PARASITE RESISTANCE IN STRAIGHTBRED AND CROSSBRED BARBADOS BLACKBELLY SHEEP 1,2

PARASITE RESISTANCE IN STRAIGHTBRED AND CROSSBRED BARBADOS BLACKBELLY SHEEP 1,2 Thomas A. Yazwinski 3, L. Goode 4, D. J. Moncol 4, G. W. Morgan 4 and A. C. Linnerud 4 North Carolina State University, Raleigh 2 7650 Summary Resistance to gastrointestinal parasites was studied in a total of 193 sheep with widely different points of origin and genetic backgrounds. Breed groups compared were Dorset (D), Barbados Blackbelly (B) and Dorset x Blackbelly (D B), Dorset X Landrace (D L) and Rambouillet Landrace (R L) crosses. In Exp. I, the sheep were carrying mixed infections of parasites, and D B sheep had lower (P<.05) total fecal egg counts (EPG, eggs/g feces) than the D, D X L and R L groups. Breed differences were not related to ewe hemoglobin type, but were primarily the result of significantly lower egg production by CTO parasites (Cooperia spp, Trichostrongylus spp, Ostertagia spp) in D x B animals. Fecal egg counts for HO parasites (H. contortus, Oesophagostomum spp) did not vary significantly among breed groups. Differences in Nematodirus spp EPG levels were not consistent among trials. In Exp. If, D x B and D ewe and ram lambs with zero EPG levels were administered standard doses of a mixed infection of larvae and maintained on concrete, and showed that breed differences in eggs per gram of feces were the result of less fecund parasites in the digestive tract of D B animals. While numbers of parasites recovered from the digestive tract did not vary significantly, egg production per CTO 1 Paper No. 5891 of the Journal Series of the North Carolina Agr. Res. Service, Raleigh. 2The use of trade names in this publication does not imply endorsement by the North Carolina AgE Res. Service of the products named, nor criticism of similar ones not mentioned. 3Present address: Dept. of Animal Science, Univ. of Arkansas, Fayetteville 72701. 4Dept. of Animal Science, Veterinary Science, Poultry Science and Experiment Statistics, respeetively. 919 parasite recovered was lower (P<.05) in D B rams and ewes. Eggs per HO parasite were lower (P<.05) in D B ewes, but not in D x B rams. Based on significantly lower EPG levels and higher hemoglobin (Hb) and mean corpuscvlar hemoglobin concentration (MCHC) levels obtained in Exp. III, B and D B ewes were shown to be more resistant to a mixed infection of parasites than D ewes. However, this was not the case when the same ewes were reinfected with a relatively pure culture of 14. contortus larvae. While EPG, Hb and usually MCHC levels consistently favored B and D B sheep, breed differences were not significant. There was no evidence that levels of circulating antibodies were involved in breed resistance. (Key Words: Sheep, Parasite Resistance, Breeds, Antibodies.) I ntrod uction Gastrointestinal parasites are a serious problem in sheep and other ruminants.' The development and use of animals possessing a high degree of parasite resistance would offer several advantages over the present situation since control is obtained entirely with anthelmintics and husbandry practices. Some breeds and strains of sheep appear to have natural 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). The initiation of a crossbreeding program in the North Carolina Agricultural Research Service flock presented an opportunity to investigate possible parasite resistance in sheep with widely different points of origin and genetic backgrounds. The primary breeds involved were the Polled Dorset, Rambouillet, Finnish Landrace and Barbados Blackbelly, a haired sheep originating in Barbados and other West Indies Islands. The objectives of this study were to deter- JOURNAL OF ANIMAL SCIENCE, Vol. 49, No. 4, 1979

920 YAZWINSKI ET AL. mine if these breeds, or their crosses, differed in their ability to resist gastrointestinal parasites, and, if so, to investigate possible physiological mechanisms responsible for the resistance. Experimental Procedure Exp. I consisted of four trials and utilized a total of 160 animals. The sheep used in each trial had been subjected to the same feed and management prior to being assigned to the experiment. At the beginning of each trial, animals were treated with standard doses of an anthelmintic. From this point, breed groups within trials were grazed together on grasslegume pastures that had been previously contaminated by grazing with sheep carrying mixed infections of parasites. No anthelmintics were administered during grazing. Fecal egg count at the end of the grazing periods was the criteria used to evaluate parasite resistance in the various breed groups. Fecal samples were examined for parasite ova according to a modified McMasters technique (Gordon and Whitlock, 1939). Numbers of parasite ova were expressed as eggs per gram of feces (EPG). Egg counts were made at frequent intervals during each trial but only the final EPG values were subjected to statistical analysis. At the final fecal examination in trials 1, 2 and 3, ova were counted and stratified according to techniques described by Dewhirst and Hansen (1961) into the following groups: (1) Haemoncbus contortus and Oesopbagostomum spp (HO group); (2) Cooperia spp, Tricbostrongylus spp and Ostertagia spp (CTO group); and (3) Nematodirus spp (NEM). A total of 37 yearling ewes were used in trial 1 and the following breed groups were compared: (1) seven Polled Dorsets (D), (2) 11 Dorset x Blackbelly crosses (D B), (3) nine Dorset x Landrace crosses (D L), and (4) 10 Rambouillet x Landrace crosses (R x L). These ewes were treated with a copper sulfate-nicotine sulfate drench on April 9 and grazed together on contaminated pasture until July 12 (94 days). During the trial the hemoglobin type of each ewe was determined by electrophoresis (Gelman Instrument Company Manual, 1969). For trials 2 and 3 a total of 69 lambs ap- 50mizole, Merck and Co., Inc., Rathway, NJ. etramisole, American Cyanamide Co,, Princeton, NJ. proximately 8 months old were treated witt thiabendazole s on July 12. After drenching, 11 non-blackbelly and 13 D B ram lambs werc assigned to trial 2, and 26 non-blackbelly anc 19 D B ewe lambs were assigned to trial 3 The non-blackbelly lambs were mainly Dorset, with a few D L lambs represented. Data or these lambs were reported under the D breed group in table 1. Ram and ewe lambs were grazed on separate contaminated pastures from July 12 to September 20 (70 days). Barbados Blackbelly (B), D and D B ewe., were compared in trial 4. Each breed group consisted of 18 mature, nongravid, ewes. All animals were treated with thiabendazole on April 10 and grazed together until July 17 (98 days). Parasite ova were not stratified in this trial. The efficacy of the anthelmintics administered in Exp. I was attested to by the fact that parasite ova were not found in the feces in any trial until at least 21 days after treatment. Exp. II consisted of two trials in which 8 month-old lambs were treated with anthelmin. tics and were reinfected with standard number., of larvae. Animals were maintained on concrete throughout the experiment. Breed groups were compared on the basis of (1) final EPG levels~ (2) numbers of parasites recovered from the digestive tract, and (3) eggs produced per para. site recovered (final EPG levels + numbers oi parasites recovered). Four D and four D B ram lambs (trial 1'~ were treated with levamisole hydrochloride 6 on October 4 and with thiabendazole on October 11. After 22 days of negative fecal egg counts (November 2), each animal was ad. ministered approximately 60,000 infective larvae (3,600 H. contortus; 29,700 Cooperia spp; 24,000 Tricbostrongylus spp and 2,70(3 Ostertagia spp). Larvae were produced by coprocuhure using sterilized peat moss and vermiculite as culture media. They were harvested by the Baermann technique (Todd et al., 1970) and were administered by stomach tube. Data on EPG levels by parasite groups were obtained as described for Exp. I. Rams were slaughtered after the final fecal collection on December 20 (48 days after infection) and parasites in the abomasum, small intestine and large intestine were collected by washing the contents of each section through a series of 850, 425, 250 and 10 /gm screens. Parasites recovered from the screens were counted, identified and stratified as to HO group, CTO group and NEM.

PARASITE RESISTANCE IN BLACKBELLY SHEEP 921 TABLE 1. MEAN FINAL EGGS PER GRAM OF FECES LEVELS (EXP. I AND 1I) Item Breed groups DX B D DX L RX L B Exp. I Trial 1 (yearling ewes) 11 7 CTO 236 a 1871 b HO 0 14 NEM 0 a 200 b Total 236 a 2085b 9 i0 2078 b 1470 b 0 0 0 a 20 a 2078 b 1490 b Trial 2 (ram lambs) 13 11 CTO 3038 a 5473 b HO 154 209 NEM 38 64 Total 3231 a 5745 b 9 ~ Trial 3 (ewe lambs) 19 26 CTO 563 a 1162 b HO 195 8O4 NEM 0 23 Total 758 a 1988 b Trial 4 (mature ewes) 18 18 Total EPG 83 a 707 b Exp. II Trial I (ram lambs) 4 4 CTO 1675 a 4450 b HO 1550 1300 NEM 0 0 Total 3225a 5750 b Trial 2 (ewe Iambs) No9 of animals 4 4 CTO 325 a 4900 b HO 1125 9625 NEM 0 0 Total 1450 a 14525 b 9 ~ 18 29 a 9 ~. a'bmeans on the same line with different superscripts differ significantly (P<.05). In trial 2 levamisole hydrochloride and thiabendazole drenches were administered to four D and four D B ewe lambs on October 11 and 18, respectively. On November 16, after 28 days of negative fecal egg counts, each animal received approximately 35,000 infective larvae (20,650 H. contortus; 350 Oesopbagostomum spp; 1,750 Cooperia spp; 9,100 Tricbostrongylus spp; and 3,150 Ostertagia spp). Ewes were slaughtered after the final fecal collection on January 4 (49 days after infection) and data on EPG levels and parasites in the digestive tract were obtained as previously described9 Six D, five B and six D x B ewes were compared in Exp III These were mature, nongravid females that had acquired mixed infections of parasites while grazing contaminated grass-legume and small grain pastures prior to the start of the experiment9 During the study

922 YAZWINSKI ET AL. (March 6 to July 1) they were maintained on concrete. The experiment consisted of three periods. In period 1 (March 6 to March 25) data were obtained while the ewes were carrying the worm burdens acquired during grazing. Ewes were bled at intervals as shown in table 4. A 10% solution of ethylenediaminetetraacetic acid was used as an anticoagulant. Hemoglobin levels (Hb), expressed as g/100 ml, were determined by the cyanmethemoglobin method (Clinical Methods Manual for the Bausch and Lomb Spectronic 20 Spectrophotometer, 1965). Mean corpuscular hemoglobin concentration (MCHC) was calculated as outlined by Coles (1967), and EPG levels were determined as described in Exp. I except that ova were not stratified. Specific antibody titers were measured in serum according to a procedure taken in part from a number of references (Stewart, 1950; Boyden, 1951; Stavitsky, 1954, 1964). The technique was one of indirect hemagglutination, wherein tanned sheep red blood cells were sensitized with an antigen source prepared by homogenizing third stage 11. contortus larvae in buffered saline. The microtitration technique was used to determine the actual titers. Period 2 (March 26 to April 25) was a transition period during which information was obtained after the ewes were drenched with levamisole hydrochloride on March 26. Data were obtained on Hb, MCHC, EPG and antibody levels as previously described. Total white blood cell levels (WBC) were also determined as outlined by Coles (1967). Period 3 extended from April 26 to July 1. A second anthelmintic, thiabendazole, was administered on April 26. Preinfection Hb, MCHC and WBC levels were obtained on May 2 and antibody titers on May 6. Immediately after bleeding on May 6 each animal was reinfected with approximately 10,000 larvae estimated to be 98% 11. contortus. Postinfection data were obtained on the variables listed above. While data on EPG levels were obtained at intervals during Experiment I and II, only the final values were subjected to statistical analysis. In trial 1 of Exp. I, EPG levels were highly variable, there were several values of zero and the distribution was not normal. Therefore, these data were transformed to log 10 and, along with all other data, were subjected to standard analysis of variance techniques (Snedecor and Cochran, 1967). Multiple mean corn- parisons were made according to Duncan's Multiple Range Test (P<.05). Results and Discussion Results of the final fecal egg counts for Exp. I and II are summarized in table 1. The data from trial 1, Exp. I, clearly shows that D x B ewes had lower (P<.05) CTO and total EPG levels than the other breed groups. Differences between D, D X L and R x L groups were not significant for these parameters. Dorset ewes had more NEM ova (P<.05) than the other groups, whereas EPG levels for HO parasites did not vary significantly. Ova from CTO parasites were most numerous in all breed groups. Gregory et al. (1939), Scrivner (1964a,b) and Rothwell et al. (1971) have also cited examples of breed resistance to gastrointestinal helminths. Data on the relationship of host hemoglobin type to EPG levels in trial 1 are shown in table 2. Evans et al. (1963), Jilek and Bradley (1969) and Radhakrishnan et al. (1972) have reported that sheep with type A hemoglobin were most resistant to H. contortus. In a later report Bradley et al. (1973) stated that sheep with type AB hemoglobin were more resistant than either type A or B. In the present study, however, D and D B ewes, which had the highest and lowest total EPG levels, were all of type B hemoglobin. Thus, hemoglobin type was not related to the difference in EPG levels observed in these groups. On the other hand, D L and R L ewes were about evenly divided between B and AB hemoglobin. In each group AB type ewes had lower CTO and total EPG levels than those with type B hemoglobin. However, numbers were limited and these differences were not statistically significant. The role of hemoglobin type in host resistance to gastrointestinal parasites is not clear and more research is needed in this area. In the present study there appears to be more than one physiological mechanism involved in parasite resistance. Data from trials 2, 3 and 4 of Exp. I also showed that D B and B sheep had lower CTO and(or) total EPG levels than non-blackbelly sheep. Differences in HO parasites and NEM were not significant in trials 2 and 3. Breed differences obtained in Exp. I could have been due to lethal or inhibitory effects which resulted in fewer or less fecund parasites in D B and B animals, or to differences in

PARASITE RESISTANCE IN BLACKBELLY SHEEP 923 TABLE 2. MEAN EGGS PER GRAM OF FECES LEVELS BY HEMOGLOBIN TYPE (EXP. I, TRIAL 1) TABLE 3. MEAN NUMBERS OF PARASITES RECOVERED AND EGGS PRODUCED PER PARASITE (EXP. II) Breed group Item D DX B DX L RX L Item DXB Breed group D Hemoglobin type B 7 11 4 5 CTO 1871 236 3725 1840 HO 14 0 0 0 NEM 200 0 0 0 Hemoglobin type AB... 5 5 CTO... 760 11 O0 HO... 0 0 NEM... 0 0 Overall mean 2085 236 2078 1490 grazing habits of such nature that D x B and B sheep ingested fewer infective larvae. The results of the final fecal egg counts obtained in Exp. II are summarized in table 1. These data support those of Exp. I in that parasites of the CTO group were mainly responsible for the lower EPG levels in D X B sheep. The results also show that differences in EPG levels among breed groups were not due to differences in grazing habits and ingestion of fewer larvae since the experimental animals were maintained on concrete throughout the study. Data on numbers of parasites recovered from the digestive tract are presented in table 3. While Nematodirus spp were lower (P<.05) in D x B rams, differences for CTO and HO parasites were not significant in either rams or ewes. On the other hand, when egg production per worm recovered was determined (table 3), CTO parasites produced fewer ova (P<.05) in D B animals in both trials. Egg production per HO parasite was significantly lower in D x t3 ewes, but not in rams. Thus, parasites in D x B animals were less fecund and this appears to be the primary cause for breed variation in EPG levels rather than a reduction in number of parasites. It was also determined that only 4.5% of the H. comortus larvae administered to D x B ewe lambs were recovered as adult parasites, whereas over 14.0% were recovered from each of the other breed groups. Data from Exp. II suggests that both breed and sex of the host animal influence parasite development and function. Parasites recovered Trial 1 (ram lambs) CTO HO NEM Total Trial 2 (ewe lambs) CTO HO NEM Total Eggs produced per parasite Trial 1 (ram lambs) CTO HO Trial 2 (ewe lambs) CTO HO 4 18925 535 5 a 19465 7615 950 0 8565 4 20590 530 80 b 21200 10850 3115 20 13985 4 4.087 a.224 b 2.034 2.956 4 4.059 a.439 b.647 a 2.905 b a'bmeans on the same line with different superscripts differ significantly (P<.05). Colglazier et al. (1968) reported that haemonchosis was more severe in male than in female sheep. The results of Exp. III are summarized in table 4. In period 1, B and D x B ewes had significantly lower EPG levels than D ewes when removed from pasture (March 6) and after 8 days on concrete (March 14). These data are in agreement with those obtained in Experiment I and II with sheep carrying mixed infections of gastrointestinal parasites. From March 6 to March 14 there was a marked rise in EPG levels. A logical explanation for these results is that removing ewes from exposure to infective larvae on March 6 reduced the immune inhibition of fourth stage larvae described by Soulsby and Stewart (1960) so that maturation of inhibited larvae was occurring in the sheep used in the present study. On March 6, B ewes had a higher (P<.05)

924 YAZWINSKI ET AL. Hb level than D ewes, The D B group was intermediate to the parent breeds. By March 21 both B and D X B groups had significantly higher Hb levels. Hb loss, which is a measure of infection severity, was.5,.75 and.92 g/100 for B, D B and D groups, respectively. Blackbelly ewes also had a higher (P<.05) MCHC level than D ewes on March 21. Based on EPG, Hb and MCHC levels in period 1, B and D x B ewes were more resistant to a mixed infection of parasites than D ewes. Antibody titers obtained on March 14 were highly variable and differences among breed groups were not significant. Differences in EPG, Hb, MCHC and WBC levels did not vary significantly among breed groups during period 2, TABLE 4. MEAN EGGS PER GRAM OF FECES, HEMOGLOBIN, MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION, WHITE BLOOD CELL AND ANTIBODY LEVELS (EXP. IID Breed groups Item B D X B D Period 1 EPG Hb MCHC Antibodies Period 2 c EPG Hb MCHC WBC 5 6 March 6 440 a 917 a March 14 2430 a 2300 a March 24 1830 3083 March 6 11.14 a 10.30 ab March 21 10.64 a 9.55 a March 6.346.336 March 21.353 a.339 ab March 14 14.40 38.67 March 28 0 0 April 18 500 70 April 2 10.92 10.55 April 25 11.44 10.73 April 2.352.341 April 25.358.335 April 2 10720 8858 April 25 10960 10093 Period 3 d EPG Preinfection May 2 140 30 Final July 1 860 1000 Hb Preinfection May 2 11.08 10.63 Final June 24 10.52 10.85 MCHC Preinfection May 2.352.340 Final June 10.349.342 WBC Preinfection May 2 10110 a 9550 a Final June 24 10460 10133 Antibodies Preinfection May 6 7.2 9.3 Final May 24 22.4 18.7 6 4000 b 5667 b 4433 8.55 b 7.63 b.334.333 b 40.67 0 0 7676 8825 150 2220 9.58 9.60.336.342 9.28 9.27 6800 b 7133.345.335 13.3 21.3 a'bmeans on same line with different superscripts differ significantly (P<.05). CAnimals drenched March 26. danimals drenched April 26 and reinfected May 6.

PARASITE RESISTANCE IN BLACKBELLY SHEEP 925 and only the initial and final values are presented in table 4. The anthelmintic treatment administered at the beginning of period 3 (April 26) did not reduce EPG levels to zero as in previous trials. Data on EPG, Hb, MCHC, WBC and antibody levels were obtained before the animals were reinfected with tl. contortus larvae on May 6. These data are presented in table 4 along with the final values for these variables. By early June there was a marked increase in EPG levels in all breed groups as a result of the larvae administered on May 6. However, EPG levels did not vary significantly among breed groups at any time after reinfection. These resuits are in marked contrast to those obtained for total EPG levels when the same sheep were carrying a mixed infection of parasites (period 1). They agree with previous data from Exp. I and II in that EPG levels for HO parasites did not vary significantly among breed groups. While antibody titers increased markedly in all breed groups after reinfection, differences were not significant and in this study there was no indication that circulating antibodies were involved in breed resistance to gastrointestinal parasites. Soulsby and Stewart (1960) were also unable to relate antibody levels with parasite resistance in sheep. Preinfection W13C levels were higher (P<.05) in B and D 13 ewes, but not at other sampling periods. All breed groups responded with elevated WBC levels after reinfection. Throughout period 3, Hb and usually MCHC levels were consistently higher in D and D B ewes than in Dorsets. However, breed differences were not significant and these data do not agree with those of period 1 when the ewes were carrying a mixed infection. The reasons for the breed differences obtained when sheep were carrying a mixed vs a relatively pure 1-1. contortus infection are not apparent at this time. The effects of mixed and pure infections could have varied with breed of host animal. It is also possible that the physiology and biochemistry of the digestive tract varied among breeds to the extent that parasite growth and development were affected. Some important relationships between the results obtained in periods 1 and 3 were observed. When breed groups were combined, the overall mean EPG level for period 3 was correlated with March 6 EPG levels (r =.75, P<.01). March 6 Hb and MCHC levels were also correlated with terminal Hb and MCHC levels of period 3 (r =.79,.61, P<.01). Therefore, animals exhibiting resistance to a naturally acquired mixed infection displayed the same relative resistance to a subsequent 11. contortus infection. This sort of repeatability would be essential in a program designed to select for parasite resistance. Jilek and Bradley (1969) also obtained repeatable results when studying both natural and artificially induced ovine haemonchosis. 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926 YAZWINSKI ET AL. The role of physiologically active amines in resistance to Trichostrongylus colubriforrnis in the guinea pig. Immunology 21:925. Scrivner, L. H. 1964a. Breed resistance to ostertagiasis in sheep. J. Amer. Vet. Med. Assoc. 144:883. Scrivner, L. H. 1964b. Transmission of resistance to ovine ostertagiasis. J. Amer. Vet. Med. Assoc. 144:1024. Snedecor, G. W, and W. C. Cochran. 1967. Statistical Methods. (6th Ed.) The Iowa State Univ. Press. Ames. Soulsby, E. J. L. and D. F. Stewart. 1960. Serological studies of the self-cure reaction in sheep infected with Haernonchus contortus. Australian J. Agr. Res. 11:595. Stavitsky, A. B. 1954. Micromethods for the study of proteins and antibodies. J. Immunol. 72:360. Stavitsky, A. B. 1964. Immunological Methods. Blackwell Scientific Publications, New York. Stewart, D. F. 1950. Studies on resistance of sheep to infestation with Haemoncbus contortus and Tricbostrongylus spp. and on the immunological reactions of sheep exposed to infestation. Australian J. Agr. Res. 1:301. Todd, K. S. Jr., Levine, N. D. and Anderson, F. L. 1970. An evaluation of the Baermann Technique using infective larvae of H. contortus. Proc. Helminthol. Soc. Wash. 37:57.