Observations on the dynamics of worm burdens in lambs infected daily with Ostertagia circumcincta

Inrernorionalfournolfor Parasitology, Vol. 17, No. 4, pp. 95 l-956,1987. Printed in Great Briroin.

0020-7519/87 $3.00 + 0.00 Pergomon Journals Ltd. 0 1987 Aucrralian Societyfor Parasitology

OBSERVATIONS ON THE DYNAMICS OF WORM BURDENS IN LAMBS INFECTED DAILY WITH OSTERTAGIA CIRCUMCINCTA C. HONG, J. F. MICHEL and M. B. LANCASTER Central Veterinary Laboratory, New Haw, Weybridge, Surrey, KT15 3NB, U.K. (Received 2 April 1986) Abstract-How C., MICHEL .I. F. and LANCASTERM. B. 1987. Observations on the dynamics of worm burdens in lambs infected daily with Ostertagia circumcincta. International Journal for Parasitology 17: 951-956. Groups of lambs were infected daily with either 250,500 or 1000 Ostertagia circumcincta larvae and the course of their worm burdens by post mortem examination at five intervals of time was studied. The number of worms appeared to be related to the rate of intake of larvae. A morphometric study of female worm lengths and observations on the incidence of reduced vulva1 flaps indicated that the population of worms turned over rapidly. The response of a small number of lambs appeared to be abnormal.

INDEX KEY WORDS: Ostertania circumcincta; pattern; host variation.

INTRODUCTION As POINTED out by Donald & Waller (1982) few studies on the iutra-host regulation of nematode populations have been undertaken with Ostertagia circumcincta in sheep and there is little evidence to show whether it conformed to the Ostertagia ostertugi/cattle system as described by Michel(l969). The results of au initial experiment in which lambs were given a single inoculum of 0. circumcincta larvae (Hong, Michel & Lancaster, 1986) suggested that a constant proportion of the worms was lost per unit of time. It therefore seems probable that, like 0. ostertagi populations in cattle, worm numbers are governed by the short mean life of the worm and that the size of the burden is approximately proportional to the rate of infection. As part of a study on the population dynamics of 0. circumcincta and also to determine whether a turnover of worms was occurring, the time course of lambs infected daily at three different rates was investigated. The problem in this type of experimentationis that it is not possible to follow the dynamics of a worm population in each animal (i.e. to kill the same animal more than once) and that therefore a high degree of uniformity in the animals must be assumed. Such an assumption was made by Gibson & Whitehead (1981) who infected lambs with 0. circumcincta and reported the course of infection

calculated from the mean worm burdens of groups of three lambs. Differences within groups, however large, were considered acceptable. Michel (1963) found that a very small number of animals behaved so differently from the rest that he could feel justified in regarding them as aberrant, excluding them from the group mean results and

length, vulval flap; population dynamics; egg count

treating them separately. Dineen, Gregg & Lascelles (1978) recognised differences in the response of sheep to Trichostrongylus columbrifomzis infection and classified their animals as either “responders” or “non-responders” on the basis of their ability to control a challenge infection after previous vaccinatioqwith irradlated larvae. They concluded that the response of lambs to vaccination was genetically determined. Earlier unpublished work by the Parasitology Department on 0. circumcincta populations indicated that lamb response to a constant infection showed a more or less continuous variation but did not justify a rigid classification resembling “responder” or “non-responder” lambs. In the present study it was decided to use the standard format of randomly selected lamb groups. MATERIALS Lambs.

AND METHODS

Sixty Dorset Horn lambs (44 females and 16 males) were bred and reared at the laboratory in conditions calculated to prevent any accidental infection by parasitic nematode worms. After weaning at 8 weeks they had free access to hay and water and were given a ration of concentrates which allowed lambs receiving the smallest infection rate to grow at the rate of 0.8-l kg per week. At the start of the experiment they were 18 weeks old and averaged 26 kg in weight. Larvae. The strain of 0. circumcincta was the same as that used in a previous experiment (Hong et al., 1986) with the exception that it had been passaged for a further generation. The third stage larvae had been stored at 4OCfor 3 months but for 3 days prior to use the infection doses were kept at room temperature. Design. The lambs were divided into three groups of 20 (Groups l-3), each of which was further subdivided into five 951

C. HONG,J. F. MICHELand M. B. LANCASTER

952

sets of four (a-e). Selection was random on a stratified liveweight basis and with regard to the sex of the animal so that all except one set contained one male and three females. On day 0 Group 1 was given an oral dose of 250 third stage larvae of 0. circumcincta. Group 2 received a dose of 500 and Group 3 a dose of 1000. This was repeated daily until the day the animal was killed. On days 30, 60, 80, 110 and 140 the lambs of one set (beginning with ‘a’) from each dose group were killed. The abomasa were processed in the manner described by Michel (1969). Worm burdens were estimated by counting total numbers present in at least a 2% aliquot of abomasal contents and digesta. The worms from each aliquot were collected and differentiated into adults (5th) developing (L4) and early fourth stage larvae (E4). In a number of instances when the adult worm burden was low the total adult population was collected. The worms were then stained and mounted on slides as semi-permanent preparations (Michel, Lancaster & Hong, 1971) and the length of each was measured using a technique based on that of Boag (198 1). The development of the female vulval flap was classified either as “flapped” (having the normal membranous structure) or “flapless” (reduced to a small solid swelling or completely absent), these being approximately equivalent to the categories 4-6 and O-3 described by Michel, Lancaster & Hong (1972) for 0. ostertagi

fourth total. One lamb in Group 2 died after 42 days from a cause not related to the experiment and therefore Group 2e contains only three animals. With

the passage of time the lambs within each set show an increased variation in worm burden. Four animals were identified as being atypical (marked with an asterisk in Table 1). Relative to their groups these lambs had abnormally large sized worm burdens comprised mainly of a population of big adults and also exhibited atypically high faecal egg counts (see below). For these reasons the four lambs

RESULTS Table 1 lists the worm burdens of the lambs of each set in their dose groups. Since the daily doses were given to the animals up to the day before they were killed, very small numbers of larvae still in the parasitic third’ stage were generally found in each aliquot counted and these were added to the early Tarus l--POST

MORTEM

WORM

COUNTS

OF LAMBS

,NFECTED

30

0

60

90

120

150

Days FIG. 1. Mean total worm burden for each dose group at five intervals of time. Group 1; *+*** Group 2; ---Group3. DAILY

WITH

250 (GROUP

I),

500 (GROUP2) OR 1000 LARVAE

(GROUP3). Group 1 Number of daily doses

5th 1600 2000 1450 1850 3850 3500

:b?

2550 2950 4800 1450

L4

Group 2

E4

Total

5th

L4

1000 1250 200 150

3300 3850 2300 2500

4700 4100 4650 3900

000 2600 900 2050 900 1300 950 2700

600 850 5300 1050 2000 6550 500 450 3900 850 1750 5150

6800 6100 4300 5500

800 500 100 750

4700 8600 6300 15650

700 600 650 500

E4

Group 3 Total

5th

L4

E4

8300 7050 6850 7550

5600 6200 6900 7550

1750 3400 2050 1150

5400 12750 4450 14050 3600 12550 3850 12550

2500 10100 2900 10500 3900 9300 5600 11850

3000 2900 8600 9300

3500 1800 900 1700

3400 9900 7900 12600 6400 15900 2500 13500

5500 10000 100 700

3300 3550 150 2000

4700 13500 3400 16950* 5600 5850 4900 7600

Total

i”p) C

4650 1850

500 600 5900 300 3000 4750 300 450 5400 100 500 2450

110 (d)

850 3350 100 1100

100 350 0 350

700 1650 900 4600 400 500 300 1750

2900 0 3450 100

1050 50 1100 400

1450 0 2200 1950

5400 50 6750 2450

1750 100 3600 1150

500 250 2800 2650

3350 750 3250 2100

140 (e)

1200 600 1925 225

550 250 225 0

1300 3050 250 1100 175 2325 425 650

3150 650 1950

1250 300 700

2150 1200 1500

6550 2150 4150

0 577 20200 0

50 550 2050 50

900 950 1800 2927 2900 25150* 650 700

* Lambs identified as “abnormal”.

600 3950 9250 850 800 10250 900 3200 10400 800 2650 19100*

6100 1100 9650* 5900

Ostertagia circumcincta in lambs

were regarded as abnormal, excluded from the following group mean results and dealt with separately. Figure 1 shows the course of the population in each of the three dose groups. Figure 2 illustrates the size range of the combined populations of adult female worms at five intervals of time for each of the three dose groups. These values were calculated from the mean percentage incidence of the four lambs in the set with the exception of those cases where the near refractory animal harboured very few female worms for measurement or none at all. The incidence of female worms with membranous vulval flaps (flapped) and those without (flapless) is shown separately. The measurements of the previously identified “abnormal” lambs which had been Group I

j

FIG. 2. The incidence

953

excluded from the mean worm burdens in Fig. 1, have been illustrated separately but on the same axes as their designated groups as pairs of shaded graphs. The pattern of faecal egg counts of the three dose groups is shown in Fig. 3. In addition, the animals previously identified in Group 3 as aberrant were placed in a separate group (Group 4) and the pattern derived from their mean egg counts illustrated. These three lambs showed a completely different response to their worm burdens as they failed to effect any control over egg output (2000-5000 eggs/g) until the final fortnight before slaughter when the sharp decline in the egg count of two of the lambs is suggestive that a significant proportion of the adult worms had been lost rather abruptly. Of the lambs identified as Group

2

Group

3

A_._,____ A I.____ A

..__._.____.

of adult female

worm

sizes in each dose group at five intervals E H‘Abnormal’ animal.

. . . . . Flapless worms.

of time. -

Flapped

worms;

954

C.

HONG,J.

F. MICHEL

and M.

B. LANCASTER

35

30

25

20 . .

12

10

8 a ; 2 fi

6

0 b & c

4

w” 9 J

2

30

60

____________

90

120

150

Days FIG.

3.

Mean

faecal eggcount. -Croup

I; . . . . . c __.._7.

responding abnormally, that in Group 3c had a final worm burden of 10000 adults and the last three weekly egg counts were 1320, 210 and 90; that in Group 3d had 3600 adults (860,570 and 10 eggs per gram) and that in Group 3e had 20200 adults (5 100, 3060 and 7960 eggs per gram). DISCUSSION The results show that the worm burdens in the three dose groups rose to levels related to the infection rate and suggest that the loss of adult worms is related to the number present in the population. As in the case of 0. osferfugi this seems to represent the primary mechanism controlling worm numbers. The consequence of such a mechanism would of course lead worm numbers to rise to and remain at a level directly related to the infection rate. That, in the present experiment, the number of adult worms declines from a peak could mean either fewer worms became

“~“u~~,---“ul”u~~,r-----r”‘uu~,.

c _^.._ ?._

_ P__.._”

established as time progressed or the rate of loss was more rapid. The somewhat lower than expected maximum values for Group 3 would mean that either resistance to development occurred faster at higher infection rates or, as found with 0. ostertagi in cattle (Michel, 1970), mean adult worm life was shorter. A turnover of the worm population can be demonstrated by the observation that the population mean worm size decreases with time and that female worms with a reduced vulva1 flap also become more prevalent. Since there is no evidence that worms shrink as they grow old or that vulval flaps can regress after development (Hong et al., 1986) it appears that the larger worms with fully developed vulval flaps are replaced by a population of smaller worms which show an increasing incidence of reduced vulval flaps. Thus the size of the worm burden can be seen to be regulated by the short mean life of the adult worm. In a study of naturally acquired burdens of in sheep Waller & Thomas 0. circumcincta

Ostertagia circumc~ncta in iambs

(1978a, b) observed that the worm burdens of both tracer and principal lambs corresponded closely to the rise and fall in pasture larval availability and that over the season tracer lambs continued to harbour worm burdens with females exhibiting prominent vulva1 gaps while the continuously grazing pinups lambs were increasingly populated with worms with reduced flaps as the grazing season progressed. They concluded that worms were constantly being replaced and that the turnover period was at approximately monthly intervals. Resistance to the establis~ent of worms evidently develops very rapidly as the present results show a quick decline from peak worm numbers. This differs from the development of a protective immunity in calves where a lengthy exposure to infection is required before they become refractory (Michel, Lancaster & Hong, 1973). Therefore a small degree of in~vidu~ variation in the slope or shape of the curve of resistance to establishment (rate) would result in increasing variation in worm burdens as the experiment progressed. This was in fact observed. The egg count pattern of the three groups develops along similar lines to that in 0. ostertugi but the results show more individual and group variation from the stereotyped curve demonstrated by Michel (1963, 1969). Once the peak egg count is reached, its subsequent decline is rather rapid, suggesting that both reduced fecundity and loss of worm numbers are operating simultaneously. These responses to an 0. circumcincta infection are not shared by the four segregated “abnormal” lambs which showed the same distinc~ve characteristics as the abnormal calves identified by Michel (1963). The worm burdens were all relatively large, comprised of big worms and the uteri of the females contained many eggs. Faecal egg counts of the lambs rose to high levels at which they remained. However, the reduced development of the vulva1 flap provided an independent indication of the age of the infection. The high percentage of flapless worms showed clear evidence that the hosts have considerable experience of infection even though they harboured large “healthy” worms and had high egg counts, both characteristics which might have suggested that the worm burdens were those from ndive animals. Michel (1968) expressed the view that the various manifestations of host resistance might be the effect of different underlying mechanisms. It seems clear that in this host/parasite system the reduction of the vulva1 flap is entirely distinct in its causation from stunting of worms or the suppression of their egg output. Because they differ from the majority in several respects and seem to represent a distinct population, their exclusion from the group mean results is justified. But while this small proportion of animals may be segregated as being grossly abnormal, there are indicatjons that there is considerable variation in the main population which is not sufficiently discontinuous to warrant the rigid division into

955

“responders” and “non-responders” employed by Dineen et al. (1978). It appears that in experimental work on the 0. circumcincta /sheep system, group means, as used among others by Gibson & Whitehead (1981) and Callinan & Arundel (1982), do not adequately describe population trends because there is a high degree of individual variation which may be discontinuous. To describe population trends by excluding a minority of animals that can be identified as quite unlike the majority, as has been done in the present work, is a valid device provided it is recognised (a) that aberrant responses may be the norm in some conditions and (b) that aberrant individuals in a flock may be of great epidemiological significance.

BOAG B. 1981. Measuring nematodes using a digitising tablet and microcomputer. Systematic Parasitology 2: 145-147. CALLINAN A. P. L. & ARUNDEL J.H. 1982. Population dynamics of the parasitic stages of Ostertagia spp. in sheep. International Journal for Parasitology 12: 53153.5. D~NEEN J. K., GREGG, P. & LASCELLES A. K. 1978. The response of lambs to vaccination at weaning with irradiated Trichostrongylus colubriformis larvae: segregation into “responders” and ‘“non-responders”. International Journalfor Parasitology 8: 59-63. DONALD A. D.& WALLER P. J. 1982. Problems and prospects in the control of helminthiasis in sheep. In: 3~010~ and Controt of~ndoparas~tes (Edited by SYUONS L. E.A., DONALD A. D. & DINEEN J. K.), pp. 157-180. Academic Press, London. GIBSON T. E. & WHITEHEAD J. D. 1981. Changes in the worm burden of lambs under continuous infection with Ostertagia circumcincta, British Veterinaty Journal 137: 192-195. HONG C., MICHEL J.F. & LANCASTER M. B. 1986. Populations of Oste~ag~a circ~mc~ncta in Iambs following a single infection. International Journal for Parasitology 16: 63-67. MICHEL J. F. 1963. The phenomena of host resistance and the course of infection of Ostertagia ostertagi in calves. Parasitology 53: 63-84. MICHEL J. F. 1968. Immunity of helminths associated with the tissues. 6th Symposium of the British Society for Parasitology, pp. 67-89. MICHEL J. F. 1969. Some observations on the worm burdens of calves infected daily with Ostertagia ostertagi. Parasitology 59: 575-595. MICHEL J. F: 1970. The regulation of populations of Ostertaaia ostertaai in calves. Parasitoloav 61: 435-447. MICHEL Jy F., LANCASTER M. B. & HoNC’C. 1971. Hostinduced effects on the size and body proportions of Ostertagia osferragi (Stiles, 1892) Ransom, 1907, a nematode parasite of cattle. Journal of Parasitology 57: I l&51189. MICHEL J. F., LANCASTER M. B. & HONG C. 1972. The development and symmetry of the vulval flap of Ostertagia ostertagi. international Journal for Parasiiology 2: 297304.

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C. HONG, J. F. MICHEL and M. B. LANCASTER

MICHEL J. F., LANCASTER M.B. & HONG C. 1973. Ostertagia ostertagi: Protective immunity in calves. The development in calves of a protective immunity to infection with Ostertagia ostertagi. Expen’mental Parasitology 33: 179-186. WALLER P. J. &THOMAS R. J. 1978a. Nematode parasitism

in sheep in North-East England: the epidemiology of Ostertagia species. International Journal for Parasitology 8: 275-283. WALLER P. J. & THOMAS R. J. 1978b. Host-induced effects on the morphology of Ostertugia circumcincta in grazing sheep. International Journalfor Parasitology 8: 365-370.