leukopenia and a mild lymphopenia. Thymus atrophy, a decreased size of spleen and enlargement of adrenal glands occurred concomitantly in infected sheep which suggested that they were caused by the stress of infection. The overwhelming change in bone marrow of infected sheep was a 4-fold increase in erythroid series cells.Primary immunization with rat erythrocytes produced similar haemagglutinating antibody responses in infected sheep and in sheep allowed to recover from infection after treatment with thiabendazole. This suggests that extant infection with H. contortus was not associated with a secondary immunodeficiency. Blastogenic responses of blood lymphocytes from infected sheep to larval antigen correlated with faecal egg counts but not with haematocrit or leukocyte values. These results suggest that the unresponsiveness of lymphocytes to worm antigen which occur during infection with H. contortus is more closely related to contact with the parasite than to the pathological effects of infection.     

Immunity acquired by sheep from an experimental infection with haemonchus contortus

Adams D.B. and Beh K.J. 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. International Journal for Parasitology11: 381–386. A primary infection of sheep with a single dose of Haemonchus contortus larvae was traced by faecal egg counts until it had substantially declined after 55 weeks. These primed sheep were then given a sequence of two reinfections with the parasite. Comparison of faecal egg counts in primed sheep and in two separate groups of previously worm-free sheep showed that primary infection conferred significant immunity. This, however, was not sufficiently protective to prevent the development of further anaemia and faecal egg counts indicative of clinical haemonchosis. It is suggested that an adaptation in the host-parasite relationship which promotes the longevity of primary infection with H. contortus may also moderate the induction of acquired immunity.The titre of haemagglutinating antibody specific for H. contortus rose in serum during the course of primary infection, but the two reinfections did not stimulate a rise in titre. Titres of haemagglutinating antibody before reinfection did not correlate with subsequent faecal egg counts.     

The response of sheep to parenteral vaccination and immunizing infections against the abomasal nematode, Haemonchus contortus

Adams D. B., Beh K. J. and Davies H. I. 1982. The response of sheep to parenteral vaccination and immunizing infections against the abomasal nematode, Haemonchus contortus. International Journal for Parasitology12: 445–449. Subcutaneous injection of relatively large amounts of unfractionated homogenates of adults plus infective larvae of Haemonchus contortus emulsified in complete Freund's adjuvant produced a degree of protective immunity when challenge infection was given eight weeks after the first or only dose of vaccine. In an attempt to improve acquired immunity, parenteral vaccination was either followed or preceded by a short immunizing infection with H. contortus, which was terminated by anthelmintic before patency. This treatment aimed at stimulating general responsiveness to worm antigens and invoking mucosal immunity in the abomasum. Disparate results were obtained; immunizing infections either increased immunity or made sheep more susceptible to challenge infection. In this latter situation, the unresponsiveness associated with primary infection with H. contortus may have been increased.     

Haemonchus contortus: Local and serum antibodies in sheep immunised with irradiated larvae

Smith W. D. and Christie M. G. 1978. Haemonchus contortus: lccal and serum antibodies in sheep immunised with irradiated larvae. International Journal for Parasitology8: 219–223. Two doses of Co⁶⁰ irradiated Haemonchus contortus protected 9-month-old sheep against a challenge infection of 10,000 normal larvae. This resistance was associated with increased concentrations of IgG antibodies in the serum as well as IgA and IgG antibodies in the abomasal mucosa.     

The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus

Martin P. J., Le Jambre L. F. and Claxton J. H. 1981. The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus. International Journal for Parasitology11: 35–41. Beginning with a mixture of 5% thiabendazole-resistant and 95% non thiabendazole-resistant Haemonchus contortus larvae, worm free sheep were artificially infected with either 10,000, 9000, 7000, 2500 or zero larvae. Four weeks later, the infected sheep were treated with 44 mg/kg of thiabendazole. Seven days after anthelmintic treatment the same sheep were infected with zero, 1000, 3000, 7500 or 10,000 larvae respectively so that each sheep was given a total of 10,000 larvae. The larvae given after thiabendazole treatment are referred to as being in refugia (defined as an area where individual members of a population can escape exposure to a drug). This was repeated for six generations of parasites, maintaining the same proportion of larvae in refugia in each generation. The egg hatch assay for resistance indicated that refugia delayed the development of resistance in Haemonchus contortus. Where none or a small proportion of larvae were in refugia, a rapid increase in resistance occurred. With an increased proportion of larvae in refugia, resistance was slower to develop. Population size, as estimated by faecal egg counts done after anthelmintic treatment and corrected for the number of larvae given in each treatment, was lower at the higher levels of refugia. This was due to a lower level of resistance in the higher refugia lines and consequently a greater effect of the drug. However, the estimate of population size, from egg counts done after the larvae introduced from refugia had matured, were similar in all lines.     

Regulation of Haemonchus contortus populations in sheep exposed to continuous infection

Groups of sheep were infected three times weekly for 15 weeks with infective larvae of Haemonchus contortus at four rates ranging from 600 to 4800 larvae per week. At 3-weekly intervals, sheep from each group were killed for total worm counts after receiving a dose of radiolabelled larvae which enabled a direct measurement of establishment of incoming larvae during the continuing infection. Peak H. contortus burdens were reached between 6 and 9 weeks of infection, and were related to infection rate. In the groups receiving 2400 and 4800 larvae per week, worm numbers then declined rapidly and by the end of the experiment were lower than those in the groups given 600 or 1200 larvae per week. Establishment of incoming larvae was not influenced by infection rate, and declined from 45% in the first 4 weeks of infection to insignificant levels during the final 6 weeks. The proportion of incoming larvae arrested in their development increased as the infection progressed. It was concluded that H. contortus numbers were regulated by development of resistance to infection and by a loss of established worms which was related to the current rate of larval intake, and to the host's previous experience of infection.     

Meiotic abnormalities in backcross lines of hybrid Haemonchus

Le Jambre L. F. and Royal W. M. 1979. Meiotic abnormalities in backcross lines of hybrid Haemonchus. International Journal for Parasitology10: 281–286. Hybrid worms from the reciprocal matings between Haemonchus contortus and H. placei were backcrossed to the parent species. Six of the possible eight backcrosses produced fertile eggs. The six backcross lines were passaged through sheep for four generations. The adults of this generation had various levels of meiotic abnormality as evidenced by abnormal variations in chromosome structure and aneuploidy. The eggs from backcross lines with a degree of meiotic abnormality had the lowest percentage hatch. Four of the six backcross lines required a higher temperature for larval development than did H. contortus. The size of the third-stage larvae varied from one with larvae larger than H. placei to one line with larvae smaller than H. contortus.     

Host-induced effects on the morphology of Ostertagia circumcincta in grazing sheep

Waller P. J. and Thomas R. J. 1978. Host-induced effects on the morphology of Ostertagia circumcincta in grazing sheep. International Journal for Parasitology8: 365–370. Morphological changes are described in adult Ostertagia circumcincta recovered from lambs slaughtered monthly from June to November. Comparisons were made between continuously grazed principal lambs exposed to infection for increasingly long periods, and tracer Iambs exposed for only four weeks. Lambs were either slaughtered immediately off pasture or held 2 weeks under worm free conditions before slaughter. Although no resistance to establishment of infection occurred, shown by comparable worm burdens of principals and tracers, a rapid parasite population turnover was observed in the principal lambs which ceased once the lambs were removed from the infected pasture. Significantly fewer adult female Ostertagia with fully developed vulval flaps were recorded in principals compared to tracers. Such differences are considered to be primarily host induced responses which rapidly come into play, evidenced by the entire populations of developing female worms in tracers slaughtered immediately off pasture showing arrested vulval flap development after they had completed development in those tracers held for a further two weeks. A progressive reduction in the proportion of adult male worms also occurred in the principal lambs. Population density was shown to have an inverse correlation with worm size. By analogy, it is suggested that seasonal change in the presence of cuticular inflations in Haemonchus contortus females with linguiform vulval flaps may be primarily host induced, rather than a change in the relative fitness of discrete genotypes.     

Control of nematode parasites by grazing management—II. Decontamination of sheep and cattle pastures by varying periods of grazing with the alternate host

Commencing in December 1970, paddocks of a uniform series of sheep pastures were grazed for 6, 12 or 24 weeks by either yearling steers is or yearling ewes. Cattle pastures were treated similarly. All ewes and steers were pre-dosed with anthelmintic.At the conclusion of alternate grazing the effectiveness of the grazing treatments was evaluated by grazing each paddock for 1 month with either ten worm-free lambs (sheep pastures) or three wormfree calves (cattle pastures). The test animals were then slaughtered for total worm counts. The grazing of sheep pastures with cattle for 6, 12 or 24 weeks from December onwards resulted in reductions in numbers of Haemonchus contortus and Trichostrongylus colubriformis in test lambs. In comparison with continuous grazing by sheep, Nematodirus spp was only reduced after 24 weeks grazing by cattle. Cattle pastures grazed by sheep for 6 weeks showed no reduction in numbers of Ostertagia ostertagi or Cooperia oncophora in test calves. However after 12 weeks with sheep, numbers of O. ostertagi though not C. oncophora were reduced and after 24 weeks of alternate grazing both these species were reduced.Calves following the 6 week sheep treatments acquired both Haemonchus contortus and Trichostrongylus colubriformis and the calves from the 12 week sheep treatment paddock also carried H. contortus. For sheep the only evidence of cross-transmission was the occurrence of small numbers of Cooperia oncophora in test lambs from the 24 week cattle grazing treatment.The results provide evidence that sequential stocking with cattle and sheep in conjunction with anthelmintic treatment is an effective management strategy for preparing parasitologically safer pastures, but further information is required to determine the optimum timing of sequential stocking in farming situations.     

Efficiency of a genetic test to detect benzimidazole resistant Haemonchus contortus nematodes in sheep farms in Quebec,Canada

Haemonchus contortus is a hemophilic nematode which infects sheep and causes anemia and death to lambs. Benzimidazole drugs are used to remove these parasites, but the phenomenon of resistance has arisen worldwide. A sensitive test to detect resistance before treatment would be a useful tool to enable farmers to anticipate the efficiency of the drug before drenching the flock. In this study, we compared a test for benzimidazole resistance based on detection of genetic markers in H. contortus before treatment with the common method of fecal egg count reduction test (FECRT). We recruited 11 farms from different regions of Quebec for this study. Fecal samples from animals were collected per rectum before and after treatment in control and treated groups (10 animals per group). The 10 sheep were treated with fenbendazole at the recommended dose rate. Among the 11 farms participating in the study, we found H. contortus in 8 of them and it was the most predominant nematode species detected by egg count. Using the genetic test, we found benzimidazole resistance in each of these 8 farms. In 5 of these 8 farms there were sufficient sheep with an egg count for H. contortus above 150 eggs per gram to allow the FECRT test to be conducted. Benzimidazole resistance was observed in each of these 5 farms by the FECRT. When we compared the results from the genetic test for samples off pasture and from individual sheep, with the results from the FECRT, we concluded that the genetic test can be applied to samples collected off pasture to estimate benzimidazole resistance levels before treatment for H. contortus infections.     

Attempts to vaccinate ewes and their lambs against natural infection with Haemonchus contortus in a tropical environment

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of grazing sheep each containing 13 ewes and their 16 lambs naturally infected with gastrointestinal nematodes. Two groups were vaccinated with either 5 or 50 μg of the antigen per immunisation, while the third, the control group, received adjuvant alone. The sheep were immunised six times at 3 week intervals, partly because the vaccine antigens are hidden and thus no immunological boost would be delivered by subsequent infection and partly because the level of Haemonchus spp. challenge was expected to be high. The vaccinated ewes, first immunised approximately 1 month before lambing, showed a circulating antibody response but no signs of reduced anaemia or Haemonchus spp. egg counts, compared with control ewes. Several ewes with severe haemonchosis in all three groups had to be given precautionary treatment with anthelmintic drugs. In contrast, vaccinating their lambs with either 5 or 50 μg of the antigen per immunisation resulted in 10 fold higher antibody titres. In the case of the lower antigen dose this was associated with significantly less anaemia, 72% reduction in the overall number of Haemonchus spp. eggs produced and significantly fewer worms compared with control lambs. It is hypothesised that the heavily pregnant or lactating ewes did not have sufficient physiological reserves to mount a protective response following vaccination in the tropical weather and high challenge conditions that prevailed. Nevertheless, the vaccine could afford useful protection for lambs against H. contortus.     

Vaccination-induced IgG response to Galα1-3GalNAc glycan epitopes in lambs protected against Haemonchus contortus challenge infection

Lambs vaccinated with Haemonchus contortus excretory/secretory (ES) glycoproteins in combination with the adjuvant Alhydrogel are protected against H. contortus challenge infection. Using glycan micro-array analysis we showed that serum from such vaccinated lambs contains IgG antibodies that recognise the glycan antigen Galα1-3GalNAc-R and GalNAcβ1-4(Fucα1-3)GlcNAc-R. Our studies revealed that H. contortus glycoproteins contain Galα1-3Gal-R as well as significant levels of Galα1-3GalNAc-R, which has not been previously reported. Extracts from H. contortus adult worms contain a galactosyltransferase acting on glycan substrates with a terminal GalNAc, indicating that the worms possess the enzymatic potential to synthesise terminal Gal-GalNAc moieties. These data illustrate that glycan micro-arrays constitute a promising technology for fast and specific analysis of serum anti-glycan antibodies in vaccination studies. In addition, this approach facilitates the discovery of novel, antigenic parasite glycan antigens that may have potential for developing glycoconjugate vaccines or utilization in diagnostics.     

Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to Trichostrongylus colubriformis and Haemonchus contortus

Resistance to an acute gastrointestinal nematode (GIN) infection is dependent on the ability of the host to recognise the parasite and mount a protective Th2 response. It is hypothesised that lambs which are genetically susceptible to GIN will differentially up-regulate Th1-type genes and therefore remain susceptible to chronic parasitism compared with genetically resistant lambs which will differentially up-regulate Th2-type genes and clear the parasite infection. Two selection flocks, in which lines of Merino sheep produced lambs genetically resistant or susceptible to GIN, were acutely challenged once or thrice with either Haemonchus contortus or Trichostrongylus colubriformis. Faecal-egg counts (FECs), and plasma and tissue anti-parasite (H. contortus or T. colubriformis) antibody isotype responses showed that resistant animals challenged three times with T. colubriformis established a protective Th2 response (negligible FEC, IgG1 and IgE) whereas susceptible animals required multiple challenges to establish a significant IgG1 response despite FECs remaining high. Trichostrongylus colubriformis elicited a more pronounced host response than H. contortus. RNA extracted from tissues at the site of each parasite infection and associated lymph nodes were interrogated by microarray and quantitative PCR analyses to correlate host gene expression to FECs and antibody responses. The IFN-γ inducible gene cxcl10 was up-regulated in the susceptible line of the Trichostrongylus selection flock sheep after a tertiary challenge with the parasites H. contortus and T. colubriformis. However, a uniform pattern of genes was not up-regulated in resistant animals from both selection flocks during both parasite infections, suggesting that the mode of host resistance to these parasites is different, although some similarities in host susceptibility were apparent.     

Infection of immunosuppressed mice with the abomasal nematode parasite of ruminants, Haemonchus contortus

Infective larvae of Haemonchus contortus established in mice either immunosuppressed with the corticosteroid, dexamethasone, or the cytotoxic drug, cyclophosphamide, or treated with the histamine H2 inhibitor, cimetidine. Infections persisted for as long as the immunosuppressive treatment (7 days) and growth of larvae was similar to that seen in sheep. Virtually no larvae survived in untreated mice. Accordingly, it would appear that adaptive immunity is an important barrier against primary infection by H. contortus in mice and is a determinant of host-range for this parasite. Antibody raised in either sheep or mice against soluble extracts of adult H. contortus precipitated with different but overlapping sets of worm antigens. This suggests that the unique antigens recognized by the mouse compared with the sheep are crucial for the rapid protective responses which prevent primary infection.     

Glutathione-S-transferase,a possible drug-metabolizing enzyme,in Haemonchus contortus: Comparative activity of a cambendazole-resistant and a susceptible strain

Kawalek J. C., Rew R. S. and Heavner J. 1984. Glutathione-S-transferase, a possible drug-metabolizing enzyme in Haemonchus contortus: comparative activity of a cambendazole-resistant and a susceptible strain. International Journal for Parasitology14: 173–175. A drug metabolizing enzyme (DME), glutathione-S-transferase, was detected in homogenates of a cambendazole-susceptible and a cambendazole-resistant strain of Haemonchus contortus. The activity was 1.5–1.8 times higher in the resistant strain. DME activation is a possible mechanism for anthelmintic resistance in H. contortus.     

Haemonchus contortus: development of a two-step, differential-display PCR to detect differential gene expression in nematodes from immune and naïve sheep

Haemonchus contortus is a blood-feeding nematode which parasitizes the abomasum of sheep and represents a serious constraint to sheep production. Anthelmintics are currently the most common method of worm control but the worldwide development of multiple-drug resistance and issues of residues in the food chain make alternatives to anthelmintics a priority. Biotechnology-driven solutions to parasitism include vaccines and silencing of genes regulating nematode development. To pursue gene targets that may be suitable for parasite control, a two stage differential-display PCR (dd-PCR) approach was developed to observe differential gene expression between Haemonchus from immune and control sheep. Twenty-four reproducible differentially-expressed bands were identified in 60 pairs of dd-PCR comparisons. The first of these cloned and sequenced corresponded to the H. contortus 60S ribosomal protein L35A. The remaining bands are being cloned and validated and may provide new targets for parasite control.     

Genetics of vulvar morph types in Haemonchus contortus: Haemonchus contortus from the Northern Tablelands of New South Wales

Le Jambre L. F. and Royal W. M. 1977. Genetics of vulvar morph types in Haemonchus contortus: Haemonchus contortus from the Northern Tablelands of New South Wales. International Journal for Parasitology7: 481–487. The inheritance of the vulvar morph types, smooth, knobbed, linguiform A-right, linguiform A-left and linguiform C-type was studied in Haemonchus contortus from the Northern Tablelands of New South Wales. The dominance hierarchy of these characters was found to be smooth > knobbed > linguiform. The linguiform phenotype was the most common in the wild type population. The modifications on the linguiform phenotype were found to be highly heritable with A-right responding most to selection. Realised heritabilities for these characters was A-right 0.37, A-left 0.15, and C-type 0.21. It appeared that the dominance of a phenotype increased following selection.     

Changes in optical path difference in the oesophageal region and the excretory cells during exsheathment in Haemonchus contortus

Davey K. G. and Sommerville R. I. 1982. Changes in optical path difference in the oesophageal region and the excretory cells during exsheathment in Haemonchus contortus. International Journal for Parasitology12: 503–507. Changes in the optical path difference (opd) between various parts of the worm and the medium in which the worms were immersed were determined by quantitative interference microscopy. The opd of the oesophagus and the excretory cells both increased upon stimulation of the worms with CO₂ at 38.5°C, suggesting a decrease in volume of those structures. The oesophagus decreased markedly in length and slightly in diameter, yielding a decrease in volume of approximately 15 to 17 pl. Desheathing the worms with NaOCl produced changes in the oesophagus but not the excretory cells. This confirms previous findings that exsheathment involves at least two parallel processes, both of which are initiated by CO₂ and only one of which is stimulated by exposure to NaOCl.