Onderstepoort J. vet. Res., 48, 229-234 (1981) STUDES ON HAEMONCHUS CONTORTUS. V. THE EFFECT OF TRCHOSTRON GYLUS AXEl AND OSTERTAGA CRCUMCNCTA ON CHALLENGE WTH H. CONTORTUS R. K. RENECKE, CHRSTEL BRUCKNER(!) and. L. DE VLLERS, Faculty of Veterinary Science, P.O. Box 1258, Onderstepoort 11 ABSTRACT RENECKE, R. K., BRUCKNER, CHRSTEL & DE VLLERS,. L., 1981. Studies on Haemonchus contortus. V. The effect of Trichostrongylus axei and Ostertagia circumcincta on challenge with. Onderstepoort Journal of Veterinary Research, 48, 229-234 (1981). Worm-free Merino yearlings were dosed with either a mixture of infective larvae of Trichostrongylus axei and Ostertagia circumcincta or with. circumcincta only, and challenged 9-93 days later with infective larvae of Haemonchus contortus. Neither of these methods protected sheep against challenge and slight protection was afforded sheep predosed with T. axei and. circumcincta and challenged with a trickle dose of. Resume ETUDES SUR HAEMONCHUS CONTORTUS. V. L 'EFFET DE TRCHOSTRONGYLUS AXEl ET DE OSTERTAGA CRCUMCNCTA S UR DES MOUTONS SOUMS A UNE EPREUVE Des Merino d'un an indemnes d'infestation aux helminthes ant lite traites avec so it, un melange de larves infectieuses de Trichostrongylus axei et d 'Ostertagia circumcincta ou seulement avec. circumcincta; its ant be eprouves 9-93 jours plus tard avec des larves infectieuses d'haemonchus contortus. Aucune de ces deux methode ne procura une protection aux moutons contre cette epreuve et une egere protection fut acquise avec des moutons infestes auparavant avec T. axei et. circumcincta et soumis a une epreuve avec une foible dose de H. contortus. NTRODUCTON t has been shown that, if dosed with infective larvae of Trichostrongylus axei. weaned Merinos were protected against subsequent ( h tllenge with Haemonchus contortus (Reinecke, Bruckner & De Villiers, 198). nfective larvae of Ostertagia circumcincta, dosed to yearling Dorper ewes, however, failed to protect them against challenge with (Reinecke, Snyman & Seaman, 1979). This paper describes 2 experiments in which Merinos were dosed with either infective larvae of. circumcincta or with a combination of. circumcincta and T. axei and subsequently or simultaneously with infective larvae of. The object of the experiments was, firstly, to test the protective effect of. circumcincta alone or in combination with T. ax ei against challenge with H. contortus ; secondly, to ascertain the effect of challenge with administered as a trickle dose over a period of 5 months. Experiment 1.- Trichostrongylus axei and Ostertagia circumcincta as a possible vaccine against Materials and Methods The experimental design is summarized in Table 1. Thirty-six 1-month-old Merinos were treated with anthelmintics, housed in worm-free pens, each labelled with an ear tag and divided into 3 equal groups of 2 sheep each. They were dosed and challenged with infective larvae and slaughtered, as summarized in Table 1. At necropsy the ingesta of the abomasum and duodenum were washed on a sieve (38 Jim apertures) and the residue on the surface of the sieve placed in a wide-mouthed 1 t jar. Formalin was added as a preservative. The muscularis and mucosal - layers of the abomasum and duodenum were digested in pepsin/hcl, sieved and preserved, as described by Reinecke (1973). TABLE 1 Experiment.- Experimental design showing the days on which infective larvae were dosed to each sheep and the day of slaughter Group A No. of infective larvae dosed to each sheep Group B Group C..... -...... -..... o_... -... --... -.-.. -- + 14.................... o_........... -. -.-........ _............................. + 9........ - -..... -.. H. contort us........ + 91................ -... H. contortus....... _.... + 92.... --.... -...... -.......... _............ _...... 5....... -....... + 119.................... Slaughter..... _...... _. T. axei+ O. circumcincta................. T. axei+ O. circumcincta............... 2 + 2.... -.-....... -... -.--................... _.... _...... _....... H. contort us... _........................ 5... -...................... Slaughter..... _... _.. _. _... _... circwncincta. circumcincta 4 H. contortus 5 Slaughter ( 1 ) Department of Health, Private Bag X63, Pretoria 1 Received 29 September 1981- Editor 229
STUDES ON HAEMONCHUS CONTORTUS. V. TABLE 2 Experiment.-Worms recovered at necropsy from Group A (controls) Stage of development L, 5 Adult Group A.-Controls: 313.................... 2 4 2 1 2 421 317................. 1 371 76 1 965 3 41 2 329....................... 92 48 4 971 6 371 33.............. 3 7 349 7 433 11 482 332....................... 2 431 2 31 2 752 337............... 1 531 242 1 572 4 345 356................. 1 33 2 1 323 378.................. 2 118 63 4 524 7272 48..................... 1 866 65 2 361 4292 423.......................... 1 274 4 967 6 241 426..................... 539 322 4 317 6 178 46.................... 1 958 72 1 35 4 28 TABLE 2 (Continued). Worms recovered at necropsy from Group B (T. axei+ O. circumcincta) T. axei. circumcincta Stage of Stage of Stage of development development development L, 5 Adult L,l 5 Adult L, 5 Adult Group B.-Day : 1 T. axei+ 1. circumcincta; Day+ 14: 1 T. axei+oooo. circumcincta: 3.................... 76 63 1 39 13 71 13 71 4 39 24 4 63 311....................... 62 24 6 63 7 49 17 156 17 156 6 23 2 897 7 147 349................... 1 84 8 8 55 1 47 16 47 16 47 7 42 82 8 24 355............. 8 4 39 5 19 12 51 12 51 4 55 1 94 6 49 359................ 82 4 86 14 63 14 63 2 64 2 2 66 363.................... 44 5 5 61 7 1 12 81 12 81 3 36 56 4 92 365............. 17 12 29 15 9 15 9 6 95 22 7 17 398................. 4 1 2 5 12 57 12 57 4 26 14 4 4 46.................... 93 4 3 35 4 32 14 21 14 21 6 78 56 8 34 418...................... 14 12 2 26 13 56 13 56 4 77 58 5 35 434......... 44 75 3 19 14 78 14 78 5 44 32 5 76 472........................ 8 2 1 14 7 14 7 97 26 23 TABLE 2 (Continued). Worms recovered from Group C (. circumcincta). circumcincta Stage of development Stage of development L, 5 Adult L, 5 Adult Group C.-Day : 2 ; Day+ 14: 2. circumcincta 36............................. 731 468 572 3 771 1 714 12 1 446 12 28 35................ 4 88 4 3 731 8 615 11 54 16 186 12 886 352.................. 526 181 1 77 12 31 1 12 32 36................ 1 24 22 86 2 32 7 919 8-2 97 1 969 362................. 34 3 343 2 971 25 2 997 368..................... 72 39 6 34 8 45 1 9 2 95 13 85 371................ 2 1 42 2 142 14 765 322 15 87 4.................. 2 21 3 463 2 3 465 412.............. 24 2 3 34 3 294 4 931 2 4 117 9 68 431..................... 54 12 2 36 3 2 7 64 4 34 944 444......................... 5 12 18 5 3 14 785 342 15 127 471........................... 3 32 4 281 3 641 11 673 181 11 854 23
R. K. RENECKE, CHRSTEL BRUCKNER &. L. DE VLLERS TABLE 3 Experiment.-Ranked worm burdens of. Only fourth stage larvae (L,) of in Group B (T. axei+. circumcincta) were significantly less (P <,1) than the controls by the Mann-Whitney U test Group A Group B Group C L. (') 5+ A (') L. 92 2 1 323 8 274 2 2 421 62 33 321 2 752 76 1 371 2 41 3 412 8 1 531 2 7 4 28 82 1 539 2 426 4 292 93 1 866 2 814 4 325 1 4 1 958 4 639 6 178 1 14 2 118 4 967 6 241 1 17 2 4 5 154 6 371 44 2 431 5 451 7272 44 3 7 7 782 11 482 1 84 P < O,OO 5+A 2 4 12 63 1 12 75 3 39 4 39 5 66 6 87 8 63 L. 5+A 1 2 21 86 24 3 343 29 34 42 1 77 39 54 18 2 142 2 5 24 181 2 32 2 26 526 321 3 2 3 19 1 72 1 8 3 294 4 32 731 2 4 3 641 5 19 2 1 2 48 3 771 7 1 3 32 3 54 5 3 7 49 4 88 3 735 8 45 1 47 5 2 6 73 8 615 (1) L 4 = 4th stage larvae (2) 5+ A = 5th stage and adult worms Results Worms recovered are set down in Table 2 and ranked and analysed by the Mann-Whitney U test in Table 3. With the exception of 4th stage larvae (L 4 ) of in Group B, which were significantly fewer than those in Group A (P<,1), the other results showed no significant difference.. circumcincta alone (Group C) was completely unsuccessful as a possible vaccine. Experiment 2.- T. axei and. ostertagia as a possible vaccine challenged with trickle doses of infective larvae of This trial differed from previous experiments in that challenge with infective larvae of to both groups of sheep was administered at irregular intervals from Day for a period of 5 months. Materials and Methods The experimental design is summarized in Table 4. This trial ran parallel with Experiment 1 and a further 24 Merinos were divided into 2 groups (D & E) of 12 sheep each. Group D served as controls and each sheep in Group E was dosed on Day with 1 infective larvae oft. axei plus 1 infective larvae of. circumcincta. This was repeated on Day + 14. All the sheep in both groups were challenged with infective larvae of from Day to Day+ 154. Larvae were dosed on different days of the week varying from 1-3 times a week. From Day to Day+ 91 the total number of larvae that were dosed in any week did not exceed 4, until each sheep had received 5 larvae. Thereafter the total number dosed per week rose to 6 per week until a further 5 larvae were dosed, i.e. from Day+ 95 - Day+ 154. Faecal samples were collected every 7 days from Day+ 21 and differential egg counts based on the identification of 1st stage larvae (L 1 ) were carried out (Whitlock, 1959). Blood samples for haematocrit (Ht) were collected from Day+ 28 onwards. All sheep were killed on Day+ l 75. Results Fluctuations in worm egg coujj.ts and Ht are presented graphically in Fig. 1 and 2. TABLE 4 Experiment 2.- Experimental design showing the days on which infective larvae were dosed to each sheep and the day of slaughter Group D No. of infective larvae dosed to each sheep G roupe......................... + 14.............................................. to+ 91............................................... + 95 to + 54............................................... + 75............................. 5 H. contortus 5 Slaughter T. axei+ O. circumcincta T. axei+ O. circumcincta 2 + 2 5 H. contortus 5 Slaughter 231
STUDES ON HAEMONCHUS CONTORTUS. V. E ~ > 4 ~ 2 V) > > w r----~------~----~-----r----~----~ 28 56 84 112 14 168 FG. 1 Variation in faecal worm egg counts of in Groups D and E. The first 3 egg counts in Group E were undifferentiated and therefore are not joined together with a line. Arrows indicate periods when infective larvae of were dosed to both groups (see Materials and Methods). Group D (Controls). Worm egg counts rose steadily from the 3rd week to reach a peak at 8 weeks, fluctuated to another peak at 12 weeks and then fell steadily to the end of the experiment at 24 weeks. The Ht, however, fell from the 4th week and rose after the 12th week. Group E (T. axei+o. circumcincta). Worm egg counts reached a peak at 1 weeks and minor peaks at 12 and 14 weeks respectively. Thereafter, as in Group D, they fell to low levels (Fig. 1). From the 4th to the 13th week Ht fell and thereafter rose to normal levels (Fig. 2). Again as worm egg counts rose Ht fell. Worm recoveries and analysis by the Mann Whitney U test are summarized in Tables 5 and 6 res pecti vel y. Group D (Controls). Worm burdens of ranged more widely than those in the controls of the previous trial (Experiment 1 Group A). Moreover there was no significant difference between Group D in the present trial and Group A (Experiment 1), although each sheep had received 1 infective larvae in the present experiment as compared with i half the number (5 larvae) dosed to sheep in Grotp A. Group E (T. axei and. circumcincta). The total worm burdens of H. contortus by the Mann-Whitney U test showed a result of 44, only 2 more than 42 for this sized group at the confidence level P <,5 (Table 6). H. T. Groeneveld (1 976, personal communication) stated this was probably significant at P < O, 1 which is not included in the tables in the reference of Siegel (1956)..4,3,2,1,4,3,2,1 28 56 84 112 Group D GroupE 14 168 FG. 2 Fluctuations in haematocrit in Groups D and E. Arrows indicate periods when infective larvae of were dosed to both groups (see Materials and Methods) DSCUSSON n a previous trial we showed that 2 doses of 1 infective larvae of T. axei dosed at an interval of 14 days was > 6/o effective in reducing the challenge by in > 6/o of the sheep (Reinecke eta/., 198). TABLE 5 Experiment 2.- Worms recovered at necropsy from the controls (Group D) Stage of development 5 Adult Group D: Controls 31................. 379.................. 381.......................... 382...................... 383........................ 385......................... 392.................... 45.......................... 427....................... 44....................... 454...................... 456..................... 432 1 478 75 1 68 3 643 2 91 359 2 11 218 283 259 85 1 14 14 1 4 14 4 964 61 7 4 3 51 2 73 89 5 58 349 447 1 51 16 1 396 1 54 1 59 1 72 7 284 5 74 1 249 7 73 567 87 1 769 285 232
TABLE 5 (Continued). Worms recovered from GroupE (T. axei+o. circumcincta) R. K. RENECKE, CHRSTEL BRUCKNER &. L. DE VLLERS L, T. axei. circumcincta Stage of Stage of Stage of development development development 5 Adult L,l 5 Adult L, 5 Adult Group E.-Day : 1 T. axei+ 1. circumcincta; Day+ 14: 1 T. axei + 1. circumcincta: 37............ 615 28 895 15 85 15 85 9 995 25 1 245 319................. 2 794 9 22 12 14 14 98 14 98 8 726 92 9 646 328................... 12 26 38 5 21 5 21 2 6 26 347............... 258 16 418 76 76 3 862 34 4 22 353....................... 478 28 758 16 69 16 69 2 742 1 14 3 882 377.......................... 4 4 13 4 13 4 6 16 19 6 35 44..................... 2 765 2 37 5 135 14 18 14 18 6 35 32 6 625 41.................... 52 4 92 1 63 1 63 2 218 12 2 338 417....................... 682 2 66 4 342 15 39 15 39 8 28 37 8 398 419................... 4 8 12 1 1 8 8 421................ 1 36 89 2 196 8 4 8 4 14 6 164 435................. 23 2 43 12 44 12 44 427 2 447 TABLE 6 Experiment 2.-The Mann-Whitney U test applied to H. contortus recovered from controls compared with the vaccinated group (Group E) Group D GroupE Group D GroupE Group D GroupE L, L, 5+A 5 + A 5 2,5 7 2 4 9 3 7 1 4 11 11 6 13 12 8 14 15 13 15,5 17 14 17 19 16 18 22 18 21 23 2 22 24 21 23 174-78 126-78 168-78 = 96 = 48 = 9 4 1 2,5 8 2 5 1 3 6 12 5 8 13 6 9 14 7 1 15 9 12 16 11 15,5 2 17 19 21 18 2 22 19 24 23 24 132-78 178-78 122-78 = 54 = 1 = 44 This analysis by the modified NPM (Reinecke, 973) is more sensitive than the Mann-Whitney test, but in Experiment, if the sheep are predosed twice with 1 T. axei plus 1. circumcincta at 4 day intervals, there is a reduction in L 4 (P< O,OO) only, and none in total worm burdens of. Moreover, 2 doses of 2. circumcincta alone had no effect on subsequent challenge with H. contortus. Thus in Group B (T. axei+ O. circumcincta) we were unable to confirm the results of Turner, Kates & Wilson (962) that these 2 species had a deleterious effect on the establishment of. n addition, Turner et a!. (1962) and Reinecke (1966) stated that simultaneous infestation with. circumcincta and H. contortus blocked particularly the establishment of and, to a lesser extent, that of. circumcincta. We were unable to confirm the deleterious effect of. circumcincta on H. contortus in Experiment if sheep were predosed with. circumcincta before challenge with. n Experiment 2 the mixture of T. axei and. circumcincta was possibly able to reduce the challenge of (P<, 5) (Groeneveld, 976, personal communication) with a trickle challenge, but tended to give the same result that a challe~ge with _ did after a period of9-93 days m Expenment. t is reasonable to assume that a mixture of T. axei and. circumcincta has less protective effect against challenge with th~n T. axei alo~e, as was shown in previous expenments by Reme~ke et al. (198). M ixing the species is not cumulative as the experiments of Turner et al. (1962) show. n these experiments lowest worm burdens of resulted if T. axei,. circumcincta and were dosed simultaneously; a better result than when either T. axei and or. circumcincta and were dosed simultaneously. The reasons for these conflicting results in the present trials are not known. ACKNOWLEDGEMENTS We wish to thank the Department of Agriculture and Fisheries for the financial assistance to enable us to carry out these experiments and the ~-i~ector, Veterinary Research nstitute, for the facihties to do the work. 233
STUDES ON HAEMONCHUS CONTORTUS. V. REFERENCES RENECKE, R. K., 1966. The value of uniform worm burdens in the larval anthelmintic test. Journal of the South African Veterinary Medical Association, 37, 133-142. RENECKE, R. K., 1973. The larval anthelmintic test in ruminants. Department of Agricultural Technical Services, Republic of South Africa Technical Communication No. 16. RENECKE, R. K., BRUCKNER, CHRSTEL & DE VL ERS,. L., 198 Studies on Haemonchus contortus.. TJtration of Trichostrongylus axei and expulsion of. Onderstepoort Journal of Veterinary Research, 47, 35-44. RENECKE, R. K., SNYMAN, MARE H. & SEAMAN, HELGA, 1979. Studies on Haemonchus contortus.. The effect of abomasal nematodes on subsequent challenge with. Onderstepoort Journal of Veterinary Research, 46, 199-25. SEGEL, S., 1956. Non-parametric statistics for the behavioural sciences. New York: McGraw Hill Book Co. nc. TURNER, J. H., KATES, K. C. & WLSON, G. 1., 1962. The interaction of concurrent infections of the abomasal nematodes Haemonchus contortus, Ostertagia circumcinta and Trichostrongylus axei (Trichostrongylidae) in lambs. Proceedings of the Helminthological Society of Washington, 29, 21-216. WHTLOCK, H. V., 1959. The recovery and identification of the first stage larvae of sheep nematodes. Australian Veterinary Journal, 35, 31-316. Printed by and obtainable from the Government Printer, Private Bag X85, Pretoria, 1 234