R.S. Green

The effect of feeding sulla (Hedysarum coronarium) or lucerne (Medicago sativa) on lamb parasite burdens and development of immunity to gastrointestinal nematodes

Two experiments investigated the efficacy of the legume Hedysarum coronarium (sulla), which contains condensed tannins (CTs), for reducing gastrointestinal nematode infections relative to lucerne. Experiment 1 was aimed to show whether the lower faecal egg count (FEC) and larval establishment previously reported in lambs grazing sulla were due to direct effects of the forage on Ostertagia circumcincta and Trichostrongylus colubriformis or were mediated through an enhanced immune response. Experiment 2 evaluated the impact of feeding sulla relative to feeding lucerne (Medicago sativa), before, at, or after larval challenge on subsequent FECs and nematode burdens. In experiment 1, 64 Romney lambs were fed either freshly cut lucerne or sulla (32 lambs per herbage) for the duration of the trial. Within each herbage there were four treatment groups (n=8 per group). Initial levels of immunity were assessed in uninfected (UN) lambs which were maintained parasite-free until challenged with 15,000 O. circumcincta and 15,000 T. colubriformis larvae on day 63, and slaughtered on day 81. The other three treatment groups were trickle-infected with each of 5000 O. circumcincta and 5000 T. colubriformis larvae three times per week from day 1 to 35. Non-steroid infected (CONTROL) and steroid-treated (STER) groups were treated with anthelmintic on day 49 and challenged with 15,000 O. circumcincta and 15,000 T. colubriformis on day 63 and slaughtered on day 81. The STER lambs were given dexamethasone trimethylacetate from day 49 to 81 to determine effects of immunity on parasite infection. From day 35 an establishment group (EST) on each herbage was fed a common pelleted lucerne diet and slaughtered on day 56 to determine nematode establishment during trickle-infection. Diet did not affect FECs but feeding lucerne increased (P<0.05) numbers of T. colubriformis in CONTROL lambs compared to those fed sulla. O. circumcincta numbers were lower (P<0.05) in UN lambs fed sulla than lucerne. The sulla diet was associated with higher (P<0.05) antibody titres against secretory-excretory antigens to adult O. circumcincta and to adult and larval T. colubriformis, so there appeared to be some immunogenic response to the sulla diet but effects upon T. colubriformis numbers were not significant. The second experiment involved 48 Romney lambs grazing conventional pasture which were infected with 10,000 each of O. circumcincta and T. colubriformis larvae either 7 days before, 7 days after, or at the time they commenced grazing either sulla or lucerne. Lambs which grazed sulla had lower (P<0.05) FEC and lower (P<0.05) O. circumcincta burdens than lambs which grazed lucerne but timing of infection had no effect on FEC or worm burdens. T. colubriformis numbers were not affected by treatment or herbage. In conclusion, the sulla diet resulted in lower O. circumcincta numbers compared to lucerne outdoors and some evidence of an immunogenic response was obtained indoors. However, neither the herbage nor the immunogenic response reduced T. colubriformis numbers in either experiment.

ASSOCIATION OF BLOOD EOSINOPHILIA WITH THE EXPRESSION OF RESISTANCE IN ROMNEY LAMBS TO NEMATODES

The dynamics of blood eosinophilia was studied in Romney lambs dosed twice weekly with 5000 infective larvae of Trichostrongylus colubriformis. A marked rise in blood eosinophil counts of the dosed lambs coincided with the start of the decline of faecal egg counts (FEC). Eosinophilia was also studied in grazing Romney lambs from a breeding programme, based on selection for high or low FEC. Comparison of the sire groups suggested that the magnitude of the eosinophil response was under genetic control. The significant negative correlations between blood eosinophil counts and FECs confirmed the association between eosinophilia and the expression of resistance in Romney lambs to nematodes. However, comparison of the mean blood eosinophil counts and FECs for both the ram and ewe lambs of the resistance sire groups suggested that the association of eosinophilia and resistance may be greater for some sire groups than others.

Discovery of quantitative trait loci for resistance to parasitic nematode infection in sheep: I. Analysis of outcross pedigrees

BackgroundCurrently most pastoral farmers rely on anthelmintic drenches to control gastrointestinal parasitic nematodes in sheep. Resistance to anthelmintics is rapidly increasing in nematode populations such that on some farms none of the drench families are now completely effective. It is well established that host resistance to nematode infection is a moderately heritable trait. This study was undertaken to identify regions of the genome, quantitative trait loci (QTL) that contain genes affecting resistance to parasitic nematodes.ResultsRams obtained from crossing nematode parasite resistant and susceptible selection lines were used to derive five large half-sib families comprising between 348 and 101 offspring per sire. Total offspring comprised 940 lambs. Extensive measurements for a range of parasite burden and immune function traits in all offspring allowed each lamb in each pedigree to be ranked for relative resistance to nematode parasites.Initially the 22 most resistant and 22 most susceptible progeny from each pedigree were used in a genome scan that used 203 microsatellite markers spread across all sheep autosomes. This study identified 9 chromosomes with regions showing sufficient linkage to warrant the genotyping of all offspring. After genotyping all offspring with markers covering Chromosomes 1, 3, 4, 5, 8, 12, 13, 22 and 23, the telomeric end of chromosome 8 was identified as having a significant QTL for parasite resistance as measured by the number of Trichostrongylus spp. adults in the abomasum and small intestine at the end of the second parasite challenge. Two further QTL for associated immune function traits of total serum IgE and T. colubiformis specific serum IgG, at the end of the second parasite challenge, were identified on chromosome 23.ConclusionDespite parasite resistance being a moderately heritable trait, this large study was able to identify only a single significant QTL associated with it. The QTL concerned adult parasite burdens at the end of the second parasite challenge when the lambs were approximately 6 months old. Our failure to discover more QTL suggests that most of the genes controlling this trait are of relatively small effect. The large number of suggestive QTL discovered (more than one per family per trait than would be expected by chance) also supports this conclusion.

Phenotypic Markers for Selection of Nematode-resistant Sheep

Nematode-resistance of an animal can be defined as an enhanced natural ability, relative to its peers, to both prevent establishment of larval nematodes and evict any that do establish. These parameters are not measurable in a practical sense and consequently nematode-resistance has usually been defined in terms of low faecal nematode egg counts (FEC). Studies in New Zealand and Australia have demonstrated that nematode-resistance, as measured by FEC, has a heritability of about 0.3 in Romney and Merino sheep. However as a selection trait FEC has practical limitations and its use may incur production penalties through withholding drench treatment for prolonged periods or from a need for artificial challenge. FEC is influenced by the level and composition of a natural nematode challenge and especially the expression of the immune response. Thus immunological parameters which reflect the underlying genetic resistance could potentially be used as phenotypic markers. Ideally, a useful phenotypic marker would be easy to sample and its assay would be inexpensive and able to be automated, in addition to being strongly correlated with nematode-resistance. Results from several New Zealand trials have indicated that antibody levels (particularly IgG1) to excretory/secretory antigens of L3 nematodes such as Trichostrongylus colubriformis may meet these criteria. Levels of antibody against L3 antigens are also independent of on-farm drenching strategies. Blood eosinophil count has also been considered for use as a selection parameter but a high degree of sample variability reduces its potential. Other immunological parameters associated with nematode-resistance which have potential as phenotypic markers include serum nematode-specific IgE and products of mucosal mast cells such as proteinases. It is likely that as the critical immune responses of sheep to nematodes become more clearly defined, new immunological parameters with potential for use as phenotypic markers will be found. The definition of these immune responses will also assist in the identification and characterization of genetic markers.

Antibody responses of sheep to challenge with Trichostrongylus colubriformis and the effect of dexamethasone treatment

Eight-month-old random bred Romney wether lambs were reared nematode-free in pens and assigned to 4 groups each of 5 lambs. Lambs in 3 groups were infected orally, twice a week, with 5000 Trichostrongylus colubriformis infective larvae (L3), a control group remained unifected throughout. Two infected groups were treated with dexamethasone (0.5 mg kg-1 bodyweight), one between days -7 and 77, the other between days 77-154. Nematode challenge infection was withheld from the third group from day 133 after anthelmintic treatment. Nematode eggs in sheep faeces (FEC) were monitored at weekly intervals. T. colubriformis-specific antibody levels were determined twice a week and specific immunoglobulin isotypes (IgA, IgG1, IgG2 and IgM) determined weekly in serum samples using ELISA. Resistance, as measured by FEC, was expressed by 35 days after L3 infection began but sheep dosed with dexamethasone did not develop resistance. Extant resistance was abrogated in sheep dosed with dexamethasone. Nematode challenge resulted in elevated serum levels of antibodies to T. colubriformis L3 excretory/secretory antigens, these consisted predominantly of IgG1 and IgM. The IgG1 response was more persistent than the IgM response. Specific serum IgA and IgG2 responses were low, but significant, in nematode-challenge sheep. Dexamethasone treatment prevented the antibody responses and resulted in a rapid reduction of extant antibody levels in resistant sheep. Weight gain was reduced by nematode challenge with or without dexamethasone treatment compared with control sheep.

Sequential cellular and humoral responses in the abomasal mucosa and blood of Romney sheep dosed with Trichostrongylus axei

Abomasal cannulae were surgically placed in 7 2-year-old New Zealand Romney sheep which had been maintained parasite-free from birth. Four of these sheep were randomly selected and dosed orally with 10,000 infective Trichostrongylus axei larvae per week for 8 weeks, while the remaining 3 sheep served as uninfected controls. Abomasal biopsy, blood and faecal samples were obtained from all sheep at regular intervals from 5 days before and until 58 days after the first infection. The sheep were then killed, worm burdens assessed and abomasal and small intestinal samples collected Faecal egg counts of all 4 dosed sheep were low and only one (No. 701) had a substantial worm burden (8400) post mortem. Overall, levels of mucosal mast cells/globule leukocytes, eosinophils, T19+ cells and larval migration inhibitory activity increased significantly in the abomasal mucosa of the dosed sheep compared to the controls. The CD4+:CD8+ cell ratio in the abomasal mucosa of the dosed sheep also increased compared to that of the controls (P = 0.06). In blood, T. axei-specific antibody (total and IgG1) and eosinophil numbers increased significantly in the dosed sheep. Mucosal cells staining for IgE (IgE+), and blood and mucosal eosinophils showed the earliest substantive increases in number followed by increases in specific serum antibody levels, numbers of mucosal cells fluorescing under UV light (UVf) and T19+ cells. The difference in the IgE+ and UVf cell responses indicated that expansion of globule leukocyte numbers lagged behind that of mucosal mast cells. The results supported the concept of CD4+ T cell help in the abomasal mucosa and defined the sequential expression of components of the immunological responses potentially mediating resistance to T. axei. In sheep No. 701, persistence of adult worms was associated with lower mucosal IgE+ cell and eosinophil responses compared with the other dosed sheep.

Genetic and phenotypic relationships among Trichostrongylus colubriformis-specific immunoglobulin E, anti-Trichostrongylus colubriformis antibody, immunoglobulin G1, faecal egg count and body weight traits in grazing Romney lambs

Variation in total and Trichostrongylus colubriformis-specific immunoglobulin E (IgE) was investigated in four- and six-month old Romney lambs reared on pasture, where they were exposed to natural challenge with nematode parasites. The lambs, which were from an experimental progeny test flock (n=64 sires), were born in spring over three years and weaned at an average age of three months (December). Data from the flock were analysed to obtain heritability estimates for IgE and genetic correlations between IgE and other immunological, parasitological and production traits. In addition, correlated responses of IgE to long-term genetic selection for high, control or low faecal nematode egg count (FEC) were investigated in a set of experimental breeding lines. Repeatabilities and heritabilities of IgE traits in the progeny-test flock were similar to those calculated previously for anti-T. colubriformis antibody (Ab), immunoglobulin G1 (IgG1) and FEC (generally >0.30). Genetic correlations of the loge transformed IgE traits (January and March samples) with loge (FEC+100) on three sampling occasions were all negative (−0.22 to −0.37). However, positive correlations (0.17 and 0.43) were found between loge IgE and dag score, indicating a tendency for there to be more severe breech soiling in lambs with elevated serum IgE. In the 1995 and 1996 selection-line lamb crops (after 17 and 18 years of selective breeding for high or low FEC), both total and T. colubriformis-specific IgE levels were higher (by between 59 and 103%) in the low than in the high line (P<0.001). IgE levels in the controls were intermediate between those of the high and low lines but closer to those of the low line. The results are in line with other evidence which suggests that greater genetic resistance to gastrointestinal nematode infection in Romney lambs is associated with an increase in activity of the TH2 arm of the immune system which mediates inflammatory responses against multicellular parasites.

The effect of corticosteroid treatment on local immune responses, intake and performance in lambs infected with Teladorsagia circumcincta

The nutritional cost of, and the sequential cellular changes associated with the developing immune response to the abomasal parasite Teladorsagia circumcincta were investigated using corticosteroid-induced immune-suppression. Six-month-old lambs with minimal nematode experience were either infected with 4000 L3 T. circumcincta per day (group IF), similarly infected and concurrently immune-suppressed with methylprednisolone acetate (group ISIF), immune-suppressed only (group IS) or remained as controls (group C). Food intake, faecal egg count (FEC) and antibody titres in plasma were recorded weekly, worm burden at necropsy on day 63 p.i. and body composition by X-ray computed tomography on days -2 and 62 p.i. Furthermore, sequential immunological changes at the site of parasite infestation in the abomasal mucosa were measured from serial biopsy tissue samples taken from additional animals that were fitted with an abomasal cannula and either infected with the same regime as IF animals above (group CnIF) or concurrently infected and immune-suppressed as above (group CnISIF). Corticosteroid treatment resulted in greater FECs (P<0.01) and worm burdens (P<0.01) in both ISIF and CnISIF compared with IF and CnIF sheep, respectively. Infection reduced feed intake by 17% between 14 and 28 days p.i. (P<0.05) and efficiency of energy utilisation by 20% (P=0.07) in IF animals but not in ISIF animals. Mast cells, globule leukocytes and IgA in tissue biopsy samples were elevated in CnIF from 42 days p.i., all of which were abrogated by corticosteroid treatment. The ability to regulate the worm population appeared to be associated with a rise in tissue IgA concentration and numbers of globule leucocytes (GL). The results support the hypothesis that a majority of the production losses that occur during infection of lambs with T. circumcincta in lambs are a consequence of the host immune response. These findings may have implications for regimes that promote the development of a strong host immune reaction to gastrointestinal parasites in lambs.

Genetic factors affecting antibody responses to four species of nematode parasite in Romney ewe lambs

Serum levels of antibodies (Ab) and immunoglobulin G1 (IgG1) to the larval (L3) stage of the internal parasites Cooperia curticei and Trichostrongylus colubriformis and levels of Ab to the L3 stages of Haemonchus contortus and Ostertagia circumcincta were determined in 1432 Romney ewe lambs which were born on one farm in 1990 and 1991 and were the progeny of 63 rams. The objectives were to estimate heritabilities of, and genetic correlations among, the serum concentrations in newly weaned lambs under commercial conditions and to estimate genetic correlations of Ab and IgG1 with production traits. Lambs were exposed to a natural parasite challenge on pasture, following an anthelmintic drench at weaning. Blood and faecal samples from 4- to 6-month-old lambs were then taken when the mean faecal nematode egg count of a monitor group reached 800-1500 eggs g-1. Heritabilities for the serum levels of the four Abs ranged from 0.25 +/- 0.05 to 0.37 +/- 0.06. Heritabilities for the level of IgG1 developed against C. curticei was 0.19 +/- 0.04 and against T. colubriformis, 0.18 +/- 0.05. Genetic correlations between Abs for the 4 species were high, averaging 0.84, and between the two IgG1s it was 0.82. The genetic correlations between Ab or IgG1 levels and weight or gain traits were negative (for the 6 significant values out of 18), with yearling fleece weight positive (for the 2 significant values out of 6), whilst those with loge faecal egg count were all negative (average -0.15).(ABSTRACT TRUNCATED AT 250 WORDS)

THE EFFECT OF CONTINUOUS DRUG EXPOSURE ON THE IMMUNE RESPONSE TO TRICHOSTRONGYLUS COLUBRIFORMIS IN SHEEP

The effect of albendazole (ABZ)-capsule (CRC) administration on parasite establishment and immunity to ABZ-resistant (RES) and -susceptible (SUS) T. colubriformis was measured in Romney lambs. During 12 weeks of twice-weekly dosing with 3000 parasite larvae (L3), eggs were observed in faeces from CRC-treated and untreated lambs given RES L3, but not CRC-treated lambs given SUS L3. Following the period of trickle challenge all lambs were drenched and, 1 week later, dosed with 20000 SUS L3. Resulting worm burdens were higher in control lambs than in those previously treated with CRCs and challenged with SUS, which in turn were higher than those in the CRC-treated or -untreated lambs previously challenged with RES L3. During the period of trickle challenge, the number of peripheral eosinophils and titres of anti-L3 and anti-adult antibody were raised only in those groups given RES L3. There was no effect of CRC administration. Following drench and challenge, antibody titres and eosinophil numbers increased in the control animals but not in those groups which had received previous trickle infection. The results demonstrate that the larval challenge alone resulted in incomplete though substantial protection against subsequent parasite challenge. The use of CRCs may potentially impact on subsequent animal performance and selection for anthelmintic resistance through a reduced level of immunity.