The biology of Hyostrongylus rubidus

J.

COMP.

PATH. 1972.Vo~.82.

THE

307

BIOLOGY IV.

OF

RESISTANCE

HYOSTRONGYLUS TO REINFECTION

RUBZDUS IN YOUNG

PIGS

BY

D.J.

BURDEN

and S. B. KENDALL

Central Veterinary Laboratory, Ministty of Agriculture, WTbridgc

INTRODUCTION

In an earlier paper (Kendall and Harding, 1970) the effects of a single and a repeated infection with Hyostrongylus rubidus in young pigs were compared. It was shown that a resistance to reinfection did occur insofar as at the expected time of patency of the second infection the total worm recovery was not greater than in those pigs that had received only the primary infection. The present paper reports the results of two further experiments on this resistance phenomenon designed to show, (a) the fate of the worms of an incoming infection in a resistant host, and (b) what previous experience of the worm is necessary to elicit this resistance mechanism. MATERIALS

AND

METHODS

The materials and methods used were essentially those already described in a previous paper of this series(Kendall, Thurley and Peirce, 1969). All the pigs used in this study were obtained from a minimal diseaseherd, which, as far as is known from regular faecesexamination of all animals, has never had experience of H. rubidus infection. Experiment (a). For this study 14 pigs were taken from 2 litters, all the piglets being either 6+ or 74 weeks old at the start of the experiment. They were infected on the same day with 4,000 H. rubidus larvae administered by stomach tube with the piglets under Trichloroethylene* anaesthesia. The infection was then allowed to run its course for 20 weeks as in the previous reinfection experiment (Kendall and Harding, 1970) and on the 141st day all the animals were reinfected with 4,000 larvae. Two uninfected piglets, 15 weeks old, were given 4,000 infective larvae to act as a control on the viability of the reinfection doses.Pigs taken at random were then killed at the following times after reinfection-& day, 1, 2, 4, 8, 12, 16, 20, 24, and 37 days. Their stomachswere examined for the numbers and state of develop ment of the worms by the methods previously described (Kendall et al., 1969). The two control pigs were killed and examined 37 days after infection. It was hoped to examine reinfected pigs at times greater than 37 days after infection, but 4 of the experimental animals died due to an allergic disorder. Throughout the experiment the egg output from each pig was followed by faecal examinations made three times a week. Experiment (b). Twenty piglets, 13 weeks old, were formed into four groups of 5, and each animal was initially infected with 4,000 H. rubidus larvae. In each group on the day of reinfection 1 pig was killed and its stomach examined for the number and state of development of the worms. The remaining pigs were reinfected with 4,000 larvae each, and were killed and examined 5 or 7, 17, 23 and 29 days after * Trikne, I.C.I.

308

Hyostrongylus

rubidus: RESISTANCE TO REINFECTION

IN PIGS

reinfection. The periods of reinfection in days after the primary infection were group 1, 2; group 2, 7; group 3, 17 and group 4, 23. These reinfection times were chosen to correspond to the various stagesof the life history of the primary infection. At 2 days the worms are still in the 3rd larval stage, at 7 days they are in the 4th stage, by 17 days they are immature adults and by 23 days they are fully mature, egg-laying adults.

RESULTS

Piglets 5 to 7 weeks old (Exp. a) Numbers of worms recovered. Table 1 shows the numbers of larvae and adult Hyostrongylus recovered from the pigs. The numbers of adult worms recovered from the pigs killed in the first 16 days of the reinfection (i.e. those worms that quite clearly must have come from the first infecting dose) were as would have been expected from a single infection of Hyostrongylus about 5 months old. The number of adult worms recovered from pigs killed after this time showed no significant increase. Pigs 199 and 205 unfortunately had most of their primary worm burdens removed by a dose of piperazine given in error. However, as this was done several weeks before the reinfection and a few worms did remain in each pig, we feel that the results from these two animals may be considered, albeit with caution. Full details of larval recoveries are not shown, but after the eighth day of reinfection the majority were in the fourth stage of development. The development to this stage, therefore, took the same time as would be expected in a primary infection (Kendall et al., 1969). The larvae having reached the fourth stage became arrested and thereafter there did not appear to be any significant drop in the number recovered throughout the experiment. No larvae were recovered from pigs 284 and 285 that were infected only on the second occasion and killed 37 days later.

TABLE 1 OF WORMS RBCOVERBD

NUMBERS

Duration of reinfection

Pig No.

197 203

12 hrs 1 day 2 days 4 39

:zi

1;

:?I 211 205

;;

:: ,*

E

z;

::

%

16

* Control

Total lame recovered 223 521 112 z 258 477 952 509 160 -

:: 3,

pigs infected

only

on the second

Total adulti recovered 427 308 574 1008 283 359 13: 195 5:; 1529 occasion.

D. J. BURDEN

AND

309

S. B. KENDALL

Numbers of worm eggs in the faeces. Fig. 1 shows the graph of the egg output of the reinfected and control pigs. As has been observed before, after the first infection this output rose to an early peak and thereafter declined slowly. At the expected time of patency of the second infection no rise in egg output was detected. The control infection in pigs 284 and 285 did become patent (Fig. 1). Pathology. As in previous experiments (Kendall and Harding, 1970) none of the pigs showed signs of clinical disease attributable to Hyostrongylus. At postmortem examination, 3 of the pigs (205, 210, 211) appeared to show some inflammatory changes in the stomach. Accordingly a more detailed examination was made. In each case a small piece of fundic tissue was taken and processed histologically by the methods previously described (Kendall et al., 1969). Pig 210 proved to be histologically normal, 211 had quite a lot of eosinophilic, and to a lesser extent lymphocytic infiltration in the interstitial tissue of the muscular coats, while 205 had moderate eosinophil infiltration of the submucosa and to a lesser extent the deep mucosa, and one patch was seen with infiltration of lymphocytes, histiocytes and eosinophils extending across most of the width of the fundic mucosa. It is considered that these changes were not significant.

-Pigs 210,211,205 --- Contt-ol pigs 284,285

50

Reinfect

0

5

IO

Weeks after Fig.

1. The

mean

weekly

egg output

of pigs in experiment

15

20

25

infection (a).

Piglets 13 Weeks Old (Exp. b) Numbers of worms recovered. Most of the pigs in this experiment had a higher worm burden than has been seen in similarly treated pigs in the past. In the control pigs of groups 3 and 4 which were killed after the primary infection had run 17 and 23 days respectively the worm recovery was well over 50 per cent. of the infecting dose. It can be seen that the numbers of adult worms recovered from pigs of groups 1 and 2 are greater than those from 3 and 4. If only those pigs killed 17, 23 and 29 days after reinfection are considered (i.e. those pigs whose worm populations would be mainly adult if the worms were developing as in a primary infection) then this difference between the groups is even greater. In groups 1 and 2 it appears that the infections are additive with mean adult worm recoveries between 3& and 4 thousand. In groups 3 and 4, however, the mean recovery of adults was of the order of 24 thousand, indicating that by the

310

h!jOStrOngYh

Yf4bidU.f:

RESISTANCE

TO

TABLE PIGS

Groufi

13

.No. &ys between 1st d 2nd infectiorrc

411+

Control

:zt 408

.No. days between reinfhion and PM

; 2 Control

Control

:

1:

:

;;

Control 17

410* 417 424 454 453

Control 23

TOid

TOor01

lanlac

adults

195 17 23 29

428* 416 422 420 406

PIGS

OLD

441:* 2; 418

IN

2

WEEKS

Pig No.

REINFECTION

-

:

4182 3474 4466

822 534 1:: -

Control

2341 43:

1: :: 17

ii 29

Control 5 17

;i 23

2

* Control pigs were killed at the time their group-mates 7 A pig of group 1 died before the experiment began.

2866 1:: 134 257 1301 iz

:%i 2361 1509 1572 3685 2724

were reinfected.

time these groups were reinfected a resistance to reinfection had developed. It seems unlikely that all the worms recovered from these two groups would have come solely from the primary infection, but in view of the large numbers of worms present in the single infection controls for these groups it is reasonable to assume that the final population was derived mainly from the primary dose. In all pigs the reinfection larvae developed to the fourth stage at the normal rate for a primary infection. The numbers of larvae recovered from pigs killed on the 17th, 23rd and 29th days after reinfection, i.e. inhibited larvae, did not show great differences between the groups. There appeared to be a slight tendency for more larvae to be arrested in groups 3 and 4 and it seems significant that of the pigs killed 29 days after reinfection, inhibited larvae were found only in those from groups 3 and 4. Numbers of worm eggs in the faeces. of each of the 4 groups (Fig. 2). Eggs 18th and 20th days after first infection was generally higher than has been seen worm burdens encountered.

There was little difference in the output first appeared in the faeces between the and quickly rose to a peak. The output in the past, probably reflecting the higher

Pathology. None of the pigs infected here showed any signs of clinical disease. The pigs were weighed at weekly intervals and no significant difference was seen in the weight gains of animals from different groups. This is consistent with the work of Castelino, Herbert and Lean (1970) who produced depressed weight gains only in pigs infected with over 200,000 larvae.

D. J. BURDEN Ah’D S. B. KENDALL

311

Weeks after initial infection Fig. 2. The mean daily egg output of pigs ip experiment (b).

Length and fecundity of female worms. Adult female worms (100) were taken at random from each pig. Each worm was then measured using a mm. scale under an illuminated magnifying glass and the number of fully developed eggs present in its uterus counted under a compound microscope. The mean group results are shown in Table 3 for pigs that were killed 23 and 29 days after reinfection. As can be seenthere was little difference between the groups in either length or fecundity. Fig 3 shows the histogram results of in utero egg counts for the pigs of each group. It is here advanced as evidence that a few worms of the second infection were maturing into adults. If group 1 is considered first, a few of the worms from pig 42 1, killed 17 days after reinfection had begun egg-laying, but the majority had not. Pig 431, killed 23 days after infection, had a population of worms showing a near normal distribution of fecundity. The same was true for pig 408 killed 29 days after reinfection. As the two worm populations in this group are but two days apart they cannot be detected by this method. In group 2 however, pig 425, killed 17 days after reinfection, had two distinct groups of females; one with 0 to 5 eggs, derived from the second infection, and one with a normal distribution of eggs (i.e. the first infection). The two groups become indistinct as reinfection worms begin to lay eggs-see pigs 407 killed at 23 days and pig 418 killed at 29 days. Thii same process can be seen in groups 3 and 4 though lessdistinctly as fewer worms of the second infection are maturing. TABLE

Group

Mean length -.

3 Mean .Na. eggs inUl%US

Hyostrongyhs rubiahs:

312

RESISTANCE

TO REINFECTION

IN PIGS

Group2

Group3 422

I

420

406

I

Group4

0Lu

102030405060700

Fig. 3.

The

number

of eggs in female

IO 2030405060700

Number worms

ofeggsin

IO 203040506070

utero

in the pigs of experiment

(b) .

DISCUSSION

These experiments follow those reported in a previous paper of this series (Kendall and Harding, 1970) in which a resistance to reinfection in young pigs sensitised by an infection of 4,000 larvae and challenged 48 months later with a similar dose was demonstrated. On that occasion 31 larvae were found in a pig killed 22 days after reinfection and none in a pig killed 11 days later. It was thought that the second infection was not maturing and that the inhibited larvae were being lost some time around the expected patency of the second infection. It can be seen that the results obtained in the first experiment were not those that were expected. Here the larvae of the reinfection developed normally until the fourth stage and then became arrested. Throughout the experiment there was no significant decline in the numbers of larvae recovered. From examination of the adult worms present, and from the egg output data it seems unlikely that any of the second infection larvae matured into adults. Whether or not they eventually mature or whether they die out without maturing is not yet known. It was apparent from the second experiment that resistance to reinfection only developed after the pig had had a certain degree of experience of the worm. The minimal length of previous stimulation required clearly lies somewhere

D. J.

BURDEN

AND

S. B.

KENDALL

313

between 7 and 17 days. Perhaps the whole phase of histotropic development must be experienced before the resistance mechanism can be elicited. In conclusion it can be said that pigs become refractory to challenge with Hyostrongylus after having experienced a sensitising infection which must last at least 7 days. Resistance seems to extend over at least 4+ months, but may be dependent on the persistence of the primary infection. Most of the larvae of an incoming second infection develop normally until the fourth stage and then become arrested, their subsequent fate not being known at the present time. The evidence from worm eggs in utero suggests that a certain number of worms do mature, although their contribution to egg output is undetectable. Castelino, Herbert and Lean (197 1) found that reinfections did become patent in colostrumdeprived pigs when challenged with large numbers of larvae, but the subsequent egg count rise was only minimal. SUMMARY

Two experiments are described which were designed to determine the fate of Hyostrongylus rubidus worms of an incoming infection in a resistant pig, and the amount of previous experience necessary to elicit this resistance mechanism. It was concluded that pigs become refractory to challenge after exposure to an initial infection of seventeen days duration and that while some worms do mature into adults the majority are inhibited in the fourth stage of development, their subsequent fate not being known. ACKNOWLEDGMENTS

We would like to thank Mr. J. D. J. Harding for carrying out the histological examinations and Messrs. J. Small and G. Snell for technical assistance.

Castelino, Res. Kendall, Kendall,

J. B., Herbert, I. V., and Lean, I. J. (1970). Brit. vet. J., 126, 579; (1971). vet. Sci., 12, 7. S. B., and Harding, J. D. J. (1970). J. camp. Path., So, 145. S. B., Thurley, D. C., and Peirce, M. A. (1969) Ibid., 79, 87. [Received for publication, September 6th, 19711