HAEMONCHUS CONTORTUS RESISTANCE IN STRAIGHTBRED AND

HAEMONCHUS CONTORTUS RESISTANCE IN STRAIGHTBRED AND CROSSBRED BARBADOS BLACKBELLY SHEEP 1 Thomas A. Yazwinski 2, L. Goode 3, D. J. Moncol 3, G. W. Morgan 3 and A. C. Linnerud 3 North Carolina State University, Raleigh 2 7650 Summary Resistance to llaemonchus contortus infection was studied in two experiments conducted with 52 lambs of widely different genetic background. Breed groups compared were: Dorset (D), Barbados Blackbelly (B), Dorset Blackbelly (D B), Suffolk Blackbelly (S B), Suffolk Dorset Blackbelly (S D B, I~ BI) and nonblackbelly (NB, D and S). In each experiment, lambs were raised on concrete from birth and were essentially parasite free until infective larvae were administered. In Exp. 1, D x B, S D B and D wether lambs approximately 3 months old were infected with a standard dose of invective larvae estimated to be 98% H. contortus. D B lambs had a longer (P<.05) prepatency period and, at necropsy 17-day postinfection, had a higher (P<.05) percentage of female parasites classified as immature than did the other breed groups. These data showed that the development of//. contortus larvae was inhibited in D B host animals. In Exp. 2, B, D X B, S B and NB ewe and wether lambs approximately 4 months old were treated with three sensitizing doses of H. contortus larvae. These were followed by a challenge infection. On the basis of fecal egg counts (eggs per gram feces, EPG), there was no evidence that the challenge infection induced a self-cure reaction in any breed group. All lambs with B breeding had significantly higher Hb levels at the end of the experiment than did NB lambs. B and S x B lambs had higher (P<.05) Hb levels than the D B lambs, and B lambs had the highest (P<.05) mean corpuscular hemoglobin concentrations. Five days after the challenge infection, B, D B and S B lambs also had significantly higher white blood cell levels than did NB lambs. Significant sex differences were also observed in Exp. 2. Ewe lambs had lower final EPG levels, higher preinfection and postinfection Hb levels and higher maximum postinfection eosinophil levels. Breed sex interactions for these parameters were not significant. (Key Words: Sheep, Parasite Resistance, Breeds, tl contortus.) Introduction Several authors have reported differences between sheep breeds in resistance to gastrointestinal parasites (Scrivner, 1964a,b; Loggins et al., 1965; Colglazier et al., 1968; Jilek and Bradley, 1969; Bradley et al., 1973; Knight, et al., 1973). Yazwinski et al. (1979) reported that straightbred and crossbred Barbados Blackbelly sheep carrying mixed infections of parasites had significantly lower fecal egg counts, higher hemoglobin levels and higher mean corpuscular hemoglobin concentrations than nonblackbelly sheep. Breed differences in fecal egg counts were the result of lower egg production by Cooperia spp, Trichostrongylus spp and Ostertagia spp. Significant breed differences for these parameters were not observed, however, when sheep were infected with a relatively pure culture of Haemonchus contortus larvae. H. contortus is regarded as one of the most serious sheep parasites in the Southeast. The objectives of the study reported herein were to further investigate possible breed differences in resistance to H. contortus and to determine the effects of breed on some physiological responses frequently used to measure ~Paper no. 6039 of the Journal Ser. of the North the severity of parasitic infections in sheep. Carolina Agr. Res. Service. Raleigh. 2 Present ~ddress: Dept. of Anim. Sci., Univ. of Arkansas, Fayetteville 72701. Experimental Procedure 3 Dept. of Anita. Sci.; Vet. Sci.; Poul. Sci., and Exp. Statist., respectively. Exp. 1 was conducted to investigate breed 279 JOURNAL OF ANIMAL SCIENCE, Vol. 51, No. 2, 1981

280 YAZWINSKI ET AL. differences in fecal egg counts (eggs per gram feces, EPG), hemoglobin (Hb) levels, peripheral and tissue eosinophilia (PEOS, TEOS) and worm development in lambs raised in a relatively parasite-free environment before being infected with tt. contortus larvae. The following breed groups, each consisting of seven wethers approximately 3 months old, were compared: Dorset (D), Dorset x Blackbelly cross (D x B) and Suffolk x Dorset x Blackbelly cross (S x D x B [~/~ B]). All lambs were maintained on concrete from birth through the end of the experiment, and all fecal egg counts made before the beginning of the experiment were negative. On June 16, preinfection Hb, PEOS and EPG levels were determined for all lambs according to procedures described by Bausch and Lomb, Inc. (1965), Moncol and Batte (1967) and Gordon and Whitlock (1939), respectively. Data on preinfection worm burdens and abomasal tissue eosinophi', levels were obtained from two lambs necropsied from each breed group on June 17. Contents of the abomasum were washed through a series of 2.0mm, 850- /am and 150-/~m screens. Parasites recovered from each abomasum were fixed in 10% formalin and were counted and characterized by sex and stage of maturity of the females. The presence or absence of uterine ova was used to designate maturity or immaturity, respectively. As each abomasum was opened, three 1.O-cm tissue sections evenly spaced along the length of the major curvature were obtained and fixed in Zenkers' solution. These tissue were sectioned and stained with hematoxylin and eosin. Slides were examined under oil immersion (x400), and the eosinophils at the base of the l,tmma propria were counted in six adjacent fields in each of the three tissue sections from each animal. The sum of the 18 fields counted was reported as the tissue eosinophil count (TEOS). On June 20, all remaining lambs were infected per os with 10,200 larvae estimated to be 98% tl. co~tortus. Two lambs from each breed group were necropsied 17 days postinfection (July 7), and the other three animals were necropsied 61 days postinfection (August 20). data on EPG, ttb, PEOS and TEOS, along with worm numbers, sex and female maturity, were obtained as previously described. Exp. 2 was designed to study the effect of breed and sex on some physiological responses in lambs given sensitizing infections of tl. contortus larvae followed by a challenge infection. Thirty-two 4-month-old lambs of Barbados Blackbelly (B), D x B, S x B and nonblackbelly (NB, D and S) breeding were used. Each breed group consisted of four wethers and four ewes. One ewe was lost from the [) x B group for reasons not related to the experiment. All lambs were maintained on concrete from birth through the end of the experiment. Preinfection levels of Hb, EPG and PEOS were determined on September 10. All animals received the first of three sensitizing doses of larvae (98% II. colltortus) on September 11. Sensitizing doses were administered per os at 2-day intervals; each dose consisted of 220 larvae/kg body weight. A challenge infection of 5,000 larvae was administered 5 weeks after the last sensitizing dose (October 22). Data on postinfection levels of Hb, EPG and PEOS were collected at regular intervals. In addition, white blood cell levels (WBC) and mean corpuscular hemoglobin concentrations (MCttC) were determined according to procedures described by Coles (1967). Data obtained in Exp. 1 and 2 were subjected to standard analysis of variance techniques (Steel and Torrie, 1960). Multiple mean comparisons were performed according to Duncan's multiple range test. Results and Discussion Data on EPG, Hb, PEOS and TEOS levels from Exp. 1 are partially summarized in table 1. Preinfection EPG levels were negative and only one parasite, a female It. contortus, was found in the six lambs necropsied on June 17. Therefore, parasitism was negligible and the lambs were essentially parasite free at the beginning of the experiment. It. contortus ova were first observed in the feces of D lambs 17 days postinfection, compared to 18 and 19 days for S x D x B and D x B lambs, respectively. The average length of prepatency period was longer (P<.05) in D x B lambs than in the other groups. Fecal egg counts made 21 days after infection (July 8) were also significantly higher in D lambs. The total number of tt. contortus recovered from the abomasum did not vary significantly among breed groups in lambs necropsied at 17 and 61 days postinfection (table 2). ttowever, at 17 days postinfection, the percentage of female

11. CONTORTUS RESISTANCE IN BLACKBELLY SIIEEP 281 TABLE 1. MEAN FECAL EGG COUNTS, PREPATENCY PERIODS, HEMOGLOBIN LEVELS, PERIPHERAL EOSINOPIIIL LEVELS AND TISSUE EOSINOPHIL LEVELS (EXP. 1) Item No. lambs per breed Date group D B Breed groups SXDXB D EPG levels c Prein fection d Prepatency period, days Itb levels, g/lo0 ml Preinfection PEOS levels e Preinfection TEOS levels f Preinfection June 16 7 0 0 0 July 6 5 0 0 0 July 7 5 0 0 40 J uly 8 3 0 a 20 a 300 b July 9 3 230 370 270 August 19 3 3,570 6,070 4,670 5 19.3 a 18.3 b 17.6 b June 16 7 12.64 12.83 11.43 August 19 3 10.93 10.57 8.77 June 16 7 136.7 128.9 119.4 June 24 5 237.6 224.4 151.8 August 19 3 304.3 168.7 51.3 June 16 2 53.5 40.0 72.0 July 7 2 1,713.0 712.5 506.5 August 20 3 474.3 154.7 175.0 a'bmeans on same line with different superscripts differ (P<.05). CEggs per gram of feces. dlambs were infected with It. contortus larvae on June 20. Necropsy dates were June 17, July 7, August 20. eperipheral eosinophil levels. ftissue eosinophil levels. parasites classified as immature was higher (P<.05) in D B animals. These data indicate greater inhibition of tt. contortus larvae in animals with B breeding. The percentage of female parasites recovered at 61 days postinfection was also lower in D B and S x D B lambs, but breed differences were not significant. From July 9, postinfection EPG levels increased markedly in all breed groups, remained high for about 4 weeks and then declined. No breed differences were significant after July 8, and only the final EPG levels are shown in table 1. The D B and S D B lambs had higher preinfection and postinfection llb levels and lost less Hb during the experiment than did the D lambs. Peripheral and tissue eosinophilia were also higher in D B and S D B lambs. However, numbers were limited and breed differences for these variables were not statistically significant. Results of Exp. 2 are summarized by breed and sex in table 3. All preinfection fecal egg counts were negative. Ova were present in the feces of all breed groups at the first postinfection examination, which was made 19 clays after administration of the first sensitizing infection. All EPG levels increased rapidly for 2 or 3 weeks and then declined until November 11, at which time they increased markedly as a result of the challenge infection administered on October 22. Breed differences were not significant at any point in the experiment, and only the final EPG levels are shown in table 3. Preinfection Hb levels did not vary significantly among breeds. However, at the end of the experiment, the B, D x B and S x B lambs had higher (P<.05) fib levels than did NB lambs. Significant breed differences in November 10 MCHC levels were also observed. At 5 days postchallenge (October 27), WBC levels were higher (P<.05) in B than in D B and S x B lambs. Both of these groups had higher (P<.05) WBC levels than NB lambs. Peripheral eosinophil levels were also highest in lambs with

282 YAZWINSKI ET AL. TABLE 2. MEAN NUMBERS BY SEX AND FEMALE STAGE OF MATURITY FOR H. CONTORTUS RECOVERED FROM TIdE ABOMASUM (EXP. 1) Item No. lambs per breed Breed group Date group D B S D B D Preinfection necropsy c No. males No. females 17-day necropsy No. males No. females Immature females % June 17 2 July 7 2 0 O.5 0 280.0 440.0 305.0 420.0 90.4 a 53.6 b 160.0 165.0 48.5 b 61-day necropsy No. males No. females Immature females % August 20 3 283.0 230.0 266.7 326.7 0 0 280.0 553.3 0 a'bmeans on same line with different superscripts differ (P<.05). CLambs were infected with II. contortus larvae on June 20. B breeding, but these differences were not significant. In a previous study, Yazwinski et al. (1979) found that D B sheep carrying mixed infections of parasites had significantly lower EPG levels than did NB sheep. Breed differences were due to lower egg counts from Cooperia spp, Tricbostrongylus spp and Ostertagia spp in D B hosts. On the other hand, breed differences for tt. contortus and Oesophagostomum spp were not significant in sheep carrying mixed infections and ewes with a relatively pure H. contortus infection. Thus, the results of the present study conducted with lambs infected with 11. contortus are in agreement with those reported earlier. However, it is not known why egg production of some parasites varies with breed of the host animal. The larvae inhibition observed in Exp. 1 suggests that the physiology of the digestive tract may vary between breeds to the extent that parasite development and function are affected. This is an area that needs further research. The increase in postchallenge EPG levels was disappointing and shows that the challenge larvae did not induce a self-cure reaction in any of the breed groups examined in Exp. 2. Stewart (1953) has shown that prechallenge sensitization must reach a certain level for the self-cure reaction to take place, a degree of sensitization the lambs in this study apparently did not have. Based on Hb and MCHC levels, the results of Exp. 1 and 2 show that lambs with B breeding are less likely to develop anemia as a result of ti. contortus infection than are NB lambs. Yazwinski et al. (1979) reported that Hb levels in mature ewes infected with tt. contortus did not vary significantly between breed groups. This discrepancy in findings may be due to the fact that age of the host animals and previous exposure to infection were different in the two studies. Although the role of leucocytes in parasite resistance is not clear, Andrews (1942), Charleston (1965) and Bradley et al. (1973) have reported that the greater the tissue and blood eosinophilia in sheep infected with H. contortus, the lower the degree of infection. In this study, lambs with B breeding showed a greater increase in leucocyte levels as a result of infection than did NB lambs. These data, along with the higher Hb and MCHC levels, show that B and B cross lambs are more resistant to 11. contortus infection than are NB lambs. These results do not agree with those reported by Todd et al. (1978), who found no evidence that B Targee lambs were more resistant to H. contortus than Targee lambs. Those authors did not evaluate the straightbred B, and our studies show that these animals are apparently more resistant than crossbreeds. The crosses evaluated in the two studies were also different, and there is evidence that sheep carrying fine wool

H. CONTORTUS RESISTANCE IN BLACKBELLY SHEEP 283 TABLE 3. MEAN FECAL EGG COUNTS, HEMOGLOBIN LEVELS, MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATIONS, PERIPHERAL EOSINOPHIL AND WHITE BLOOD CELL LEVELS (EXP. 2) Breed group Sex group Item Date B D X B S )( B NB Ewe Wether No. lambs 8 7 8 8 15 16 Final EPG levels d Nov. 11 925.0 671.0 988.0 1,200.0 347.0 x 1,525.0Y Hb levels, g/loo ml Preinfection Sept. 10 11.96 11.33 11.76 11.33 12.14 x 11.05Y Final Nov. 10 12.39 a 11.34 b 12.35 a 10.39 c 12.17 x 11.12Y MCHC level~ e Nov. 10.354 a.340 b.346 ab.335b.344.344 PEOS levels" Preinfection Sept. 10 242.0 189.8 173.3 118.3 213.8 147.8 Maximum postinf~ctiong 841.5 667.9 467.5 455.1 780.3 x 442.8Y WBC levels" Oct. 27 13,701.0 a 8,440.0 b 7,930.0 b 5,370.0 c 8,590.0 9,130.0 a'b'cbreed groups only, means on same line with different superscripts differ (P<.05). deggs per gram feces. emean corpuscular hemoglobin concentrations. fperipheral eosinophil levels. gmean of maximum responses by individual animals. hwhite blood cell levels, 5 days postinfection. X'YSex group only, means on same line with different superscripts differ (P<.05). breeding such as the Targee are more resistant to gastrointestinal parasites than are British breeds (Warwick et al, 1949; Colglazier et al., 1968). Sex differences were also observed in Exp. 2. Ewe lambs had significantly lower final EPG levels and higher preinfection and postinfection Hb levels and displayed greater peripheral eosinophilia than wether lambs. Breed x sex interactions were not significant for these variables. Our data support results reported by Colglazier et al. (1968), who found haemonchosis to be more severe in male than in ewe lambs. To date, the only research basis for greater resistance in ewes than wethers is the study reported by Hogarth-Scott (1969), who demonstrated that homocytropic IgGla antibody increased markedly in response to a parasite infection. He also presented a model in which the capacity to produce lggla was linked to the X chromosome, thereby accounting for its greater production in ewes than in males. It should also be noted that host endocrine levels may have contributed to the sex differences observed in this study. The relationship of host sex to parasite resistance needs to be investigated. Literature Cited Andrews, J. S. 1942. Stomach worm (Haernoncbus contortus) infection in lambs and its relationship to gastric hemorrhage and general pathology. J. Agr. Res. 65:1. Bausch and Lomb, Inc. 1965. Clinical Methods Manual, Spectronic 20. Bausch and Lomb, Inc., New York. Bradley, R. W., C. V. Radhakrishnan, V. G. Patil-Kulkami and P. E. Loddins. 1973. Responses in Florida Native and Rambouillet lambs exposed to one and two oral doses of Haemoncbus contortus. Amer. J. Vet. Res. 34:729. Charleston, W.A.G. 1965. Pathogensis of experimental haemonchosis in sheep, with special reference to the development of resistance. J. Comp. Pathol. 75:55. Coles, E. H. 1967. Veterinary Clinical Pathology. W. B. Saunders Co., Philadelphia. Colglazier, M. G., I. L. Lindahl, J. H. Turner, G. 1. Wilson, G. El Whitanore and R. L. Wilson. 1968. Effect of management systems on the growth of lambs and development of internal parasitism. J. Parasit. 56:89. Gordon, H. M. and H. V. Whitlock. 1939. A new technique for counting nematode e~s in sheep feces. J. Council Sci. and Ind. Res. (Australia) 12: 50. Hogarth-Scott, R. S. 1969. Homocytotropic antibody in sheep. Immunology 16: 543. Jilek, A. F. and R. E. Bradley. 1969. Hemoglobin types and resistance to Haernoncbus contortus in sheep. Amer. J. Vet. Res. 30:1773. Knight, R. A., H. H. Vegors and H. A. Glimp. 1973.

284 YAZWINSKI ET AL. Effects of breed and date of birth of lambs on gastrointestinal nematode infections. Amer. J. Vet. Res. 34:323. Loggins, P. E., L. E. Swanson and M. Koger. 1965. Parasite levels in sheep as affected by heredity. J. Anim. Sci. 24:286. Moncol, D. J. and E. G. Batte. 1967. Peripheral blood eosinophilia in porcine ascariasis. Cornell Vet. 62:96. Scrivner, L. H. 1964a. Breed resistance to ostertagiasis in sheep. J. Amer. Vet. Med. Assoc. 144:883. Serivner, L. H. 1964b. Transmission of resistance to ovine ostertagiasis. J. Amer. Vet. Med. Assoc. 144:1024. Steel, R.G.D. and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Stewart, D. F. 1953. Studies on resistance of sheep to infestation with Haernoncbus contortus and Tricbostrongylus spp. and on the immunological reactions of sheep exposed to infestation. V. Australian J. Agr. Res. 4:100. Todd, K. S. Jr., N. D. Levine and F. L. Anderson. 1978. An evaluation of the Baermann Technique using infective larvae of H. contortus. Proc. Helminthol. Soc. Washington 37:57. Warwick, B. L., R. O. Berry, R. D. Turk and C. O. Morgan. 1949. Selection of sheep and goats for resistance to stomach worms, Haemoncbus contortus. J. Anita. Sci. 8:609. Yazwinski, T. A., L. Goode, D. J. Moncol, G. W. Morgan and A. C. Linnerud. 1979. Parasite resistance in straightbred and crossbred Barbados Blackbelly sheep. J. Anita. Sci. 49:919.