GENETIC RESISTANCE TO H a e m o n c h u s c o n t o r t u s in R o m a n o v SHEEP

G. LUFFAU (1), T.C. NGUYEN (2), Philippa CULLEN (3), Jacqueline VU TIEN KHANG (4), J. BOUIX (4), G. RICORDEAU (4).

(1) Groupe de Laboratoires de Pathologie Animale, I.N.R.A., Route de Thiverval, 78850 THIVERVAL-GRIGNON, FRANCE.

(2) Laboratoire des Groupes Sanguins, C.N.R.Z., Domaine de Vilvert, 78350 JOUY-EN-JOSAS, FRANCE.

(3) Laboratoire de G6n6tique Biochimique, C.N.R.Z., Domaine de Vilvert, 78350 JOUY-EN-JOSAS, FRANCE.

(4) Station d'Amelioration Genetique des Animaux, I.N.R.A. Toulouse, B.P. 27, 31326 CASTANET-TOLOSAN CEDEX, FRANCE.

S U M M A R YThe responses of sheep to an injection with human serum

albumin (HSA) and to an infection with the parasite H a e m o n c h u s c o n t o r t u s were compared in relation to haemoglobin genotype. The subjects were 51 female lambs of the R o m a n o v breed, and were the offspring of 8 sire-families. The 51 animals fell into 3 groups of 17, representing the three haemoglobin genotypes, Hb AA, AB and BB ; they were also tested for markers of the major histocompatibility system, OLA. Information was obtained on the development of immunity following exposure to the para­site, and its subsequent partial loss following drenching. The response to HSA did not show any positive correlation with resistance to H. c o n t o r t u s . Resistance to H. c o n t o r t u s appeared to be independent of Hb genotype. Association with OLA genotype was not excluded.

KEY WORDS : sheep, R o m a n o v breed, immune response, human serum albumin, parasitism, H a e m o n c h u s c o n t o r t u s , resis­tance, haemoglobin type, OLA system.

683

INTRODUCTIONDuring the past twenty years, there have been several

reports suggesting that sheep homozygous for the haemoglo­bin A allele are more resistant to the parasite H a e m o n c h u s con- tortus than are homozygotes for haemoglobin B (EVANS et al., 1963 ; JILEK and BRADLEY, 1969 ; ALLONBY and URQUHART, 1976 ; ALTAIF and DARGIE, 1976, 1978a, 1978b, ; PRESTON and ALLONBY, 1979). It has also been suggested this increased resistance might not only be to H a e m o n c h u s contor t u s , but could be linked to a generally improved immune response (CUPERLOVIC et al.,1978).

In the experiments described below, we have looked at the relationship between haemoglobin genotype and resistance to H a e m o n c h u s c o n t o r t u s on the one hand, and also to the response to immunisation with aggregated human serum albumin (HSA). In addition we were able to study the development of parasite immunity following inoculation, and its loss following oral drenching. The animals in the study were retrospectively tested for antigens of the sheep major histocompatibility system (OLA), and the relevance of these genetic markers was conside­red in our analysis.MATERIALS AND METHODSAnimals

The experiments were performed on 51 lambs of the Rom a n o v breed. Only females were used as preliminary studies had shown that females can be more easily immunised than males against H a e m o n c h u s co n t o r t u s (LUFFAU et al., 1981a). These animals were the offspring of 8 sires which were of haemoglobin genotype AB ; and the 51 lambs fell into three groups of 17, each repre­senting the three haemoglobin genotypes, Hb AA, Hb AB, and Hb BB. The three genotypes were selected evenly from the different sire families to reduce the influence of any sire effect upon the effect of haemoglobin genotype under study. The 51 lambs and most of their dams were typed for antigens of the OLA sys­tem ; the sires were not typed but their genotype could be inferred and transmission of markers determined in many cases. The animals were maintained on a grass free diet from birth to avoid environmental exposure to H a e m o n c h u s contortus.

Experimental ProtocolAt the age of 6 months all the lambs had been immunised

with aggregated human serum albumin (HSA) ; their serum was tested for haemagglutinating antibodies to HSA after two weeks. Experimental inoculation with H a e m o n c h u s c o n t o r t u s larvae began at the age of 8 months, a previous study having shown that effective immunisation can be achieved from the age of about 7 months (URQUHART et al., 1966).

684

Three parasite infection experiments were performed suc­cessively on the same group of animals. Three weeks old L_ lar­vae artificially incubated were used throughout for infection.

In experiment 1, the 51 animals were divided into three treatment groups :a) 18 animals were given 5.000 larvae on day 0, followed by

10.000 larvae on day 31 and 20.000 larvae on day 64 (group 1).

b) 18 animals received the above treatment but on days 31 and 64 only (group 2).

c) 15 animals received the day 64 treatment only (group 3). Parasite egg excretion was monitored in all animals.

Experiments 2 and 3 each consisted of an infection of10.000 larvae on day 0. In each experiment the sheep were divi­ded into two groups ; one drenched before day 0, and the other not so treated. Egg excretion was monitored as in experiment 1.Variables

The 26 animals which produced no detectable antibodies to HSA are scored as zero for this response, while the inverse of the titre detected (1/2 - 1/512) is used to denote the level of response in the remaining 25 animals. Immunity to the parasite is denoted by mean egg count per gramme of faeces (epg) - and log (epg) - at a given time following inoculation (the animals with better immunity being those which excrete the fewer eggs). Figure 1 shows these variables.RESULTS AND DISCUSSION

Experiment 1 demonstrated the effectiveness of immunising by inoculation of larvae (LUFFAU et al., 1981b). The animals which had been exposed to the parasite on day 0 mounted a much greater secondary response when given a second (larger) dose, than did control animals given the same dose as a primary ino­culation (figure 2).

In experiment 2 and 3 it was clear that drenching animals with pre-existing immunity to the parasite markedily reduced their resistance to a subsequent inoculation with larvae (figu­re 2).

With regard to the influence of haemoglobin genotype, no effect was noted in our conditions (namely in R o m a n o v breed) on the development of either parasite immunity or the response to HSA. These results disagree with those of a number of authors (see introduction) but agree with the findings of RIFFKIN and YONG (1984) who found no detectable relationship between Hb genotype and resistance to H a e m o n c h u s c o n t o r t u s .

685

No correlation was found between the intensity of the immune response to the parasite followed in experiment 1, and the antibody titre to HSA. On the other hand, in experiments 2 and 3, those with highest antibody titres were among those least resistant to the parasite. This finding was significant, and while contrary to the hypothesis of CUPERLOVIC et al.(1978), it is in agreement with ALBERS et al. (1984) who found a negative correlation between epg excreted after inoculation and the immune response to chicken red cells. It should be no­ted that a negative correlation between antibody response and helminth immunity has also been found in the mouse (BLUM and CIOLI, 1978 ; DEELDER et al., 1978 ; PERRUDET-BADOUX et al., 1978 ; WAKELIN, 1978).

Several OLA markers are significantly correlated with the variables in experiments 2 and 3. The transmission of paternal OLA haplotypes within each sire family could be compatible with a hypothesis that there exist, in or near the sheep major his­tocompatibility complex, genes influencing resistance to Hae- m o n c h u s c o n t o r t u s . These results would accord with those of OUTTERIDGE et al= (1984) who found an association between OLA markers and the response to vaccination against the parasite T r i c h o s t r o n g y l u s c o l u b r i f o r m i s .

Analysis of the variability in genetic resistance to H a e ­m o n c h u s c o n t o r t u s, based on epg measurements at different times considered as the same trait showed a strongly significant "individual" effect ; the repeatability, calculated by the coefficient of intra-class correlation, was between 0.35 and 0.43. There were also several indications that a "sire-effect" was operating, although larger numbers of animals would be necessary to confirm such a hypothesis. However, our results would agree with those of ALBERS et al. (1984) who found a heritability of 0.3 for resistance to H a e m o n c h u s c o n t o r t u s in A u s t r a l i a n Merinos.

CONCLUSION

This study of experimental injection with H a e m o n c h u s c o n ­tortus has demonstrated that immunity can be acquired by deli­berate exposure to the parasite but may subsequently be des­troyed by drenching. There are several indications of a genetic component in resistance to H a e m o n c h u s c o n t o r t u s which could offer a basis for selective breeding. For this purpose it would be useful to have a genetic marker for increased resistance. We have found that haemoglobin genotype and humoral immunity to HSA are not very useful indicators of resistance, but the pos­sibility that OLA markers could be valuable in this respect certainly merits further investigation.

686

LITERATURE CITED

ALBERS G.A.A., BURGESS S.E., ADAMS D.B., BARKER J.S.F., LE JAMBRE L.F., PIPER L.R., 1984. Breeding H a e m o n c h u s con- t o r t u s resistant sheep - problems and prospects. In Immu- nogenetic approaches to the control of endoparasites with particular reference to parasites of sheep. Proceedings of a workshop held at Sydney University, October 22-23 1983, on behalf of the Australian Wool Corporation. East Melbourne, Australia ; CSIRO, 41-52.

ALLONBY E.W., URQUHART G.M., 1976. A possible relationship bet­ween haemonchosis and haemoglobin polymorphism in M e r i n o sheep in Kenya. Res. Vet. S c i . , 20 (2), 212-214.

ALTAIF K.I., DARGIE J.D., 1976. Genetic resistance of sheep to H a e m o n c h u s contortus. In Nuclear techniques in animal pro­duction and health. Proceedings of a symposium, Vienna,2-6 Feb. 1976, jointly organized by the IAEA and FAO. Vienna, Austria ; International Atomic Energy Agency, 449-461.

ALTAIF K.I., DARGIE J.D., 1978a. Genetic resistance to hel­minths. The influence of breed and haemonglobin type on the response of sheep to primary infections with H a e m o n ­c h u s co n t o r t u s . Parasitol., 77 (2), 161-175.

ALTAIF K.I., DARGIE J.D., 1978b. Genetic resistance to hel­minths. The influence of breed and haemoglobin type on the response of sheep to re-infection with H a e m o n c h u s c o n t o r ­tus. P a r a s i t o l ., 77 ,(2) 177-187.

BLUM K., CIOLI D., 1978. Paradoxical behaviour of Biozzi high and low responder mice upon infection with S c h i s t o s o m a man s o n i . Eur. J. Immunol., 8, 52-56.

CUPERLOVIC K., ALTAIF K.I., DARGIE J.D., 1978. Genetic resis­tance to helminths : a possible relationship between hae­moglobin type and the immune responses of sheep to non- parasitic antigens. Res. Vet. Sci., 25 (1), 125-126.

DEELDER A.M., CLASS F.H.J., DE VRIES R.R.P., 1978. Influence of the mouse H2 gene complex on experimental infection with S c h i s t o s o m a mansoni. Trans. R. S o c . Trop. Med. H y g . , 72, 321.

687

EVANS J.V., BLUNT M.H., SOUTHCOTT W.H., 1963. The effects Of infection with H a e m o n c h u s c o n t o r t u s on the sodium and po­tassium concentrations in the erythrocytes and plasma, in sheep of different haemoglobin types. R u s t r . J. Agric. Res., 14, 549-558.

JILEK A.F., BRADLEY R.E., 1969. Haemoglobin types and resistan­ce to H a e m o n c h u s c o n t o r t u s in sheep. Am. J. Vet. Res., 30 1773-1778.

LUFFAU G., PERY P., CHARLEY J., 1981a. R^ponse immunitaire chez les ovins infest6s exp6rimentalement par H a e m o n c h u s c o n ­tortus. Etude comparative chez le male et chez la femelle. Ann. Rech. V6t. , 12 (2), 173-181.

LUFFAU G., PERY P., PETIT A., 1981b. Self-cure and immunity following infection and reinfection in ovine haemonchosis. Vet. P a r a s i t o l ., 9, 57-67.

OUTTERIDGE P.M., WINDON R.G., DINEEN J.K., DAWKINS H.J.S.,1984. Associations between lymphocyte antigens in sheep and responsiveness to vaccination against intestinal para­sites. In Immunogenetic approaches to the control of endo- parasites with particular reference to parasites of sheep. Proceedings of a workshop held at Sydney University, Octo­ber 22-23 1983, on behalf of the Australian Wool Corpora­tion. East Melbourne, Australia ; CSIRO, 103-111.

PERRUDET-BADOUX A., BINAGHI R.A., BOUSSAC-ARON Y., 1978. Tri- c h t n e l l a spi r i l i s infection in mice : mechanism of the resistance in animals genetically selected for high and low antibody production. Immunology, 35, 519-522.

PRESTON J.M., ALLONBY E.W., 1979. The influence of haemoglobin phenotype on the susceptibility of sheep to H a e m o n c h u s c o n t o r t u s infection in Kenya. Res. Vet. Sci, 26, 140-144.

RIFFKIN G.G., YONG W.K., 1984. Recognition of sheep which have innate resistance to Trichostrongylid Nematode parasites. In Immunogenetic approaches to the control of endoparasi- tes with particular reference to parasites of sheep. Pro­ceedings of a workshop held at Sydney University, October 22-23 1983, on behalf of the Australian Wool Corporation. East Melbourne, Australia, CSIRO, 30-40.

URQUHART G.M., JARRETT W.F.H., JENNINGS F.W., Me INTYRE W.I.M., MULLINGAN W., 1966. Immunity to H a e m o n c h u s c o n t o r t u s in­fection. Relationship between age and successful vaccina­tion with irradiated larvae. Am. J. Vet. Res., 27 1645-1648.

WAKELIN D., 1978. Genetic control and resistance to parasiticinfection. Adv. P a r a s i t o l . , 16, 219-308.

688

Figure 1Variables describing immunity to Haemonchus contortus

in infection experiments 1, 2 and 3EXPERIMENT 1

Group 1

D O (5000 L3) D 3 ! ( 10000 L3) D 6 4 (20000 L3)

Group 2

Group 3

\I Ml ! iK~“i —►

EXPERIMENT 2

D O (10000 L3)IEXPERIMENT 3

D O (10000 L3)1(D3I.D34.D38)

(D33.D36.D40)

- H o r i z o n t a l axes re p re s e n t the tim e ( in da ys).- V e r t ic a l a r r o w s indicate e x p e rim e n t a l infe ctions ( i n b ra c k e ts : num be r o f la r v a e L 3 ) .- V a r ia b l e s a r e mean egg counts per gramm (e p g ) o f faeces d u r i n g indicated pe riods.

►

689

Figure 2Faecal egg output in in fe c tio n exp erim en ts I , 2 and 3

w i th Haem onchus con to r t us

EXPERIMENT I

EXPERIMENT 2

EXPERIMENT 3

100000

<♦ O % \ W'y m/

0' '■ >\jVw

■

I

* ' Group 1

'' Group 2

•• Group 3

/

20 40 60 80 100 120 140 160 Day

10000

1000

Epg 100

10

1 1--- .-------------------•----10 10 20 30 40 50 60 Day

10000

1000

Epg 100

10

/Drenched group

Not drenched group

/I — ♦-<♦*----•--------------*---->----10 10 20 30 40 50 60 70 Day

690