Seeders. Faecal worm egg counts. R. K. REINECKE' '', I. L. DE VILLIERS 111 and GERDA JOUBERT' 21

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1 Onderstepoort J. vet Res.. 5 I, (1984) STUDIES ON HAEMONCHUS CONTORTUS. X. THE EFFECT OF TRICHOSTRONGY LUS AXEIIN MERINOS ON ARTIFICIAL PASTURES R. K. REINECKE' '', I. L. DE VILLIERS 111 and GERDA JOUBERT' 21 ABSTRACT REINECKE, R. K., DE VILLIERS, I. L & JOUBERT, GERDA, Studies on Haemonhus ontortus. X. The effet of Trihostrongylus a.xei in Merinos on artifiial pasture. Onderstepoort Journal of Veterinary Researh, 51, (1984). A reently established Eragrostis urvula pasture I, I ha in size at Hennops River in the Transvaal Highveld was ontaminated from 6 Otober 1976 until 2 May 1977 by 3 weaned Merinos, eah infested with 7 infetive larvae of. On 2 November groups of weaned Merinos and again on lo January 1977 a further group of Merinos were infested with 4 infetive larvae of Trihostrongylus a.xei. From 19 November a single group of sheep, predosed with T. a.xei, and a ontrol group grazed with the seeders for 8 weeks. After 6 weeks another group of sheep dosed with T. a.xei grazed with them, thus ensuring a 2-week overlap. This ontinued until autumn, when the last groups were removed. Effiay against hallenge reahed Class C (>SO % effetive against total worm burdens of in >SO% of sheep) in early summer and autumn, but only reahed signifiant levels of either P<O,Ol or P<O,OS in summer. Peak worm burdens in ontrols were reorded in early autumn. INTRODUCTION Horak ( 198) and Horak & Louw ( 1977) arried out extensive trials on irrigated pastures on an experimental farm at Hennops River in the Transvaal Highveld. Traers, plaed on these pastures at regular intervals from Otober 1968 to May 1973, aquired worm burdens of Haemonhus ontortus onsiderably in exess of those reported on natural pastures at Tonteldoos in the eastern Highveld (Horak, 1978). The present trial was an attempt to protet Merinos against hallenge with aquired naturally on artifiial pastures on the same farm by predosing them with Trihostrongylus axei. MATERIALS AND METHODS A flok of month-old weaned Merino wethers was purhased, transferred to the University of Pretoria's (UP) Experimental Farm, treated with anthelmintis, ear-tagged and vainated with bluetongue, quarter evil and enterotoxaemia vaines on Day -58. One hundred and three sheep were set aside for the trial at Hennops River and the balane were used in another trial on the UP Experimental Farm. The experimental design is summarized in Table I. Seeders Eah of lo sheep was given 7 infetive larvae of on Day - 51, another I sheep were similarly dosed on Day -44 and a further I on Day -37 respetively. Controls and groups dosed with T. axei Groups HI and HII onsisted of 12 sheep eah, and. HIII of 13 sheep. On day -44 eah sheep was dosed with 4 infetive larvae of. The 36 undosed ontrols were divided into 3 equal groups, Groups HI, H2 and H3. On day +47 a further 25 Merinos of the same age arrived at the UP Experimental Farm, were ear-tagged, treated with mebendazole and vainated with bluetongue and enterotoxaemia vaine. On Day +52 these were divided into 12 undosed ontrols (Group H4) and 13 sheep (Group HIV) were eah dosed with 4 infetive larvae of. Neither ontrols nor sheep dosed with grazed until the alloated periods mentioned in Table I but remained in onrete-floored kraals whih were swept daily with brooms and leaned with a old "'Faulty of Veterinary Siene, University of Pretoria, P.O. Box 1258, Onderstepoort 11 '~' 359 Cliff Avenue, WaterkloofRidge 181 Reeived 25 November Editor water hose. All the sheep were kept in these pens from 15h overnight until 9h the following morning. Chaffed luerne, hay and water ad lib. were provided in steel troughs in the pens. Grazing A newly established Eragrostis urvula paddok (I, 1 ha) at Hennops River (25 5 S; E; alt. ± 1 28 m) was used. From Day -37 (13 Otober 1976) the seeders were plaed on the pasture and grazed there every day until Group H4 and Group HIV were removed from the pasture on Day + 18 (May 2, 1977), when the seeders were returned to the UP Experimental Farm. At first the seeders ontaminated the entire pasture, but on Day the amp was divided into 4 equal strips with Bonox fening, eah overing an area approximately I X 25m. Thereafter, all the sheep grazed in the first of the strips until the vegetation had been depleted and were then moved to an adjaent strip until the entire amp had been grazed. Thereafter they were transferred to the first strip and the proess was repeated. Irrigation In months when less than 6 mm of rain was reorded, the pastures were spray-irrigated to provide preipitation in exess of 6 mm/month (Table 4 ). The ontrol groups (Groups H 1, H2 and H3) and the groups (Groups HI, HII and HIII) were transferred to Hennops River on Day and Groups H4, and HIV on Day +56. Exposure to infestation and slaughter The various groups grazed with the seeders on the pasture every day from 9h to 15h as indiated in Table I. Groups H4 and HIV grazed on infested pasture for 54 days. All the other groups grazed on pastures for 56 days. Grazing was arranged so that there was an overlap of 14 days between suessive groups. Three to 5 days after removal from grazing half of eah group was and the balane 14 or 15 days later. Worms were reovered as desribed by Reineke (1973). Faeal worm egg ounts Faees were olleted from eah "seeder" from Day -9 (I November) every week for the duration of the experiment. One either egg ounts exeeded 3 epg or sheep were very anaemi, Y, of the therapeuti dose of thiabendazole or mebendazole was administered. This was neessary one only in 19 sheep and twie in I sheep. 33

2 STUDIES ON HAEMONCHUS CONTORTUS. X TABLE 1 Hennops River (H) Experimental design showing the days on whih weaned Merino sheep were divided into groups and dosed either with infetive larvae of (seeders) or of and plaed on artifiial pasture with worm-free ontrols Day Merino wethers 5 months of age arrive at the University of Pretoria's (UP) Experimental Farm on 2 September and were housed in onrete-floored pens -58 Flok treated with mebendazole at 18 mglkg, vainated against enterotoxaernia and bluetongue, ear-marked and 13 sheep seleted for this trial infetive larvae of dosed to eah of 1 sheep (seeders) infetive larvae of dosed to eah of 1 sheep (seeders). 4 infetive larvae of dosed to eah of 37 sheep in Groups HI, HII and Hili infetive larvae of dosed to eah of 1 sheep (seeders). Thirty seeders transferred to Hennops River and plaed on artifiial pastures from 9h-15h and housed in worm-free onrete-floored kraals from 15h overnight until 9h the following day 19 November Eah of 36 sheep in Groups HI, HII and Hili and a further 36 undosed ontrols: Groups Hl. H2 and H3 'transferred to worm-free kraals at Hennops River. Group HI (ontrols) and Group HII () plaed on artifiial pastures +42 Groups H2 (ontrols) and HII () plaed on artifiial pastures sheep arrived from Amersfoort, treated, vainated against enterotoxaernia, bluetongue and ear-tagged and housed in worm-free kraals +52 Eah of 13 sheep dosed with 4 infetive larvae of (Group HIV) +56 Groups Hl (ontrols) and HI () removed from pastures +59 Half of Groups Hl (ontrols) and HI () Balane of Groups Hl (ontrols) and HI () +84 Groups H3 (ontrols) and Hili () plaed on pastures +98 Groups H2 (ontrols) and HII () removed from pastures + 11 Half of Groups H2 (ontrols) and HII () Balane of Groups H2 (ontrols) and HII () Groups H4 (ontrols) and HIV () plaed on pasture + 14 Groups H3 (ontrols) and Hill () removed from pasture Half of Group 3 (ontrols) and 7 sheep in Group III () Balane of Groups 3 (ontrols) and III () + 18 Groups 4 (ontrols) and IV () removed from pastures Half of Group 4 (ontrols) and 7 sheep in Group IV () June 1977, Balane ofgroup4 (ontrols) and IV () Analysis of data The square roots of the mean faeal worm egg ounts an~ the range (i.e. the upper and lower limits) were estimated. The weekly variation is depited graphially in Fig. I. and differential worm ounts were ranked and either analysed by the non-parametri method NPM (Clark, ited by Reineke, 1973) or by the Mann-Whitney U test (Siegel, 1956). RESULTS Seeders Flutuations in worm egg ounts are illustrated in Fig. I. The highest egg ount of 74 8 eggs per gram (epg) in 169 was reorded on 25 November 1976 (Day + 7). Mean egg ounts flutuated and fell gradually until mid February, then rose steadily to a minor peak in early Marh 1977 as a result of good rains in February (2 I mm) (Table 4). Thereafter egg ounts slowly fell to stabilize in April and May (Fig I). It is lear that pastures were being ontinually ontaminated with eggs of H. ontortus at a high level throughout the experimental period. Controls and groups predosed with The number of worms reovered at neropsy are summarized in Table in Group H1 died on Day +2, but no worms were ounted post-mortem. The mean total worm burdens of and the perentage of the total worm load retarded as 4th stage larvae (L 4 ) for the various groups are summarized in Table 3. Despite the high rate of ontamination by the seeders (Fig. 2), initial pasture infestation was very low from November-January, but rose slowly in February to show an almost fivefold inrease from February to early April (Groups H3 and Hill). It then tapered off rapidly to a low level in May. Until the end of February, L 4 of H. ontort us formed <1% of the total worm burden. As the worm burdens inreased in the autumn, L 4 represesented 78,6 % and 79,2 % of Group H3 and H4, and 72, % and 72,5 % of Group Hill and HIV respetively. 34

3 R. K. REINECKE, I. L. DE VILLIERS & GERDA JOUBERT epg 24 E ~ ~ 18..._ <ll a. I~ ~ 12 6 Nl J F M A M FiG. I. Variations in the faeal worm, egg ounts (epg) of the seeders. The meanj, upper and lower limit are onverted to the square root of the eggs per gram TABLE 2 Improved pastures: worms reovered at neropsy Group HI Controls L.tll 5(2) AI3J II I *33 35' * * I I 737 * I L3 5 A & A Median X,5 = 173,5 5 x o,5 = 174 Group HI 4 L3 of on Day -44 L.oJ 5(2) AI3J L3 L. 5 A 5 * * * I '' 1 Fourth stage i2l Fifth stage 1 Adults * An additional 1 I 1 aliquot ounted 3/ 11 > 173.3/11 >174 35

4 STUDIES ON HAEMONCHUS CONTORTUS. X TABLE 2 (ontd.) Group H2 Controls L. 5 A Feb I Feb I Feb * Feb Feb Feb I Mar I Mar Mar Mar Mar Mar I I L3 L. 5 A Group HII 4 L, of on Day -44 Median X,5 = 1218 X,5 = 1344,5 5 A L3 5 A 1 28 Feb 142 **51 28 Feb 35 **83 28 Feb Feb Feb Feb Mar Mar Mar Mar Mar Mar I I * Inluding 18 L3 of ** An additional 1 / 1 aliquot ounted Group H3 Controls P<O,OI P<,1 5 A L3 5 A 4 12 Apr I Apr Apr II Apr Apr I Apr Apr I 965 *3 25 Apr Apr *78 25 Apr Apr II Apr I Group Hili 4' L, of on Day - 44 Median 1949 X,5 = X,5 = A L, L. 5 A Apr Apr Apr Apr Apr 6 23 * Apr Apr Apr Apr Apr Apr Apr Apr I I II II * An additional 1 I 1 aliquot ounted 4113> P<,5

5 TABLE 2 (ontd.) Group H4 Controls R. K. REINECKE, I. L. DE VILLIERS & GERDA JOUBERT 5 A L3 5 A May June May June May *272 6 June May June * May June May 29 6 June I 11 I I I I I & A Median X,4 = 239,6 6 X,5 = 1377 Group H IV 4 L 3 of on day +52 L. 5 A L, L. 5 A May June May *262 6 June May June May June * May June May June * May I 151 I 13 ** I I I 64 II II 128 I I ! I * An additional 1 / 1 aliquot ounted ** Inluding 8 L3 3/ 13 > 239 B 3/13 > 1377 Effiay of groups dosed with Worm burdens on ontrols and in groups predosed with are listed in Table 2, together with the results analysed by the NPM or Mann Whitney U test. worm burdens of H. ontortus in groups predosed with were signifiantly less in Group HII (P<O,O I) and in Group III (P<,5) but rose to Class C ( >5 % effetive in >5 % of sheep) in Groups I and IV respetively. The highest effiay was reorded against 5th stages and adults in Group IV, when it rose to >6% in >6% of sheep (Class B). This group grazed on infested pastures from 25 Marh-2 May. The monthly rainfall and supplementary spray irrigation figures are reorded in Table 4. The dry months were January, April and May, during whih an additional 2-5 mm of moisture was supplied by irrigation. TABLE 3 The perentage of L 4 of expressed as a perentage of the mean total worm burdens Group Period on grazing Mean total L.'7'o worm burden HI 19 November ,2 HI - 14 January , H2 31 Deember ,8 HII -25 February 1977 I 298 9,2 H3 II February ,6 Hill -8 April , H4 25 Marh ,2 HIV - 2 May 97 72,5 37 TABLE 4 Rainfall and irrigation on Eragrostis urvula grazing at Hennops River Month and year Rainfall (mm) Irrigation (mm) Otober November ,5 Deember January ,5 4 February Marh ,5 Aprill May 1977 II 5 TABLE 5 The mean monthly total worm burdens L 4 expressed as a perentage of the total worm burdens of 127 traer lambs whih grazed on improved pastures at Hennops River from Otober May 1973 (Horak & Louw, 1977) H. ontortus Month of sheep Mean total L.% worm burden January ,8 February ,2 Marh 1 I 3 61,6 April ,5 May ,9 June ,6 July ,1 August ,6 September ,8 Otober ,2 November ,1 Deember ,6 DISCUSSION Horak & Louw (1977) grazed 2-3 "traers" for periods of 33 days on improved pastures on the same farm used in the present trials.

6 STUDIES ON HAEMONCHUS CONTORTUS. X TABLE 6 A omparison of estimated mean total wonn burdens on data from Horak & Louw (1977) ompared with data from ontrols in the present trials H1 H2 H3 H4 Group Estimated mean total wonn burden based on data from Horak & Louw(1977) Atual mean wonn burdens in the ontrols in the present trials The pastures in their experiment were spray-irrigated on 2-4 oasions eah month, approximately 37 mm of water being supplied on eah oasion. When no rain was reorded from June to August or September, they were irrigated on 3 or 4 oasions ( mm), and even from Deember to Marh, when it rained every month, pastures were irrigated at least twie (2 X 37 = 74 mm). This irrigation was onsiderably in exess of the 2-5 mm applied in dry months in the present trials. The lambs in the trials of Horak & Louw ( 1977) grazed from 7h-17h every day, i.e. for 1 h/day for 33 days, or 33 h. The sheep in our trials only grazed from 9h-15h every day, i.e. for 6 h/day for either 54 days or 324 h (Groups 4 and IV) or for 56 days or 336 h (Groups 1 and I; 2 and II and 3 and III). There was almost no differene in the atual times spent on pastures. However, it has been shown that indiret light enourages migration on to herbage, but in bright sunlight larvae migrate to the humus layer or "mat" on the ground surfae and are thus less available to grazing sheep (Crofton, 1948). Sine this ours only in the presene of moisture, the earlier grazing from 7h-9h and in the late afternoon from 15h-17h, partiularly in the autumn, provides the neessary periods of indiret light. Indiret light plus additional irrigation would suggest that the pastures in the trials reported on by Horak & Louw (1977) were onsiderably more favourable for infetive larvae of than the onditions in the present trials. The ontrols of these trials (Table 3) are ompared with the data summarized in Table 5 abstrated from Horak & Louw (1977). If the mean total worm burdens of eah month in Table 5 are divided by 33, i.e. the number of days the sheep grazed, the mean number of infetive larvae that were infetive enough to develop to parasiti stages in the host are: November 4/day, Deember 14/day, January 21/day, February 35/day, Marh 3/day, April116/day and May 125/day respetively. The mean total worm burdens of the ontrols in the present trial are listed above. We have also indiated the exat period during whih eah group was on the pasture. By simple multipliation of figures extrated from Horak & Louw (1977), given above, estimates an be made of the worm burdens the sheep would have aquired. In Table 6 these estimates are ompared with the atual worm burdens of the sheep in our trial. Both the estimated and the atual worm burdens show a rise from Group HI to H2. The fat that the mean worm burdens in the present trials, i.e in Group H2, exeed the estimate of in Group H3 indiates a rapid build up of pasture infestation in our trails. There is a subsequent explosive inrease in Group H3 followed by a dramati fall in Group H4 in ontrast with the marked inrease between the estimated burdens in Group H3 and Group H4, based on Horak & Louw (1977). 38 A simple explanation for these differenes is: (i) In February 21 mm and in Marh 11,5 mm of rain was reorded, but rain plus irrigation was only 7 mm and 61 mm in April and May 1977 respetively in the present trials. (ii) Horak & Louw (1977) reorded a major peak of 7 21 epg in Marh 197, whereas in the present trials mean faeal worm egg ounts ranged from epg during the period Groups H3 and Hill were exposed to grazing. (iii) In our trials the stoking rate was very high from February, i.e. there were 54 sheep infested with (3 seeders+ 12 sheep in Group H2 and 12 in Group HII) and an additional 25 sheep ( 12 in Group H3 and 13 in Group Hill) making a total of 79 sheep/ha. After the removal of 24 sheep ( 12 in Group H2 and 12 in Group HII), the stoking rate fell to 55 sheep/ha, but for the last 5 of the 8 weeks the 25 sheep in Groups H3 and Hill aquired and this also ontributed to pasture ontamination. By the time the last 25 sheep (12 in Group H4 and 13 in Group HIV) were added to those already on the pasture, boosting the numbers to 8 sheep/ha, half the pasture was so heavily overgrazed that it ould no longer be used and grazing was limited to the other,5 ha for the balane of the trial. In their trials Horak & Louw ( 1977) never reahed the state of overgrazing on the grass lover pastures at Hennops River that we reahed on the E. urvula pasture by the end of Marh 1977 in our trials. Their average stoking rate at Hennops River is 3 sheep/ha aording to J.P. Louw (personal ommuniation, 1981). We deliberately set out to expose sheep in these trials to maximum hallenge with regardless of whether the pastures were abused by overrowding. The effiay of 4 infetive larvae oft. a.xei in proteting sheep in Group Hill against 5th stage and adult H. ontortus by >5 % in >5% of sheep (Class C) and the signifiant redution (P<,5) in total worm burdens during this period of maximum hallenge, onfirm the favourable results in natural pastures at the UP Experimental Farm onduted at the same time (Reineke, De Villiers & Brukner, 1984). Effiay in Group HIV improved to >6 % against 5th stage and adult in >6 % of sheep and Class C against total worm burdens. This is interesting, beause these animals were dosed with 4 infetive larvae of T. a.xei on Day +52 and exposed to hallenge on the grazing from Day Day + 18 or days later (25 Marh to 2 May). Reineke, De Villiers & Brukner (1984) had similar results in UP Group IV predosed with whih grazed on natural pastures. In their trial the latter group was >6 % effetive against 5th stage, adult and total worm burdens of in >6 % of sheep (Class B). This group had been dosed on Day (November ) with T. a.xei and exposed to hallenge for more than 7 months before being removed from pastures on 6 June It seems that the longer the sheep were exposed to H. ontortus after dosing with the greater their resistane. The present trial, however, shows that sheep dosed with T. a.xei in Group HIV are more effiient in resisting hallenge in autumn (25 Marh-2 May) than in late summer and early autumn. This was the ase in Group Hill when exposed to hallenge from 14 February to 8 April. This raises the interesting possibility that predosing with T. a.xei in November may be followed by treatment with disophenol in January, a hemoprophylati

7 effetive against only. Disophenol still reahes Class C against hallenge with 3 months after treatment (Reineke, Brukner & De Villiers, 1981). ACKNOWLEDGEMENTS We wish to thank MSD (SA Pty Ltd) for providing failities and grazing at the Hennops River Researh Centre and the University of Pretoria for finanial assistane to omplete these trials. REFERENCES CROFfON, H. D., The eology of immature phases oftrihostrongyle nematodes. I. Vertial distribution of infetive larvae of Trihostrongylus retortaeformis in relation to their habitat. Parasitology, 39, HORAK, I. G. & LOUW, J. P., Parasites of domesti and wild animals in South Afria. IV. Helminths in sheep on irrigated pasture on the Transvaal Highveld. Onderstepoort Journal of Veterinary Researh,44, HORAK, I. G., Parasites of domesti and wild animals in South Afria. V. Helminths in sheep on dryland pasture on the Transvaal Highveld. Onderstepoort Journal of Veterinary Researh, 45, 1-6. HORAK, I. G., 198. The inidene of helminths in pigs, sheep, attle, impala and blesbok in the Transvaal. Ph.D. Thesis, University of Natal, Pietermaritzburg. REINECKE, R. K., The larval anthelminti test in ruminants. Tehnial Communiation, Department of Agriultural Tehnial Servies, Republi of Sq)Jth Afria, 16. REINECKE, R. K., BRUCKNER, CHRISTEL & DE VILLIERS, I. L., A residual anthelminti 2, 6-diiodo-4 nitrophenol (disophenol). Methods of testing its anthelminti effiay. Journal of the South Afrian Veterinary Medial Assoiation, 52, 195-2QO. REINECKE, R. K., DE VILLIERS, I. L & BRUCKNER, CHRISTEL, Studies on Haemonhus ontortus. IX. The effet of Trihostrongylus a.xei in Merinos on natural pastures. Onderstepoort Journal of Veterinary Researh, 51, SIEGEL, S., Non-parametri stlltistis for the behavioral sienes. New York: MGraw-Hill. 39