P.G.C. Douch

Improved bioassay for estimation of inhibitory effects of ovine gastrointestinal mucus and anthelmintics on nematode larval migration.

A bioassay based on the ability of substances present in ovine gastrointestinal mucus or anthelmintics to paralyse third stage larval (L3) nematodes and inhibit their passage through 20 microns nylon mesh sieves [larval migration inhibition (LMI) activity] is described. Factors influencing the reproducibility of the bioassay were examined using exsheathed L3 of the sheep gastrointestinal nematode parasite Trichostrongylus colubriformis. Levamisole, morantel tartrate and piperazine were shown to inhibit L3 migration in the bioassay. The bioassay was used to demonstrate that gastrointestinal mucus from nematode-resistant sheep possessed greater L3 inhibitory activity than mucus from nematode-susceptible sheep. LMI activity of mucus used in this bioassay was significantly correlated with LMI activities obtained using two previously described similar bioassays. The action of mucus components on nematode larvae was shown to be reversible in the bioassay. The modified assay has advantages over other bioassays as it avoided the use of temperature-dependent agar blocks, reduced the number of L3 required to a more manageable size, and the whole experiment could be performed on a 48-well culture plate. The reproducibility, high correlation with other bioassays, ease of performance, suitability for testing a large number of samples and low cost make this modified assay the method of choice for determining antiparasitic activity of gastrointestinal mucus components and as a screen for potential anthelmintics.

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.

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.

Quantification of total sheep IgE concentration using anti-ovine IgE monoclonal antibodies in an enzyme immunoassay

Monoclonal antibodies (mAbs) which recognize separate epitopes on ovine immunoglobulin E (IgE) have been used to develop a non-competitive antibody sandwich enzyme immunoassay (EIA) for quantitating ovine IgE. Purified anti-IgE mAb (YD3) coated onto polystyrene microtitre plates was used to capture IgE in serum samples. Biotinylated anti-IgE mAb (XB6) followed by streptavidin conjugated with horseradish peroxidase were used to detect captured IgE. Tetramethylbenzidine and H2O2 were used as enzyme substrate. A reference serum was prepared by pooling sheep sera containing elevated IgE levels. This reference serum was assigned a value of 100 units ml-1 and used to prepare standard curves for the EIA. The linear region of log-log transformed standard curve data covered a range of 0.05-0.8 units ml-1. The equation of a linear regression line fitted to this curve was used to determine sample concentrations. Using purified IgE, 1 unit of reference serum was equivalent to 0.86 micrograms ml-1 IgE. Maximum intra- and inter-assay coefficients of variation for the EIA were 4.6% and 9.7%, respectively. Subjecting serum samples to 15 freeze/thaw cycles, storage at room temperature for 16 days or incubation at 37 degrees C for 8 h resulted in minimal loss of IgE detection. Incubation of serum at 56 degrees C resulted in rapid reduction in detection of IgE by the EIA. The assay was used to determine IgE levels in adult sheep monospecifically infected with weekly doses of the nematode Trichostrongylus axei. Serum IgE levels increased from 9 to 16 days following first infection and reached maximum levels by days 35-58. Serum IgE responses closely followed IgE positive cell responses in the abomasal mucosa.

The effect of age on the response of Romney sheep to gastrointestinal nematodes during grazing

Three groups of Romney sheep, reared worm-free in pens from birth until 4, 16 or 28 months of age, were permitted to graze nematode-infected pasture for 4 weeks after which they were returned to pens and slaughtered 4 weeks later. Three groups of three Romney sheep reared and maintained worm-free for similar periods served as uninfected control groups. Faecal egg counts (FECs) 8 weeks after grazing commenced were significantly greater in 4-month-old sheep than the 28-month-old animals. At slaughter, no difference in nematode burdens among these three age groups was found. Histological examination of the abomasal and small intestine mucosa, taken from sheep at slaughter, revealed significantly greater numbers of globule leukocytes/mucosal mast cells (GL/MMC) bur fewer connective tissue type mast cells (CTMC) in sheep aged 16 and 28 months than in those aged 4 months. The numbers of eosinophils in the gastrointestinal mucosal tissues of the three groups of infected sheep were not significantly different. In uninfected sheep the numbers of eosinophils, GL/MMC and CTMC in the mucosal tissues did not differ between the three age groups and the numbers of GL/MMC and CTMC were similar to those in 4-month-old infected sheep. Eosinophil numbers in the mucosal tissue of the infected groups were significantly higher than in the uninfected groups. In the infected groups, FECs but not worm burdens were significantly negatively correlated with GL/MMC numbers.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and characterisation of monoclonal antibodies recognising ovine IgE

Two monoclonal antibodies (mAbs), XB6 and YD3, which recognise ovine immunoglobulin E (IgE) were produced. Mast cells isolated from ovine intestinal mucosa were used as a source of IgE to immunize mice. Culture supernatants of hybridomas were screened by immunoassays on small-intestine tissue sections, isolated mucosal cells, and dot blots of lysed mast cell homogenate. Two mAbs were chosen for their specific binding to mast cells. Antigen bound by these mAbs was purified by immunoaffinity chromatography using XB6 mAb, and this produced two bands consistent with IgE heavy chain (86,000 Daltons) and immunoglobulin light chain (28,000 Daltons) when run under reducing conditions on SDS-PAGE gels. Purified IgE was shown on dot blots to react weakly with mAb to chimeric ovine IgE and strongly to polyclonal anti-sheep antibodies. The two mAbs induced an immediate hypersensitivity-like reaction when injected into the skin of sheep. The mAbs bound to mast cells and other mononuclear cells, presumably IgE-secreting B-cells in mesenteric lymph node sections. These mAbs proved useful for detecting IgE-bearing cells in various ovine tissues, for purifying mast cells from cell isolates by panning and immunomagnetic bead separation, for purifying serum IgE using immunoaffinity chromatography and for detecting IgE in an ELISA. Competitive binding assays showed that the two mAbs bind to different epitopes on IgE. These mAbs will be useful in research applications and in diagnostic assays.

Secretion of anti-parasite substances and leukotrienes from ovine gastrointestinal tissues and isolated mucosal mast cells

The presence of larval migration inhibitory (LMI) compounds in the gastrointestinal mucus of nematode resistant sheep has been shown previously to be associated with increased numbers of gastrointestinal mucus of nematode resistant sheep has been shown previously to be associated with increased numbers of gastrointestinal mucosal mast cells (MMC) and globule leukocytes (GL). This experiment was designed to determine if LMI compounds were secreted by MMC/GL in response to nematode antigenic challenge and if so, could secretion account for levels observed in mucus. Romney sheep were immunized by repeated cycles of infection with Trichostrongylus colubriformis or Haemonchus contortus larvae and anthelmintic treatment. After slaughter, gastrointestinal tissue was taken for examination of histology and mucus anti-parasite activity. Segments of small intestine were ligatured to form sacs which were incubated with exsheathed nematode larvae or larval excretory/secretory antigens. Tissue slices from small intestine or abomasum were also incubated with nematode larvae or antigens. After homologous challenge, levels of leukotrienes secreted into small intestinal tissue sacs were significantly higher than levels in heterologously challenged sacs or unimmunized sheep intestinal sacs challenged with larvae of any nematode species (279.4 +/- 33.7, 141.0 +/- 27.8 and 39.5 +/- 15.2 ng h-1 respectively). Tissue slices gave a similar pattern of leukotriene secretion. LMI activity was also significantly elevated in intestinal sacs from immunized sheep challenged homologously with nematode larvae or antigen (64 +/- 10 and 68 +/- 14% respectively cf. heterologous challenge 32 +/- 10% and unimmunized sheep sacs 15 +/- 6%). Histological examination of abomasal and small intestinal sections showed that immunized sheep had significantly greater numbers of MMC/GL than unimmunized sheep. MMC/GL isolated and purified from immunized sheep secreted leukotrienes and compounds having LMI activity when cultured with homologous nematode larvae or antigens. Secretion of leukotrienes and molecules having LMI activity from MMC/GL could account for the levels of these substances observed in small intestine mucus.