Increased establishment and longevity of Nippostrongylus brasiliensis in immune rats given repeated small infections

Increased establishment and longevity of Nippostrongylus brasiliensis in immune rats given repeated small infections

International Journal for Parasitology, 1972, Vol. 2, pp. 105-l 11. Pergamon Press, Printed in Great Britain INCREASED ESTABLISHMENT AND LONGEVITY O...

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International Journal for Parasitology,

1972, Vol. 2, pp. 105-l 11. Pergamon Press, Printed in Great Britain

INCREASED ESTABLISHMENT AND LONGEVITY OF NIPPOSTRONG YLUS BRASILIENSIS IN IMMUNE RATS GIVEN REPEATED SMALL INFECTIONS D. CONWIL

JENKINS

The Research Laboratories,

and R. F. PHILLIPSON

May & Baker, Ltd., Dagenham,

(Received 25 May 1971; in revisedform

Essex, England

26 August 1971)

Abstract R. F., 1972. Increased establishment and longevity of Nippostrotzgylus brasiliensis in immune rats given repeated small infections. International Journal for Parasitology, 2: 105-l 11. Rats that were previously immunized with a heavy primary infection of Nippostrongylus brasiliensis acquired a moderately heavy secondary worm burden when they were exposed to a low-level trickle infection over a period of weeks. The secondary worms were morphologically distinct from ‘normal’ primary infection worms and were able to remain within the host for over 3 months. The presence or absence of residual primary infection worms had little effect on the rate of establishment of the secondary worm burden. INDEX KEY WORDS: Nippostrongylus brasiliensis: trickle infections; immunity; increased establishment and longevity. JENKINS D.

C. and

PHILLIPSON

INTRODUCTION

(Sarles & Taliaferro, 1936; Mulligan et al., 1965; Ogilvie, 1965a) showed that when rats were exposed to a heavy challenge infection with the nematode Nippostrongylus brasiliensis, the worms which developed to the adult stage were stunted and were expelled earlier than those which developed from a primary infection. Jenkins & Phillipson (1971) showed that when clean rats were exposed to a series of small daily infections over a period of weeks, the worms that developed after the first 14 days, though stunted, were able to remain within the host and remain patent for over 3 months. The aim of the present work was to establish whether a similar pattern of infection could be produced in rats that had previously been immunized with a heavy primary infection. The experiments were also designed to indicate whether the age of the rats or the size of the primary infection influenced the course of the ensuing ‘trickle’ infection. PREVIOUS workers

MATERIALS

A previous paper parasite. It described parasite.

(Jenkins methods

AND

METHODS

& Phillipson, 1971) gave details of the strain of host and for maintaining the parasite and for infecting rats with the

Recovery and examination of worms The rats were killed with chloroform, their small intestines removed, split along their length and incubated in a Baermann apparatus containing a 0.85 per cent saline solution at 40°C. The worms that accumulated at the base of the apparatus were removed after 2 h incubation into Petri dishes and counted under a low power binocular microscope. The sex and condition of each individual worm was carefully noted. 105

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Egg counts

Egg counts were taken from the pooled faeces of all the rats and expressed as the number of eggs per gramme (e.p.g.) whole faeces. Details of the techniques involved are given in a previous publication (Jenkins & Phillipson, 1971). Anthelmintic

treatment

Some rats were treated with thiabendazole (Merck, Sharp & Dohme Ltd). The drug was administered as an oral suspension at the rate of 1000 mg/kg bodyweight. This treatment was given in order to remove any residual worms remaining from the primary infection. This was done in all the experiments to facilitate the subsequent identification of secondary worms as opposed to primary worms and in experiments 1 and 2 to determine whether the persistence of primary infection worms affected the establishment of the secondary worms. DESIGN

Experiment

OF EXPERIMENTS

AND

RESULTS

I

Sixty 6-week-old male rats were each given a primary infection of 1000 larvae on day 1 of the experiment. The rats were then divided into 6 equal groups and treated as follows: Group f. KilIed and individual worm burdens examined on day 10 to estabhsh the level of the primary infection. Group 2. Killed and worm burdens examined on day 21 to confirm that a host immune response had occurred and most of the primary worm burden had been expelled. Groups 3, 4, 5 and 6. Infected with 20 larvae on each weekday (Monday-Friday) from days 22-50 inclusive. Groups 3 and 4 were killed and their worm burdens were examined on day 51 while groups 5 and 6 were killed and examined on day 78. Groups 4 and 6 were treated with thiabendazole on day 20 in order to remove the residual primary worm burden. Egg counts were taken weekly from the pooled faeces of the rats in groups 5 and 6 from day 21 onwards. The results of the experiment are given in Tables 1 and 2. The mean worm burden of the rats on day 10 was 461, their sex ratio being typical of a normal primary infection. By day 21, the worm burden was down to a mean of 79 worms per rat and the sex ratio was strongly biased in favour of male worms indicating that a host TABLE ~-WORMS RECOVERED FROMRATSIN EXPERIMENT 1. (Each rat received a primary infection of 1000 larvae on day 1 of the experiment.)

Rat group No.

Day when anthelmintic treatment given

1 2 3 4 5 6

20 20

No. of larvae given at rate of 20 per weekday from day 22 onwards 400 400 400 400

Mean No. worms recovered

Day killed 10 21 51 51 78 78

Males 175 61 143 134 97 111

Females 286 18 109 159 110 151

Immatures 0 0 13 12 0 0

Total i standard deviation 461 79 265 305 207 262

Lt 51 i: 32 f 97 & 52 & 127 f 26

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TABLE2--EXPERIMENTS. (Egg output of secondary infection. Twenty, 6-week-old rats each received a primary infection of 1000 infective larvae on day 1, followed by a secondary trickle infection given at the rate of 20 larvae per weekday for 4 weeks from day 22 of the experiment. Rats in Group 6 were treated with thiabendazole on day 20.) Eggs per gramme faeces Day of experiment 23 29 36 43 50 57 64 71 78

Day of secondary infection

Group 5

Group 6 ~_

1 7 14 21 28 35 42 49 56

110 86 850 2500 1830 2950 2460 2080 1830

0 0 1130 2530 8350 1110 1980 2630 2550

immune response had occurred. The administration of 20 larvae per weekday for 4 weeks resulted in the establishment of a secondary infection, the percentage take of which was approximately 61 per cent in the thiabendazole treated rats and somewhat less in the untreated rats. These secondary worms were stunted in size (males 2-5-4-0 mm; females 29-3-5 mm) but produced fertile eggs as shown by the egg counts obtained from the rats in Groups 5 and 6. The sex ratio of the worms in the thiabendazole treated rats was different from that in the untreated rats on day 51 in that there were more females than males in the former and more males than females in the latter. The larger number of males in the untreated rats can be accounted for if it is assumed that a number of residual primary infection males were present in these rats. The egg output of the secondary worms remained fairly consistent for the duration of the experiment and there was no significant decrease in the size of the worm burden. Experiment 2

In this experiment the rats were older and on this occasion a number of acute-challenge control groups were included. Seventy &week-old male rats were each given a primary infection of 1000 larvae on day 1. The rats were then divided into 7 groups and treated as follows: Group 1. Killed and individual worm burdens examined on day 10. Group 2. Killed and worms examined on day 21. Groups 3 and 4. Challenged with 1000 larvae on day 21, killed and worms examined on day 31 to test the host’s response to acut challenge. Group 4 was treated with thiabendazole 2 days before challenge. Group 5. Treated with thiabendazole on day 21. Challenged with 400 larvae on day 24, killed and worms examined on day 34 in order to test the host’s response to an acute challenge which corresponded in size to the sum of the larvae given to groups 6 and 7 as a trickle infection. Groups 6 and 7. Infected with 20 larvae on each weekday (Monday-Friday) from days 23-51 inclusive. Killed and worms examined on day 52. Group 7 was treated with thibendazole on day 20.

108

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Egg counts were taken weekly from the pooled faeces of the rats in Groups 6 and 7 from day 23 onwards. The results are given in Tables 3 and 4. The mean worm burden on day 10 was 452, but by day 21 it had fallen to 36 indicating that an acute host immune response had occurred. The administration of 20 larvae per weekday for 4 weeks resulted in the establishment of a secondary worm burden of 131 worms in the thiabendazole treated rats and 159 worms in the untreated rats. The percentage take of the secondary infection was approximately 32 per cent and the sex ratio (male TABLE ~---WORMS RECOVEREDFROM RATS IN EXPERIMENT2. (Each rat received a primary infection of 1000 larvae on day 1 of the experiment.)

Rat group No. 1

Day when anthelmintic treatment given

2 3

-

4

21

-

21

20

No. of larvae given at rate Day and no. of 20 per of larvae weekday from given as day 23 challenge onwards

21 1OOOL 24 1000 L 24 400L -

Mean No. worms recovered Total f standard deviation

Day killed

Males

Females

-

10 21 31

208 35 19

244 1 12

0 0 4

452 z!=41 36 f 32 35 f 31

-

34

4

12

1

17 rt 1.5

-

34

1

2

5

400

52 52

55 36

68 58

36 37

-

Immatures

8f9 159 f 102 131 & 86

TABLE &-EXPERIMENT 2. (Egg output of secondary infection. Twenty S-week-old rats each received a primary infection of 1000 infective larvae on day 1, followed by a secondary trickle infection given at the rate of 20 larvae per weekday for 4 weeks from day 23 of the experiment. Rats in Group 7 were treated with thiabendazole on day 20.)

Eggs per gramme faeces Day of experiment 23 30 37 44 51

Day of secondary infection

I 8 15 22 29

Group 6 27 17 483 533 1130

Group 7 0 0 250 263 800

to female) of the worms was close to 3 : 4. Approximately 27 per cent of the worms were immature. The egg counts on day 51 were around 1000 e.p.g. in both groups. Both the anthelmintic treated and untreated rats in Groups 3 and 4 resisted a challenge infection of 1000 larvae, the percentage take of the challenge being less than 2 per cent. Challenge with 400 larvae (Group 5) was also strongly resisted the percentage take being less than 3 per cent.

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Experiment

109

INFECTIONS

3

This experiment was carried out in order to confirm the observations made in the previous experiments and in addition to determine whether the size of the primary infection influenced the course of the subsequent trickle infection. Two groups A and B each consisting of fifty 5-week-old male rats were given a primary infection of 2500 and 500 larvae respectively on day 1. Both groups were then divided into 5 sub-groups each of 10 rats and treated as follows: Sub-groups Al and Bl. Killed and worms examined on day 10. Sub-groups A2 and B2. Killed and worms examined on day 21. Sub-groups (A3 and B3) (A4 and B4) and (A5 and B5). Treated with thiabendazole on day 20. Infected with 20 larvae on each weekday (Monday-Friday) from day 23-51 inclusive. Killed and worms examined on days 52, 108 and 135 respectively. Weekly egg counts were taken from the rats in Groups A7 and B7 from day 23 onwards. Similar groups not treated with thiabendazole were not included in this experiment since the results obtained in experiments 1 and 2 indicated that the presence or absence of residual primary infection worms had little effect on the establishment of the secondary worm population. The results obtained are shown in Tables 5 and 6. TABLE ~-WORMS

3.

RECOVEREDFROMRATSINEXPERIMENT

No. of larvae

No. of larvae given as Day when Rat primary anthelmintic infection treatment subon day 1 given group Al A2 A3 A4 A5 Bl B2 B3 B4 B5

2500 2500 2500 2500 2500 500 500 500 500 500

20 20 20 20 20 20

given at rate of 20 per weekday from day 23 onwards 400 400 400 400 400 400

Mean No. worms recovered

Day killed 10

21 52 108 135 10 21 52 108 135

Males 405 123 52 51 35 99 85 89 79 62

Females 425 15 73 74 56 148 29 121 115 83

Immatures 0

0 11 0 0 0 0 12 0 0

Total i standard deviation 830 f 72 138 + 84 136 rt 38 125 j, 85 91 f 83 237 & 39 114+52 222 f 77 194 + 49 145 rt 39

The mean primary worm burden of the rats on day 10 was 830 and 237 in Groups A and B respectively. By day 21 however the numbers had fallen to 138 in the former and 114 in the latter. The sex ratio was strongly biased in favour of the male worms indicating that a host immune response had occurred but the ratio of worms expelled to those remaining in the rats was not as great as that seen in the previous experiments. The administration of 20 larvae per weekday from day 23-51 resulted in the establishment of a secondary worm burden, the percentage take of which was approximately 32 per cent in the rats of Group A and 55 per cent in those of Group B indicating that rather more worms established in the rats that had received the smaller primary infection. The number of worms present on day 108 was similar to that of day 52 indicating that no worm expulsion had occurred but by day 135 the numbers had fallen to 91 worms in A and 145 in B. More females than males were present for the duration of the secondary infection.

D. CONWIL JENKINSand

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TABLE ~-EXPERIMENT 1. (Egg output of secondary infection. Rats in Sub-groups A5 and B5 each received primary infections of 2500 and 500 larvae respectively on day 1, followed by a secondary trickle infection given at the rate of 20 iarvae per weekday for 4 weeks from day 23 of the experiment. All the rats were treated with thiabendazole on day 20.)

Day of experiment 30 2 51 59 66 73 80 87 94 101 108 115 122 129

Eggs per grammefaeces Day of ___secondary infection Sub-group Sub-group A5 BS

7 14 21 28 35 42 49 56 63 70 77 84 91 98 105

0 183 700 1030 1010 1080 2000 1820 1830 2050 980 2200 820 1270 370

0 317 2330 1650 980 1200 1880 1430 3070 1860 1680 1750 2230 1730 1210

DISCUSSION Jenkins & Phillipson (197 1) showed that when previousIy uninfected rats were subjected to a trickle infection over a period of weeks, the worms that developed after the first 14 days were mo~hologically distinct from those found in a normal primary infection. Further, they showed that these worms did not elicit an acute host immune response and were able to remain within the rat intestine for a number of months. The present work shows that relatively stable secondary worm burdens can develop in previously immunized rats providing the secondary infection is acquired gradually over a number of weeks. The fact that 5-week-old rats developed a heavier secondary infection than S-week-old rats indicates that the age of the rats was of some importance. It is known (Jarrett et al., 1966; Kassai & Aitken, 1967) that neonatal rats do not expel a primary infection as efficiently as adult rats. The rats used in the present experiments however were not behaving as true neonates since all of them had developed an adult-type acute immune response to the primary infection before trickle infection began. The results obtained in experiment 3 indicate that the size of the primary infection slightly affected the take of the subsequent trickle infections since rather more secondary worms established in the rats that had received a primary infection of 500 larvae than in those that had received 2500 larvae. It is known (Sarles & Taliaferro, 1936; Mulligan et al., 1965; Ogilvie, 1965a) that worms which deveIop in rats as the result of a heavy challenge infection are stunted and are expelled more rapidly than worms derived from a primary infection. In our experiments however, many of the worms that developed from the trickle infections though stunted, were able to remain within the host for as long as 3 months. Furthermore, they continued to produce fertile eggs for the duration of this period indicating that their reproductive systems could not have been severely damaged. It is possible that these worms were able to remain within

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INFECTIONS

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the host because they did not stimulate the host’s immunity sufficiently. The fact that all the worms were stunted and that their egg production was not as high as normal primary infection worms however, indicates that they were subjected to certain factors within the host which are not apparent in non-immune rats. It is also of interest that female worms were not preferentially expelled from these infections. This is in contrast to the situation in primary infections where females are more immunogenic and are more rapidly expelled by the host immune response (Ogilvie, 1965b). It is obvious that the worms established by trickle infection were suited to life in an immune host and it seems likely that this was because they were physiologically adapted in some way, this resulting in mutual tolerance between host and parasite. Further work is required to investigate the nature of the changes involved. In conclusion, it is worth mentioning that these results may be of importance in the context of nematode vaccines, since they show at least in the Nippostrongylus-rat system, that resistance acquired from a heavy primary infection does not preclude the possibility of the establishment of a moderately heavy and persistent secondary worm burden. REFERENCES JARRETTE. E. E., JARRETTW. F. H. & URQUHARTG. M. 1966. Immunological unresponsiveness in adult rats to the nematode Nippostrongylus brasiliensis induced by infection in early life. Nature 221: 1310-1311. JENKINSD. C. 8c PHILLIPSONR. F. 1971. The kinetics of repeated low-level infections of Nippostrongylus brasiliensis in the laboratory rat. Parasitology 62: 457-465. KASAI T. & AITKENI. D. 1967. Induction of immunological tolerance in rats to Nippostrongylus brasiliensis infection. Parasitology 57: 403418. MULLIGANW., URQUHARTG. M., JENNINGSF. W. & NEILSONJ. T. M. 1965. Immunological studies on Nippostrongylus brasiliensis infection in the rat: the self-cure phenomenon. Experimental Parasitology 16: 341-347.

OGILVIEB. M. 1965a. Use of cortisone derivatives to inhibit resistance to Nippostrongylus brasiliensis and to study the fate of parasites in resistant hosts. Parasitology 55: 723-730. OGILVIEB. M. 196513.Role of adult worms in immunity of rats to Nippostrongylus brasiliensis. Parasitology 55: 325-335.

SARLESM. P. & TAUAFERROW. H. 1936. The local points of defense and the passive transfer of acquired immunity to Nippostrongylus muris in rats. Journal of Infectious Diseases 59: 207-220.