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The biology of Hyostrongylus rubidus
J.
(:OMP.
PATH.
1970.
VOL.
THE
BIOLOGY II.
80.
145
HYOSTRONGYLUS
OF
REPEATED
INFECTION
RUBIDUS
IN YOUNG
PIGS
B) S. B. KENDALL Central
~‘eterinary
Laboratory,
and
J. D. J. HARDING
Afinistry
of .&pdture.
it’rybri&a
INTRODUCTION and In an earlier paper (Kendall, Thurley and Peirce, 1969) the parasitological pathological effects of primary infection with Hyostrongylus rubidus were described. The present paper compares the effects of a single and a repeated infection.
MATERIALS
.4ND
METHODS
‘I’he materials used and the techniques employed were essentially those described earlier (Kendall et al:, 1969). The experimental design is recorded in Table 1. Of twelve 4-month-old prgs received from a minimal diseases herd nine were initially infected each with 4,000 larvae of H. rubidus. All nine were excreting H. rubidus eggs in their faeces 21 days later and thereafter daily records of the numbers of worm eggs from all pigs were made. At 143 days after the first infection, three of the previously infected pigs (group C) together with the three pigs which had been kept uninfected (group B) were each given 4,000 larvae of H. rubidus. At about the same time (143 to 175 days after infection) the other six of the previously infected pigs (group A) were killed and their stomachs examined either for the presence of worms or for evidence of pathological change. All the remaining pigs were killed and examined about a month after the time of the second infection which should by then have been patent. At post-mortem exa.mination, in an effort to distinguish between worms which might be lying superficially on the mucous membrane or more deeply, the stomachs were first placed in warm normal saline for about half an hour, any worm which emerged being counted separately from those recovered after pepsin digestion (Kendall et al., 1969). A very few worms were, in addition, recovered from the stomach contents. Random samples of worms recovered by the saline extraction were identified as to sex and lueasured. In one instance a record was made of the distribution of the worms in the four regions of the stomach by the method previously described (Kendall et al., 1969). Pathological examinations of stomachs were made in the same way as previously, five pigs used for this purpose being consequently unavailable for parasitological examination.
RESULTS
Nu,mbers of Worms Recovered Table 1 indicates the total numbers of adult and larval Hyostrongylus from seven pigs from which, as far as possible, all worms were recovered. Group A had received an infection on the first occasion only. Group B received a single infection on the second occasion and group C received the double infections.
146
Hyostrongylus
rubidus:
REPEATED
INFECTION
IN YOUNG
PIGS
As no larvae were recovered from the initial saline extraction and there was no obvious relationship betwen the numbers of adult worms recovered respectively by the initial saline extraction and by the pepsin digest, the numbers obtained by the two processes were pooled. Male and female worms appeared to occur in approximately equal numbers. It will be seen (Table 1) that the greatest number of worms was recovered soon after the patency of a single infection (group B).
TABLE EXPER~~NTAL
DESIGN
AND
1
RECOVERIES
OF Hyostrongyh Worm
Group
(A)
Infected
W
Infection
(C)
Infected
Adults
143 165 175 143 143 175
222 112 263 * * *
0
532 711 *
4 0 -
302 183 *
31 0 -
on day 143
on day 0 and day 143
for pathological
recovered
Day when killed
on day 0
* Pigs reserved
rubidu
165 176 176
LWkZ
i -
examination.
Later examination of pigs which had received a single infection showed a considerable reduction in numbers (group A). The numbers recovered after the repeated infection were lower than might have been expected (group C). Sizes of worms. Larvae were recovered only from the two pigs killed at 22 days after an infection, i.e. the first patency. The average lengths of adult male and female worms were in the range of O-61 to 0.68 mm. and O-83 to 0.90 mm. respectively. There did not seem to be any appreciable difference in the sizes of worms from the three groups.
Regional
Distribution
of Worms
in the Stomach
Table 2 records the numbers of worms and their distribution in the stomach of the pig from group A killed 143 days after infection. Having regard to the labour involved it was not possible to treat more than one stomach in this way.
S. B.
KENDALL
AND
J.
TABLE TOTAL
RECOVERY
OF WORMS
FROM
D. J.
2
ONE PIG T O SHOW THE
147
HARDING
THE
REGIONAL
I)I’I’KIBI’TION
IN
STOMACH
Stomach contents and washings __--~~
----.--.
Saline retraction
Pepsin cligwt Total
Numbers
~-
Cardiac Oesophageal Pyloric E‘undic
1’; 11)
Cardiac Oesophageal Pyloric Fundic recovery
11 ‘I ,I I 2 108
-.-2
i
-.--__.
__7 9 23 19
..--
13 9 26 11:
of H’orm Eggs in the Faeces
..\fter the first appearance of worm eggs about the 21.51 day after infection there was a rise in the numbers over the first few days and thereafter a fluctuating output of the order of 50 eggs/g. of faeces (Fig. 1). There was, however, a considerable variation between individual pigs and between the numbers from an individual pig on different days. The significance of these variations is not apparent. Fig.
0 Record Group Group
2
4
6
of worm eggs in faeces. A infected at day 0 B infected at day 143
8
IO
1.
12 Weeks Group
14
C infected
16
18
20
at day 0 and at day
22
24
143
Detailed records are on file at Weybridge. By the time of the second infection the egg output from the previously infected pigs (groups A and C) had decreased as indicated in Fig. 1. The second infection had no effect on the egg count of group C, but became patent in group B.
148
Hyostrongylus
rubidus:
REPEATED
INFECTION
IN YOIING
PIGS
Numbers of Eggs per Worm Representative samples of about 25 female worms were collected at the timt. oi post-mortem examination of five pigs and the numbers of mature eggs in thG; uteri counted. These averaged 39, 60 and 52 respectively for three pigs which had been infected on one occasion and 57 and 56 for two pigs which rccei\,ed tiic. repeated infection. There was apparently no reduction which could be related to an immunological effect in the twice infected pigs. The apparently lower average of 39 was obtained from worms from a pig infected once and killed 22 days lat<,r,. Pathology There was no evidence of clinical disease which could be attributed LO Hyostrongylus. As indicated in Table 1 stomachs from representative pigs front each of the three groups were examined for evidence of pathological change which could be associated with the infection. There did not appear to be any striking differences between those pigs which had been infected on one or on two occasions. There were fairly numerous lymphoid nodules particularly in the cardiac mucosa, but probably not markedly more than in uninfected pigs. There was a slight diffuse submucosal eosinophil infiltration, particularly in the fundic region. At the junction of the cardiac and fundic mucosa there was an area with quite pronounced eosinophilic and histiocytic infiltration between the glands. On section no larvae were detected within the mucosa, but in one of the onct infected pigs there were a few pieces of worm on the surface of the fundic mucosa. In the submucosa under the cardio-fundic junction of another pig from tht reinfected group there was a mass of degenerate eosinophils encapsulated by young granulation tissue which was infiltrated with eosinophils. DISCUSSION
The object of this experiment was to look for resistance to reinfection as evidenced by tissue reaction and by such manifestations as a reduction in worm numbers, size and fecundity. Table 1 shows that the number of worms recovered at about the time of patency after a single infection with 4,000 larvae was of the same order (group B, 536 and 711 worms) as previously observed (Kendall et al., 1969). At the 22nd day after the infection most worms were adult and judging by the egg output in Fig. 1 were fully productive within a few days. As has been previously observed H. rubidus is not, however, a prolific egg-layer compared with, for example, Oesophagostomum (Jacobs, 1967). By about the fifth month it seems that the numbers of worms had decreased considerably (group A-263, 222 and 112) and this is reflected in a reduction in the numbers of worm eggs recovered from the faeces. In those pigs which received the double infection (group C) and were killed at the expected time of patency of the second infection fewer worms were recovered than might have been expected from consideration of the numbers recovered from groups A and B and the numbers were of the same order as in group A which had received the first infection only. On the available evidence it is not possible to determine the reason for the reduction. Observations on the
S.
B.
KENDALL
AND
J.
D.
J.
149
HARDING
sizes and egg content of the worms recovered from the twice infected pigs and the evidence of the uniformly fluctuating, if declining, numbers of worm eggs in the faeces all tend to indicate the persistence of the worm population of the first infection. It seems likely that the second infection failed to remain established, although the recovery of 31 larvae from one twice infected pig 22 days aft(‘r the second infection indicates that reinfection did occur. Pathological changes were not striking, but even if there had been a markrd reaction at the time of the second infection this might not have persisted until the time of post-mortem. The necessary limitation on the numbers of minimal clisease pigs and the need to reserve some pigs for pathological examination resulted in \cry few pigs being available in each ,group for parasitological cxaminatioi~. Further work in progress may clarify our present views on the course of c*\~nts jollo~ving a repeated infection with Hyostro?7g?ks ruhidnts.
;\bout a month after a primary infection in young pigs with 4,000 larvae of rubidus nearly all womls were adult and there was a recovery of the order of 600 from each pig. Four months later the number had declined to about 200. Reinfection at this time apparently led to the establishment of some larvae, but at their expected time of patency the total worm recovery was not greater than in pigs which had received the primary infection only. Hyosfrongylus
ACKNOWLEUGXIENTS
Ch thanks are due to Mrs. .4nn Rogers and to Messrs. J. Small and G. Snell for rrchnical assistance. REFERENCES
Jacobs.D. E. (1967). Nord. Vet-Med., 19, 457. ‘KeIldall. S. H.. Thurley, D. C., and Peirce, M. A. (1969). J. camp. Path., 79, 87. [Received
for publication,
May
29th,
19691
(:OMP.
PATH.
1970.
VOL.
THE
BIOLOGY II.
80.
145
HYOSTRONGYLUS
OF
REPEATED
INFECTION
RUBIDUS
IN YOUNG
PIGS
B) S. B. KENDALL Central
~‘eterinary
Laboratory,
and
J. D. J. HARDING
Afinistry
of .&pdture.
it’rybri&a
INTRODUCTION and In an earlier paper (Kendall, Thurley and Peirce, 1969) the parasitological pathological effects of primary infection with Hyostrongylus rubidus were described. The present paper compares the effects of a single and a repeated infection.
MATERIALS
.4ND
METHODS
‘I’he materials used and the techniques employed were essentially those described earlier (Kendall et al:, 1969). The experimental design is recorded in Table 1. Of twelve 4-month-old prgs received from a minimal diseases herd nine were initially infected each with 4,000 larvae of H. rubidus. All nine were excreting H. rubidus eggs in their faeces 21 days later and thereafter daily records of the numbers of worm eggs from all pigs were made. At 143 days after the first infection, three of the previously infected pigs (group C) together with the three pigs which had been kept uninfected (group B) were each given 4,000 larvae of H. rubidus. At about the same time (143 to 175 days after infection) the other six of the previously infected pigs (group A) were killed and their stomachs examined either for the presence of worms or for evidence of pathological change. All the remaining pigs were killed and examined about a month after the time of the second infection which should by then have been patent. At post-mortem exa.mination, in an effort to distinguish between worms which might be lying superficially on the mucous membrane or more deeply, the stomachs were first placed in warm normal saline for about half an hour, any worm which emerged being counted separately from those recovered after pepsin digestion (Kendall et al., 1969). A very few worms were, in addition, recovered from the stomach contents. Random samples of worms recovered by the saline extraction were identified as to sex and lueasured. In one instance a record was made of the distribution of the worms in the four regions of the stomach by the method previously described (Kendall et al., 1969). Pathological examinations of stomachs were made in the same way as previously, five pigs used for this purpose being consequently unavailable for parasitological examination.
RESULTS
Nu,mbers of Worms Recovered Table 1 indicates the total numbers of adult and larval Hyostrongylus from seven pigs from which, as far as possible, all worms were recovered. Group A had received an infection on the first occasion only. Group B received a single infection on the second occasion and group C received the double infections.
146
Hyostrongylus
rubidus:
REPEATED
INFECTION
IN YOUNG
PIGS
As no larvae were recovered from the initial saline extraction and there was no obvious relationship betwen the numbers of adult worms recovered respectively by the initial saline extraction and by the pepsin digest, the numbers obtained by the two processes were pooled. Male and female worms appeared to occur in approximately equal numbers. It will be seen (Table 1) that the greatest number of worms was recovered soon after the patency of a single infection (group B).
TABLE EXPER~~NTAL
DESIGN
AND
1
RECOVERIES
OF Hyostrongyh Worm
Group
(A)
Infected
W
Infection
(C)
Infected
Adults
143 165 175 143 143 175
222 112 263 * * *
0
532 711 *
4 0 -
302 183 *
31 0 -
on day 143
on day 0 and day 143
for pathological
recovered
Day when killed
on day 0
* Pigs reserved
rubidu
165 176 176
LWkZ
i -
examination.
Later examination of pigs which had received a single infection showed a considerable reduction in numbers (group A). The numbers recovered after the repeated infection were lower than might have been expected (group C). Sizes of worms. Larvae were recovered only from the two pigs killed at 22 days after an infection, i.e. the first patency. The average lengths of adult male and female worms were in the range of O-61 to 0.68 mm. and O-83 to 0.90 mm. respectively. There did not seem to be any appreciable difference in the sizes of worms from the three groups.
Regional
Distribution
of Worms
in the Stomach
Table 2 records the numbers of worms and their distribution in the stomach of the pig from group A killed 143 days after infection. Having regard to the labour involved it was not possible to treat more than one stomach in this way.
S. B.
KENDALL
AND
J.
TABLE TOTAL
RECOVERY
OF WORMS
FROM
D. J.
2
ONE PIG T O SHOW THE
147
HARDING
THE
REGIONAL
I)I’I’KIBI’TION
IN
STOMACH
Stomach contents and washings __--~~
----.--.
Saline retraction
Pepsin cligwt Total
Numbers
~-
Cardiac Oesophageal Pyloric E‘undic
1’; 11)
Cardiac Oesophageal Pyloric Fundic recovery
11 ‘I ,I I 2 108
-.-2
i
-.--__.
__7 9 23 19
..--
13 9 26 11:
of H’orm Eggs in the Faeces
..\fter the first appearance of worm eggs about the 21.51 day after infection there was a rise in the numbers over the first few days and thereafter a fluctuating output of the order of 50 eggs/g. of faeces (Fig. 1). There was, however, a considerable variation between individual pigs and between the numbers from an individual pig on different days. The significance of these variations is not apparent. Fig.
0 Record Group Group
2
4
6
of worm eggs in faeces. A infected at day 0 B infected at day 143
8
IO
1.
12 Weeks Group
14
C infected
16
18
20
at day 0 and at day
22
24
143
Detailed records are on file at Weybridge. By the time of the second infection the egg output from the previously infected pigs (groups A and C) had decreased as indicated in Fig. 1. The second infection had no effect on the egg count of group C, but became patent in group B.
148
Hyostrongylus
rubidus:
REPEATED
INFECTION
IN YOIING
PIGS
Numbers of Eggs per Worm Representative samples of about 25 female worms were collected at the timt. oi post-mortem examination of five pigs and the numbers of mature eggs in thG; uteri counted. These averaged 39, 60 and 52 respectively for three pigs which had been infected on one occasion and 57 and 56 for two pigs which rccei\,ed tiic. repeated infection. There was apparently no reduction which could be related to an immunological effect in the twice infected pigs. The apparently lower average of 39 was obtained from worms from a pig infected once and killed 22 days lat<,r,. Pathology There was no evidence of clinical disease which could be attributed LO Hyostrongylus. As indicated in Table 1 stomachs from representative pigs front each of the three groups were examined for evidence of pathological change which could be associated with the infection. There did not appear to be any striking differences between those pigs which had been infected on one or on two occasions. There were fairly numerous lymphoid nodules particularly in the cardiac mucosa, but probably not markedly more than in uninfected pigs. There was a slight diffuse submucosal eosinophil infiltration, particularly in the fundic region. At the junction of the cardiac and fundic mucosa there was an area with quite pronounced eosinophilic and histiocytic infiltration between the glands. On section no larvae were detected within the mucosa, but in one of the onct infected pigs there were a few pieces of worm on the surface of the fundic mucosa. In the submucosa under the cardio-fundic junction of another pig from tht reinfected group there was a mass of degenerate eosinophils encapsulated by young granulation tissue which was infiltrated with eosinophils. DISCUSSION
The object of this experiment was to look for resistance to reinfection as evidenced by tissue reaction and by such manifestations as a reduction in worm numbers, size and fecundity. Table 1 shows that the number of worms recovered at about the time of patency after a single infection with 4,000 larvae was of the same order (group B, 536 and 711 worms) as previously observed (Kendall et al., 1969). At the 22nd day after the infection most worms were adult and judging by the egg output in Fig. 1 were fully productive within a few days. As has been previously observed H. rubidus is not, however, a prolific egg-layer compared with, for example, Oesophagostomum (Jacobs, 1967). By about the fifth month it seems that the numbers of worms had decreased considerably (group A-263, 222 and 112) and this is reflected in a reduction in the numbers of worm eggs recovered from the faeces. In those pigs which received the double infection (group C) and were killed at the expected time of patency of the second infection fewer worms were recovered than might have been expected from consideration of the numbers recovered from groups A and B and the numbers were of the same order as in group A which had received the first infection only. On the available evidence it is not possible to determine the reason for the reduction. Observations on the
S.
B.
KENDALL
AND
J.
D.
J.
149
HARDING
sizes and egg content of the worms recovered from the twice infected pigs and the evidence of the uniformly fluctuating, if declining, numbers of worm eggs in the faeces all tend to indicate the persistence of the worm population of the first infection. It seems likely that the second infection failed to remain established, although the recovery of 31 larvae from one twice infected pig 22 days aft(‘r the second infection indicates that reinfection did occur. Pathological changes were not striking, but even if there had been a markrd reaction at the time of the second infection this might not have persisted until the time of post-mortem. The necessary limitation on the numbers of minimal clisease pigs and the need to reserve some pigs for pathological examination resulted in \cry few pigs being available in each ,group for parasitological cxaminatioi~. Further work in progress may clarify our present views on the course of c*\~nts jollo~ving a repeated infection with Hyostro?7g?ks ruhidnts.
;\bout a month after a primary infection in young pigs with 4,000 larvae of rubidus nearly all womls were adult and there was a recovery of the order of 600 from each pig. Four months later the number had declined to about 200. Reinfection at this time apparently led to the establishment of some larvae, but at their expected time of patency the total worm recovery was not greater than in pigs which had received the primary infection only. Hyosfrongylus
ACKNOWLEUGXIENTS
Ch thanks are due to Mrs. .4nn Rogers and to Messrs. J. Small and G. Snell for rrchnical assistance. REFERENCES
Jacobs.D. E. (1967). Nord. Vet-Med., 19, 457. ‘KeIldall. S. H.. Thurley, D. C., and Peirce, M. A. (1969). J. camp. Path., 79, 87. [Received
for publication,
May
29th,
19691