Circumcincta Larvae Research Articles

Characterisation of a niche-specific excretory\u2013secretory peroxiredoxin from the parasitic nematode Teladorsagia circumcincta

BackgroundThe primary cause of parasitic gastroenteritis in small ruminants in temperate regions is the brown stomach worm, Teladorsagia circumcincta. Host immunity to this parasite is slow to develop, consistent with the ability of T. circumcincta to suppress the host immune response. Previous studies have shown that infective fourth-stage T. circumcincta larvae produce excretory–secretory products that are able to modulate the host immune response. The objective of this study was to identify immune modulatory excretory–secretory proteins from populations of fourth-stage T. circumcincta larvae present in two different host-niches: those associated with the gastric glands (mucosal-dwelling larvae) and those either loosely associated with the mucosa or free-living in the lumen (lumen-dwelling larvae).ResultsIn this study excretory–secretory proteins from mucosal-dwelling and lumen-dwelling T. circumcincta fourth stage larvae were analysed using comparative 2-dimensional gel electrophoresis. A total of 17 proteins were identified as differentially expressed, with 14 proteins unique to, or enriched in, the excretory–secretory proteins of mucosal-dwelling larvae. One of the identified proteins, unique to mucosal-dwelling larvae, was a putative peroxiredoxin (T. circumcincta peroxiredoxin 1, Tci-Prx1). Peroxiredoxin orthologs from the trematode parasites Schistosoma mansoni and Fasciola hepatica have previously been shown to alternatively activate macrophages and play a key role in promoting parasite induced Th2 type immunity. Here we demonstrate that Tci-Prx1 is expressed in all infective T. circumcincta life-stages and, when produced as a recombinant protein, has peroxidase activity, whereby hydrogen peroxide (H2O2) is reduced and detoxified. Furthermore, we use an in vitro macrophage stimulation assay to demonstrate that, unlike peroxiredoxins from trematode parasites Schistosoma mansoni and Fasciola hepatica, Tci-Prx1 is unable to alternatively activate murine macrophage cells.ConclusionsIn this study, we identified differences in the excretory–secretory proteome of mucosal-dwelling and lumen-dwelling infective fourth-stage T. circumcincta larvae, and demonstrated the utility of this comparative proteomic approach to identify excretory–secretory proteins of potential importance for parasite survival and/or host immune modulation.

Niche-specific gene expression in a parasitic nematode; increased expression of immunomodulators in Teladorsagia circumcincta larvae derived from host mucosa

Metazoan parasites have to survive in many different niches in order to complete their life-cycles. In the absence of reliable methods to manipulate parasite genomes and/or proteomes, identification of the molecules critical for parasite survival within these niches has largely depended on comparative transcriptomic and proteomic analyses of different developmental stages of the parasite; however, changes may reflect differences associated with transition between developmental stages rather than specific adaptations to a particular niche. In this study, we compared the transcriptome of two fourth-stage larval populations of the nematode parasite, Teladorsagia circumcincta, which were of the same developmental stage but differed in their location within the abomasum, being either mucosal-dwelling (MD) or lumen-dwelling (LD). Using RNAseq, we identified 57 transcripts which were significantly differentially expressed between MD and LD larvae. Of these transcripts, the majority (54/57) were up-regulated in MD larvae, one of which encoded for an ShKT-domain containing protein, Tck6, capable of modulating ovine T cell cytokine responses. Other differentially expressed transcripts included homologues of ASP-like proteins, proteases, or excretory-secretory proteins of unknown function. Our study demonstrates the utility of niche- rather than stage-specific analysis of parasite transcriptomes to identify parasite molecules of potential importance for survival within the host.

Transcriptional profiling of the ovine abomasal lymph node reveals a role for timing of the immune response in gastrointestinal nematode resistance

Gastrointestinal nematodes are a serious cause of morbidity and mortality in grazing ruminants. The major ovine defence mechanism is acquired immunity, with protective immunity developing over time in response to infection. Nematode resistance varies both within and between breeds and is moderately heritable. A detailed understanding of the genes and mechanisms involved in protective immunity, and the factors that regulate this response, is required to aid both future breeding strategies and the development of effective and sustainable nematode control methods. The aim of this study was to compare the abomasal lymph node transcriptome of resistant and susceptible lambs in order to determine biological processes differentially expressed between resistant and susceptible individuals.Scottish Blackface lambs, with divergent phenotypes for resistance, were challenged with 30,000 Teladorsagia circumcincta larvae (L3), and abomasal lymph nodes recovered at 7 and 14days post-infection (dpi). High-throughput sequencing of cDNA from the abomasal lymph node was used to quantitatively sample the transcriptome with an average of 32 million reads per sample. A total of 194 and 144 genes were differentially expressed between resistant and susceptible lambs at 7 and 14 dpi respectively. Differentially expressed networks and biological processes were identified using Ingenuity Pathway Analysis. Genes involved in the inflammatory response, attraction of T lymphocytes and binding of leukocytes were more highly expressed in resistant animals at 7 dpi and in susceptible animals at 14 dpi indicating that resistant animals respond to infection earlier than susceptible animals. Twenty-four Single Nucleotide Polymorphisms (SNP) within 11 differentially expressed genes, were tested for association with gastrointestinal nematode resistance in the Scottish Blackface lambs. Four SNP, in 2 genes (SLC30A2 and ALB), were suggestively associated with faecal egg count.In conclusion, a large number of genes were differentially expressed in the abomasal lymph node of resistant and susceptible lambs responding to gastrointestinal nematode challenge. Resistant Scottish Blackface lambs appear to generate an earlier immune response to T. circumcincta. In susceptible lambs this response appears to be delayed. SNP in 2 differentially expressed genes were suggestively associated with faecal egg count indicating that differentially expressed genes may be considered candidate loci for mediating nematode resistance.

Breed differences in humoral and cellular responses of lambs to experimental infection with the gastrointestinal nematode Teladorsagia circumcincta.

While Texel lambs have increased resistance to infection with the gastrointestinal nematode Teladorsagia circumcincta compared to Suffolk lambs, the underlying resistance mechanisms are still unknown. The aim of this study was to compare parasitological, humoral and cellular responses of Texel and Suffolk lambs over time following a single experimental infection with T. circumcincta. Gastrointestinal nematode free (but not naïve) lambs received a single oral dose of 3 × 104 infective T. circumcincta larvae. The variables examined included worm burden, mucosal and serum IgA, abomasal mast cells and eosinophils, haematological parameters and plasma pepsinogen. Texel lambs had significantly lower worm burden on day 14 and lower plasma pepsinogen concentration from day 14 onwards than Suffolks and their response in mucosal IgA to infection occurred earlier. The results from the study suggest that an earlier local IgA response in the Texel contributes to the resistant characteristics of the breed, while the increased level of plasma pepsinogen in the Suffolk lambs implies greater abomasal tissue damage arising from the nematode infection.

Response to Teladorsagia circumcincta infection in Scottish Blackface lambs with divergent phenotypes for nematode resistance

The objective of this study was to identify Scottish Blackface lambs that were at the extremes of the spectrum of resistance to gastrointestinal nematodes and characterise their response to an experimental nematode challenge. Lambs (n=90) were monitored for faecal egg count (FEC) (2 samples from each of 2 independent natural infections). The most resistant (n=10) and susceptible (n=10) individuals were selected and challenged with 30,000 Teladorsagia circumcincta larvae (L3) at 9 months of age. Response to infection was monitored by measuring FEC, plasma pepsinogen, serum antibodies against nematode larval antigens and haematology profile, until necropsy at 71 days post infection. Worm burden, worm fecundity and the level of anti-nematode antibodies in abomasal mucosa were determined at necropsy.FEC was consistently higher in susceptible animals (P<0.05), validating the selection method. Worm fecundity was significantly reduced in resistant animals (P=0.03). There was also a significant correlation (r=0.88; P<0.001) between the number of adult worms and FEC at slaughter. There was no effect of phenotype (resistance/susceptibility) on plasma pepsinogen or on haematology profile. Phenotype had a significant effect on the level of anti-nematode IgA antibodies in serum (P<0.01), reflecting a higher peak in resistant animals at day 7 post infection.It is concluded that significant variation in the response to gastrointestinal nematode challenge exists within the Scottish Blackface population with resistant animals displaying significantly lower FEC, lower worm fecundity and higher concentration of anti-nematode IgA antibodies in serum.

Effect of the consumption of heather on incoming larvae and established population of Teladorsagia circumcincta in experimentally infected Cashmere goats

This study was performed in Cashmere goats that were experimentally infected with Teladorsagia circumcincta to investigate the effects of heather consumption on the establishment of incoming infective larvae (experiment 1) and on an adult nematode population (experiment 2). In experiment 1, 24 non-lactating goats were divided into 2 groups: heather-supplemented vs. non-supplemented. After 2 weeks of adaptation to the diet, all of the goats were experimentally infected with 6000 infective larvae of T. circumcincta. Twelve animals (6 controls and 6 supplemented with heather) were slaughtered at 6 days and at 3 weeks post-infection (pi). After slaughter, the worms were counted and the female worm fecundity and development were determined. Heather consumption was associated with a significant reduction in larval establishment at 6 days (P=0.033) and at 3 weeks (P=0.041) pi. No differences in worm counts between the slaughter times were found. In the goats slaughtered at 3 weeks pi, the number of eggs in utero and length of the female worms were significantly (P<0.001) lower than those of control group. In experiment 2, 24 non-lactating goats were experimentally infected with 10,000 T. circumcincta infective larvae daily for 5 consecutive days (total infection of 50,000 larvae). After 3 weeks, 2 groups were established: control and heather-supplemented. The faecal egg output of each animal was measured at 2-days interval during the experimental period. The goats were slaughtered after 3 weeks of heather administration for parasitological studies. Heather administration was associated with a significant decrease in egg excretion between 25 and 29 days pi. The worm counts were similar in both groups, but the female length and fecundity were significantly (P<0.001) lower in supplemented goats. These results show that heather consumption reduces the establishment of T. circumcincta larvae in goats and the development and fecundity of female adult parasites.

Sensitivity of periparturient breakdown of immunity to parasites to dietary protein source1

Effects of increased MP supply on the degree of periparturient relaxation of immunity (PPRI) in sheep may be dependent on quality of supplied MP. Here we tested the hypothesis that additional MP supply from rations based on xylose-treated soybean meal would be more effective than from rations based on faba beans in reducing the degree of PPRI, as indicated by nematode egg excretion. Twenty-four multiple-bearing ewes were trickle infected with Teladorsagia circumcincta larvae from d -56 to d 31 relative to start of lactation (d 0). From d -26 onwards, ewes were fed at either 0.8 (LP) or at 1.2 times their respective calculated MP requirements using either xylose-treated soybean (HPS) or faba beans (HPB). Litter size was adjusted to 2 lambs at parturition. Feeding treatments did not affect nematode egg excretion, ewe BW or BCS during late pregnancy (P > 0.10), but HPS and HPB ewes had reduced plasma pepsinogen concentrations (P = 0.003). During lactation, HPS and HPB feeding increased ewe BW gain (P < 0.001) and BCS (P = 0.017), and reduced plasma pepsinogen concentrations (P = 0.008) to the same extent, compared with LP feeding. However, only HPS feeding increased litter weight gain (P = 0.017) and reduced nematode egg excretion (P = 0.015), which were both similar between HPB and LP (P > 0.10). The results support the view that extra MP supply from xylose-treated soybean based rations is more effective in reducing parasitism than MP from faba bean-based rations, suggesting that protein source and/or quality are important factors to consider for the nutritional control of parasitism.

Attempts to immunize sheep against Teladorsagia circumcincta using fourth‐stage larval extracts

A ConcanavilinA (ConA)-binding fraction of a detergent-soluble membrane extract from Teladorsagia circumcincta (formerly Ostertagia circumcincta) fourth-stage larvae was isolated, and two vaccine trials were conducted with this preparation in groups of 7 worm-free sheep. All groups were challenged with a total of 5000 T. circumcincta larvae from 1 week after the final immunization and protection assessed by comparing the egg and worm counts, and length of developing worms, of the immunized groups with their respective controls. Immunization with the ConA-binding antigen induced high-titre serum antibody responses in both trials. However, no significant reduction in either egg count or worm burdens was observed in the vaccinated groups in either trial. It was concluded that detergent-soluble, ConA-binding extracts prepared from T. circumcincta fourth-stage larvae did not contain significantly protective antigens, despite the fact that an extract prepared in a similar manner from Ostertagia ostertagi had previously significantly protected calves against homologous challenge.

Does milk supply have long-term benefits for resistance and resilience to nematode parasites in sheep?

The long-term benefit of suckling for the resistance and resilience of lambs to Teladorsagia circumcincta infection was investigated in a 20-week after-lambing trial period; during which time the immune response was anticipated to be strengthening. In a two by two factorial arrangement, one factor was ‘suckling’ with one twin lamb from each of 34 pairs being either weaned (W−) at 39 days of age or allowed to continue suckling (S−) until necropsy at mean ages of either 84, 112 or 140 days. The second factor was ‘parasite infection’ with lambs in each ‘suckling’ treatment group either artificially challenged with an equivalent of 1000 L 3 T. circumcincta larvae d −1 (−I) from day 42 of age until necropsy or not artificially challenged (−N). All lambs and their dams grazed ryegrass pastures throughout the trial; these were intended to be relatively parasite safe, although worm burdens of SN and WN groups suggested there was a considerable parasite challenge from pasture from day 84 onwards. Despite this, suckled lambs consistently exhibited lower faecal egg counts (FECs; P < 0.05) and worm burdens at slaughter on days 84 and 112 ( P < 0.05 for both). Worms from suckled lambs appeared to have reduced egg production indicating either a direct effect of milk proteins on parasite fecundity or an indirect benefit of suckling through the provision of nutrients for enhance immune capacity. Lamb growth rate was reduced by weaning but not by artificial challenge ( P > 0.05). It was concluded that while the enhanced protein supply from suckling will facilitate greater growth rates in young lambs, weaning may not necessarily be associated with reduced resilience to T. circumcincta infection. Furthermore, suckling appears to assist in reducing worm burdens and parasite fecundity either through a direct anti-parasitic effect or through hastening the acquisition of immunity when lambs are exposed to larvae early in life.

Reduction of faecal worm egg count, worm numbers and worm fecundity in sheep selected for worm resistance following artificial infection with Teladorsagia circumcincta and Trichostrongylus colubriformis

We examined the changes to populations of Trichostrongylus colubriformis and Teledorsagia circumcincta in mature sheep selected for reduced faecal worm egg count (WEC). Worm resistant (n=19) and control (n=10) genotype sheep were penned and dosed with a total of 10,000 T. colubriformis and 10,000 T. circumcincta per week for 18-weeks. Sheep genotypes were from lines previously bred over 15 years for either low WEC (resistant) or non-selected (control). Weekly WEC and the proportion of larvae from each species in faecal culture were measured during weeks 3-17. Egg hatchability was assessed on a pooled faecal sample from worm resistant or control genotype sheep at weeks 7, 9, 10, 13, 14 and 17. At week 18 the number of adult and immature worms (early and late 4th, and 5th stage), and indicators of worm fecundity (female worm length and number of eggs in utero) were assessed at necropsy. Results show that resistant sheep had reduced mean WEC to only 18% of the control (P<0.05) and increased the proportion of T. circumcincta larvae in faecal culture during weeks 8-17 (P<0.10). Egg hatch assays indicated a slight reduction in the viability of eggs from worm resistant genotypes at weeks 14 (P<0.05) and 17 (P<0.10). At necropsy, resistant animals had 93% fewer adult T. colubriformis, 44% fewer adult T. circumcincta and had reduced indicators of fecundity in T. circumcincta by up to 40% (P<0.05). We observed no change in the number T. circumcincta worms but an 11% increase in the proportion of early 4th stage T. circumcincta larvae in resistant animals (P<0.05). There were different temporal patterns in WEC and different prediction equations for WEC from necropsy traits for the two sheep genotypes (P<0.05). Thus, our results suggest a changed host-parasite relationship in sheep selected for low WEC. We conclude that lower WEC is achieved through reduced number of adult worms for both species and reduced fecundity for T. circumcincta. These results support the hypotheses that worm resistant sheep with a strong immune function can regulate T. colubriformis by rejecting adult worms but that T. circumcincta is regulated through a combination of suppressed development and reduced female fecundity.

Merino ewes bred for parasite resistance reduce larval contamination onto pasture during the peri-parturient period

The peri-parturient period is crucial for controlling worms as the acquired immunity of ewes is disrupted, resulting in an increase in faecal worm egg counts. Two hypotheses were tested in this experiment – that ewes bred for worm resistance would have lower faecal worm egg counts than unselected control ewes, during late pregnancy and lactation, under similar but separate grazing areas; and also that numbers of infective nematode larvae would be lower on pastures grazed by resistant ewes than pastures grazed by unselected control ewes. Faecal samples were collected from resistant and unselected ewes in late pregnancy and early lactation, during the winter rainfall season, and analysed for numbers of Trichostrongylus colubriformis and Teladorsagia circumcincta. Pasture samples were taken 1 week before and 7 weeks after lambing started and analysed for infective larvae. In all sheep, worm egg counts rose 2 weeks prior to lambing and continued into lactation. Worm egg counts were significantly lower in the resistant ewes from 1 week before lambing to 2 weeks after lambing. There were no differences in egg counts between single- and twin-bearing ewes in the resistant line. However, twin-bearing control ewes had significantly higher egg counts than single-bearing control ewes. Following lactation, plots grazed by resistant ewes had substantially less contamination with T. colubriformis larvae, but there were no differences in numbers of T. circumcincta larvae. Our results demonstrate that sheep bred for worm resistance has lower worm burdens during the peri-parturient phase and that lambs born to resistant ewes face a lower larval challenge during their introduction to grazing. In our environment, selection for low worm egg counts has produced sheep highly resistant to T. colubriformis, but has had less impact on resistance towards T. circumcincta.

Development and validation of real-time PCR methods for diagnosis of Teladorsagia circumcincta and Haemonchus contortus in sheep

Parasitic gastroenteritis, a disease caused by parasitic nematodes, is of major concern to the sheep industry and threatens sustainability. Traditional methods for diagnosis of the type and level of infection in a sheep flock require laborious laboratory extraction, culture and microscopic examination of eggs or larvae from faecal samples. Advances in molecular technology offer the potential for more efficient and reliable methods. This study aimed to develop and test a real-time PCR method for routine diagnosis of infection by Teladorsagia circumcincta and Haemonchus contortus in sheep. Primer/probe sets were designed around the ribosomal internal transcribed spacer 2 (ITS2) region as sequence data was available from other studies and so tests used published primer/probe sets, as well as those designed at the Food and Environment Research Agency (Fera). Different primer/probe combinations were tested for specificity against DNA extracted from T. circumcincta larvae or H. contortus DNA. All sets were tested for cross reactivity against four other closely related species, using real-time PCR technology. Reactions were optimised with the best primer/probe combination for each species and then tested for sensitivity against samples containing different T. circumcincta or H. contortus DNA concentrations. Faecal samples were collected from sheep infected with T. circumcincta or H. contortus alone and the eggs harvested, counted and DNA extracted. Serial dilutions were prepared to give a range of concentrations between approximately 3000 and 50 eggs per sample and real-time PCR reactions were carried out for each and mean cycle time (Ct) values were calculated. These Ct values were plotted against the sample egg concentration to produce a standard curve. Regression analysis was carried out using the generated data. Eggs were then harvested from faecal samples collected in the field from sheep carrying natural mixed infections, DNA extracted and Ct values recorded as before. Results were compared to the standard curve data in order to calculate the approximate number of T. circumcincta and H. contortus eggs in each field sample. These values were compared to the number of eggs determined using the traditional laboratory methods and Pearson product-moment correlation coefficients calculated. Results showed a strong correlation between numbers of eggs determined using the traditional and novel molecular methods suggesting that the developed molecular tools adequately predict egg numbers in this range. Further development and validation work should allow practical use of the method. This assay has significant advantages over the traditional methods currently used routinely at Fera and other parasitology laboratories and these are discussed.

Consequences of infection pressure and protein nutrition on periparturient resistance to Teladorsagia circumcincta and performance in ewes

The consequences of protein nutrition on the degree of periparturient relaxation of immunity to nematode parasites in sheep may be more pronounced at higher levels of infection pressure. Here, we investigated interactive effects of metabolizable protein (MP) nutrition and infection pressure on resistance and lactational performance of ewes. Twin-rearing ewes were trickle infected with either 1000, 5000 or 10,000 infective Teladorsagia circumcincta larvae and fed either at 0.8 (low protein, LP) or 1.3 (high protein, HP) times their estimated MP requirement. Expected interactions between feeding treatment and infection pressure were not observed. Periparturient relaxation of immunity, as indicated by variation in faecal egg counts, was higher in LP ewes than in HP ewes and FEC showed an inverse relationship with infection pressure indicating possible density dependency effects on worm fecundity. Plasma pepsinogen concentration linearly increased with infection pressure. Daily total nematode egg excretion, assessed at week three of lactation, was not significantly affected by infection pressure but was reduced by 65% in HP ewes compared to LP ewes. MP supplementation improved lamb performance but had little effect on ewe body weight and plasma protein concentrations, whilst lactational performance, as judged from lamb performance, tended to be reduced with increased infection pressure. The results suggest periparturient MP supplementation to ewes reduces nematode egg excretion independent of infection pressure and improves lactational performance of parasitized ewes even in the presence of moderate MP scarcity.

Kinetics of the local cellular response in the gastric lymph of immune and susceptible sheep to infection with Teladorsagia circumcincta

Groups of yearling sheep were trickle infected with Teladorsagia circumcincta for 8 weeks, then the infection cleared with anthelmintic and both these animals and a group of parasite naïve sheep were challenged with 50 000 infective T. circumcincta larvae. The previously infected sheep demonstrated acquired immunity to the parasite, manifested by reduced worm burdens which were evident as early as 2 days after challenge. Cannulation of the common efferent gastric lymph duct allowed the kinetics of their local cell traffic to be monitored, and the phenotype of these lymphocytes was analysed. A blast cell response, consisting of both T and B lymphocytes, was observed in both groups of sheep, however this occurred more rapidly in the previously infected, immune animals. CD4+, CD8+ and CD25+ blast cell output peaked at day 3 in the previously infected animals, whereas CD21+ blast cell output peaked slightly later at day 5. In the control group the peak output of all phenotypes of blast cells occurred more slowly, peaking 10 days after infection.

Changes in food intake and circulating leptin due to gastrointestinal parasitism in lambs of two breeds1

A reduction in food intake is a prominent feature of many infectious diseases. However, the underlying mechanisms of parasite-induced anorexia in sheep are poorly understood. Here, we tested the hypotheses (a) that the degree of parasite-induced anorexia in lambs is influenced by their growth potential and (b) that nematode infection results in elevated plasma leptin concentration in lambs. The hypotheses were tested with Suffolk x Greyface (S) and Scottish Black-face (B) lambs that are known to differ in their growth potential (S lambs are of greater growth potential than B lambs). During a primary parasite infection, 24 out of 48 lambs per breed were trickle-infected with 7,000 infective Teladorsagia circumcincta larvae per day, 3 d/wk, for a period of 12 wk (experiment I). The lambs were then dewormed, and after a 2-wk interval, half of the 24 lambs per breed that were previously infected were reinfected for another 12 wk with the same parasite and dose as used in the primary infection (experiment II). In both experiments, infected lambs were fed grass pellets for ad libitum intake, whereas noninfected lambs were fed grass pellets for either ad libitum or restricted intakes. The S lambs were more susceptible than B lambs to nematode infection, as judged from the differences in fecal egg counts (P = 0.007). Parasitized lambs of the more susceptible breed (S) showed anorexia [i.e., a decrease in intake of 13% compared with uninfected controls (P = 0.01)], whereas no significant reduction in food intake was observed in lambs of the more resistant breed (B). Reexposure to nematode infection of previously infected animals tended to result in renewed anorexia in S lambs but not in B lambs (P = 0.08) in a similar extent as during primary infection. Plasma leptin concentrations did not differ between ad libitum-fed infected and control lambs but were greater in infected than in noninfected lambs at a similar level of food intake during both the primary (P = 0.02) and the secondary parasitic infection (P = 0.004) in both breeds. The results show that leptin may be involved in the response of lambs to infection but that it is unlikely that leptin alone is responsible for the parasite-induced anorexia in lambs.

Parasite loss and inhibited development of Teladorsagia circumcincta in relation to the kinetics of the local IgA response in sheep

Groups of yearling sheep, which had been trickle infected with Teladorsagia circumcincta for 8 weeks and then drenched, were challenged with 50 000 T. circumcincta larvae together with groups of worm-free controls. Fewer parasites and a greater proportion of early fourth stage larvae were recovered from previously infected sheep compared to controls. Worm loss and arrested development were evident by 5 days after challenge whereas growth retardation of developing worms was observed by day 10. In the previously infected sheep a secondary IgA response was observed in the efferent gastric lymph from 5 days post-infection. Western blot analysis showed the lymph IgA to be predominantly dimeric and nonsecretory in nature and that the somatic antigens recognized were predominantly in the 100-250 kDa range. The concentration of IgA in lymph was always higher than in blood and in the previously infected sheep increased fivefold 8 days post-challenge in contrast to blood where IgA levels were unchanged. The timing of the response suggested that it occurred too late to have been the cause of worm loss or arrested development, though it may have retarded the growth of developing parasites.

A short-term divergent selection for resistance to Teladorsagia circumcincta in Romanov sheep using natural or artificial challenge

This experiment was conducted to assess the efficiency of selection on the basis of response to artificial challenges in order to breed sheep resistant to natural infection. A short-term divergent selection process was designed to estimate the genetic parameters of these two traits. Two flocks, including 100 Romanov ram lambs each, were challenged in 1990 when they were 6 months old. One flock received three artificial infections with 20 000 third-stage Teladorsagia circumcincta larvae, at intervals of 7 weeks. Faecal egg counts (FEC) were performed on Days 22, 25 and 28 post infection (p.i.) and the animals were drenched on Day 28 p.i. The other flock was grazed for 5 months on a pasture contaminated with the same species. Faecal samples were taken from the lambs at similar ages. About 5 rams with the lowest FEC and 5 with the highest FEC were selected in each flock and mated with unselected ewes. Their offspring (200 animals) were challenged in 1992, half in the same way as their sires, and the other half by the other method. Because of a drought in the summer of 1990, it was necessary to repeat part of the experiment, and in 1992 the 5 and 8 rams with the lowest and highest FEC, respectively, were selected from the offspring challenged on the pasture in 1992 and were mated with unselected ewes. Their progeny (about 80 animals) were challenged in 1994, half by natural infection, half by artificial infection. The mean FEC of the flock increased from the first to the third artificial infection. The natural infection was highly variable in different years, reflecting the difficulty of assessing resistance using this mode of challenge. Genetic parameters were estimated using animal models and REML solutions. The repeatabilities of the FEC following artificial and natural infection were 0.49 and 0.70 respectively within a period of one week, and 0.22 and 0.41 respectively for periods separated by intervals of 7 weeks; the heritabilities of the single egg count were 0.22 and 0.38 respectively. The genetic correlation was 0.87: the FEC recorded under natural or artificial infection appear to depend on the same genetic potential.

Milk and milk components reduce the motility of Ostertagia circumcincta larvae in vitro

AIM: To examine the effects in vitro of bovine milk and milk products and soymilk on the motility of sheathed and ex- sheathed L3 Ostertagia circumcincta (also known as Teladorsagia circumcincta) as a measure of larval viability and infectivity. METHODS: L3 were exsheathed in 0.2% sodium hypochlorite, resuspended in Hank’s Balanced Salt Solution (HBSS) pH 7.4 and incubated with test solutions at 37°C for up to 48 h. The motility of 50 larvae from each incubate was assessed at selected times using a McMaster slide. Larvae were considered immotile only if straight and not moving. Fresh bovine milk, homogenised milk (3.3% fat), low-fat milk (0.2% fat) and lamb milk replacer were diluted with HBSS pH 7.4 to concentrations from 1.6–100%, and incubated with exsheathed L3 for 1, 24 or 48 h. Bovine whey protein was tested in concentrations of 5–15% at pH 2.5–6.5, casein at 5 or 7.5%, and skim milk powder from 5–15% at pH 5.5 or 6.5, all for 2, 4 or 24 h. Soymilk was tested in concentrations of 1.6–100% for 1, 2, 24 or 48 h. HBSS was used as the control solution. Sheathed L3 were incubated in HBSS pH 7.4, 50% homogenised milk in HBSS, or 50% soymilk in HBSS. Each solution was incubated for 1, 2, 24 or 48 h. RESULTS: The motility of exsheathed L3 was reduced by fresh bovine milk, homogenised milk, low-fat milk, lamb milk replacer, whey, casein and skim milk solutions, but not by soymilk. The mean percentage (and SE) immotile at 48 h were: fresh milk 38% (SE 20); homogenised milk 65% (SE 7); low-fat milk 57% (SE 5); lamb milk replacer 43% (SE 7); and soymilk 7% (SE 0.5). Larval immotility increased in whey protein solutions from 5–15%, from pH 2.5–6.5 and from 2 to 24 h (all p<0.001); in skim milk from 5–15% (p<0.001), and was greater at pH 6.5 than at pH 5.5 (p<0.001); in casein from 5–7.5% (p<0.001), but was no different at pH 5.5 and 6.5. The motility of sheathed L3 was reduced at 24 h (p=0.009) and 48 h (p<0.001) by 50% homogenised milk, but not by 50% soymilk or HBSS. CONCLUSIONS: Bovine milk proteins, or components associated with the proteins, reduced the motility of both sheathed and exsheathed L3 O. circumcincta. Soymilk had no effect on nematode motility. Lower larval motility may reduce worm establishment and be a contributing factor to the smaller burdens of gastrointestinal nematodes in milk-fed animals compared with animals after weaning.

The effect of pasture species on parasitism and performance of lambs grazing one of three grass—white clover pasture swards

Lamb parasitism and performance were compared on three pasture treatments: browntop/white clover, Yorkshire fog/white clover, and ryegrass/white clover swards maintained at a sward height of 5 cm in each of two years by using continuous variable stocking management. In year 1, lamb performance (both suppressively drenched (SD) and trigger drenched (TD)) was similar on ryegrass and browntop swards but was poorer ( P<0.05) on Yorkshire fog swards. In year 2, lamb performance (both SD and TD) was markedly better on ryegrass than on Yorkshire fog or browntop swards. This coincided with an increase in white clover content in the ryegrass/white clover swards and a decrease in white clover content in the browntop/white clover swards. Lamb performance was related to the white clover content of the swards ( R 2=85.4, P<0.001 and R 2=77.5, P<0.001 for SD lambs and TD lambs, respectively). Differences among pasture treatments in faecal egg count (FEC) of TD lambs were greater in year 2 than in year 1 when no significant pasture effects were observed. In year 2, FEC was lowest in lambs which grazed ryegrass/white clover, intermediate in those which grazed Yorkshire fog/white clover and highest in lambs which grazed browntop/white clover, despite herbage allowance (kg herbage mass/kg lamb mass) being greater on the browntop/white clover and Yorkshire fog/white clover swards than the ryegrass/white clover swards. Circulating antibodies to gastrointestinal parasites were measured at the end of year 2. Antibody titres were highest ( P<0.05) in the plasma of lambs which grazed ryegrass/white clover swards, and lowest in lambs which grazed browntop/white clover swards due to differences in levels of antibody to ( P<0.05) Haemonchus contortus, Ostertagia circumcincta larvae and adults, but not Trichostrongylus colubriformis larvae or adults. These findings indicate that pasture composition, especially, the proportion of white clover, can help alleviate the production losses due to gastrointestinal parasitism. This information can provide a quick and readily adoptable practice for farmers who aim to reduce drench reliance and could become a key component of any integrated parasite control programme.

Reduced Ostertagia circumcincta burdens in milk-fed lambs

Aims: To compare the susceptibility to parasitism by Ostertagia circumcincta of lambs fed entirely with bovine milk or weaned on to solid feed at 3 weeks of age. In addition, the effect of a single daily feed of milk on worm burdens was assessed. Methods: Eight lambs were assigned to each of the 3 diets: milk (M), milk plus solid feed (cereal-based pellets and lucerne chaff) (MS), or solid feed only (S). Those to be fed solid feed were converted from complete milk feeding to the designated diet during their third week of life. From 3 weeks of age, all lambs were infected with 1000 O. circumcincta larvae twice weekly for 6 weeks; 4 lambs from each diet group were given normal sheathed L3 and another 4 were infected with exsheathed larvae. Faecal egg counts (FEC) and serum gastrin and pepsinogen concentrations were monitored from Day 17 after first infection, and worm burdens and abomasal pH and morphology were determined at necropsy. Results: Total worm burdens and FEC were significantly lower in the M than MS and S groups, whereas there was no significant difference between those receiving sheathed and exsheathed larvae. The milk-fed lambs had a smaller reticulo-rumen and omasum and a more acidic abomasal pH. Serum gastrin and pepsinogen were increased in all groups, irrespective of diet or type of larvae used for infection. Conclusions: The cause of the lower worm establishment in lambs fed only milk was probably not failure to exsheath in the immature gastro-intestinal tract, as there were similar worm burdens in lambs whether sheathed or exsheathed larvae were administered. The lower pH of the abomasal contents of the preruminant lambs may have been a factor, as the parasites have previously been shown to die more rapidly in vitro at low pH. Alternatively, the milk itself had adverse effects on the parasites, but was ineffective when combined with solid feed. There was no benefit from feeding a milk plus solid diet over a solid diet.