The responses of a tropical breed of domestic rabbit, Oryctolagus cuniculus, to experimental infection with Trichostrongylus colubriformis

Clinical, parasitological and pathological responses of a tropical out-bred domestic rabbit to experimental Trichostrongylus colubriformis infection were used to evaluate its suitability as a laboratory host and model for studying the host-parasite relationships of T. colubriformis. In the first experiment, three groups each of 16, predominantly juvenile male, 8- to 10-week-old rabbits were given a single pulse infection with 500, 5000 or 25000 infective larvae (L3) of T. colubriformis, to represent low, medium and high levels of infection, respectively. A fourth group of 16 rabbits of similar age formed the uninfected controls. In the second experiment, two groups of 10 juvenile (8- to 10-week-old) and 10 adult (8- to 10-month-old) rabbits were similarly infected with 20000 L3, with appropriate naive controls. Prepatency was 14 and 16 days and peak faecal egg counts occurred on days 24 and 20 after infection in young and adult rabbits respectively. Peak worm counts occurred on day 14 in both age groups and at all levels of infection. Subsequently, parasite burdens declined in a highly significantly dose- and age-dependent manner. At low and moderate levels of infection, approximately 83-98% of worms were recovered from the first 60 cm of the small intestine. Worm fecundity was also significantly influenced by host age and larval dose. Host age also had a significant effect on worm length. Infections with T. colubriformis were associated with a highly significant loss of body weight, accompanied by anorexia, diarrhoea and 25% mortality at high dose levels during the patent period of infection. There were no significant changes in packed cell volume and eosinophil counts at all ages and levels of infection but significant lymphocytosis occurred at the high dose level between days 7 and 21. Parasite-specific serum IgG responses were not related to worm burden. Overall, data showed that this miniature, docile and relatively inexpensive breed of rabbit is a potentially valuable laboratory host for studying T. colubriformis infections. The larval dose, duration of infection and host age were major determinants of host responsiveness to primary infections in this rabbit genotype.     

In vivo and in vitro egg production by Nematospiroides dubius during primary and challenge infections in resistant and susceptible strains of mice

Experiments were conducted to examine adult worm burdens, fecal egg output, and in vitro fecundity of Nematospiroides dubius in resistant LAF1 and susceptible CBA mice 12, 15, 18, and 21 days following primary and challenge infections. A strong correlation was obtained on the number of eggs produced by worms cultured in vitro and the egg production as assessed by fecal egg count. Worm counts, fecal egg counts, and in vitro fecundity were similar on all days studied following a primary infection in both mouse strains. However, after challenge infection, LAF1 mice showed lower worm burdens, fecal egg output, and in vitro egg production when compared to CBA mice. Although the egg production of surviving female worms from immune LAF1 mice was decreased, it never fell below a threshold of 100 eggs/day. The reduced fecundity may be a manifestation of a general anti-worm response rather than responses directed specifically at worm reproduction.     

Response to re-infection with Brachylaima cribbi in immunocompetent and immunodeficient mice

The course of infection in C57BL/6J mice re-infected with Brachylaima cribbi was assessed by comparing faecal egg excretion of re-infected mice with age- and sex-matched mice receiving a primary infection only. For both male and female mice there was a significant reduction in the mean number of eggs per gram of faeces at the peak of infection 4 weeks after the challenge infection compared with mice receiving a primary infection only. There was no significant difference in the duration of the infection. This experiment was repeated using age-matched male mice but on this occasion all mice were killed and dissected 4 weeks after the challenge infection and mean eggs per gram of faeces, worm burden and fecundity determined. There was no significant difference in the worm burdens of the re-infected mice compared with age-matched animals receiving a primary infection only. However, there were significant differences in the mean faecal eggs per gram and worm fecundity with the challenge infection group having lower egg counts and reduced fecundity. An enzyme-linked immunosorbent assay using whole worm antigens was developed and used to determine mouse anti-B. cribbi serum antibody levels during the course of infection. Anti-B. cribbi serum antibody absorbance ratios increased six- to sevenfold by 4 weeks after a primary infection beyond which a constant level was maintained. The course of challenge infection in non-obese diabetic severe combined immunodeficient mice showed no significant differences in egg excretion, worm burden or fecundity when primary and challenge infections were compared. These results indicate that the immune response invoked by a previous B. cribbi infection in immunocompetent mice affects fecundity but does not affect the establishment or duration of infection.     

Population dynamics of the parasitic stages of Oesophagostomum dentatum in pigs in single and trickle infections

A mathematical model was developed to describe the dynamics of the parasitic stages of Oesophagostomum dentatam in pigs. An immigration-death model with constant establishment, development and death rates was fitted to L3, L4 and adult worm burdens observed in a single-infection experiment. Female worm length was modelled by a function of worm age and total worm burden, while worm egg production (eggs per gram faeces per female worm) was modelled by a function of worm age and worm length. The model was then used to predict worm burdens observed in a trickle-infection experiment. The predicted worm burdens were much higher than those observed, suggesting that worm death rates were higher during the trickle infection. After increusing worm death rates to fit the observed worm burdens, female worm lengths and egg production in the trickle infection were predicted. At the medium- and high-dose rates, predicted worm lengths and, thus, egg preduction were lower than observed, while at the low-dose rate predicted egg production was too high. It appeared that in the trickle infections, total worm burden had less influence on observed female worm length and egg production than in the single infections. The results suggest that the demography of O. dentatum in pigs differs between single and trickle infections.     

Is the establishment rate and fecundity of Haemonchus contortus related to body or abomasal measurements in sheep?

The relationship among parasitological parameters, abomasal size and body size measurements was investigated in lambs following an experimental infection with Haemonchus contortus. In total, 100 lambs from five different genotypes (German Merino (GM), Texel × GM, Suffolk × GM, German Blackhead Mutton × GM and Ile de France × GM) were experimentally infected with 5000 infective third stage larvae of H. contortus at the time of weaning at 12 weeks of age. Four and six weeks after infection, individual faecal samples were collected for estimation of faecal egg counts (FECs). Furthermore, wither height, shoulder width, heart girth, loin girth and body length were taken at 18 weeks of life. Lambs were slaughtered and necropsied 7 weeks post-infection, and worm counts, abomasal volume and surface area were determined. Positive correlations were found between different body size parameters, body weight and abomasal sizes. FEC and worm counts were not significantly correlated either with body size parameters or with abomasal size. The mean worm burden was higher in GM than in crossbred lambs. There was no significant difference in abomasal size between GM and crossbred lambs. The results suggest that the variations between animals in worm burden following an experimental infection with H. contortus (worm resistance) are not influenced by body size parameters or abomasal sizes. Therefore, other factors, including genetic-based differences in resistance, must cause these findings between and within breeds.     

Retention and egg production of Microphallus pygmaeus in mice: the influence of the adrenal cortex

Fewer adult Microphallus pygmaeus are recovered from the small intestine of adrenalectomized male mice than from sham-operated counterparts. Preinfection intraperitoneal injection of 2 IU or 4 IU ACTH daily for 7 days increases the initial primary worm burden in male mice. Preinfection intramuscular injection of 325 micrograms corticosterone daily for 7 days increases the initial worm burden and alters the subsequent rate of decline and duration of the primary infection. However, egg production is unaffected. The changes in parasite numbers are attributed to the immunosuppressive action of corticosterone.     

A mechanistic model of developing immunity to Teladorsagia circumcincta infection in lambs

Acquired immunity influences the severity of parasitic disease, but modelling the effects of acquired immunity in helminth infections has proved challenging. This may be due to a lack of suitable immunological data, or to the perceived complexity of modelling the immune response. We have developed a model of T. circumcincta infection in domestic sheep that incorporates the effects of acquired immunity on parasite establishment and fecundity. A large data set from commercially managed populations of Scottish Blackface sheep was used, which included relationships between IgA activity and worm length, and between worm length and fecundity. Use was also made of a recently published meta-analysis of parasite establishment rates. This realistic but simple model of nematode infection emulates observed patterns of faecal egg counts. The end-of-season faecal egg counts are remarkably robust to perturbations in the majority of the parameters, possibly because of priming of the immune system early in the season, reducing parasite establishment and growth and, therefore, faecal egg counts. Lowering the amount of early infection leads to higher end-of-season egg counts. The periparturient rise in egg counts in ewes appears to have an important role in supplying infection for the priming of the immune response. This feedback in the immune priming suggests that nematode infections may be difficult to eliminate.     

Faecal egg counts provide a reliable measure of Trichostrongylus tenuis intensities in free-living red grouse Lagopus lagopus scoticus

The reliability of different egg counting methods for estimating the intensity of Trichostrongylus tenuis infections in red grouse, Lagopus lagopus scoticus, was investigated in the autumn, when grouse may harbour high parasite intensities. Possible limitations to the use of these methods were also examined. Faecal egg counts were found to accurately estimate T. tenuis worm intensities, at least up to an observed maximum of c. 8000 worms. Two egg counting methods (smear and McMaster) gave consistent results, although the exact relationship with worm intensity differed according to the method used. Faecal egg counts significantly decreased with increasing length of sample storage time, but egg counts were reliable for estimating worm intensity for three weeks. The concentration of eggs in the caecum was also found to reliably estimate worm intensity. However, egg counts from frozen gut samples cannot be used to estimate worm intensities. These results conclude that, despite some limitations, faecal and caecum egg counts provide useful and reliable ways of measuring T. tenuis intensities in red grouse.     

Parasitological and immunological responses of genetically resistant Merino sheep on pastures contaminated with parasitic nematodes.

One hundred and twenty lambs were grazed continuously from weaning until 9 months of age on 12 plots contaminated with larvae of three nematode species (Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta). The lambs were sired by either a genetically resistant ram or susceptible rams (determined by the response of previous progeny to artificial H. contortus infection). Half the resistant and half the susceptible lambs were given strategic anthelmintic treatment and the remainder remained untreated. Faecal egg counts and blood packed cell volume were measured frequently in all animals. One and 5 months after weaning, two lambs from each plot were slaughtered, and worm burdens and larval establishment rates of the three species of nematode were estimated. At the second slaughter, leukotriene levels and larval migration inhibitory (LMI) activity were measured in mucus collected from the small intestine. The dominant species in all faecal samples and the gastrointestinal tract was T. colubriformis. Lambs of the resistant genotype had lower faecal worm egg counts, lower worm burdens and higher levels of resistance to larval establishment. There were no differences in larval migration inhibition (LMI) activity, but resistant lambs had higher levels of the leukotriene LTC4/D4/E4. Further, the resistant genotype, identified on responsiveness to artificial infections with H. contortus, was more resistant to infections of three important species acquired naturally from contaminated pastures. All these genetic differences were maintained while the lambs were subject to strategic anthelmintic treatment.     

Experimental infection of pigs with three dose levels of Trichuris suis

The objective of the study was to follow the course of Trichuris suis infection in pigs given infective eggs at low (400 eggs), medium (4,000 eggs) and high inoculation dose (40,000 eggs), respectively. Interestingly, despite a 100-fold difference in dose level no significant difference was found in either blood parameters, total faecal egg excretion, fecundity or worm burdens at necropsy 12 weeks post inoculation. The highest and lowest median faecal egg output was found in the medium and high dose group, respectively. With increasing dose level, worm size, establishment and prevalence of T. suis positive pigs decreased while worms were dislocated aborally. In addition there was a highly significant correlation between female worm burden and faecal egg excretion.     

Detection of a quantitative trait locus associated with resistance to Ascaris suum infection in pigs

Helminths almost invariably have an over-dispersed distribution in the host population. Human and animal studies have provided evidence suggesting that a large part of this variation is due to host genetic factors. Recently, the heritability for roundworm (Ascaris suum) infection levels in pigs was estimated to be 0.45. We used single nucleotide polymorphism markers to perform a whole-genome scan on 195 pigs experimentally infected with A. suum. A putative quantitative trait locus for worm burden on chromosome 4 covering 2.5 Mbp was identified by measured genotype analysis, although none of the SNPs reached genome-wide significance. To validate the putative quantitative trait locus, we genotyped two of the SNPs within the region in unrelated, informative animals exposed to experimental or natural infections and from which we had worm counts and/or faecal egg counts; the validation studies showed that one of the SNPs (TXNIP) was associated with total worm burden (P < 0.001) and adult worm burden(P < 0.0001), whereas the other SNP (ARNT) was associated with adult worm burden (P < 0.025) in these populations. We were thus able to confirm the existence of the quantitative trait locus on chromosome 4.This is to our knowledge the first report of a quantitative trait locus associated with helminth burden in pigs.     

Class I antigens of the bovine major histocompatibility system are weakly associated with variation in faecal worm egg counts in naturally infected cattle

Three bulls selected for high faecal worm egg counts and three bulls selected for low faecal worm egg counts were mated to Africander-Hereford cross cows. Faecal worm egg counts were taken on four occasions from the 132 offspring. Also, each animal was typed for 32 class I antigens of the bovine major histocompatibility system (BoLA). Least squares analysis of variance showed that line, sex and some of the antigens were associated with differences in worm egg output in the faeces. After adjusting for the effects of line and sex, cattle with antigen W9 had about twice as many worm eggs in their faeces as cattle without W9; cattle with antigen CA45 had about half the concentration of faecal worm eggs as cattle without CA45. However, the antigen associations were of borderline significance at the 5% level and more work in additional populations is necessary to confirm these associations.     

Is the allocation of metabolisable protein prioritised to milk production rather than to immune functions in Teladorsagia circumcincta-infected lactating ewes?

It has been suggested that the periparturient breakdown of immunity to parasites has a nutritional basis. Our overall hypothesis is that it results from a prioritised scarce nutrient allocation to reproductive functions (e.g. milk production) rather than to immune functions. We tested this hypothesis by offering five levels of dietary metabolisable protein, ranging from 0.65 to 1.25 times their assumed requirements, for 4 weeks post-parturition to twin-rearing Greyface ewes, experimentally infected with Teladorsagia circumcincta. We hypothesised that the initial increments of metabolisable protein supply would increase milk production without affecting the degree of breakdown of immunity whilst later increments would reduce the degree of breakdown of immunity. The first two increments of metabolisable protein supply indeed increased milk production and did not affect final worm burdens, but in contrast to the expectation, reduced faecal egg counts and total egg output. The last two increments of metabolisable protein supply did not further affect milk production and egg output, but resulted in reduced final worm burdens. Metabolisable protein supply did not affect plasma IgG and IgE antibody against somatic L(3) antigen but the first three increments reduced plasma pepsinogen and plasma IgA antibody. The last increment did not further reduce plasma pepsinogen but increased plasma IgA. Metabolisable protein supply did not systematically affect abomasal mucosal mast cell, globule leukocyte and eosinophil counts. Our results support the view that the priority of scarce metabolisable protein allocation to milk production over immune functions may be gradual rather than absolute. The contrast between effects of metabolisable protein supply on faecal egg count and final worm burden points towards the possibility that if different effector responses regulate fecundity and worm expulsion, then they would differ in their sensitivity towards changes in the degree of nutrient scarcity.     

Rapid improvement of immunity to Teladorsagia circumcincta is achieved through a reduction in the demand for protein in lactating ewes

Protein supplementation can improve the resistance to parasites of periparturient ewes, as indicated by reduced nematode egg excretion and worm burdens. However, the rate at which this improvement can occur is largely unknown. We investigated the rate of improvement by assessing temporal changes in faecal egg counts after we experimentally reduced nutrient demand. Three groups of nine pregnant ewes each were trickle infected with Teladorsagia circumcincta from day(-70) to day(16) (parturition is day0). Two groups of twin-rearing ewes were fed at 0.8 (L22) or 1.2 (H22) times their assumed metabolizable protein requirements, and a third group was fed the same daily food allowances as L22 ewes, but one of their lambs was removed on day10 (L21). Ewes were slaughtered on day21 to assess worm burdens, in vitro larval establishment on abomasal explants, and mucosal inflammatory cells. Faecal egg counts of L22 ewes were higher than H22 ewes throughout lactation. After the removal of one lamb, faecal egg counts of L21 ewes decreased within 5 days to levels similar to H22 ewes. Relative to L22 ewes, L21 and H22 ewes had lower worm burdens, parasite per capita fecundity and in vitro establishment rates of both T. circumcincta and Haemonchus contortus. Mucosal mast cell and eosinophil counts were similar for all ewes, but H22 ewes had higher globule leukocyte counts than L22 and L21 ewes. The data suggest that a reduction in protein demand can rapidly improve periparturient immunity to T. circumcincta. This may be associated with increased parasite expulsion, reduced fecundity and non-parasite specific reduction of in vitro larval establishment.     

Population dynamics of Trichostrongylus colubriformis in sheep: the effect of infection rate on the establishment of infective larvae and parasite fecundity

The establishment of Trichostrongylus colubriformis was estimated in helminthologically naive 20-week-old Merino sheep given third stage infective larvae (L3) at rates of 2000, 632 or 200 L3 per day, 5 days per week. After varying periods of continuous L3 intake, a levamisole-susceptible strain of T. colubriformis was replaced with a highly resistant strain for 1 week. The animals were then treated with levamisole to remove the susceptible population, and establishment of the cohort of resistant worms was estimated. In previously uninfected sheep, approximately 65% of the L3 given in the first week became established as adults. This fell to low levels (less than 5%) after 7, 10 and 14 weeks of continuous L3 intake for the high, medium and low infection rates, respectively. At the low infection rate, establishment remained at maximum levels for the first 4 weeks, but then fell at a rate similar to that observed for the higher infection rates. This implied that a threshold of worm exposure was required before resistance to establishment developed. Parasite egg production, expressed as eggs per gram of faeces, was proportional to infection rate and is explained by higher worm burdens occurring at high infection rates. However, estimates of fecundity in eggs per female per day showed the opposite relationship with rate of infection. Fecundity stayed high (approximately 600) for 5 weeks at the low infection rate but only maintained this level for 3 weeks and 1 week at the medium and high rates, respectively. This suggests that fecundity, like establishment, was similarly affected at threshold levels of immunological recognition.     

Variation in resistance to haemonchosis: selection of female sheep resistant to Haemonchus contortus.

Seventy female lambs (6-7 months old) which were exposed to natural infections of Haemonchus contortus were designated as responders or non-responders on the basis of 10 weekly cumulative faecal egg counts. Selected responder and non-responder lambs were treated with ivermectin, housed separately and 6 weeks post-housing, seven lambs from each group were given a trickle infection of Haemonchus contortus at 1000 L3 daily for 5 days per week up to 2 weeks and examined weekly for 10 weeks after first infection. Analysis of data revealed significantly lower mean faecal egg counts and non-significantly less weight loss in responder than non-responder lambs. Mean values of haemoglobin, packed cell volume, total serum protein and peripheral eosinophil counts were significantly higher in responders than non-responders. In contrast, serum pepsinogen concentration was significantly less in responders than in non-responders. At 10 weeks post-infection, there were fewer pathological lesions and significantly lower worm burdens in responders than in non-responders. These results demonstrate a distinct resistance in responders to Haemonchus contortus infection.     

Formulation of a strategy for the application of Duddingtonia flagrans to control caprine parasitic gastroenteritis

Experiments on the influence of egg density and varying quantities of chlamydospores on the nematode-trapping ability of Duddingtonia flagrans, influence of D. flagrans on the larval translation of gastrointestinal nematodes, doses of chlamydospores required for the effective control of gastrointestinal nematodosis and the epidemiology of nematode parasites were conducted in goats, which generated baseline data required for strategic application of the biocontrol agent. The nematode-trapping ability of D. flagrans, measured by numerical enumeration of infective third-stage larvae developed in the faecal culture, revealed that the efficacy is dependent on both nematode egg and chlamydospore density. Pasture plot studies revealed that D. flagrans, if deposited at the same time as nematode eggs, prevents translation of third-stage larvae of caprine nematodes from the faecal pats onto the grass blades. Feeding of 1 x 106 chlamydospores per kg body weight and above to goats virtually eliminated larvae from the pasture. Application of as few as 1 x 104 and 1 x 105 chlamydospores per kg body weight had a profound impact on larval recovery. The effect persisted as long as the chlamydospores were fed. Monthly faecal worm egg counts of adult goats maintained under a semi-intensive management system on the Chhattisgarh plain and pasture larval burden revealed that June to August were the months of high risk for nematodosis. Haemonchus was the dominant species recorded throughout the year. The present data can best be utilized by formulating a strategic control measure when the larval challenge to the animal is maximum (June to August), so as to prevent establishment of patent infection. The observations reinforced the strategy to be adopted for nematode parasite control in goats by applying the biocontrol option at the onset of the monsoon.     

Experimental infections with Cooperia oncophora (Railliet, 1918) in calves. Results of single infections with two graded dose levels of larvae.

Two experiments were carried out in which calves reared parasite-free were infected with a single dose of 3rd-stage larvae of Cooperia oncophora. In the first experiment the calves received 20 000 or 200 000 infective larvae and they were autopsied 28 or 56 days after the infection. In the second experiment the doses were the same but the animals were killed 14, 84 or 140 days after infection. If a dose of 20 000 was given, clinical signs were never observed, while at a dose level of 200 000 the weight gain was less on 56 and 84 days after the infection compared with the low-infected groups or the control animals. After 170 days the differences in weight gain were compensated. Faecal egg output was higher in the 200 000 groups only in the first period of patency, thereafter the calves in the 20 000 groups produced more. No obvious differences between the two infection levels were observed with regard to the haematological data. In the low-infected groups worm counts were only slightly lower when the results after 28 days were compared with those after 56 days. Also, the worm numbers after 14 days were almost equal to those after 84 days, while at 140 days 1 animal still had the same number, the other one had lost its worm burden. In the high-infected groups the worm loss was much quicker. After 28 days a great part of the population had already been lost. Obviously, at the 200 000 level the reaction of the host against the parasite was much stronger. Adult worms were expelled at a higher rate than early 4th-larval stages. In the first experiment worm measurements revealed differences between the length of females, males and spicules of males, these being significantly longer in the low-infected groups. Analysis of the distribution of worms over the small intestine showed that in the low-infected groups worms were mainly restricted to the first 6 metres. In the high-infected groups the worm population was distributed more evenly over the whole small intestine.     

Congenital transmission of Schistosoma japonicum in the rabbit

Fourteen pregnant rabbits were each infected with 300 cercariae of Schistosoma japonicum and divided into two groups. Group M (n = 8) was infected during mid-gestation (the organogenetic stage) and group L (n = 6) was infected during late-gestation (the post-organogenetic stage). Mother rabbits and rabbit kittens were killed 45-60 days after infection and perfused in order to obtain worm counts. Furthermore, faecal egg counts and tissue egg counts from livers were obtained from the mother rabbits as well as the rabbit kittens. All mother rabbits became infected harbouring 207.6 +/- 20.2 and 220.0 +/- 27.5 adult worms in group M and L, respectively. In groups M and L, 13.5% and 46.7% of the kittens were infected, respectively. In 12 of 14 litters at least one kitten was infected. The infected kittens harboured between one and three adult S. japonicum. The livers of the kittens infected with a worm pair displaced lesions as a result of egg deposition. The results, therefore, show that congenital transmission of S. japonicumcan occur in rabbits. The close anatomical resemblance between the rabbit and human placenta may be indicative of the presence of congenital transmission of S. japonicum infection in humans.     

Schistosoma japonicum: day to day variation in excretion and hatchability of parasite eggs in the domestic pig, Suis suis

The aim of the present study was to describe the course of an Schistosoma japonicum infection in individual pigs over time, with special regards to fecal egg counts and egg hatchability, emphasizing the extent of variation during and between days. Five specific pathogen free Danish Landrace/Yorkshire/Duroc crossbred male pigs were each given 3500 cercariae intramuscularly. From day 36 to 62 post infection, fecal samples were collected from each pig, morning and evening. Pigs were perfused 62 days post infection. Actual fecal egg counts, miracidial counts, and worm burdens were determined. The trend of fecal egg excretion was adequately described with a third order polynomial and logarithmic link function. Miracidial counts were related directly to morning fecal egg counts through a simple linear function. The study revealed a fairly similar overall pattern of egg excretion for all pigs, showing an increase in egg excretion until week 8, followed by a marked reduction to almost zero. In general, large fluctuations around this trend were seen for all pigs, as revealed in variations in egg counts within the same day as well as between days. However, as revealed by the estimated model lines, these fluctuations are due to random variation in egg density in stools rather than being caused by biologically determined cycles. An exact time for both first occurrence of eggs in feces (days 35-38 post infection) and for peak egg counts (days 44-48 post infection) was predicted for each individual pig. Furthermore, the model revealed that miracidial counts can be related directly to the expected fecal egg counts of the same day through a parameter, which represents hatchability. This hatchability parameter was found to be independent of time post infection for each pig, but large individual differences were seen in hatchability between the pigs.