Resistance of young lambs to Haemonchus contortus infection, and its loss following anthelmintic treatment

1988. Resistance of young lambs to Haemonchus contortus infection, and its loss following anthelmintic treatment. International Journal for Parasitology 18: 1107–1109. A comparison of worm burdens of grazing Merino lambs that were untreated, treated once and allowed to graze for 2 weeks, or given suppressive anthelmintic treatment until their final 2 weeks on pasture, showed that resistance to reinfection with Ostertagia circumcincta and Trichostrongylus colubriformis did not develop until the lambs were about 8 months old. By contrast, there was clear evidence that untreated lambs, but not lambs given one or more treatments, had acquired substantial resistance to infection with Haemonchus contortus by an age of 4 months.     

Persistence of acquired immunity to Trichostrongylus colubriformis in sheep after termination of infection

Lambs were infected with 6000 Trichostrongylus colubriformis L3 per week for 18, 12 or 6 weeks, beginning at ages 14, 20 and 26 weeks, respectively. At the end of the primary infection subgroups had geometric mean adult worm burdens of 5000, 15,000 and 18,000, respectively. In the remaining sheep the worm population was removed with anthelmintic, and sheep had no further larval intake until challenged with T. colubriformis 1,6, 12, 18 or 24 weeks later. In the groups given 18 or 12 weeks primary infection, establishment of challenge doses was low (less than 25% of establishment in helminth naive controls) in most animals at all challenge times. However, for the groups given 6 weeks primary infection, establishment was low only at the first two challenge times. Thereafter it had similar mean to control groups, but much greater variance. Other subgroups were challenged with T. colubriformis, Ostertagia circumcincta and Haemonchus contortus 1 week after worm removal. In these animals T. colubriformis establishment was not different to animals challenged at the same time with T. colubriformis alone, however immunity to T. colubriformis afforded little protection against the other species. The results of this experiment were incorporated into a simulation model of the population dynamics of T. colubriformis.     

The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae

1988. The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae. International Journal for Parasitology 18: 1035–1038. Groups of weaned Romney ewe lambs were immunized with two doses of 28,000, 35,000 or 42,000 (2000 kg⁻¹) infective larvae of Trichostrongylus colubriformis at 8, 12 or 16 weeks of age, respectively. Each group and helminthfree control lambs of similar age were challenged with T. colubriformis at the same dose rate as used for immunization. Faecal egg counts (FEC) and haematological observations were made during the experiment, and at slaughter, 42 days after challenge, worm burdens were determined and small intestinal histology was examined.

Lambs in each immunized group were identified as ‘responders’ or ‘non-responders’ on the basis of both FEC and worm burdens. A significant (P<0.001) decrease in the worm burdens recovered as a proportion of the challenge infections in both unimmunized and immunized lambs with increasing age was observed.

Globule leukocyte numbers increased with the age of lambs. In addition, within each age group globule leukocyte numbers reflected individual responsiveness to immunization, significantly (P<0.01) greater numbers being present in ‘responders’ than ‘non-responders’ or unimmunized lambs. No difference in haematological responses were found in relation to the lambs' responsiveness to immunization.     

The response of immune sheep to challenge with Trichostrongylus colubriformis: enteric plasma loss and secretion of biogenic amines

Sheep were challenged with a single large dose of larvae after vaccination with irradiated Trichostrongylus colubriformis larvae. Worm counts performed on vaccinated sheep 6 or 7 days after challenge (DAC) showed that they were solidly immune and only retarded L3 larvae were recovered at this time. Enteric plasma loss (EPL) in vaccinated animals increased immediately after challenge to peak 4–6 DAC and then decreased to pre-challenge levels. In contrast, a substantial rise in EPL did not occur in unvaccinated sheep until 10 DAC. Secretion of histamine into the duodenum of immunized sheep increased significantly from 2 to 7 DAC with the highest value at 6 DAC which corresponded with a lower duodenal tissue histamine level at this time. Histamine and 5HT secretion into the small intestine of previously uninfected sheep gradually increased during 12 weeks of a trickle infection of 3000 normal T. colubriformis larvae per week. The results indicate that rejection of incoming larvae by immune sheep is accompanied by an intestinal inflammatory response involving secretion of biogenic amines and a concurrent plasma loss.     

The molecular size of nematode acetylcholinesterases and their separation from nematode allergens

The molecular size of nematode acetylcholinesterases, and their separation from nematode allergens. International Journal for Parasitology 3: 735–741. Acetylcholinesterases and allergens were derived from two parasitic nematodes, Nippostrongylus brasiliensis which parasitises rats and Trichostrongylus colubriformis which infects sheep and guinea pigs.

Chromatographic studies indicated that the mol. wt of nematode acetylcholinesterases lies between 65,000 and 75,000. The acetylcholinesterases of both species were separated from nematode allergens whose mol. wt is in the range of 10,000–50,000.

γG binding antigens of T. colubriformis were located in fractions with a mol. wt range of 30,000–150,000. γE binding activity was confined to allergenic material with a mol. wt of less than 70,000.     

The effect of specific immunization or infection with Trichostrongylus colubriformis on production of eosinophil differentiation factors in guinea pigs.

The cultivation of bone marrow was used to quantitate the levels of eosinophil differentiation factors (EDF) produced in conditioned medium (CM) by incubation of mesenteric lymph node cells (MLNC) with mitogens or specific antigens from the intestinal nematode parasite, Trichostrongylus colubriformis. In liquid cultures with 20 units ml⁻¹ recombinant murine interleukin-5 (IL-5), bone marrow cells (BMC) from either normal or infected donors contained <5% eosinophils and differentiated to> 50% eosinophils over 2–3 weeks. Conditioned medium from 3–4 week infected donors produced between 20 and 50% eosinophils when donor MLNC were stimulated with the specific antigen preparation SP3, but macrophages predominated when using CM from MLNC incubated with Concanavalin A (ConA). CM from MLNC of challenged donors incubated with SP3 produced 30–70% eosinophils in BMC assays, with highest levels induced by CM from high responder (HR) donors. Marrow from parasitized or normal donors gave rise to comparable proportions of eosinophils. CM was also produced from LNC of donors given protein or parasite antigens in adjuvant where between 28 and 35% eosinophils were produced in culture. There were no differences between activities attributable to the antigen, but Freund's complete adjuvant induced earlier differentiation of BMC than alum-induced CM. The results confirm that high levels of EDF activity are specifically induced by parasitic infection, and can also be produced by intraperitoneal and subcutaneous inoculation of adjuvanted antigens. Consistent with the greater eosinophilia exhibited by HR guinea pigs to infection with T.colubriformis L3, their MLNC also produced the highest levels of EDF activity.     

Response to helminth infection of sheep selected for resistance to Haemonchus contortus

Lines of Merino sheep selected for increased (IRH) and decreased (DRH) resistance to Haemonchus contortus were compared with an unselected (CH) line, after approximately four generations of selection. Measurements were recorded on 69 IRH, 47 DRH and 84 CH animals. Following artificial challenge with H. contortus, the IRH line had significantly (P < 0.001) lower faecal egg counts than the CH and DRH lines (2730,12,720 and 17,400 epg, respectively). Significant differences (P<0.05) were found between all lines in the minimum packed cell volumes during artificial infection (25.7, 22.0 and 20.3%) and in faecal egg counts after natural infection (140, 3590 and 8750 epg). Differences were also recorded (P<0.05) following artificial challenge with Trichostrongylus colubriformis (490, 840 and 1340 epg). On a percentage basis, faecal egg counts in the IRH line deviated less from the CH line following artificial infection with T. colubriformis (42%) than with H. contortus (79%). The reverse was true for the DRH line (60 and 37%, respectively). Differences in egg output of this magnitude should have a marked effect on requirements for anthelmintic treatment, rate of development of drug resistance and level of pasture contamination when the lines are grazed separately.     

Effects of changes in temperature and saturation deficit on the survival of eggs of Trichostrongylus colubriformis (Nematoda: Trichostrongylidae)

and 1972. Effects of changes in temperature and saturation deficit on the survival of eggs of Trichostrongylus colubriformis (Nematoda: Trichostrongylidae) International Journal for Parasitology, 2: 439–447. In conformity with a hypothesis relating to survival of the developing T. colubriformis egg exposed to desiccation, samples of eggs initially at the early blastomere stage of development showed decreased mortality during development with increasing temperature of incubation up to 25°C, for approximately constant rates of evaporation. At 30°C there was a higher percentage mortality for fixed evaporation rate than at 20° or 25°C. It is suggested that at 30°C there may be an abrupt increase in the initial rate of water loss from the developing embryo resulting from a change in the permeability to water of the lipid layer of the egg envelope.

Fully embryonated T. colubriformis eggs were obtained by incubation at 20°C in the presence of a moderate saturation deficit during development. When such eggs were transferred to 30° and 40°C there was no mortality at the higher temperature, providing that the saturation deficit was substantially increased. A hypothesis proposed for survival at high temperature is based on analogy with water loss through the arthropod cuticle and is attributed to a decrease in permeability of the protein-chitin layer of the egg envelope under conditions of high evaporation rate, even though permeability of the lipid layer might be increased by high temperature.     

Population dynamics of Haemonchus contortus and Trichostrongylus spp. in sheep

Twenty-five lambs grazing a parasite-contaminated pasture were divided into four groups. One group of seven lambs were left untreated and three groups of six lambs each were treated with levamisole at 2 week, 3 week or 1 month intervals respectively. Body weights, fecal egg counts and packed cell volumes were determined every 2 weeks for 8 weeks. Lambs were then necropsied on the day they would have received another treatment, thus the maximum accumulation of parasites at each treatment frequency was measured. Treatment at 2 and 3 week intervals markedly suppressed fecal egg counts, but egg counts of sheep treated every month rose between treatments and untreated animals continually shed eggs. Treatment at all three frequencies provided only slight protection from blood loss, but it lessened weight loss. There was little difference between treatment groups in burdens of adult H. contortus, but burdens of T. axei and T. colubriformis and arrested larvae of H. contortus increased with decreasing frequency of treatment. The results support the findings of other workers that Trichostrongylus spp. have a long adult life span and continue to accumulate with time, whereas H. contortus may have a short adult life span and a rapid population turnover. In the face of constant reinfection, repeated anthelmintic treatment gives more protection from the effects of the long-lived parasites than from those with a rapid population turnover.     

Bile and bile salts and exsheathment of the intestinal nematodes Trichostrongylus colubriformis and Nematodirus battus

1972. Bile and bile salts and exsheathment of the intestinal nematodes Trichostrongylus colubriformis and Nematodirus battus. International Journal for Parasitology, 2: 433–438. Exsheathment of T. colubriformis was potentiated at physiological levels of CO₂ by bile and bile salts. Lamb bile and crude sodium taurocholate preparations produced the greatest increase in exsheathment while rabbit bile, sodium glycocholate and deoxycholate had less pronounced effects. Exsheathment in bile and taurocholate occurred at a pH above 4 and it is therefore suggested that infective larvae which fail to exsheath in the abomasum could well do so in the proximal part of the small intestine. Pure sodium taurocholate was found to greatly potentiate exsheathment of N. battus in vitro but this occurred at a pH below 3 and thus the action of this bile salt could not affect exsheathment in the host.     

Cryopreservation of the first-stage larvae of trichostrongylid nematode parasites

First stage (L1) larvae of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta can be cryopreserved in the presence of DMSO using a two-step freezing protocol involving an initial period at −80°C prior to transfer to liquid nitrogen. Thawed L1 larvae continue development in vitro producing third stage (L3) larvae that are infective to sheep when dosed per os. Establishment rates for L3 larvae grown from thawed L1 larvae were 40 and 80% for H. contortus and T. colubriformis, respectively. There was no difference in survival or infectivity between benzimidazole (BZ)-susceptible and BZ-resistant H. contortus isolates and cryopreservation caused no shift in their BZ-resistance status as indicated in an in vitro larval development assay. Cryopreservation also had no effect on the sensitivity of these isolates to the avermectins or levamisole in vitro. High survival rates (60–70%), good levels of establishment and the stability of anthelmintic resistance status of isolates indicate that little if any selection occurs during the cryopreservation process. L1 larvae of all 3 species have been successfully recovered after 16 months storage in liquid nitrogen, cultured to the L3 stage and established in sheep.     

Experimental American leishmaniasis and Chagas'' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi

, and 1988. Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi. International Journal for Parasitology 18: 1053–1059. Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) previously infected with either Leishmania braziliensis braziliensis or L. b. panamensis were challenge infected with blood-form trypomastigotes of Trypanosoma cruzi (Brazil strain). Monkeys previously infected with T. cruzi were challenged with stationary phase promastigote forms of L. b. braziliensis. Monkeys were examined during the course of challenge for evidence of infection, electrocardiographic alterations and parasite-specific antibody responses. T. cruzi epimastigotes were cultured from the blood of monkeys up to 3 months after challenge with this parasite. Unulcerated cutaneous lesions appeared and persisted in monkeys challenged with L. b. braziliensis. The formation of satellite lesions was observed in one monkey. Increased QRS intervals were not observed in T. cruzi challenged monkeys without prior cardiac irregularities and QRS left axis shifts were observed in only two of these monkeys. Elevated titers of parasite binding IgM and IgG specific for both T. cruzi and L. braziliensis were observed in all monkeys following challenge. These results indicate that prior infection with T. cruzi or L. braziliensis does not protect against heterologous challenge infection with these organisms. However, prior infection with Leishmania parasites may provide some protection against chagasic cardiopathies.     

Development and validation of an in vitro Trichostrongylus colubriformis motility assay

Development and validation of an in vitro Trichostrongylus colubriformis motility assay. International Journal for Parasitology 17: 1441–1444. An in vitro Trichostrongylus colubriformis motility assay involving the use of a micromotility meter has been developed and validated. Four commercially available ruminant anthelmintics (albendazole, ivermectin, levamisole hydrochloride, and coumaphos) and an investigational hydrazone compound (p-toluoyl chloride phenylhydrazone) were evaluated in this assay at four concentrations each. At 100 μg ml^-1, all five treatments significantly (P 0.05) reduced the motility of ensheathed L-3 T. colubriformis larvae, thereby indicating anthelmintic activity. At this concentration, coumaphos was significantly less active than any of the other four treatments. At 10 μg ml^-1 albendazole, ivermectin, levamisole hydrochloride and the hydrazone compound were active, but coumaphos was not. At 1 μg ml^-1 albendazole, ivermectin and levamisole hydrochloride remained significantly active, but neither coumaphos nor the hydrazone compound showed significant activity. At all three of the higher concentrations (1,10 and 100 μg ml^-1), levamisole hydrochloride indicated greater activity than any of the other treatments. This difference was statistically significant at the 1 and 10 μg ml^-1 concentrations. None of the five treatments showed significant activity at the lowest concentration (0.1 μg ml^-1). The in vitro T. colubriformis motility assay proved to be sensitive, accurate, rapid, and repeatable. This assay system should be another valuable addition to the tests used to identify potential anthelmintics, monitor helminth resistance to drugs, and define the kinetics and mode of action of drugs.     

The development of Taenia pisiformis in various definitive host species

In pups experimentally infected with T. pisiformis evagination and attachment of cysticerci occurred in the small intestine, proglottization was initiated 3–5 DAI and gravid cestodes were recovered on and after 35 DAI. Establishment in fox cubs (Vulpes vulpes) was similar but only a few worms had become gravid by 70 DAI. T. pisiformis developed slowly in kittens and although reaching sexual maturity 21 DAI, infections were lost before worms became gravid. Cysticerci fed to ferrets (Mustela putorius) evaginated in the small intestine but failed to attach and passed rapidly into the colon. No significant host-induced effects on morphological features of the parasites, apart from their sizes and rates of development, were detected.     

High levels of congenital transmission of Toxoplasma gondii in a commercial sheep flock.

Our current understanding of congenital transmission of Toxoplasma gondii from ewe to lamb dictates that infection frequently results in abortion and the death of the developing foetus, that the birth of live infected lambs occurs rarely and that the cat is the predominant source of infection in ewes. Using direct polymerase chain reaction detection of T. gondii, we report high levels of congenital transmission occurring in a commercially managed sheep flock. We sampled foetal-derived placental tissue and tissues from aborted lambs and showed that congenital transmission was detected in these tissues from 61% of all pregnancies. Where pregnancies resulted in the death of one or more lambs, T. gondii was detected in the lamb tissue for all but one of 18 (94%) pregnancies. Of the successful pregnancies resulting in the birth of live lambs we were able to detect T. gondii in foetal-derived placental tissue from 37 of 70 (42%) pregnancies. These results show that congenital transmission is occurring in a high percentage of lambings including normal healthy lambings, at this farm, suggesting that this route of transmission from generation to generation may be much more significant than that reported previously. These results may have implications for sheep husbandry and future epidemiological studies of T. gondii.     

The pathogenic importance of Chabertia ovina (Fabricius, 1788) in experimentally infected sheep

Pathogenicity studies in sixteen 4-month-old Merino cross wethers maintained on a low plane of nutrition and given 400, 1000 and 2000 C. ovina larvae showed fifth stage and adult worms to be more pathogenic than third and fourth stage larvae. Clinical signs were first detected 26–28 days after infection, a time which coincides with the first attachment of fifth stage larvae to the colonie mucosa. There was no evidence that the parasite caused anaemia at these levels of infection, but a protein-losing enteropathy associated with hypoalbuminaemia and weight loss was demonstrate at 6 weeks in sheep infected with 2000 larvae. The sheep's resistance appeared to be unimpaired by the low plane of nutrition and a marked eosinophilic response was recorded in both blood and tissues.     

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.     

Observations on the cross-immunity between Theileria lawrencei (Serengeti) and Theileria parva (Muguga) in cattle

Observations on the cross-immunity between Theileria lawrencei (Serengeti) and Theileria parva (Muguga) in cattle. Internationaljournal for Parasitology 3: 723–728. Cattle immunized against Theileria parva (Muguga) showed little resistance to Theileria lawrencei (Serengeti) stabilate challenge, while cattle immune to T. lawrencei (Serengeti) were fully resistant to challenge with T. parva (Muguga) stabilate. Cattle inoculated with cultured lymphoid cells infected with T. lawrencei (Serengeti) macroschizonts survived a subsequent T. lawrencei (Serengeti) stabilate challenge.     

Synergism and antagonism between two trematode species in the snail Lymnaea rubiginosa

Synergism and antagonism between two trematode species in the snail Lymnaea rubiginosa. Internationaljournal for Parasitology 3: 729–733. Sporocysts of Trichobilharzia brevis in the snail exerted a synergistic effect on sporocysts of Echinostoma hystricosum: The rate of infection with E. hystricosum was much higher in snails harboring T. brevis than in control snails with no other infection. Rediae of E. hystricosum and sporocysts of T. brevis were antagonistic, the predatory rediae consuming the sporocysts and ultimately eliminating T. brevis from the snail. Once a snail was occupied by E. hystricosum it could not be superinfected by T. brevis.     

Nippostrongylus brasiliensis: changes in plasma levels of gastrointestinal hormones in the infected rat

Plasma concentrations of gastrointestinal hormones were measured by radioimmunoassay in fasted rats 9 days after infection with a range of doses of Nippostrongylus brasiliensis. Values for infected rats fed ad libitum were compared with those of weight matched, pair fed, uninfected rats to control for the possible effects of dose-dependent reductions in food intake associated with infection. The plasma concentrations of some of the gastrointestinal hormones in infected rats were very different from those of their pair fed partners. The magnitude and direction of the changes varied according to the hormone being examined. Plasma concentrations of gastrin and pancreatic polypeptide were similar in pair fed and infected rats at all doses used. For the other hormones assayed, infection was associated with dose-related changes. The plasma concentrations of cholecystokinin and insulin were slightly but significantly reduced in infected rats. In contrast, secretin, enteroglucagon, and pancreatic glucagon concentrations were markedly increased. At the highest dose given (52 larvae/g body wt), the plasma levels of secretin and enteroglucagon in infected rats were elevated 9 X and 15 X, respectively. A comparison of the changes seen in N. brasiliensis-infected rats with those reported for other helminth infections revealed striking differences. The possible etiology of alterations in plasma gastrointestinal hormone concentrations and their contribution to the pathological changes seen in animals infected with helminths are discussed.