The biology of Hyostrongylus rubidus

J.

COMP.

PATH.

1973.

THE VI.

THE

\.oL.

71

83.

BIOLOGY

OF

H2’-OSTRONGYLUS

PERI-PARTURIENT

FAECAL

D. J. BURDEN Central

Veterinary

Laboratop,

EGG

and S. B. Ministry

RUBIDUS OUTPUT

IN

PIGS

KENDALL

of Agriculture,

W’tvbridge,

Surr~v

INTRODUCTION

A post-parturient rise of faecal nematode egg production has been described in sows by Barnett (1966), Connan (196710) and Thomas and Smith (1968). The rise was associated with Oesophagostomum spp, but it was agreed by all these workers that Hyostrongylus rubidus probably played a part. The form of the rise was similar to that which had been reported under similar conditions in the ewe. In pigs the rise began some 2 weeks before farrowing and egg output reached a maximum about 6 weeks after the litter had been born. There was a close association with lactation, since once the litter had been weaned egg production fell to its pre-parturient level, and this occurred whether weaning was early or late. These observations were all carried out in the field and no evidence was available to suggest whether the rise was caused by increased fecundity of the female worms present or by an increase in adult worm numbers caused either by a greater susceptibility to new infection or the resumption of development of inhibited larvae. The experiment reported here was an attempt to demonstrate whether a peri-parturient rise of faecal egg production occurs in pigs infected with Hyostrongylus under controlled laboratory conditions and to elucidate the mechanism behind it.

MATERIALS

AND

METHODS

Thirty, 9 months old female pigs were bought from a commercial producer. They were housed in pairs in open stieswithin a large building and on faecal examination 2 pairs were found to have patent Ascarissuuminfections, 3 pairs had Trichuris spp. whilst 7 pairs had patent strongyle infections. After faecal culture and examination of the infective larvae the strongyle infections were all shown to be Oesophagostomum spp., and no H. rubiduslarvae were seen. The animals infected with Ascariswere dosed at the approved rate with piperazine and thereafter no eggsof this parasite were seen. All the pigs were dosed with Thiabendazole” at the recommended rate of 100 mg./kg. and subsequent strongyle egg production was completely suppressed. As a precaution the pigs were dosed again with Thiabendazole 2 weeks later. Egg production from all pigs then remained completely suppressed until the experiment began 9 weeks later. At weekly intervals throughout the experiment faeces from each animal were cultured with peat by the method previously described (Kendall, Thurlry and Peirce, 1969) and the infective larvae were examined. Only 5 showed * ‘Thibenzole-hfercke,

Sharpe

& Dohme

Ltd.

72

D.

J.

BURDEN

AND

S.

B.

KENDALL

Oesophagostomum spp. larvae and the egg count data for these animals were not used in the final results. The experiment began 9 weeks after the last anthelmintic dose. Each pig was infected every day with 100 infective H. rubidus larvae. This was done using proprietary brand sow nuts (B.O.C.M. Hialac Jumbo Sow Nuts). Each nut had a hole drilled in it large enough to accommodate 0.5 ml. of larval suspension containing the required number of infective larvae. A small amount of the powder obtained from the drillings was poured on top to soak up the fluid and form a plug. The nut was fed to the pig by hand. The larvae were added to the nuts immediately before administration, which was carried out at about 2 p.m. (midway between morning and evening feeds). The pigs quickly learnt to take the nuts in this way and, as they generally swallowed them whole, very few larvae could have been lost before they reached the stomach. Faeces were collected from the rectum of all pigs 3 times a week and the worm egg production recorded. The basic plan was that once a stable worm population had been achieved, half the pigs would be made pregnant. Any subsequent changes in the worm populations would be investigated by killing 2 pregnant and 2 empty control pigs at monthly intervals through pregnancy, then at farrowing time and finally at weaning time. Both early (5 weeks) and late (8 weeks) weaning systems were employed. Some of the breeding and empty control animals were killed as soon as the litters had been weaned, and other a few weeks later. There were a few minor departures from this basic plan due to technical difficulties and these will be explained later. Eighteen weeks after the infections began, 1 pig was killed and examined for worms by methods already described (Kendall et al., 1969) to check the level of infestation and this procedure was repeated at 23 weeks. The worm recoveries from the 2 pigs, together with the level of worm egg production at this time, considered in relation to previous experimental work, suggested that the worm populations had reached a steady state. The oestrus cycles of the pigs were synchronized by the oral administration of Aimax* at the approved rate for 20 consecutive days, followed by a subon the 21st day, and intramuscular cutaneous injection of 1000 I.U. of Foligont injection of 500 I.U. of ChorulonS on the 25th day. This treatment was designed to ensure that all the pigs ovulated on the 26th day, when 20 of them were artificially inseminated in the hope that 15 would become pregnant. The insemination was repeated on the following day. Eleven became pregnant and these formed experimental group 1: 13 empty pigs formed group 2. As only 11 pigs became pregnant at least 2 more pregnant animals were required to satisfy the experimental plan outlined above. The plan to kill the pigs at 36 weeks when the pigs would have been 3 months pregnant was therefore abandoned in this part of the experiment. Two pregnant pigs from group 1 were killed 1 month after insemination and examined for worms by the methods described. Two empty pigs from group 2 were also killed and examined at this time to act as controls. This was repeated with 2 pigs from each group when the pregnancy had run for 2 months. One week before farrowing was due the daily infection was stopped, i.e. at the 4th week of the experiment. This was designed to match the conditions in the field where the pregnant SOWS would be brought into clean quarters shortly before farrowing. It was thought that under normal conditions in a regularly cleaned farrowing house little or no infection would be picked up. The first 2 pigs of group 1 that farrowed were immediately killed and examined for worms together with 2 control pigs from group 2. The 5 remaining inpig animals were allowed to farrow and suckle their litters, and were divided into 2 sub-groups. The first (la) consisted of 3 pigs that had their litters removed and weaned after 5 weeks, when 1 pig was killed and examined along with a group 2 control. The remaining 2 were kept alone for a further 2 weeks before they were killed along with 2 group 2 controls. The second sub-group (lb) consisted of 2 pigs that had their * Methalibure-I.C.I. t Pregnant mares serum-Intervet. : Folicular stimulating hormone-Intervet.

Hyostrongylus

rubidus:

VI.

PERI-PARTURIENT

FAECAL

EGG

73

OUTPUT

litters weaned at 8 weeks, when one of them was killed and examined with a control. The last pig and its control were examined after a further week. Two group 2 pigs were killed after a further 3 weeks, i.e. 53 weeks after the start of the experiment. The omission from group 1 of pigs killed when 3 months pregnant was compensated by re-inseminating 4 pigs, 2 of which became pregnant and formed group 3. The other 2 remained empty and formed group 4 which acted as a control group. The inseminations were carried out during the 34th week of the main experiment and all 4 pigs were killed and examined in the 46th week when the in-pig animals were 3 months pregnant. These pigs were infected continuously until they were killed and thus had 46 weeks of infection compared with the 40 weeks of groups 1 and 2. Thr rrsults are considered separately. Table 1 shows the experimental procedure employcad. TABLE

1

EXPERIMENTAL

PROCEDURE

Killed 2 i Killed 2 1

Pregnant PigsGroup

1 0

I

I

I

1

2345678-F

I

1

I

I

Killed 1 2

Killed 2 4

I

11

__--_ -__ I

t 11 made pregnant

13

months

liilled 11 1 c I 12

/ 13

months

Eariy Weaning

I

I

t InfeAtions began

I

12

I 11

f 10 Infections stopped I Farrowed

Late weanin,q

Pregnant

PigsGroup

I 3

0 ,+ Jnfeitions began

I

I

I

f

I

I

1

2

3

4

5

I

I

I

I

I

6

7

8

9

10

11

empty

control

Each pregnant

animal

was killed

with

a corresponding

I

I

12 1 Killed 2

13

-

mortths

pig.

RESULTS

Worm Populations Tables 2, 3 and 4 show the detailed constitution of the worm populations recovered. In the pregnant pigs of group 1 and the empty control pigs of group 2, third stage and early fourth stage larvae were recovered in comparable numbers until administration of larvae was stopped in the 40th week. Thus there was a continual initial establishment of new worms, the level of which appeared to be the same in both groups. When administration had stopped after the 40th week, the numbers of early larval stages that were recovered declined to zero, demonstrating that the husbandry methods employed during the experiment did not allow significant auto-infection. Fourth stage larvae

74

D.

J.

BURDEN

AND TABLE

WORM

RECOVERIES

Weeks after initial infection when killed

Stage of breeding cycle when killed

558 559 540 541

28

548 555 543

40 40.5 46

553

48

preg. preg. preg. 22 months preg. Infections Just farrowed Just farrowed Litter just weaned at 5 weeks 2 weeks after weaning at

556

48

547

49

554

50

Pig No.

FROM

KENDALL

2 GROUP

1 PREGNANT

Worm LS

1 month

‘3: 32

S. B.

EL,

10 10 113 stopped

:: i2” after 40 weeks

5 weeks

Adult 3’s

LA 750 2060 632 478

Adult 9’s

975 1,430 1,263 1,405

515

1,550 1,050 1,024

30

830 530 880

240

865 605 300

-

110

120

150

_-

-

490

340

540

-

-

230

:; 10

2 weeks after weaning at 5 weeks Litter just weaned

Recoveries

7

545

575

1,490

1,070

at8weeks

1 week after weaning at 8 weeks

210

360

680

L, = third stage larvae EL, = early fourth stage larvae L, = late fourth stage larvae

WORM

Pig No.

RECOVERIES

Weeks after initial infection when killed

FROM

-4

542 560 568 551 550 545 549 562 564

L,, EL,

2 NONPREGNANT

-

and L, see table z

CONTROLS

Recoveries

EL,

L4

Adult 3’s

8

789 793 599

2,470

2,583

1,094 1,306

1,213 1,491

a21 540 412

1,362 1,315 962

1,734 1,375 1,214

1; 24

-

Infections

For

3

Worm

12 9 13 12

546 567 565 545 561 563

TABLE GROUP

stopped 70 40

after

40 weeks 650 630 390 240 890 290 260 410 580

680 690 430

100 560 320 760

Adult

0’s

720 880 710 200 920 390

690

1,020 980

120

240

H~ostrongylus

rubidus:

VI.

PERI-PARTURIENT

FAECAL

EGG

75

OUTPUT

were recovered from all pigs. Before administration of larvae was stopped the numbers of these larvae were fairly constant in both groups 1 and 2, i.e. no large inhibition of larval development was seen. Once the dosing was stopped, fourth stage larvae continued to be recovered, even after 13 weeks, though their numbers tended to be a little depleted. Thus a majority of the fourth stage larvae present were, or subsequently became, inhibited when the administration stopped. There were no significant differences between the larval recoveries from pigs in groups la, lb and control group 2. TABLE !VORM

Pig No.

TVeeks after initial infection when killed

Group 3 (pregnant 1 539 46 552 46 G’t-oup 4 (non 557 544

For

I,,, EL,

pregnant 46 46

RECOVERIES

Stfge of breedzng cvcle when killed

3 months 3 months

preg. preg.

controls) Control Control

and L, see table

Infections

FROM

4 GROUP

3

AND

GROUP

4

worm

Recoveries

.----. .Idult

3

Adult

,

L3

EL,

LA

10 -

130 90

790 480

141 480

151 820

190 240

130 300

1 IO ‘, 10

continued

until

10 death

1

Adult worm recovery indicated a general pattern of decrease in numbers as the experiment progressed. Earlier work showed that in young pigs infected daily with 100 larvae a stable population of worms was reached after about 10 weeks and was maintained for at least a further 7 weeks. The results from the present experiment confirm that the decline in worm numbers begins bctween the 18th and 23rd weeks after infection and is caused by a loss of adult worms. The rate of loss was similar in both groups 1 and 2 until the 40th week when group 1 pigs farrowed. The control group 2 pigs continued this trend for decreasing adult worm numbers until week 46 after which there remained residual populations of about 1000 adult worms per pig. This continued until the end of the experiment at week 53, although there was much variation between individual pigs. Group la pigs that had their litters weaned at 5 weeks after farrowing, showed similar adult worm recoveries to those of the control group. Group 1b pigs that were weaned at 8 weeks were slightly different. The pig killed just after its litter had been removed, showed a high adult worm burden (about 2500 adults) and a low larval population (230) as compared with the pigs of the same group that were killed just after farrowing, though their total worm burdens were comparable. It was thought possible that some of the larvae in this pig had resumed their development to adults towards the end of the extended lactation period. The pig killed a week after weaning showed a lower level of adults, but a normal proportion of larvae, suggesting that this pig had not experienced a maturation of its inhibited larvae.

76

D.

J.

BURDEN

AND

S. B.

KENDALL

Worm recoveries from the pigs of group 3 and 4 are shown in Table 4. The numbers of 3rd and 4th stage larvae recovered from the pigs of group 3 (pregnant) were of the same order as those recovered during the infection period in group 1 and 2, i.e. initial establishment of new worms was taking place. In group 4 (controls) very few early larval stages were recovered, indicating that newly introduced worms were not becoming established. This was also reflected in the number of 4th stage larvae recovered, group 3 pigs yielding more than twice as many as group 4 pigs. In both groups the total worm burden had decreased from the level seen in the group 2 pigs killed during the 40th week of continuous infection. Thus it seemed to make little difference to the final worm burden whether infection was continued or not, and in fact the control pigs of group 4 ended up with smaller worm burdens than control pigs of group 2 killed at the same time and in which daily infection stopped 6 weeks previously. Worm Eggs in the Faeces

Fig. 1 shows the mean weekly worm egg output of the pigs in groups 1 and 2. Egg production began during the 4th week of daily infection and rose, more or less logarithmically, to a peak of some 150 eggs/g. of faeces by the 1 lth week. There then followed a decline to a level of about 60 e.p.g. by week 20 and thereafter a fluctuating, but very gradual, decrease in egg production took place. The administration of Aimax to the pigs during the oestrus synchronization procedure had no marked effect upon egg output and after insemination there was little difference between the output of pregnant or empty pigs, at least until the time the pigs farrowed. The graph shows that 4 to 5 weeks after farrowing the egg output from the suckling sows began to rise and that this rise was reversed when the litters were weaned, whether at 5 or 8 weeks. However, this rise was fairly small and was probably not significant.

InfectIon

stopped

Weanmg

I I I,,,

Time

Fig.

1. Mean

weekly

egg output

after

from

InfectIon

5weeks

I I,lllllllllrlilll

(weeks)

pigs of groups

1 (-----)

and 2 (- - - -).

Week

18

Week

23

-. :

40 30

Week

46

Week

48

Week

49

Week

50

Week

53

568

20 IO 3

0 i 40 30 20 IO 0

0

Gg. 2. Distribution

of in-utero

0

IO 2030405060 No eggs

IO 2030405060 No. eggs

eggs in groups

1 and 2. Dotted

line indicates

arithmetic

mez

78

Hyostrongylus

rubidus:

VI.

PERI-PARTURIENT

FAECAL

Week

40

Week

46

Week

4%

EGG

OUTPUT

542 560

568

Lengih

Fig.

of worm5

3. Distribution

of female

worm

lengths

in groups

1 and

2.

D.

J.

BURDEN

AND

S. B.

KENDALL

79

For the pigs of groups 3 and 4, the pattern of egg production was less distinct due to the smaller number of pigs, and large fluctuations occurred. However, there was no great difference between the groups, production rising to a maximum and then slowly declining. Final egg output was similar to group 1 and 2 pigs at this time despite the extra 6 weeks of daily infection. Xumbers oJ‘ Eggs in Female l~orms Fig. 2 shows the distribution and arithmetic mean of the number of eggs in tllc uteri of female worms taken from each pig in groups 1 and 2. Where 2 pigs were kilied on the same date, their results have been combined, and the earlv and late weaned groups are shown together. The group 2 results show no significant differences in either distribution or arithmetic mean throughout the observation period. This was expected, as the egg production remained in a steady state during this time. The results from the group 1 pregnant pigs were similar. Presumably the decline of in-utero eggs had occurred before the 18th week when the first pig was killed and by this time a basal level of around 2.5 eggs per uterus had been reached. Similarly with groups 3 and 4, no significant differences were seen. i,ength of Female H.lvorms Fig. 3 shows the distribution and arithmetic mean of female worm lengths horn the pigs of groups 1 and 2. In both groups worm length had a wide distribution, in some cases the longest worms being twice the size of the shortest. Roth groups showed a slight tendency for a decrease in worm length throughout the experiment but, as with the in-utero eggs, it was thought that a more or less basal level had been reached by the time observations began in the 18th week of infection. Similar results were obtained with the pigs of groups 3 and 4. DISCUSSION

No significant post-parturient rise of faecal egg production was seen. A small rise did occur towards the end of lactation which may have been the start of a larger rise, had the suckling period been extended. The worm burden from the pig that was the main contributor to this rise suggested that it was initiated by inhibited larvae resuming their development. Apart from this case there was little difference in the size or constitution of the worm burdens in pregnant and non-pregnant pigs. The number of adult worms in each pig declined slowly from a peak some time before the 18th week of infection to a basal Ievel of abou t 1000 worms per pig by the 46th week after infections began. This was seen to be independent of whether infection was continued until death or stopped at the 40th week. There was a little evidence to suggest that in more mature infections the pregnant animals would be more susceptible to new infection than non-pregnant ones. The larval populations of both pregnant and empty pigs remained constant throughout the experiment, i.e. there was no build-up of inhibited 4th stage larvae, and it appeared that these larvae represented normally developing

80

D.

J.

BURDEN

AND

S. B.

KENDALL

forms. However, when the administration of larvae was stopped there was no appreciable decrease in the numbers of these larval stages that were recovered post mortem, i.e. the larvae had either become inhibited after the dosing had stopped or they had been inhibited all the time. As it would seem most unlikely for worms that had been developing at a normal rate to suddenly become inhibited when daily dosing was stopped it must be concluded that these larvae were inhibited in some way all the time. This raises the problem of why larval recoveries should have remained constant throughout the infection period, for clearly, if a percentage of each infection was becoming inhibited, then a larval build up would have occurred. The post mortem worm recoveries from the pigs killed during the infection period yielded 3rd and 4th stage larvae, thus demonstrating that a few worms from each daily infection were becoming established. It is possible that these newly established worms could have been killed off and expelled before they had reached the 4th stage, but there is no evidence to support this. If it is accepted that newly established worms were not being eliminated immediately, it must follow that there was a continual turnover of “inhibited” 4th stage larvae. A system would have to be visualized whereby there is a maximum number of inhibited larvae that can exist in a pig at any one time and if new worms become established they must be replacing older larvae maturing into adults. Since the maturation of inhibited larvae ceased after infection was stopped it could be concluded that the establishment of new worms was the stimulus for the resumption of development of some of the inhibited larvae; however, this is difficult to believe. It would seem more likely that new worms could establish themselves only if there were a “space” left for them by the maturation of older larvae. By the time the first pig of this experiment had been killed and examined it appeared that the rate of acquisition of new worms had been drastically reduced. The number of 3rd stage larvae recovered from pigs killed between the 18th and 28th week of infection averaged 12. As it takes at least 2 days for the 3rd stage larvae to reach the 4th stage, the mean number of worms establishing themselves per day was 6. Pig 558 killed after 18 weeks of infection had an adult worm population of over 5000. To attain such a population an average of at least 45 infective larvae per day must have become established. Even if allowance is made for the difficulties in recovering these early larval stages it is clear that a reduction in establishment rate must have occurred during the infection period. The results from group 4 would indicate that this establishment rate tended towards zero, since very few 3rd stage larvae were recovered from these pigs. It is conceivable, therefore, that by the time the infections in groups 1 and 2 were stopped the maturation rate of 4th stage larvae and consequently the establishment rate of new 3rd stage larvae had almost reached zero. Thus the size of the inhibited larval population remained constant for the next 13 weeks until the end of the experiment. The subsequent maturation of these inhibited stages was not seen and the conditions under which this occurs must be the subject for further work. A peri-parturient rise of nematode egg production has been observed in pigs by Barnett (1966), Connan (1967b) and Thomas and Smith ( 1968). All these

Hyostrongylus

rubidus:

VI.

PERI-PARTURIENT

FAECAL

EGG

OUTPUT

81

authors likened the rise to that seen previously in sheep by such workers as Taylor (1935), Connan (1967a), Arundel and Ford (1967), Gibbs (1967) and Jansen (1968). The rise in pigs, which in all the published caseshas been produced by a mixed infection of H. rubidus and Oesophagostomum spp. began a few weeks before farrowing and increased during the suckling period until the litter had been weaned. Egg production then fell to its peri-parturient level. Connan 1’1967b) demonstrated the dependence of the rise on lactation for if the litter from a pig were weaned off early, then the worm egg production declined early. The work of Connan (1968) has suggested that the acquisition of new infection contributes to the peri-parturient rise in sheep and O’Sullivan and Donald ( 1970) have demonstrated that the rise can occur in the absence of any inhibited larvae. This would tend to suggest an immunological mechanism and one, which as O’Sullivan and Donald (1970) propose, is brought about by endocrine changes associated with lactation. There are, therefore, numerous possible mechanisms by which a peri-parturient rise in nematode egg production may be elicited. SUMMARY

An experiment was designed to investigate a reported peri-parturient rise of nematode egg production in sows. Thirty young femaIe pigs were infected daily over a period of several months with Hyostrongylusrubidus. After synchronization of the oestrus cycles of all the pigs, half were inseminated and the effect of pregnancy and lactation on the worm populations was determined by periodic killing of breeding and non-breeding animals. No peri-parturient rise was seen and there was little difference between the worm burdens of breeding and control pigs. Possible factors controlling the development of populations of H. rubidus in breeding sows are discussed. ACKNOWLEDGMENTS

We would

like to thank MessrsJ. A. Small, G. Snell and P. Phipps for technical

assistance. This paper contains data submitted by D. J. Burden requirements for the Ph.D. degree of London University.

in part

fulfilment

of the

REFERENCES

Arundel, J. H., and Ford, G. E. (1967). Aust. vet. J., 45, 89. Barnett, S. F. (1966). Vet. Rec., 79, 156. Connan, R. M. (1967a). Ibid., 80,401; (I967bj. 1&d., 424; (1968).J. He&r&&., 42, 9. Gibbs, H. C. (1967). Prod. 3rd int. Conf. World Ass. Ad Vet. Parasit. Lyons, in C’et. :\&d. Rev., p. 160. Jansen. ( 1968). Tijdschr. voor Diergeneeskunde, 93, 422. Kendall, S. B., Thurley, D. J., and Peirce, M. A. (1969). 3. camp. Path., 79, 87. O’Sullivan, B. M., and Donald, A. D. (1970). Parasitology, 61, 301. Taylor, E. L. ( 1935). 3. Par&t., 21, 175. Thomas, R. J,, and Smith, W. C. (1968). Vet. Rec., 83, 489. [Received for publication,

February 24th, 19721