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Veterinary Parasitology 61 (1996) 249-263

Nematode burdens and immunological responses following natural challenge in Romney lambs selectively bred for low or high faecal worm egg count S.A. Bisset *, A. Vlassoff, P.G.C. Douch, W.E. Jonas, C.J. West, R.S. Green AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand

Received 29 December 1994; accepted 10 April 1995

Abstract Breeding lines of Romney sheep, selected as lambs for consistently low or high faecal nematode egg count (FEC) following periods of natural challenge, have been maintained at Wallaceville for some years. In order to determine the extent to which FECs in low and high genotypes reflected their ability to resist the establishment of gastro-intestinal nematode burdens, we investigated the infection status and immune responses in 8- to 9-month-old progeny of selected rams from low and high FEC breeding lines following a period of grazing without antheimintic treatment in autumn/early winter. In each of the 2 years of the study, outcross male progeny of the two lowest FEC (LFEC) (i.e. most 'resistant') and two highest FEC (HFEC) (i.e. most 'susceptible') rams from the divergent lines were slaughtered shortly after autumn/early winter FECs had been analysed. Post-mortem worm counts and examination of intestinal histology were then undertaken. Blood samples collected before slaughter in the second year of the study were assayed to measure serum levels of Trichostrongylus colubriformis-speciflc antibody and immunoglobulins (IgG 1 and IgM), and numbers of circulating eosinophils. Overall, correlations between pre-slaughter FEC and total trichostrongyle burdens in the lambs proved to be very high (0.91 and 0.85, respectively, for the 2 years studied). In the first year, LFEC lambs, which were shedding only 28.6% as many strongyle eggs as their HFEC counterparts at slaughter, were found to harbour 37.6% as many adult trichostrongyle worms, while in the second year, LFEC lambs, which were shedding 16.1% as many strongyle eggs as their HFEC counterparts at slaughter, were found to harbour 33.5% as many adult trichostrongyle worms. Results, particularly in the second

* Corresponding author. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0304-4017(95)00836-5

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S.A. Bisset et al./ Veterinary Parasitology 61 (1996) 249-263

year, confirmed that significantly fewer worms of most of the important abomasal and small intestinal nematode species which infest lambs in New Zealand (i.e. Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Nematodirus spathiger, T, colubriformis, and Trichostrongylus vitrinus) had established in the LFEC genotypes than in their HFEC counterparts. In addition, in utero egg counts of female intestinal Trichostrongylus spp. were significantly lower in LFEC lambs than in their HFEC counterparts, indicating a reduction in fecundity of those worms which did establish. There was also some evidence of an effect of host response on the developmental composition of burdens in the case of some worm species. In relation to host responses, numbers of globule leucocytes/mucosal mast cells in the intestinal mucosa were significantly higher ( P < 0.01) in LFEC lambs than in HFEC lambs in both years of the study. Numbers of connective tissue type mast cells and eosinophils in the intestinal mucosa were also significantly higher in LFEC lambs but only in the second year of the study ( P < 0.01 and P < 0.05, respectively). Numbers of circulating eosinophils did not differ significantly between the genotypes. T. colubriformis-specific antibodies, IgG 1 and IgM to both L 3 and adult worm antigens were all significantly higher (P < 0.01 or P < 0.05) in LFEC lambs than in HFEC lambs. Keywords: Sheep-Nematoda; Selective breeding genetics; Resistance; tmmunity-Nematoda

1. Introduction Divergent breeding lines of Romney sheep, selected as lambs for high or low faecal worm egg counts (FEC) when exposed to successive periods of untreated natural challenge with nematode parasites, have been established at Wallaceville Animal Research Centre for some years (Baker et al., 1990; Bisset et al., 1991). The lines were initially set up on a small scale by one of the authors (A.V.) in 1979, after screening approximately 300 field-grazed ewe lambs for FEC. O f these, 30 were allocated to a 'resistant' line and 30 to a 'tolerant' line on the basis of low or high FEC, respectively. Initially, they were mated to unscreened rams, but subsequently, both ewe and ram replacements for the lines (about 2 0 - 3 0 % of the total flock per annum in the case of ewes), were selected from line-bred progeny on the basis of a combination of consistently low or high FEC and above average growth rate when left untreated. The lines were maintained in this way until 1988 when they were merged with a second set of divergent 'resistant' and 'susceptible' lines (comprising 9 0 - 9 5 ewes each) established in 1986 (Bisset et al., 1991). Selection in these later lines was based on consistently low or high FEC following each of three successive periods o f natural poly-generic nematode challenge, between weaning (early December) and a u t u m n / e a r l y winter ( M a y / J u n e ) . Each test period was terminated by an anthelmintic treatment. This procedure has since been followed for the merged lines. It was confidently anticipated that selective breeding for divergence in FEC following natural challenge would result in lambs which were divergent in their abilities to resist the establishment of trichostrongyle worm burdens. FECs have been used for many years as an ante-mortem means of indicating the size of trichostrongyle nematode burdens in lambs. While their use as a diagnostic tool has limitations insofar as they are influenced by a variety of factors such as total faecal output, composition of infection and resistance status of the host, correlations of 0.74 and 0.82 between individual FECs


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and strongyle worm counts (excluding Nematodirus spp.) in lambs were found by McKenna (1981) and Douch et al. (1984), respectively. Considerable progress has been made in developing the lines for experimental use and in understanding the genetics of FEC as a selection trait. Results of genetic analyses from the Wallaceville and other similar selection flocks in New Zealand have been published in several papers, which have recently been reviewed by Morris et al. (1995). FEC has proved to be a moderately heritable trait in lambs grazed under New Zealand conditions with heritability estimates averaging about 0.23 + 0.02. Divergence between the selection lines has developed to the extent where 1993-born lambs differed in FEC by a factor of 9.2. However, despite the widely differing FEC levels between individual sire progeny groups and between the breeding lines themselves when grazed together, there has proved to be no consistent relationship between FEC and breech soiling or liveweight gain in lambs under nematode challenge (Morris et al., 1995), raising the possibility that differences in FEC did not reflect differences in actual worm burdens in field-grazed lambs. Although there has been a limited amount of work carried out to examine the establishment of experimental doses of selected nematode species in sheep breeding lines showing high or low FEC under field conditions in New Zealand (e.g. Watson et al., 1989; WiUiamson et al., 1994), there is little information on naturally acquired mixed worm burdens in continuously challenged grazing lambs. In this paper we examine the nematode parasite status and immunological responses of progeny of selected Romney rams, bred for genetic divergence in FEC, following an extended period of exposure to naturally contaminated pasture.

2. Materials and methods 2.1. Experimental animals and study design

The study was conducted during 1989 and 1990. In both years animals involved were 8- 9-month-old spring-born (mainly September) outcross male progeny of the two lowest FEC (LFEC) (i.e. most 'resistant') and the two highest FEC (HFEC) (i.e. most 'susceptible') rams from Wallaceville Animal Research Centre's divergent Romney breeding lines. In the first year a total of 26 lambs (13 sired by LFEC rams and 13 sired by HFEC rams) were used, while in the second year 39 lambs (21 sired by LFEC rams and 18 sired by HFEC rams) were used. In both years the lambs were grazed together from birth as part of a larger 'progeny test' flock being used to verify the genetic merit of selected divergent line rams. Each of the rams tested had been mated to 20 randomly allocated mixed age ewes and the performance of their progeny with respect to FEC and production parameters assessed. As part of the progeny testing procedure, FECs were recorded for each of the lambs at weaning in early December (approximately 3 months old), in January, in February (Year 1) or March (Year 2), and in early June (Year 1) or May (Year 2). Following each of the first three sampling times all lambs received anthelmintic treatment (Year h oxfenda-

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zole (Systamex; Coopers Animal Health NZ Limited); Year 2: levamisole (Nilverm; Coopers Animal Health NZ Limited)), at the manufacturer's recommended dose rate, to remove existing worm burdens. Faecal egg count reduction tests were used to check anthelmintic efficacy. There was no indication of anthelmintic resistance. Thus, each successive FEC represented a new infection. Immediately after the final faecal sample had been taken (June in Year 1; May in Year 2) all male lambs in selected sire progeny groups were transferred from pasture to pens where they were kept for 24 h without food to reduce gut fill before slaughter. All animals were then slaughtered humanely using a captive bolt pistol followed by exsanguination in accordance with the requirements of the Wallaceville Animal Research Centre Animal Ethics Committee.

2.2. Faecal egg counts Faecal egg counts were carried out using a modified McMaster technique in which each egg counted represents 100 worm eggs per gram of faeces.

2.3. Postmortem processing and worm counting procedures Following slaughter, the gastrointestinal tract of each lamb was removed, and the abomasum, small intestine and caecum/large intestine were ligatured and separated. Recovery and counting of worms from the abomasum and small intestine were carried out using similar techniques to those described by Brunsdon (1972). Briefly, the contents of the abomasum and the total length of the small intestine of each lamb were thoroughly washed out and respective washings made up to 8 I. A one-tenth aliquot of this material was screened over a 4 5 / z m mesh sieve and preserved in 10% formalin. All the worms present in a one-tenth sub-sample of the preserved material from each of these o~gans were identified, differentiated by developmental stage, and counted using stereo- or compound microscopes as appropriate. Species composition of Trichostrongylus and Ostertagia burdens was estimated by examination of the spicule structure in up to 30 adult male worms. Worm counts for each animal were estimated by multiplying the numbers of each species counted by the aliquot factor (100). In the case of the caecum/large intestine, the total contents were thoroughly washed out and screened over a 635/xm mesh sieve, examined over a light box, and nematodes removed for differentiation and counting. In the second year in utero egg counts were carried out on 30 randomly selected female Trichostrongylus and Cooperia from each host where this number of worms was available. This was impractical in most other species due to the low numbers of adult worms in many of the lambs, particularly those in the LFEC genotypes.

2.4. Host immune responses Immediately after slaughter, in both years of the study, a small sample of intestinal tissue was taken from each lamb approximately 1 m distal to the bile duct to assess numbers of globule leucocytes/mucosal mast cells ( G L s / M M C s ) , connective tissue type mast cells (CTMCs) and eosinophils present in the mucosa. Histological and cell


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counting procedures were as previously described by Douch and Morum (1993). In the second year, in addition to the above tissue sample, a short length (10-15 cm) of small intestine from an adjacent position was tied off and removed to assess numbers of globule leucocytes present in the intestinal lumen. Procedures and results have been published elsewhere (Stankiewicz et al., 1993). Following removal of a small aliquot of the fluid contents for cell counting the balance of the material was returned to the appropriate post-mortem sample before processing for worm counting. In addition, in the second year two separate blood samples were collected from each lamb by jugular venepuncture, one approximately 1 week before slaughter using 15-ml serum separation tubes and the other at the time of slaughter using 10-ml heparinized tubes. Levels of Trichostrongylus colubriformis-specific antibody and specific immunoglobulin isotypes IgG 1 and IgM were determined in the first sample by ELISA using secretory/excretory antigens derived from both adult worms and infective larvae (L3). Procedures used were as described previously by Douch et al. (1994). Fifty microlitres of blood from the second sample were placed in 450/xl of Carpentier's stain and the eosinophils counted in a haemocytometer.

2.5. Statistical analysis Between-genotype differences in FECs, nematode establishment, developmental composition, in utero egg counts, and host cellular and antibody responses were examined using Kruskal-Wallis non-parametric tests. As is appropriate with such tests, data are presented as medians. Spearman's rank correlation coefficients were used in correlation analyses.

3. Results

3.1. Year 1 Table 1 summarises FEC data from lambs in the first year of the study. Although there was considerable variation in individual FECs as shown by the range, in February

Table 1 Faecal egg counts (FECs) of outcross male progeny of selected low FEC and high FEC Romney rams from divergent breeding lines, following successive periods of exposure to natural challenge during summer and autumn (1989) (values are given as median with range in parentheses) Sire selection line Low FEC Sample size FEC

13 23 January 26 February 6 June

1700 (450-8100) 1500 (500-5100) 1400 (200-7100)

* P < 0.05; * * P < 0.01; NS, not significant.

High FEC 13 2650 (1000-5100) NS 3300 (800-9400) * 4900 (1900-12 000) * °

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Table 2 Post-mortem worm burdens ~ of outcross male progenyof selected low FEC and high FEC Romneyrams from divergent breeding lines, following exposure to natural challenge without anthelmintic treatment during autumn/early winter (1989) (values are given as median with range in parentheses) Sire selectionline Sample size

Low FEC

High FEC

13

13

Haemonchus contortus Ostertagia circumcincta b Trichostrongylus axei Cooperia curticei Nematodirus spp. c Strongyloides papillosus Trichostrongylus colubriformis Trichostrongylus vitrinus Chabertia ovina Oesophagostomum venulosum Trichuris ovis

500 (100-4000) 4100 (500-21500) 2500 (600-5700) 200 (0-15 700) 500 (0-4200) 900 (100-2800) 10716 (2800-23328) 3150 (0-9792) 57 (17-198) 130 (32-498) 34 (2-234)

400 (0-4000) NS 11 300 (800-19 800) NS 4300 (900-9500) NS 5800 (100-19 600) " * 1100 (0-17100) NS 400 (0-4700) NS 21912 (3000-30448) * ° 7080 (0-12 635) * 120 (8-204) NS 272 (0-518) NS 24 (2-74) NS

Total adult trichostrongylidsd

18600 (7300-64 300)

49 500 (14100-80100) * °

All developmentalstages includedexcept where stated. b Small numbersof O. trifurcata present in this total. c Both N. filicollis and N. spathiger present. Adult stages only of Haemonchus sp., Ostertagia sp., Cooperia sp., Trichostrongylus spp.; excludes Nematodirus spp. * P < 0.05; * " P < 0.01; NS, not significant.

and June progeny of the LFEC rams were shedding significantly fewer worm eggs than were their HFEC counterparts. In June, median FECs differed by a factor of 3.5. Post-mortem worm burdens in the lambs are shown in Table 2. As can be seen total adult trichostrongylid burdens differed markedly between LFEC and HFEC lambs ( P < 0.01), with LFEC lambs harbouring only slightly more than one-third (37.6%) of the adult trichostrongylids of their HFEC counterparts. While there was a tendency for this difference to be reflected at the individual species level, differences were statistically significant only in the case of the intestinal species Cooperia curticei, T. colubriformis, and Trichostrongylus vitrinus. However, in the case of Ostertagia circumcincta, LFEC lambs were carrying a significantly higher proportion of fourth stage larval worms than were HFEC lambs (median 0.39 cf. 0.23, P < 0.05). Overall, total strongyle burdens (excluding Nematodirus spp.) were strongly correlated with the final FEC before slaughter (June) ( r = 0.91, P < 0.01). Correlations with earlier FECs, which due to intervening drench treatment represented different infections, were not as strong but nevertheless significant in the case of the February sample ( r = 0.31, NS and r = 0.47, P < 0.05 for January and February samples, respectively). Correlations between total strongyle burdens and June FEC, when calculated for LFEC and HFEC lambs separately, were similar to each other and not substantially different from that calculated for the overall (combined) data set ( r = 0.79 and r = 0.84 for LFEC and HFEC lambs, respectively). Histological examination of tissue samples from

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Table 3 Faecal egg counts (FECs) of outcross male progeny of selected low FEC and high FEC Romney rams from divergent breeding lines, following successive periods of exposure to natural challenge during summer and autumn (1990) (values are given as median with range in parentheses) Sire selection line Low FEC Sample size FEC

21 23 January 6 March 9 May

600 (100-2200) 700 (200-3900) 700 (0-2100)

High FEC 18 1850 (900- 8800) * * 3700 (1000-9100) * " 4350 (400-15000) * *

* * P < 0.01.

the small intestine revealed significantly higher numbers of G L s / M M C s in the intestinal mucosa of LFEC lambs than in their HFEC counterparts (median count 43.0 cf. 21.7 cells per standard field scanned, P < 0.01). Numbers of CTMCs and eosinophils in the intestinal mucosa did not differ significantly between the groups (median counts 16.7 cf. 11.7 cells per field, and 64.7 cf. 59.7 cells per field, respectively). Nevertheless, overall there were significant negative correlations between the numbers of both G L s / M M C s and CTMCs and the total number of small intestinal nematodes present (r = - 0 . 5 3 and r = - 0 . 5 1 , respectively; both P < 0.01).

3.2. Year 2 Table 3 summarises FEC data from lambs in the second year of the study. Significantly fewer nematode eggs were shed by progeny of LFEC rams at all sampling dates. In May, median FECs differed by a factor of more than six. Post-mortem worm burdens in the lambs after autumn grazing are shown in Table 4. Overall, LFEC lambs were carrying only one-third of the adult trichostrongyle burden (33.5%) of their HFEC counterparts ( P