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Genetic control of resistance to helminths in sheep
Veterinary immunology and immunopathology
Veterinary Immunology and lmmunopathology 54 (1996) 245-254
Genetic control of resistance to helminths in sheep R.G. Windon CSIRO Diuision
ofAnimal
Production.
Pastoral
Research Lnhorcctory.
Priuute
Mail Bug. Amid&.
NSW
2350. Austruliu
Abstract
Uncertainties over the continued effectiveness of currently available anthelmintics and the massive costs associated with development of new drugs have provided an impetus to search for alternative measures to control gastrointestinal nematodes in sheep. One option is to exploit the genetically determined variability in resistance existing within host populations. A number of selection experiments, comprising divergent and control lines, have been initiated to investigate the nature of this genetic regulation. It was found that the heritability of worm-egg counts in faeces after infection ranges from 0.2 to 0.4, indicating that worthwhile genetic gains can be achieved in commercial breeding programmes. Immune responses directed against parasites are under genetic control and appear to be the major factor responsible for the interline differences. Consequently, selection for increased resistance to gastrointestinal nematodes has resulted in an enhanced reactivity across a broad range of immunological functions (humoral, cellular and effector responses). These mechanistic studies have relevance to the development of vaccines and vaccination strategies, as well as for the application of phenotypic and genetic markers to measure resistance more accurately or to identify genetically resistant animals independently of infection.
1. Introduction The introduction
of modem
broad-spectrum
anthelmintics
promised
a safe,
reliable
and economical means to control gastrointestinal nematodes in sheep. However, the appearance of parasite strains resistant to the single mode of action of these chemicals and the enormous costs and long lead times required to develop new drugs have brought into question current practices which rely heavily on chemotherapy. Furthermore, there are increasing environmental concerns about chemical residues in meat and on pasture resulting from anthelmintic usage. For these reasons, attention has focused on the need for alternative control measures to either reduce or eliminate the current dependence on chemotherapy. The immune response plays an integral role in determining the outcome of a host/parasite interaction (Dineen, 1978). However, despite intensive efforts over a 0165-2427/96/$15.00
Published by Elsevier Science B.V.
P/f SOl65-2427(96)05710-S
number of years, the complex interactions between the various diverse components of the immunological repertoire which act to cause worm expulsion remain poorly defined (Rothwell. 1989). A knowledge of the mechanisms of resistance is required to optimize vaccination strategies. Thus, manipulation of the route of administration and type of adjuvant for protective antigens may enhance the generation of appropriate effector responses. A constraint on vaccination occurs. however. in that the immune response is modulated by a number of intrinsic and extrinsic factors (Dineen, 1978). Of these, genetic constitution exerts a major influence over individual host resistance to helminth infections (Wakelin. 1985). A commercially successful vaccine will therefore have to overcome these modulating influences and, in particular. the genetic restriction imposed by susceptible genotypes. There has been an increasing interest in breeding parasite-resistant sheep. In contrast to the immediate effect chemotherapy has on the parasite burden. the implementation of genetic resistance is long term in nature but has the potential to offer low cost control throughout an animal’s life (Windon et al.. 1993; Woolaston and Eady, 1995). However, it has yet to be unequivocally shown that there are no adverse consequences associated with selection for resistance (for example. production parameters or responses to non-parasite pathogens). This paper will focus on work being carried out in Australia with and New Zealand to investigate genetic control of resistance to helminths, particular reference to gastrointestinal nematodes. Consideration will also be given to studies into the mechanisms of resistance using the experimental selection flocks.
2. Evidence
for genetic
control
of resistance
Evidence for genetic control of resistance to gastrointestinal nematodes in sheep comes from comparisons carried out between and within breeds. It is of interest to note that, from a survey of the literature. Gray et al. (1987) concluded that variability in resistance within breeds may be as boreat as the variability between breeds. However. Gray (1991) and (Gray and Gill. 1993) highlight the need to exercise caution in the interpretation of results comparing different populations (for example, breeds, strains and bloodlines). They consider that the designs of the majority of these experiments do not take into account any confounding effect relating to the undue influence of particular sires. These sample groups, therefore, may not be representative of the populations being studied.
It has been known for many years that some breeds are more resistant to gastrointestinal nematodes than others (for reviews see Gruner and Cabaret. 1988: Gray, 1991). Although reports of these differences have been largely empirical in nature. there is a consistent pattern of responsiveness between breeds that is associated with different production characteristics (Gray. I99 I ). Thus. in general. the exotic hair-type breeds (such as the Red Maasai. Florida Native. Barbados Blackbelly and St. Croix) are more resistant than European breeds, which in turn are more resistant than breeds primarily
R.G. Windon / Vrterinury Immunology und Immuru,pathobgy
54 f 1996) 245-254
23-l
maintained for their fine-wool production (such as the Merino and Rambouillet). The value of between-breed variation will come from the substitution of a susceptible breed with one having enhanced resistance. However, this would have to be carried out with due regard to the appropriate production characteristics (meat or wool) required in particular localities (Gray, 1991). In most experiments, the mechanistic basis of breed differences has not been defined. However, Windon et al. (1993) reported the results of an experiment which compared responses of Romney and fine-wool Merino lambs to the intestinal nematode Trichostrongylus cofubriformis. These lambs were reared and maintained in pens to standardize environmental influences and ensure acquired responses were generated to a defined parasite load. No differences occurred between breeds in the unvaccinated controls, but Romney lambs had significantly lower worm-egg counts in faeces (82% protection) than Merino lambs (43% protection) after vaccination with irradiated T. colubriformis larvae and challenge with normal larvae. Although this experiment may not have satisfied the criteria of Gray and Gill (1993) for between-breed comparisons, it does suggest that acquired (immunological) responses rather than innate resistance play a role in the differences observed between Romney and Merino lambs. 2.2. Within breeds A number of distinct populations can exist within breeds. For example, in the Australian Merino a number of strains have developed in response to particular environments (Massey, 1990). Furthermore, the stud hierarchy within strains has resulted in the formation of a number of bloodlines. A study carried out recently by Woolaston and Eady (1995) examined the degree of variation in resistance to gastrointestinal nematodes within Merino populations. In this work, lambs from over 57 bloodlines, representing each of the major strains, were artificially infected with either the abomasal nematode Haemonchus contortus or T. colubriformis at six different locations across Australia. The results showed that most of the genetic variation (85%) occurred within flocks, whereas variation between strains and between bloodlines within strains was small (4% and 9%. respectively). The authors concluded that within-flock selection offers the greatest potential for the genetic improvement of this trait within the constraints of the existing production system. 2.3. Selection experiments Genetic variability within flocks has been utilized and manipulated by selection in a number of experimental programmes in Australia (Windon, 1990; Windon et al., 1993; Woolaston and Eady, 1995), New Zealand (Bisset et al., 1991; Morris et al., 1995) and elsewhere (Sreter et al., 1994). These long-term selection programmes have common aims: ( 1) to estimate genetic parameters and the correlated responses between resistance to nematodes and other economically important (production) traits; (2) to understand the mechanisms of resistance under genetic control; (3) to identify phenotypic and genetic markers with resistance which facilitate incorporation of parasite resistance into commercial breeding programmes; (4) to assess the specificity of selection with regard to
other parasite and non-parasite pathogens. Of the flocks undergoing selection for resistance, the Australian flocks located at Armidale, NSW. are described here as an example of the results being achieved. 2.3. I. The Trichostronyylus selection flock The Trichostrongylus selection flock has been described by Windon and colleagues (Windon and Dineen. 1984; Windon. 1990. 1991a; Windon et al.. 1993). This flock of medium wool Peppin Merinos was commenced in 1975 with the testing of parent generation lambs. and subsequent selection and assortative matings have been used to establish lines in which lambs are either immunologically responsive (high responders) or susceptible (low responders). An unselected line provides animals as controls for the selection and also parasitological controls. Lambs from this flock have usually been reared helminthologically naive in pens from birth and subjected to a testing regime which incorporates vaccination with irradiated 7: colubriformis larvae and challenge with normal larvae. Individual responsiveness in the pen testing regime is based on the mean of five faecal egg counts carried out after challenge. Lambs from the high ~(1 counts about 10% of those for low responders. responder line currently have faecal ebb and the estimated heritability (h’) for response to vaccination and challenge in this flock is 0.37 + 0.04 (Woolaston and Eady. 1995). More recently. responses of pasture-reared lambs from this selection flock have been evaluated after each of two artificial infections with T. cohbri’ormis. Lambs were weaned at I4 weeks of age. given a primary challenge at I7 weeks, treated with anthelmintic at 22 weeks, and given a secondary challenge at 23 weeks. Although ~0 counts were apparent between the high and low significant differences in faecal ekt. responder lines after each challenge. these were greater in the second challenge period. The heritability estimate for faecal egg count during the first challenge period was low. but during the second challenge period this was at comparable levels (0.39 _t 0.11) to those calculated for the pen studies (see above) (Woolaston and Eady. 1995). Genetic correlations between faecal egg counts from both paddock infection periods and the pen testing were high, suggesting that essentially the same trait was being measured. 2.3.2. The Haemonchus selection ,flock This fine-wool Merino flock was established in 1978 (Piper. 1987: Woolaston, 1990: Woolaston et al.. 1990: Woolaston and Eady. 1995). Divergent selection lines. together with an unselected control line. have been established in which lambs on pasture exhibit increased and decreased resistance to a single artificial infection with H. contortus at 5-6 months of age. Individual responsiveness is based on maximal faecal egg counts between 3 and 6 weeks after infection. After IS years of selection, mean worm-egg counts in the increased resistance line are 20-30s of those in the decreased resistance line. The heritability estimates for faecal ebb 0~ counts after infection is 0.29 + 0.03 (Woolaston and Eady. 1995). 2.3.3. The Haemonchus resistance jlock (UNE ‘Golden Ram’ jlock) This flock was established at the University of New England, Armidale, in 1980 (Albers et al.. 1987; Woolaston and Eady. 1995) after a trial designed to investigate the
R.G. Windon / Vererinury Immunology and lmmumpathobgy
54 11996) 245-254
249
degree of genetic variability in resistance to H. contortus. This work identified a ram (the ‘Golden Ram’) whose progeny exhibited extreme levels of resistance. Subsequent backcross matings between relatives of this ram have been used to establish a line of animals with increased resistance to H. conrorrus, and these genetically resistant lambs are compared with control (unselected) animals. Progeny testing consists of monitoring faecal egg counts in pastured lambs given a single infection of H. contortus at about 4-5 months of age. The heritability of faecal egg counts in this flock is 0.24 f 0.04 (Woolaston and Eady, 1995). Genetic analyses have failed to find any evidence of a putative major gene being responsible for the extreme expression of resistance in the founding sire.
3. Mechanisms
of resistance
The selection flocks is a valuable research resource to study the mechanisms of resistance to parasites. By comparing the defined extremes of responsiveness existing between lines, intensive investigations into the underlying mechanisms have been carried out in the Trichostrongylus selection flock and the Haemonchus resistance (‘Golden Ram’) flock (Windon, 1991a; Gill et al., 1991). However, to date, little information is available on the mechanistic basis of resistance in the Haemonchus selection flock. A number of immunological responses have been shown to be under genetic control in rodent/parasite models (Wakelin, 1985). Resistance in the Trichosrrongylus selection flock is overtly immunologically based, and the Haemonchus resistance flock also has a strong immunological basis. Selection for increased resistance is therefore associated with heightened reactivity across a broad range of immunological functions (Windon, 1991 a; Gill et al., 1991). In the Trichostrongylus selection flock, high responder animals have greater antigen recognition (parasite specific cellular and antibody responses) and effector responses (mast cells/globule leukocytes, circulating eosinophils and mediator release) (reviewed by Windon, 1990, 1991a,b; Windon et al., 1993). Similarly, with the Haemonchus resistance flock, resistance is associated with increased antibody responses (local and circulating) (Gill et al., 1993b,1994), cellular responses (Gill et al., 1993a; Gill, 19941, and effector responses (reviewed by Gill et al., 1991). Recent studies in the selection flocks have been focused on T helper (CD41 cells which appear to play a central role in the regulation and control of appropriate effector responses. Haig et al. (1989) showed that CD4 cells were activated during infection with H. conforfus, and Gill et al. (1993~) were able to induce susceptibility in genetically resistant lambs by depletion of CD4 cells by monoclonal antibodies. These latter workers also found that CD4 cell depletion was associated with reduced numbers of mucosal mast cells, globule leukocytes and eosinophils in tissue. Rodent T helper cells can be divided into two groups, ThI and Th2, on the basis of the cytokines they produce (Mossmann and Coffman, 1989). Furthermore, in mice, resistance to nematodes has been associated with a Th2 response, whereas susceptibility was associated with a Thl response (Else and Grencis, 1991; Else et al., 1994; Robinson et al., 1995). The immune responses under Th2 control include the induction of IgE, mastocytosis and eosinophilia
(Finkelman and Urban. 1992). Although. as yet, there IS no evidence for a ThI/Th2 dichotomy in sheep. the availability of an increasing number of ovine cytokine reagents has allowed the initiation of studies directed towards identifying the cytokine profiles associated with resistance and susceptibility to gastrointestinal nematodes in the selection flocks. It is anticipated that these profiles will allow greater definition of the factors controlling the immune response and identify possible candidate genes for analysis as genetic (DNA) markers. Work carried out with lymphocytes isolated from helminthologically naive high and low responders (Trichosrron~ylus selection flock) has demonstrated that genetically resistant lambs are inherently able to produce more colony stimulating factors than low responders. This was assessed by colony growth and differentiation of bone marrow cells in soft agar cultures. Furthermore. bone marrow cells from high responders were able to respond more vigorously to these differentiation factors (Windon. Haig. Seow and Rothel. unpublished results). Of particular interest for the practical application of genetically determined resistance is whether selection for one parasite species will also result in improved protection against other related and unrelated parasite species. and indeed other non-parasite pathogens. Some circumstantial evidence is accumulating that mechanistic differences exist in the responses provoked by infection with 7’. colubriformis and H. contortus (see Windon. I99 I b). This corroborates results which show that resistance to infection with the heterologous nematode species ( H. corttortu.s or T. cnluhr~fbrn~is) in the Trichostrongylus and Hnemonchus selection flocks is not as great as that for the homologous infection against which each selection is based (see Windon. 1990). It is anticipated that knowledge of the mechanisms of resistance, and particularly the cytokine profiles, will allow prediction of the susceptibility of parasite-resistant sheep to nonparasite pathogens (Windon et al., 199.3).
4. Markers for resistance A number of immunological and physiological parameters, or the genes controlling them, have been investigated as markers for resistance (see Windon, 1990). To be of most benefit. markers should either provide a more accurate measure of resistance (relative to the currently used faecal e gg counts). or provide a predictive indication of resistance which is independent of infection and environmental factors. 4.1. Phenotypic
nudwrs
Phenotypic markers are those that have a functtonal basis and are usually expressed during infection. Eady (1995) and Douch et al. (1996) have reviewed phenotypic markers based on parasitological, immunological and physiological traits, their characteristics and the requirements for inclusion in breeding programmes. Faecal egg counts are used to measure individual responsiveness in the experimental selection programmes. This parameter itself is an indirect measure of the host’s worm burden. However. faecal egg count may not necessarily be a good indicator because a number of factors can affect egg production by female worms or faecal consistency
R.G.
Windon
/ Veterinctry
Immunology
und Immunopurholo~y
54 f 1996)
245-254
251
(Douch et al., 1996). A study of the population dynamics of T. colubrifot-mis infection has shown that worm-egg production is influenced by immunological responses earlier than adult worms are expelled (Dobson et al., 1990). In contrast, egg counts more accurately reflect H. contorfus worm burdens (Barger et al., 1985). Faecal egg counts can, however, directly influence the level of larval contamination on pasture (Eady, 1995). Circulating eosinophils are associated with resistance under strictly controlled conditions in penned Merinos (Dawkins et al., 1989) and in penned and naturally infected Romneys (Buddle et al., 1992). On the basis of these and other studies, Hohenhaus and Outteridge (1995) have suggested that eosinophils, alone or in conjunction with ovine lymphocyte antigen (OLA) typing. may be a suitable selection criterion by which to include parasite resistance in breeding programmes. However, this was not confirmed by Woolaston et al. (1996), who concluded that circulating eosinophil numbers were less effective than faecal egg counts during infection with either H. contorrus or T. colubriformis. Thus, although eosinophil counts have relevance to immunological studies, the nature of the eosinophil response reduces their suitability as a trait for selection. In this regard, it has been found that circulating eosinophil counts are significantly correlated with the acquisition of resistance, but highly resistant animals that have expelled their worm burden as well as susceptible animals have low eosinophil counts (Windon, unpublished results). There are also differences in the association between circulating eosinophil counts and the acquisition of resistance to different gastrointestinal nematode species. This association is strong for T. colubriformis but weak for H. contortus (see Windon et al., 1993). A phenotypic marker receiving particular attention in New Zealand is serum antibody levels against excretory/secretory parasite antigens determined by ELISA procedures (reviewed by Douch et al., 1996). In this work, antibody levels (predominantly IgGl) were found to be moderately heritable and genetically correlated with faecal egg count. A selection experiment has subsequently been established to further investigate the association. Early results have shown that selection for antibody response was successful but no differences occurred in faecal egg counts between lines (high antibody. low antibody, control). The authors consider that this observation may be a result of low nematode infection. 4.2. Genetic markers The potential advantage of genetic markers is that, by definition, they are based on genes or DNA sequences which can be used to identify resistant or susceptible animals independently of infection. A number of genetic markers have been evaluated for associations with resistance to internal parasites. Of these, some success has been reported by Hohenhaus and Outteridge (1995) using OLA typing, but other workers using human MHC class II restriction fragment length polymorphism probes did not find a statistically significant association with resistance to T. colubriformis (Hulme et al., 1993). An approach using linkage analysis to identify genetic markers for resistance to gastrointestinal nematodes has been recently described by Blattman and Beh (1995) and Beh and Maddox (1996). There have been three resource flocks established in Australia
and New Zealand to carry out this work. A flock at the CSIRO Division of Animal Production was based on the crossing of high and low responder progeny from the Trichosfrongylus selection flock to produce six sires heterogeneous for resistance to T. colubrifornzis. Each of these sires is being used to produce 200 half sib progeny. The AgResearch (New Zealand) resource flock has similarly been established by crossing lines of Romney sheep bred for high and low faecal egg counts. The University of Melbourne flock was established by crossing low responder rams from the Trichosrrongylus selection flock with AgResearch resistant Romney ewes to produce offspring which will subsequently be used as parents of an intercross second generation. In each case. microsatellite markers are being used to scan the entire genome. In addition, other techniques are being used which focus on microsatellite-associated candidate genes.
5. Conclusions Substantial genetic variation exists in resistance to gastrointestinal nematodes both between and within breeds of sheep. Within breeds, experimental selection programmes have demonstrated that resistance is a heritable trait, and substantial gains can be achieved in commercial breeding enterprises. The selection flocks are also a valuable research resource for studies into the mechanisms of resistance to nematodes as they provide animals having defined extremes of responsiveness. An understanding of resistance mechanisms. which appear to be immunologically based. is crucial for the optimization of vaccination strategies. Furthermore. these studies may facilitate the identification of phenotypic and genetic markers. The acceptance of parasite resistance as a trait for incorporation into commercial breeding programmes will depend on the development of a cost-effective. accurate and repeatable measure and. in particular, one which is independent of infection and environmental influences. As the parasite population itself exhibits heterogeneity and has shown a capacity to adapt in the face of strong selection pressure (for example, anthelmintics), successful control programmes should not rely on only one procedure. Future parasite control, therefore. may well involve an integrated approach using genetically resistant animals in conjunction with anthelmintic treatment. vaccination and pasture management (including nutrition).
Acknowledgements The Armidale based selection flocks referred to in this paper have been supported by Australian woolgrowers and the Australian Government through the Australian Wool Research and Promotion Organisation.
References Albers.
G.A.A..
Parasitol.. Bargrr,
Gray.
A.D.,
Piper.
L.R..
Bar!er.
J.S.F..
Lc Jamhrc,
L.F.
and
Barger,
I.A..
17: 1355.
[.A., Le Jamhrr,
L.F.. Grorp~.
J.R. and Davw,
H.I..
lUX5. Int. J. Parasitol.,
IS- 529.
1987.
Int. J.
R.G.
Windon
/ Veterincrry
Immunology
und Immunoputhology
54 (1996)
245-254
253
Beh, K.J. and Maddox, J.N., 1996. Int. J. Parasitol., in press. Bisset, S.A., Vlassoff, A. and West, C.J.. 1991. Proceedings 21st Seminar of the Sheep and Beef Society. New Zealand Veterinary Society, p. 83. Blattman, A.N. and Beh, K.J.. 1995. In: G.D. Gray, R.R. Woolaston and B.T. Eaton (Editors), Breeding for Resistance to Infectious Diseases in Small Ruminants. ACIAR, Canberra, p. 237. Buddle, B.M., Jowett, G., Green, R.S., Douch, P.G.C. and Risdon, P.L., 1992. Int. J. Parasitol., 22: 955. Dawkins, H.J.S., Windon, R.G. and Eagleson, G.K., 1989. Int. J. Parasitol., 19: 199. Dineen, J.K., 1978. In: A.D. Donald, W.H. Southcott and J.K. Dineen (Editors), The Epidemiology and Control of Gastrointestinal Parasites of Sheep in Australia. CSIRO Division of Animal Health, Melbourne, p. 121. Dobson, R.J., Wailer, P.J. and Donald, A.D., 1990. Int. J. Parasitol., 20: 347. Douch, P.G.C., Green, R.S., Morris, C.A., McEwan, J.C. and Windon, R.G., 1996. Int. J. Parasitol., in press. Eady, S.J., (1995. In: G.D. Gray, R.R. Woolaston and B.T. Eaton (Editors), Breeding for Resistance to Infectious Diseases in Small Ruminants. ACIAR, Canberra, p. 219. Else, K.J. and Grencis, R.K., 1991. Parasitol. Today, 7: 313. Else, K.J., Finkelman, F.D., Maliszewski, C.R. and Grencis, R.K., 1994. J. Exp. Med., 179: 347. Finkelman, F.D. and Urban, J.F., 1992. Parasitol. Today, 8: 311. Gill, H.S., 1994. Int. J. Parasitol., 24: 749. Gill. H.S., Gray, G.D. and Watson, D.L., 1991. In: G.D. Gray and R.R. Woolaston (Editors), Breeding for Disease Resistance in Sheep. Australian Wool Research and Development Corporation, Melbourne, p. 67. Gill, H.S., Colditz, LG. and Watson, D.L., 1993a. Res. Vet. Sci., 54: 361. Gill, H.S., Gray, G.D., Watson, D.L. and Husband, A.J.. 1993b. Parasite Immunol., 15: 61. Gill, H.S., Watson, D.L. and Brandon, M.R., 1993~. Immunology, 78: 43. Gill, H.S., Husband, A.J., Watson, D.L. and Gray, G.D., 1994. Res. Vet. Sci., 56: 41. Gray, G.D., 1991. In: R.F.E. Axford and J.B. Owen (Editors), Breeding for Disease Resistance in Farm Animals. CAB International, Wallingford, p. 139. Gray, G.D. and Gill, H.S., 1993. hit. J. Parasitol., 23: 485. Gray, G.D., Presson, B.L., Albers, G.A.A., Le Jambre, L.F., Piper, L.R. and Barker, J.S.F., 1987. In: B.J. McGuirk (Editor), Merino Improvement Programs in Australia. Australian Wool Corporation, Melbourne, p. 365. Gruner, J. and Cabaret, 1988. In: E.F. Thomson and F.S. Thomson (Editors), Improving Small Ruminant Productivity in Semi-Arid Areas. Kluwer, Dordrecht, p. 257. Haig, D.M., Windon. R.G., Blackie, W., Brown, D. and Smith, W.D., 1989. Parasite Immunol., 11: 463. Hohenhaus, M.A. and Outteridge, P.M., 1995. Br. Vet. J., 151: 119. Hulme, D.J., Nicholas, F.W.. Windon, R.G., Brown, S.C. and Beh, K.J., 1993. J. Anim. Breed. Genet., I IO: 459. Massey, C.J., 1990. The Australian Merino. Viking O’Neil, Melbourne. Morris, C.A., Watson, T.A., Bisset, S.A., Vlassoff, A. and Douch, P.G.C., 1995. In: G.D. Gray, R.R. Woolaston and B.T. Eaton (Editors), Breeding for Resistance to Infectious Diseases in Small Ruminants. ACIAR, Canberra, p. 53. Mossmann, T.R. and Coffman, R.L., 1989. Adv. Immunol., 46: Ill. Piper, L.R., 1987. In: B.J. McGuirk (Editor), Merino Improvement Programs in Australia. Australian Wool Corporation, Melbourne, p. 35 1. Robinson, K., Bellaby, T. and Wakelin, D., 1995. Parasitology, 110: 71. Rothwell, T.L.W., 1989. Int. J. Parasitol., 19: 139. Stiter, T., Kassai, T. and Takacs, E., 1994. Int. J. Parasitol., 24: 871. Wakelin, D., 1985. Parasitol. Today, 1: 17. Windon, R.G., 1990. Rev. Sci. Tech. Off. Int. Epizoot., 9: 555. Windon, R.G., 1991a. In: G.D. Gray and R.R. Woolaston (Editors), Breeding for Disease Resistance in Sheep. Australian Wool Research and Development Corporation, Melbourne, p. 77. Windon, R.G., 199lb. In: R.F.E. Axford and J.B. Owen (Editors), Breeding for Disease Resistance in Farm Animals. CAB International, Wallingford, p. 162. Windon, R.G. and Dineen, J.K.. 1984. In: J.K. Dineen and P.M. Outteridge (Editors), Immunogenetic
Approaches Division Windon,
to the Control
of Animal
of Endoparas~tc~ -with
Health,
Melbourne.
R.G.. Gray. G.D. and Wool&on,
Woolaston,
R.R.,
19X). Wool Technol.
Woolaston.
R.R.. End?. S.J..
( 1995).
G.D. Gray. R.R. Woolaston
p.
to the Parasites of Sheep. CSIRO
in Breeding
Woolaston,
R.R.. Barger. I.A. and Piper, L.R.. R.R., Manurli.
for Resistance
to Infecuous
Canberra),
1990. Int. J. Parasltol..
P.. Eady. S.J.. Barger.
17. 37.
I.
and B.T. Eaton, Eds. (ACIAR,
in press.
Rrferrncc
R.R.. 1993. Proc. Sheep Vet. Sot..
Sheep Breed., 38.
Woolaston,
1996. Int. J. Parasitol.,
Partmular
Ii.
1.A.. Le Jambre,
Diseases in Small Ruminants,
p. 53.
20: 1015. L.F.,
Banks,
D.J.D.
and Windon,
R.J..
Veterinary Immunology and lmmunopathology 54 (1996) 245-254
Genetic control of resistance to helminths in sheep R.G. Windon CSIRO Diuision
ofAnimal
Production.
Pastoral
Research Lnhorcctory.
Priuute
Mail Bug. Amid&.
NSW
2350. Austruliu
Abstract
Uncertainties over the continued effectiveness of currently available anthelmintics and the massive costs associated with development of new drugs have provided an impetus to search for alternative measures to control gastrointestinal nematodes in sheep. One option is to exploit the genetically determined variability in resistance existing within host populations. A number of selection experiments, comprising divergent and control lines, have been initiated to investigate the nature of this genetic regulation. It was found that the heritability of worm-egg counts in faeces after infection ranges from 0.2 to 0.4, indicating that worthwhile genetic gains can be achieved in commercial breeding programmes. Immune responses directed against parasites are under genetic control and appear to be the major factor responsible for the interline differences. Consequently, selection for increased resistance to gastrointestinal nematodes has resulted in an enhanced reactivity across a broad range of immunological functions (humoral, cellular and effector responses). These mechanistic studies have relevance to the development of vaccines and vaccination strategies, as well as for the application of phenotypic and genetic markers to measure resistance more accurately or to identify genetically resistant animals independently of infection.
1. Introduction The introduction
of modem
broad-spectrum
anthelmintics
promised
a safe,
reliable
and economical means to control gastrointestinal nematodes in sheep. However, the appearance of parasite strains resistant to the single mode of action of these chemicals and the enormous costs and long lead times required to develop new drugs have brought into question current practices which rely heavily on chemotherapy. Furthermore, there are increasing environmental concerns about chemical residues in meat and on pasture resulting from anthelmintic usage. For these reasons, attention has focused on the need for alternative control measures to either reduce or eliminate the current dependence on chemotherapy. The immune response plays an integral role in determining the outcome of a host/parasite interaction (Dineen, 1978). However, despite intensive efforts over a 0165-2427/96/$15.00
Published by Elsevier Science B.V.
P/f SOl65-2427(96)05710-S
number of years, the complex interactions between the various diverse components of the immunological repertoire which act to cause worm expulsion remain poorly defined (Rothwell. 1989). A knowledge of the mechanisms of resistance is required to optimize vaccination strategies. Thus, manipulation of the route of administration and type of adjuvant for protective antigens may enhance the generation of appropriate effector responses. A constraint on vaccination occurs. however. in that the immune response is modulated by a number of intrinsic and extrinsic factors (Dineen, 1978). Of these, genetic constitution exerts a major influence over individual host resistance to helminth infections (Wakelin. 1985). A commercially successful vaccine will therefore have to overcome these modulating influences and, in particular. the genetic restriction imposed by susceptible genotypes. There has been an increasing interest in breeding parasite-resistant sheep. In contrast to the immediate effect chemotherapy has on the parasite burden. the implementation of genetic resistance is long term in nature but has the potential to offer low cost control throughout an animal’s life (Windon et al.. 1993; Woolaston and Eady, 1995). However, it has yet to be unequivocally shown that there are no adverse consequences associated with selection for resistance (for example. production parameters or responses to non-parasite pathogens). This paper will focus on work being carried out in Australia with and New Zealand to investigate genetic control of resistance to helminths, particular reference to gastrointestinal nematodes. Consideration will also be given to studies into the mechanisms of resistance using the experimental selection flocks.
2. Evidence
for genetic
control
of resistance
Evidence for genetic control of resistance to gastrointestinal nematodes in sheep comes from comparisons carried out between and within breeds. It is of interest to note that, from a survey of the literature. Gray et al. (1987) concluded that variability in resistance within breeds may be as boreat as the variability between breeds. However. Gray (1991) and (Gray and Gill. 1993) highlight the need to exercise caution in the interpretation of results comparing different populations (for example, breeds, strains and bloodlines). They consider that the designs of the majority of these experiments do not take into account any confounding effect relating to the undue influence of particular sires. These sample groups, therefore, may not be representative of the populations being studied.
It has been known for many years that some breeds are more resistant to gastrointestinal nematodes than others (for reviews see Gruner and Cabaret. 1988: Gray, 1991). Although reports of these differences have been largely empirical in nature. there is a consistent pattern of responsiveness between breeds that is associated with different production characteristics (Gray. I99 I ). Thus. in general. the exotic hair-type breeds (such as the Red Maasai. Florida Native. Barbados Blackbelly and St. Croix) are more resistant than European breeds, which in turn are more resistant than breeds primarily
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maintained for their fine-wool production (such as the Merino and Rambouillet). The value of between-breed variation will come from the substitution of a susceptible breed with one having enhanced resistance. However, this would have to be carried out with due regard to the appropriate production characteristics (meat or wool) required in particular localities (Gray, 1991). In most experiments, the mechanistic basis of breed differences has not been defined. However, Windon et al. (1993) reported the results of an experiment which compared responses of Romney and fine-wool Merino lambs to the intestinal nematode Trichostrongylus cofubriformis. These lambs were reared and maintained in pens to standardize environmental influences and ensure acquired responses were generated to a defined parasite load. No differences occurred between breeds in the unvaccinated controls, but Romney lambs had significantly lower worm-egg counts in faeces (82% protection) than Merino lambs (43% protection) after vaccination with irradiated T. colubriformis larvae and challenge with normal larvae. Although this experiment may not have satisfied the criteria of Gray and Gill (1993) for between-breed comparisons, it does suggest that acquired (immunological) responses rather than innate resistance play a role in the differences observed between Romney and Merino lambs. 2.2. Within breeds A number of distinct populations can exist within breeds. For example, in the Australian Merino a number of strains have developed in response to particular environments (Massey, 1990). Furthermore, the stud hierarchy within strains has resulted in the formation of a number of bloodlines. A study carried out recently by Woolaston and Eady (1995) examined the degree of variation in resistance to gastrointestinal nematodes within Merino populations. In this work, lambs from over 57 bloodlines, representing each of the major strains, were artificially infected with either the abomasal nematode Haemonchus contortus or T. colubriformis at six different locations across Australia. The results showed that most of the genetic variation (85%) occurred within flocks, whereas variation between strains and between bloodlines within strains was small (4% and 9%. respectively). The authors concluded that within-flock selection offers the greatest potential for the genetic improvement of this trait within the constraints of the existing production system. 2.3. Selection experiments Genetic variability within flocks has been utilized and manipulated by selection in a number of experimental programmes in Australia (Windon, 1990; Windon et al., 1993; Woolaston and Eady, 1995), New Zealand (Bisset et al., 1991; Morris et al., 1995) and elsewhere (Sreter et al., 1994). These long-term selection programmes have common aims: ( 1) to estimate genetic parameters and the correlated responses between resistance to nematodes and other economically important (production) traits; (2) to understand the mechanisms of resistance under genetic control; (3) to identify phenotypic and genetic markers with resistance which facilitate incorporation of parasite resistance into commercial breeding programmes; (4) to assess the specificity of selection with regard to
other parasite and non-parasite pathogens. Of the flocks undergoing selection for resistance, the Australian flocks located at Armidale, NSW. are described here as an example of the results being achieved. 2.3. I. The Trichostronyylus selection flock The Trichostrongylus selection flock has been described by Windon and colleagues (Windon and Dineen. 1984; Windon. 1990. 1991a; Windon et al.. 1993). This flock of medium wool Peppin Merinos was commenced in 1975 with the testing of parent generation lambs. and subsequent selection and assortative matings have been used to establish lines in which lambs are either immunologically responsive (high responders) or susceptible (low responders). An unselected line provides animals as controls for the selection and also parasitological controls. Lambs from this flock have usually been reared helminthologically naive in pens from birth and subjected to a testing regime which incorporates vaccination with irradiated 7: colubriformis larvae and challenge with normal larvae. Individual responsiveness in the pen testing regime is based on the mean of five faecal egg counts carried out after challenge. Lambs from the high ~(1 counts about 10% of those for low responders. responder line currently have faecal ebb and the estimated heritability (h’) for response to vaccination and challenge in this flock is 0.37 + 0.04 (Woolaston and Eady. 1995). More recently. responses of pasture-reared lambs from this selection flock have been evaluated after each of two artificial infections with T. cohbri’ormis. Lambs were weaned at I4 weeks of age. given a primary challenge at I7 weeks, treated with anthelmintic at 22 weeks, and given a secondary challenge at 23 weeks. Although ~0 counts were apparent between the high and low significant differences in faecal ekt. responder lines after each challenge. these were greater in the second challenge period. The heritability estimate for faecal egg count during the first challenge period was low. but during the second challenge period this was at comparable levels (0.39 _t 0.11) to those calculated for the pen studies (see above) (Woolaston and Eady. 1995). Genetic correlations between faecal egg counts from both paddock infection periods and the pen testing were high, suggesting that essentially the same trait was being measured. 2.3.2. The Haemonchus selection ,flock This fine-wool Merino flock was established in 1978 (Piper. 1987: Woolaston, 1990: Woolaston et al.. 1990: Woolaston and Eady. 1995). Divergent selection lines. together with an unselected control line. have been established in which lambs on pasture exhibit increased and decreased resistance to a single artificial infection with H. contortus at 5-6 months of age. Individual responsiveness is based on maximal faecal egg counts between 3 and 6 weeks after infection. After IS years of selection, mean worm-egg counts in the increased resistance line are 20-30s of those in the decreased resistance line. The heritability estimates for faecal ebb 0~ counts after infection is 0.29 + 0.03 (Woolaston and Eady. 1995). 2.3.3. The Haemonchus resistance jlock (UNE ‘Golden Ram’ jlock) This flock was established at the University of New England, Armidale, in 1980 (Albers et al.. 1987; Woolaston and Eady. 1995) after a trial designed to investigate the
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degree of genetic variability in resistance to H. contortus. This work identified a ram (the ‘Golden Ram’) whose progeny exhibited extreme levels of resistance. Subsequent backcross matings between relatives of this ram have been used to establish a line of animals with increased resistance to H. conrorrus, and these genetically resistant lambs are compared with control (unselected) animals. Progeny testing consists of monitoring faecal egg counts in pastured lambs given a single infection of H. contortus at about 4-5 months of age. The heritability of faecal egg counts in this flock is 0.24 f 0.04 (Woolaston and Eady, 1995). Genetic analyses have failed to find any evidence of a putative major gene being responsible for the extreme expression of resistance in the founding sire.
3. Mechanisms
of resistance
The selection flocks is a valuable research resource to study the mechanisms of resistance to parasites. By comparing the defined extremes of responsiveness existing between lines, intensive investigations into the underlying mechanisms have been carried out in the Trichostrongylus selection flock and the Haemonchus resistance (‘Golden Ram’) flock (Windon, 1991a; Gill et al., 1991). However, to date, little information is available on the mechanistic basis of resistance in the Haemonchus selection flock. A number of immunological responses have been shown to be under genetic control in rodent/parasite models (Wakelin, 1985). Resistance in the Trichosrrongylus selection flock is overtly immunologically based, and the Haemonchus resistance flock also has a strong immunological basis. Selection for increased resistance is therefore associated with heightened reactivity across a broad range of immunological functions (Windon, 1991 a; Gill et al., 1991). In the Trichostrongylus selection flock, high responder animals have greater antigen recognition (parasite specific cellular and antibody responses) and effector responses (mast cells/globule leukocytes, circulating eosinophils and mediator release) (reviewed by Windon, 1990, 1991a,b; Windon et al., 1993). Similarly, with the Haemonchus resistance flock, resistance is associated with increased antibody responses (local and circulating) (Gill et al., 1993b,1994), cellular responses (Gill et al., 1993a; Gill, 19941, and effector responses (reviewed by Gill et al., 1991). Recent studies in the selection flocks have been focused on T helper (CD41 cells which appear to play a central role in the regulation and control of appropriate effector responses. Haig et al. (1989) showed that CD4 cells were activated during infection with H. conforfus, and Gill et al. (1993~) were able to induce susceptibility in genetically resistant lambs by depletion of CD4 cells by monoclonal antibodies. These latter workers also found that CD4 cell depletion was associated with reduced numbers of mucosal mast cells, globule leukocytes and eosinophils in tissue. Rodent T helper cells can be divided into two groups, ThI and Th2, on the basis of the cytokines they produce (Mossmann and Coffman, 1989). Furthermore, in mice, resistance to nematodes has been associated with a Th2 response, whereas susceptibility was associated with a Thl response (Else and Grencis, 1991; Else et al., 1994; Robinson et al., 1995). The immune responses under Th2 control include the induction of IgE, mastocytosis and eosinophilia
(Finkelman and Urban. 1992). Although. as yet, there IS no evidence for a ThI/Th2 dichotomy in sheep. the availability of an increasing number of ovine cytokine reagents has allowed the initiation of studies directed towards identifying the cytokine profiles associated with resistance and susceptibility to gastrointestinal nematodes in the selection flocks. It is anticipated that these profiles will allow greater definition of the factors controlling the immune response and identify possible candidate genes for analysis as genetic (DNA) markers. Work carried out with lymphocytes isolated from helminthologically naive high and low responders (Trichosrron~ylus selection flock) has demonstrated that genetically resistant lambs are inherently able to produce more colony stimulating factors than low responders. This was assessed by colony growth and differentiation of bone marrow cells in soft agar cultures. Furthermore. bone marrow cells from high responders were able to respond more vigorously to these differentiation factors (Windon. Haig. Seow and Rothel. unpublished results). Of particular interest for the practical application of genetically determined resistance is whether selection for one parasite species will also result in improved protection against other related and unrelated parasite species. and indeed other non-parasite pathogens. Some circumstantial evidence is accumulating that mechanistic differences exist in the responses provoked by infection with 7’. colubriformis and H. contortus (see Windon. I99 I b). This corroborates results which show that resistance to infection with the heterologous nematode species ( H. corttortu.s or T. cnluhr~fbrn~is) in the Trichostrongylus and Hnemonchus selection flocks is not as great as that for the homologous infection against which each selection is based (see Windon. 1990). It is anticipated that knowledge of the mechanisms of resistance, and particularly the cytokine profiles, will allow prediction of the susceptibility of parasite-resistant sheep to nonparasite pathogens (Windon et al., 199.3).
4. Markers for resistance A number of immunological and physiological parameters, or the genes controlling them, have been investigated as markers for resistance (see Windon, 1990). To be of most benefit. markers should either provide a more accurate measure of resistance (relative to the currently used faecal e gg counts). or provide a predictive indication of resistance which is independent of infection and environmental factors. 4.1. Phenotypic
nudwrs
Phenotypic markers are those that have a functtonal basis and are usually expressed during infection. Eady (1995) and Douch et al. (1996) have reviewed phenotypic markers based on parasitological, immunological and physiological traits, their characteristics and the requirements for inclusion in breeding programmes. Faecal egg counts are used to measure individual responsiveness in the experimental selection programmes. This parameter itself is an indirect measure of the host’s worm burden. However. faecal egg count may not necessarily be a good indicator because a number of factors can affect egg production by female worms or faecal consistency
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(Douch et al., 1996). A study of the population dynamics of T. colubrifot-mis infection has shown that worm-egg production is influenced by immunological responses earlier than adult worms are expelled (Dobson et al., 1990). In contrast, egg counts more accurately reflect H. contorfus worm burdens (Barger et al., 1985). Faecal egg counts can, however, directly influence the level of larval contamination on pasture (Eady, 1995). Circulating eosinophils are associated with resistance under strictly controlled conditions in penned Merinos (Dawkins et al., 1989) and in penned and naturally infected Romneys (Buddle et al., 1992). On the basis of these and other studies, Hohenhaus and Outteridge (1995) have suggested that eosinophils, alone or in conjunction with ovine lymphocyte antigen (OLA) typing. may be a suitable selection criterion by which to include parasite resistance in breeding programmes. However, this was not confirmed by Woolaston et al. (1996), who concluded that circulating eosinophil numbers were less effective than faecal egg counts during infection with either H. contorrus or T. colubriformis. Thus, although eosinophil counts have relevance to immunological studies, the nature of the eosinophil response reduces their suitability as a trait for selection. In this regard, it has been found that circulating eosinophil counts are significantly correlated with the acquisition of resistance, but highly resistant animals that have expelled their worm burden as well as susceptible animals have low eosinophil counts (Windon, unpublished results). There are also differences in the association between circulating eosinophil counts and the acquisition of resistance to different gastrointestinal nematode species. This association is strong for T. colubriformis but weak for H. contortus (see Windon et al., 1993). A phenotypic marker receiving particular attention in New Zealand is serum antibody levels against excretory/secretory parasite antigens determined by ELISA procedures (reviewed by Douch et al., 1996). In this work, antibody levels (predominantly IgGl) were found to be moderately heritable and genetically correlated with faecal egg count. A selection experiment has subsequently been established to further investigate the association. Early results have shown that selection for antibody response was successful but no differences occurred in faecal egg counts between lines (high antibody. low antibody, control). The authors consider that this observation may be a result of low nematode infection. 4.2. Genetic markers The potential advantage of genetic markers is that, by definition, they are based on genes or DNA sequences which can be used to identify resistant or susceptible animals independently of infection. A number of genetic markers have been evaluated for associations with resistance to internal parasites. Of these, some success has been reported by Hohenhaus and Outteridge (1995) using OLA typing, but other workers using human MHC class II restriction fragment length polymorphism probes did not find a statistically significant association with resistance to T. colubriformis (Hulme et al., 1993). An approach using linkage analysis to identify genetic markers for resistance to gastrointestinal nematodes has been recently described by Blattman and Beh (1995) and Beh and Maddox (1996). There have been three resource flocks established in Australia
and New Zealand to carry out this work. A flock at the CSIRO Division of Animal Production was based on the crossing of high and low responder progeny from the Trichosfrongylus selection flock to produce six sires heterogeneous for resistance to T. colubrifornzis. Each of these sires is being used to produce 200 half sib progeny. The AgResearch (New Zealand) resource flock has similarly been established by crossing lines of Romney sheep bred for high and low faecal egg counts. The University of Melbourne flock was established by crossing low responder rams from the Trichosrrongylus selection flock with AgResearch resistant Romney ewes to produce offspring which will subsequently be used as parents of an intercross second generation. In each case. microsatellite markers are being used to scan the entire genome. In addition, other techniques are being used which focus on microsatellite-associated candidate genes.
5. Conclusions Substantial genetic variation exists in resistance to gastrointestinal nematodes both between and within breeds of sheep. Within breeds, experimental selection programmes have demonstrated that resistance is a heritable trait, and substantial gains can be achieved in commercial breeding enterprises. The selection flocks are also a valuable research resource for studies into the mechanisms of resistance to nematodes as they provide animals having defined extremes of responsiveness. An understanding of resistance mechanisms. which appear to be immunologically based. is crucial for the optimization of vaccination strategies. Furthermore. these studies may facilitate the identification of phenotypic and genetic markers. The acceptance of parasite resistance as a trait for incorporation into commercial breeding programmes will depend on the development of a cost-effective. accurate and repeatable measure and. in particular, one which is independent of infection and environmental influences. As the parasite population itself exhibits heterogeneity and has shown a capacity to adapt in the face of strong selection pressure (for example, anthelmintics), successful control programmes should not rely on only one procedure. Future parasite control, therefore. may well involve an integrated approach using genetically resistant animals in conjunction with anthelmintic treatment. vaccination and pasture management (including nutrition).
Acknowledgements The Armidale based selection flocks referred to in this paper have been supported by Australian woolgrowers and the Australian Government through the Australian Wool Research and Promotion Organisation.
References Albers.
G.A.A..
Parasitol.. Bargrr,
Gray.
A.D.,
Piper.
L.R..
Bar!er.
J.S.F..
Lc Jamhrc,
L.F.
and
Barger,
I.A..
17: 1355.
[.A., Le Jamhrr,
L.F.. Grorp~.
J.R. and Davw,
H.I..
lUX5. Int. J. Parasitol.,
IS- 529.
1987.
Int. J.
R.G.
Windon
/ Veterincrry
Immunology
und Immunoputhology
54 (1996)
245-254
253
Beh, K.J. and Maddox, J.N., 1996. Int. J. Parasitol., in press. Bisset, S.A., Vlassoff, A. and West, C.J.. 1991. Proceedings 21st Seminar of the Sheep and Beef Society. New Zealand Veterinary Society, p. 83. Blattman, A.N. and Beh, K.J.. 1995. In: G.D. Gray, R.R. Woolaston and B.T. Eaton (Editors), Breeding for Resistance to Infectious Diseases in Small Ruminants. ACIAR, Canberra, p. 237. Buddle, B.M., Jowett, G., Green, R.S., Douch, P.G.C. and Risdon, P.L., 1992. Int. J. Parasitol., 22: 955. Dawkins, H.J.S., Windon, R.G. and Eagleson, G.K., 1989. Int. J. Parasitol., 19: 199. Dineen, J.K., 1978. In: A.D. Donald, W.H. Southcott and J.K. Dineen (Editors), The Epidemiology and Control of Gastrointestinal Parasites of Sheep in Australia. CSIRO Division of Animal Health, Melbourne, p. 121. Dobson, R.J., Wailer, P.J. and Donald, A.D., 1990. Int. J. Parasitol., 20: 347. Douch, P.G.C., Green, R.S., Morris, C.A., McEwan, J.C. and Windon, R.G., 1996. Int. J. Parasitol., in press. Eady, S.J., (1995. In: G.D. Gray, R.R. Woolaston and B.T. Eaton (Editors), Breeding for Resistance to Infectious Diseases in Small Ruminants. ACIAR, Canberra, p. 219. Else, K.J. and Grencis, R.K., 1991. Parasitol. Today, 7: 313. Else, K.J., Finkelman, F.D., Maliszewski, C.R. and Grencis, R.K., 1994. J. Exp. Med., 179: 347. Finkelman, F.D. and Urban, J.F., 1992. Parasitol. Today, 8: 311. Gill, H.S., 1994. Int. J. Parasitol., 24: 749. Gill. H.S., Gray, G.D. and Watson, D.L., 1991. In: G.D. Gray and R.R. Woolaston (Editors), Breeding for Disease Resistance in Sheep. Australian Wool Research and Development Corporation, Melbourne, p. 67. Gill, H.S., Colditz, LG. and Watson, D.L., 1993a. Res. Vet. Sci., 54: 361. Gill, H.S., Gray, G.D., Watson, D.L. and Husband, A.J.. 1993b. Parasite Immunol., 15: 61. Gill, H.S., Watson, D.L. and Brandon, M.R., 1993~. Immunology, 78: 43. Gill, H.S., Husband, A.J., Watson, D.L. and Gray, G.D., 1994. Res. Vet. Sci., 56: 41. Gray, G.D., 1991. In: R.F.E. Axford and J.B. Owen (Editors), Breeding for Disease Resistance in Farm Animals. CAB International, Wallingford, p. 139. Gray, G.D. and Gill, H.S., 1993. hit. J. Parasitol., 23: 485. Gray, G.D., Presson, B.L., Albers, G.A.A., Le Jambre, L.F., Piper, L.R. and Barker, J.S.F., 1987. In: B.J. McGuirk (Editor), Merino Improvement Programs in Australia. Australian Wool Corporation, Melbourne, p. 365. Gruner, J. and Cabaret, 1988. In: E.F. Thomson and F.S. Thomson (Editors), Improving Small Ruminant Productivity in Semi-Arid Areas. Kluwer, Dordrecht, p. 257. Haig, D.M., Windon. R.G., Blackie, W., Brown, D. and Smith, W.D., 1989. Parasite Immunol., 11: 463. Hohenhaus, M.A. and Outteridge, P.M., 1995. Br. Vet. J., 151: 119. Hulme, D.J., Nicholas, F.W.. Windon, R.G., Brown, S.C. and Beh, K.J., 1993. J. Anim. Breed. Genet., I IO: 459. Massey, C.J., 1990. The Australian Merino. Viking O’Neil, Melbourne. Morris, C.A., Watson, T.A., Bisset, S.A., Vlassoff, A. and Douch, P.G.C., 1995. In: G.D. Gray, R.R. Woolaston and B.T. Eaton (Editors), Breeding for Resistance to Infectious Diseases in Small Ruminants. ACIAR, Canberra, p. 53. Mossmann, T.R. and Coffman, R.L., 1989. Adv. Immunol., 46: Ill. Piper, L.R., 1987. In: B.J. McGuirk (Editor), Merino Improvement Programs in Australia. Australian Wool Corporation, Melbourne, p. 35 1. Robinson, K., Bellaby, T. and Wakelin, D., 1995. Parasitology, 110: 71. Rothwell, T.L.W., 1989. Int. J. Parasitol., 19: 139. Stiter, T., Kassai, T. and Takacs, E., 1994. Int. J. Parasitol., 24: 871. Wakelin, D., 1985. Parasitol. Today, 1: 17. Windon, R.G., 1990. Rev. Sci. Tech. Off. Int. Epizoot., 9: 555. Windon, R.G., 1991a. In: G.D. Gray and R.R. Woolaston (Editors), Breeding for Disease Resistance in Sheep. Australian Wool Research and Development Corporation, Melbourne, p. 77. Windon, R.G., 199lb. In: R.F.E. Axford and J.B. Owen (Editors), Breeding for Disease Resistance in Farm Animals. CAB International, Wallingford, p. 162. Windon, R.G. and Dineen, J.K.. 1984. In: J.K. Dineen and P.M. Outteridge (Editors), Immunogenetic
Approaches Division Windon,
to the Control
of Animal
of Endoparas~tc~ -with
Health,
Melbourne.
R.G.. Gray. G.D. and Wool&on,
Woolaston,
R.R.,
19X). Wool Technol.
Woolaston.
R.R.. End?. S.J..
( 1995).
G.D. Gray. R.R. Woolaston
p.
to the Parasites of Sheep. CSIRO
in Breeding
Woolaston,
R.R.. Barger. I.A. and Piper, L.R.. R.R., Manurli.
for Resistance
to Infecuous
Canberra),
1990. Int. J. Parasltol..
P.. Eady. S.J.. Barger.
17. 37.
I.
and B.T. Eaton, Eds. (ACIAR,
in press.
Rrferrncc
R.R.. 1993. Proc. Sheep Vet. Sot..
Sheep Breed., 38.
Woolaston,
1996. Int. J. Parasitol.,
Partmular
Ii.
1.A.. Le Jambre,
Diseases in Small Ruminants,
p. 53.
20: 1015. L.F.,
Banks,
D.J.D.
and Windon,
R.J..