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Inhibition of development: Variation within a population of Ostertagia ostertagi
J.
COMP.
PATH.
1973.
‘~'OL.
83.
351
INHIBITION OF DEVELOPMENT: VARIATION WITHIN OSTERTAGI A POPULATION OF OSTERTAGIA
J. F. MICHEL,
M. B. LANCASTER
and C. HONG
Central Veterinary Laboratory, Ministry oJ‘dgriculture, Fisheries and Food, Wcvbridge, Surrey
INTRODUCTION
Armour, Jennings and Urquhart (1967, 1969) pointed out that some strains of Ostertagia ostertagi showed a greater propensity for arrested development than others since under similar conditions 4 times as many larvae of a population from a farm in Ayrshire became inhibited as of the Weybridge strain. There is evidence that the culture of 0. ostertagi maintained at Weybridge since 1957 has altered with the passage of time. In comparable experimental infections approximately 24 per cent. of the larvae became inhibited in 1961, 13 per cent. in 1964,8 per cent. in 1965, 6 per cent. in 1967 and 5 per cent. in 1970. This change has occurred as a result of what appears to have been a relatively mild selection pressure. The possibility must be considered that there are not 2 or more stable regional strains, but that there is considerable variation within populations so that they can be readily modified by a change in management. The experiment described here suggests that this is so. The capacity for interrupted development of larvae which were the progeny of inhibited worms that had overwintered in the host was compared with that of larvae which had overwintered on the pasture. MATERIALS
AND
METHODS
puslures. Six paddocks, A, B, C, D, E and F’, each of 8 acres were laid out on a sward in which perennial ryegrass and SlOO white clover predominated, but which contained an appreciable admixture of cocksfoot and some creeping bent. This pasture had not previously been grazed by cattle. Animals. Ayrshire calves and yearlings were used to contaminate these paddocks. They were purchased from local breeders and reared in conditions calculated to prevent accidental infection with strongyloid nematodes. The ability of the resulting larvae to interrupt their parasitic development was determined by means of test Jersey calves. These were collected and reared in the same manner as the Ayrshire calves and exposed to infection on the paddocks at au average age of 110 days. Post-mortem worm counts were made as described by Michel (1969a). Regular examinations of the herbage were made by the technique of Lancaster (1970). Design of exjeriment. Two preparatory steps were taken in 1968. A group of 5 calves was infected with 0. ostertagi on June 21st, each receiving 10 000 larvae of the Weybridge strain. These calves contaminated paddock A from July 15th to September 19th and were then removed from the experiment. A further 5 calves were exposed to infection on a pasture carrying an infestation of larvae of the Weybridge strain
352
J. F. MICHEL
et. d.
throughout the summer of 1968 and until January 6th when they were housed. As group 3 these animals were used to contaminate paddocks D and E during 1969. The movements of animals in 1969 are summarized in Fig. 1. On April 28th 4 calves (group 1) were put on paddock A on which a residual infestation had persisted through the winter. These calves, in their turn, contaminated paddock A from about May 21st until June 25th when they were housed. They were turned out on paddock B on August 5th and contaminated it until September 16th when they were removed from the experiment.
A second group of 4 calves (group 2) grazed paddock A from July 24th until August 5th. They were then transferred to paddock C which they contaminated until September 16th. The 5 yearlings of group 3 contaminated paddock D from April 28th until May 30th when they were housed. They were put in paddock E on August 5th and con-
tinued to contaminate this until September 16th. Four calves constituting group 4 were exposed to infection on paddock D from July
15th until
August
5th. They were then transferred
to paddock
F which
they
contaminated until September 16th. On a particular paddock the proportion of the larvae which failed to develop beyond the early 4th stage when ingested by a susceptible host was determined by grazing 2 calves on the paddock for a short period, housing them for a fortnight and then killing them so that worm counts could be made. Paddock A was grazed by a pair of test calves in this way from April 28th until May 15th. All the paddocks were grazed by pairs of test calves from October 15th until October 22nd. They were grazed by further pairs of calves from December 3rd to December 17th and finally paddocks B and E were grazed from March 9th to March 16th. RESULTS
The egg output of the yearlings of group 3 was low and consequently the infestation which arose on the herbage of paddocks D and E was also low. However, here as on all the paddocks the infestation on the herbage tended to rise through the winter. Egg output in the faeces of groups 1 and 3 reached the usual maximum level and followed the normal course (Michel, 1969b). Clinical ostertagiasis developed in the calves of group 2 of which 3 died. Herbage infestations on paddock A were heavier than on paddocks D and E, but not as heavy as on F or C. There was a fairly close co-relation between the herbage infestations and the number of worms acquired by the test calves. These worm burdens are shown in Table 1 which also indicates the percentage of worms arrested at the early 4th stage. It will be seen that a greater proportion of worms picked up in December interrupted their development than of those acquired in October. By March the percentage that became arrested at the early 4th stage had fallen markedly from the December level. It will also be seen that on paddocks D, E and F the development of a greater proportion of worms was interrupted than on paddocks A, B and C (a mean of 10-l per cent. as against 2.3 per cent. in October and of 27.1 per cent. as against 10.7 per cent. in December). The worms on paddocks D, E and F were derived from eggs passed by group 3 during 1969. Many of the worms picked up by these animals during the autumn and winter of 1968 would have accumulated in them as arrested early 4th stage larvae. The adult worms that they also carried when housed would have been lost by the spring and replaced by the resumed development of arrested forms. The larvae on paddocks D, E and F
A
4512 4527 1 Mean
Calf no.
2100 1900
100 0
2:3
4.5
Lab 4th and 5lh Early o/o 4th inhistage worms stage bited 4622 4630 Mean 4633 4639 Mean 4621 4631 Mean 4618 4634 Mean 4625 4638 Mean 4611 4636 Mean
Cacf no.
Mean 4650 4656 Mean 4651 4653 Mean 4667 4668 Mean
22.: 0 12.5 12.6 1’0.: 3.8 11.1 7.5
600 250 150 600
4150 2950 4800
150 200 50 0
0 50
100 1050
4647 4654 Mean 4649 4661 Mean 4665 4657
0” 0
Calf no.
CALVES
5900 4200 1400 900 450 150 1900 1000 5100 4100
8900 8800 600 500 3100 2650 33 400 18 500
150 450
stage _-_
E;$F
27 900 39 200
4700 3600
Latz 4th and 5th stage
OF TEST
;:; 4.7 4q5
300 400
0”
5300 9500
2200 1500
1
COUNTS
TABLE WORM
Late 4th Early y. inhiand 5th 4th stage stage bited
POSTMORTEM
42.9 23.1 33.0 38.0 27.4 32.7 13.3 17.8 15.6
12
13.6 13.6
4726 4787 Mean
13 500 12 700
17 100 1400
1Y.f 1:.: 9.7
4742 4777 Mean
Late 4th and 5th stage
%
inhibited
6.3 5.9 6.1
800
1.7 0.7 1.2
y. inhibited
900
300 100
Earb 4th stage
3.
2! 3 2 GN, P 2: R a
g
g
E =j
354
Fig. 1.
J. F. MICHEL
et. al.
How the 6 paddocks were grazed in 1969 by 4 groups of cattlewhich acted as a source of infection and by 15 pairs of test calves. Paddock A had been contaminated by calves in the previous year. Group 3 had grazed an infected pasture during the previous summer and Group 3; q Group 4; n Test calves. autumn and until January : q Group I ; q Group 2 :
were, therefore, the progeny of worms whose development had been interrupted for a time. It may be concluded that as a result of this selection there was an inherent difference between the worms on paddocks D, E and F and those on paddocks A, B and C. However, when on paddock F, the worms completed a further generation without any selection, the proportion of larvae capable of interrupting their development appears to have been reduced. Meanwhile it is evident that the proportion of worms that were arrested was not affected by whether contamination of the pasture occurred before the end of June or after the beginning of August. DISCUSSION
It is becoming clear that in a number of host-nematode systems the inhibition of development is associated with some reversible change in the larvae brought about by experience of appropriate external conditions. Armour (1970) induced 0. ostertagi to become inhibited by exposing larvae to conditions of light and temperature equivalent to those obtaining in the autumn and Michel, Lancaster and Hong (unpublished observations) have caused inhibition by storing the larvae in the dark. In the present experiment a greater proportion of the larvae picked up in December were arrested than of those picked up in October. The proportion of Cooperia oncophora that interrupt their development when ingested by calves increases through the autumn and early winter (apparently to the shortest day) and then decreases again (Michel, Lancaster and Hong, 1970). Temperature may be involved as well as light. Fernando, Stockdale and Ashton (1971) have shown that a greater proportion of Obeliscoides cuniculi become inhibited if the larvae have been stored at 4 “C. than at 17 “C.
INHIBITION
OF &dZ@Z
O~tertdgi
355
If larvae are stimulated to interrupt their development by exposure to appropriate conditioning treatment, then it is to be expected that there will be some variation between individuals in the facility with which they respond to that treatment. The present experiments suggest that variation within populations of 0. ostertagi may be considerable. The changes that have occurred in the Weybridge strain during the 35 generations for which it has been maintained in the Laboratory have been the result of a relatively mild selection pressure. The procedure employed has been to infect calves with a single dose of 30 000 larvae and to make cultures from faeces passed during the period of i.e. early in the infection. This tended to select maximum egg production, against worms whose development was interrupted, but since no conditioning treatment was given, their numbers were not large. Where the larvae are exposed to conditioning treatment there is a possibility of much more effective selection. Thus, in the present experiment it was possible in a single generation to go far towards reversing changes which have occurred over 12 years and 28 generations. The properties of a population of worms may be expected to depend on the manner in which it is managed and to this extent it may be misleading to speak of a Weybridge strain. On the basis of the work reported here it would be reasonable to expect, for example, that where calves graze every year on pastures which were contaminated by calves in the previous year, the worms would have a smaller propensity for interrupted development than where the calves habitually acquire their infections from pastures contaminated by yearlings. There are no good grounds for believing that different strains of 0. ostertagi are to be found in different parts of the country. Rather, the population on one farm is likely to have different attributes from that on the next. Similarly, the fact that 2 laboratories maintain cultures which were originally derived from the same source, is no guarantee that they are working with identical material. It cannot be assumed that a culture of worms maintained in one laboratory will exactly correspond with that maintained in any other or indeed with any naturally occurring population. This is not to say that such laboratory maintained cultures are not useful for experimental work, but caution is needed in their use and in the interpretation of results. An attempt is at present being made in this Laboratory to develop a strain of 0. ostertagi with a very pronounced aptitude for interrupted development. Larvae are given conditioning treatment and are then administered to a calf. The worms that develop beyond the early 4th stage within a few weeks are then removed by anthelmintic treatment and cultures are made when the arrested forms have resumed their development. It is recognized, nevertheless, that the strain that may result will be a somewhat artificial creation in that it is likely to respond uncommonly well to the particular conditioning treatment employed and to develop very promptly when adult worms are removed. SUMMARY
A total of 47 calves on 6 paddocks was used to study variation within a strain of Ostertagia ostertagi in respect of the inherent capacity of worms to interrupt their development at the early fourth stage. It was shown that the
356
J. F. MICHEL
et.
d.
progeny of worms whose development had been arrested had a greater propensity for interrupted development than the progeny of worms whose development had not been arrested. REFERENCES
Armour, J. (1970). Vet. Rec., 86, 184. Armour, J., Jennings, F. W., and Urquhart, Res. vet. Sk.,
Fernando, Lancaster, Michel, J. Michel, J.
G. M. (1967). I&d., 80, 208; (1969).
10, 238.
M. A., Stockdale, P. H. G., and Ashton, G. C. (1971). Parasitology, 63, 21. M. B. (1970). J. Helminth., 44, 219. F. (1969a). Parasitology, 59, 575 ; ( 1969). Ibid., 829. F., Lancaster, M. B., and Hong, C. (1970). Brit. vet. J., 126, 35. [Received for publication,
September 4th, 19721
COMP.
PATH.
1973.
‘~'OL.
83.
351
INHIBITION OF DEVELOPMENT: VARIATION WITHIN OSTERTAGI A POPULATION OF OSTERTAGIA
J. F. MICHEL,
M. B. LANCASTER
and C. HONG
Central Veterinary Laboratory, Ministry oJ‘dgriculture, Fisheries and Food, Wcvbridge, Surrey
INTRODUCTION
Armour, Jennings and Urquhart (1967, 1969) pointed out that some strains of Ostertagia ostertagi showed a greater propensity for arrested development than others since under similar conditions 4 times as many larvae of a population from a farm in Ayrshire became inhibited as of the Weybridge strain. There is evidence that the culture of 0. ostertagi maintained at Weybridge since 1957 has altered with the passage of time. In comparable experimental infections approximately 24 per cent. of the larvae became inhibited in 1961, 13 per cent. in 1964,8 per cent. in 1965, 6 per cent. in 1967 and 5 per cent. in 1970. This change has occurred as a result of what appears to have been a relatively mild selection pressure. The possibility must be considered that there are not 2 or more stable regional strains, but that there is considerable variation within populations so that they can be readily modified by a change in management. The experiment described here suggests that this is so. The capacity for interrupted development of larvae which were the progeny of inhibited worms that had overwintered in the host was compared with that of larvae which had overwintered on the pasture. MATERIALS
AND
METHODS
puslures. Six paddocks, A, B, C, D, E and F’, each of 8 acres were laid out on a sward in which perennial ryegrass and SlOO white clover predominated, but which contained an appreciable admixture of cocksfoot and some creeping bent. This pasture had not previously been grazed by cattle. Animals. Ayrshire calves and yearlings were used to contaminate these paddocks. They were purchased from local breeders and reared in conditions calculated to prevent accidental infection with strongyloid nematodes. The ability of the resulting larvae to interrupt their parasitic development was determined by means of test Jersey calves. These were collected and reared in the same manner as the Ayrshire calves and exposed to infection on the paddocks at au average age of 110 days. Post-mortem worm counts were made as described by Michel (1969a). Regular examinations of the herbage were made by the technique of Lancaster (1970). Design of exjeriment. Two preparatory steps were taken in 1968. A group of 5 calves was infected with 0. ostertagi on June 21st, each receiving 10 000 larvae of the Weybridge strain. These calves contaminated paddock A from July 15th to September 19th and were then removed from the experiment. A further 5 calves were exposed to infection on a pasture carrying an infestation of larvae of the Weybridge strain
352
J. F. MICHEL
et. d.
throughout the summer of 1968 and until January 6th when they were housed. As group 3 these animals were used to contaminate paddocks D and E during 1969. The movements of animals in 1969 are summarized in Fig. 1. On April 28th 4 calves (group 1) were put on paddock A on which a residual infestation had persisted through the winter. These calves, in their turn, contaminated paddock A from about May 21st until June 25th when they were housed. They were turned out on paddock B on August 5th and contaminated it until September 16th when they were removed from the experiment.
A second group of 4 calves (group 2) grazed paddock A from July 24th until August 5th. They were then transferred to paddock C which they contaminated until September 16th. The 5 yearlings of group 3 contaminated paddock D from April 28th until May 30th when they were housed. They were put in paddock E on August 5th and con-
tinued to contaminate this until September 16th. Four calves constituting group 4 were exposed to infection on paddock D from July
15th until
August
5th. They were then transferred
to paddock
F which
they
contaminated until September 16th. On a particular paddock the proportion of the larvae which failed to develop beyond the early 4th stage when ingested by a susceptible host was determined by grazing 2 calves on the paddock for a short period, housing them for a fortnight and then killing them so that worm counts could be made. Paddock A was grazed by a pair of test calves in this way from April 28th until May 15th. All the paddocks were grazed by pairs of test calves from October 15th until October 22nd. They were grazed by further pairs of calves from December 3rd to December 17th and finally paddocks B and E were grazed from March 9th to March 16th. RESULTS
The egg output of the yearlings of group 3 was low and consequently the infestation which arose on the herbage of paddocks D and E was also low. However, here as on all the paddocks the infestation on the herbage tended to rise through the winter. Egg output in the faeces of groups 1 and 3 reached the usual maximum level and followed the normal course (Michel, 1969b). Clinical ostertagiasis developed in the calves of group 2 of which 3 died. Herbage infestations on paddock A were heavier than on paddocks D and E, but not as heavy as on F or C. There was a fairly close co-relation between the herbage infestations and the number of worms acquired by the test calves. These worm burdens are shown in Table 1 which also indicates the percentage of worms arrested at the early 4th stage. It will be seen that a greater proportion of worms picked up in December interrupted their development than of those acquired in October. By March the percentage that became arrested at the early 4th stage had fallen markedly from the December level. It will also be seen that on paddocks D, E and F the development of a greater proportion of worms was interrupted than on paddocks A, B and C (a mean of 10-l per cent. as against 2.3 per cent. in October and of 27.1 per cent. as against 10.7 per cent. in December). The worms on paddocks D, E and F were derived from eggs passed by group 3 during 1969. Many of the worms picked up by these animals during the autumn and winter of 1968 would have accumulated in them as arrested early 4th stage larvae. The adult worms that they also carried when housed would have been lost by the spring and replaced by the resumed development of arrested forms. The larvae on paddocks D, E and F
A
4512 4527 1 Mean
Calf no.
2100 1900
100 0
2:3
4.5
Lab 4th and 5lh Early o/o 4th inhistage worms stage bited 4622 4630 Mean 4633 4639 Mean 4621 4631 Mean 4618 4634 Mean 4625 4638 Mean 4611 4636 Mean
Cacf no.
Mean 4650 4656 Mean 4651 4653 Mean 4667 4668 Mean
22.: 0 12.5 12.6 1’0.: 3.8 11.1 7.5
600 250 150 600
4150 2950 4800
150 200 50 0
0 50
100 1050
4647 4654 Mean 4649 4661 Mean 4665 4657
0” 0
Calf no.
CALVES
5900 4200 1400 900 450 150 1900 1000 5100 4100
8900 8800 600 500 3100 2650 33 400 18 500
150 450
stage _-_
E;$F
27 900 39 200
4700 3600
Latz 4th and 5th stage
OF TEST
;:; 4.7 4q5
300 400
0”
5300 9500
2200 1500
1
COUNTS
TABLE WORM
Late 4th Early y. inhiand 5th 4th stage stage bited
POSTMORTEM
42.9 23.1 33.0 38.0 27.4 32.7 13.3 17.8 15.6
12
13.6 13.6
4726 4787 Mean
13 500 12 700
17 100 1400
1Y.f 1:.: 9.7
4742 4777 Mean
Late 4th and 5th stage
%
inhibited
6.3 5.9 6.1
800
1.7 0.7 1.2
y. inhibited
900
300 100
Earb 4th stage
3.
2! 3 2 GN, P 2: R a
g
g
E =j
354
Fig. 1.
J. F. MICHEL
et. al.
How the 6 paddocks were grazed in 1969 by 4 groups of cattlewhich acted as a source of infection and by 15 pairs of test calves. Paddock A had been contaminated by calves in the previous year. Group 3 had grazed an infected pasture during the previous summer and Group 3; q Group 4; n Test calves. autumn and until January : q Group I ; q Group 2 :
were, therefore, the progeny of worms whose development had been interrupted for a time. It may be concluded that as a result of this selection there was an inherent difference between the worms on paddocks D, E and F and those on paddocks A, B and C. However, when on paddock F, the worms completed a further generation without any selection, the proportion of larvae capable of interrupting their development appears to have been reduced. Meanwhile it is evident that the proportion of worms that were arrested was not affected by whether contamination of the pasture occurred before the end of June or after the beginning of August. DISCUSSION
It is becoming clear that in a number of host-nematode systems the inhibition of development is associated with some reversible change in the larvae brought about by experience of appropriate external conditions. Armour (1970) induced 0. ostertagi to become inhibited by exposing larvae to conditions of light and temperature equivalent to those obtaining in the autumn and Michel, Lancaster and Hong (unpublished observations) have caused inhibition by storing the larvae in the dark. In the present experiment a greater proportion of the larvae picked up in December were arrested than of those picked up in October. The proportion of Cooperia oncophora that interrupt their development when ingested by calves increases through the autumn and early winter (apparently to the shortest day) and then decreases again (Michel, Lancaster and Hong, 1970). Temperature may be involved as well as light. Fernando, Stockdale and Ashton (1971) have shown that a greater proportion of Obeliscoides cuniculi become inhibited if the larvae have been stored at 4 “C. than at 17 “C.
INHIBITION
OF &dZ@Z
O~tertdgi
355
If larvae are stimulated to interrupt their development by exposure to appropriate conditioning treatment, then it is to be expected that there will be some variation between individuals in the facility with which they respond to that treatment. The present experiments suggest that variation within populations of 0. ostertagi may be considerable. The changes that have occurred in the Weybridge strain during the 35 generations for which it has been maintained in the Laboratory have been the result of a relatively mild selection pressure. The procedure employed has been to infect calves with a single dose of 30 000 larvae and to make cultures from faeces passed during the period of i.e. early in the infection. This tended to select maximum egg production, against worms whose development was interrupted, but since no conditioning treatment was given, their numbers were not large. Where the larvae are exposed to conditioning treatment there is a possibility of much more effective selection. Thus, in the present experiment it was possible in a single generation to go far towards reversing changes which have occurred over 12 years and 28 generations. The properties of a population of worms may be expected to depend on the manner in which it is managed and to this extent it may be misleading to speak of a Weybridge strain. On the basis of the work reported here it would be reasonable to expect, for example, that where calves graze every year on pastures which were contaminated by calves in the previous year, the worms would have a smaller propensity for interrupted development than where the calves habitually acquire their infections from pastures contaminated by yearlings. There are no good grounds for believing that different strains of 0. ostertagi are to be found in different parts of the country. Rather, the population on one farm is likely to have different attributes from that on the next. Similarly, the fact that 2 laboratories maintain cultures which were originally derived from the same source, is no guarantee that they are working with identical material. It cannot be assumed that a culture of worms maintained in one laboratory will exactly correspond with that maintained in any other or indeed with any naturally occurring population. This is not to say that such laboratory maintained cultures are not useful for experimental work, but caution is needed in their use and in the interpretation of results. An attempt is at present being made in this Laboratory to develop a strain of 0. ostertagi with a very pronounced aptitude for interrupted development. Larvae are given conditioning treatment and are then administered to a calf. The worms that develop beyond the early 4th stage within a few weeks are then removed by anthelmintic treatment and cultures are made when the arrested forms have resumed their development. It is recognized, nevertheless, that the strain that may result will be a somewhat artificial creation in that it is likely to respond uncommonly well to the particular conditioning treatment employed and to develop very promptly when adult worms are removed. SUMMARY
A total of 47 calves on 6 paddocks was used to study variation within a strain of Ostertagia ostertagi in respect of the inherent capacity of worms to interrupt their development at the early fourth stage. It was shown that the
356
J. F. MICHEL
et.
d.
progeny of worms whose development had been arrested had a greater propensity for interrupted development than the progeny of worms whose development had not been arrested. REFERENCES
Armour, J. (1970). Vet. Rec., 86, 184. Armour, J., Jennings, F. W., and Urquhart, Res. vet. Sk.,
Fernando, Lancaster, Michel, J. Michel, J.
G. M. (1967). I&d., 80, 208; (1969).
10, 238.
M. A., Stockdale, P. H. G., and Ashton, G. C. (1971). Parasitology, 63, 21. M. B. (1970). J. Helminth., 44, 219. F. (1969a). Parasitology, 59, 575 ; ( 1969). Ibid., 829. F., Lancaster, M. B., and Hong, C. (1970). Brit. vet. J., 126, 35. [Received for publication,
September 4th, 19721