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Host-induced effects on the morphology of Ostertagia circumcincta in grazing sheep
InternationalJournalfor Parasitology.1978 Vol. 8, pp. 365-370. Pergamon Press. Printed in
Great
Britain.
HOST-INDUCED EFFECTS ON THE MORPHOLOGY OF OSTERTAGIA CIRCUMCINCTA IN GRAZING SHEEP P. J. WALLER* and R. J. THOMAS? *CSIRO Division of Animal Health, McMaster Laboratory, Private Bag No. 1, P.O. Glebe, N.S.W. 2037, Australia and TDepartment of Agriculture, The University of Newcastle-upon-Tyne, England (Received 23 November
1977)
AbSfIW&--WALLER P. J. and THOMAS R. J. 1978.Host-induced effects on the morphology of Ostevrugia circumcincta in grazing sheep. Internutianal Journal for Parasitology 8: 365-370. Morphological changes are described in adult Ostertugia circumcincfa recovered from lambs slaughtered monthly
from June to November. Comparisons were made between continuously grazed principal lambs exposed to infection for increasingly long periods, and tracer lambs exposed for only four weeks. Lambs were either slaughtered immediately off pasture or held 2 weeks under worm free conditions before slaughter. Although no resistance to establishment of infection occurred, shown by comparable worm burdens of principals and tracers, a rapid parasite population turnover was observed in the principal lambs which ceased once the lambs were removed from the infected pasture. Significantly fewer adult female Ostertqqia with fully developed vulva1 flaps were recorded in principals compared to tracers. Such differences are considered to be primarily host induced responses which rapidly come into play, evidenced by the entire populations of developing female worms in tracers slaughtered immediately off pasture showing arrested vulva1 flap development after they had completed development in those tracers held for a further two weeks. A progressive reduction in the proportion of adult male worms also occurred in the principal lambs. Population density was shown to have an inverse correlation with worm size. By analogy, it is suggested that seasonal change in the presence of cuticular inflations in Huemonchus contortus females with linguiform vulva1 flaps may be primarily host induced, rather than a change in the relative fitness of discrete genotypes. INDEX KEY WORDS: sex-ratio; length, width.
Morphology;
Ostertagiu
INTRODUCTION INTRASPECIFIC variability in morphological characters of trichostrongylid nematodes has received considerable attention since Taliaferro & Sarles (1939) reported that adult worms were larger in fully susceptible hosts than in animals which had developed resistance following initial infection. The source of this variation appears to be genetic polymorphism in the parasite and/or host-induced effects on parasite development. The relative importance of these two sources may vary with the species concerned.
Among the nematodes of ruminants a number of morphological types of Haernonchus contortus has been described based on the form of the vulva1 flap (Das & Whitlock, 1960). The three main morph types namely linguiform, knobbed and smooth are distinct genotypes (Daskalov, 1971; Le Jambre & Ractliffe, 1971) and the frequency of these forms does not show seasonal variation in any one environment (Le Jambre & Whitlock, 1968; McKenna, 1971~). However within the linguiform complex, types A and B show seasonal fluctation (Le Jambre
circumcincta
host induced effects; vulva1 flap;
& Whitlock, 1968; Le Jambre & Ractliffe, 1971, 1976; McKenna, 19716). Le Jambre & Ractliffe (1971) consider these types to be genetic variants which exhibit different relative fitness to the free living and host environment resulting in a shift in the prevalent morph type from spring to autumn based on the premise that several parasite generations occur during this time. This change in frequency in vulva1 flap types is analagous to that reported by Michel and co-workers for the cattle nematode, Ostertagia ostertagi. However, in this nematode, variations in the vulva1 flap have been shown to be developmental stages (Michel, Lancaster & Hong, 1972~) and an increase in the frequency of reduced flaps is primarily due to host induced effects associated with resistance (Michel, Lancaster & Hong, 19726), with genetic factors of the parasite playing only a minor role (Michel, Lancaster & Hong, 1976). All observations on 0. ostertagi are based on experimental infections, not on naturally acquired worm populations and no information is available on 0. circumcincta in sheep apart from a description of common flap types by Denham (1969). This paper describes observations made during an epidemio365
366
P. J.
WALLFR
and R.J.
logical study of parasitism in North-East England, in which 0. circumcincta was the dominant species. The design of the experiment enables comparisons to be made between Ostertagia populations in tracer lambs and lambs continuously exposed to larval pickup, i.e. between susceptible lambs and potentially resistant lambs, through the course of seasonal exposure to natural infection. This made it possible to identify changes in morphology, associated with previous experience and intensity of infection under field conditions, and to relate these changes to manifestations of resistance. MATERIALS
AND METHODS
Four one acre plots of previously ungrazed pasture were each stocked with six ewes and twin lambs in April 1973. The lambs were weaned on 23 July by removing the ewes. Each month from June to November two continuously grazed ‘principal’ lambs per plot were randomly selected for slaughter. In addition, from July to November two worm-free ‘tracer’ lambs of the same breed and age as principals were added to each plot and allowed to graze for four weeks before removal on the same day as principals. Of the two principals and tracers per plot removed each month, one was slaughtered immediately and the other after 2 weeks grazing on wormfre: pasture to allow time for development of immature forms to adults. Abomasal worm counts were performed by the methods described by O’Sullivan and Donald (1970). Species differentiation showed over 90% of the Ostertagia population were 0. circunxincta. From the abomasal contents of each lamb, 100 adult Ostertagia females were classified according to vulva1 flap structure into four types: Type l-vulva1 flap entirdly absent; Type 2-rudimentary flao: Tvoe 3-small. knob like flap; Type &fully f&me&‘pr&nent flap. From each lamb taken off one plot, a further 100 adult female Ostertagia were drawn under a camera lucida and measurements of length, and width immediately anterior to the genital orifice were made using an opisometer. RESULTS Analysis of total Ostertagia spp. worm counts showed no significant difference between lambs slaughtered immediately and those slaughtered after 2 weeks for either principal or tracer lambs at each slaughter month. These results are therefore pooled for each lamb type and are shown in Table 1, together with the percentage inhibition estimated from those lambs held 2 weeks before slaughter. The pattern of a rising worm burden to September, followed by a sharp and significant decline (P < O-05) in October and November associated with the onset of accelerating inhibition, is common to both principal and tracer lambs. In addition the numbers of Ostertagia present in principals and tracers were not significantly different for each individual month. Examining the adult female Ostertagia morphology it was found that of the four classes of vulva1 flap, Types 2 and 3 were very infrequent and
TABLE
I.J.P. VOL. 8. 1978
THOMAS
I-TOTAL Ostertagia WORM BURDENS CENTAGE LARVALINHIBITION
Slaughter month June July August September October November
Principal lambs 0, /0 Total Inhibition 0 658 3 4258 5 6399 14,396 4 6362 38 4221 78
AND
PER-
Tracer lambs _ % Total Inhibition 8660 4 2 12,525 3 12,751 9488 48 4427 84 _____.
never accounted for more than 5 y0 of the adult females. These two types have therefore been excluded from further consideration. Analysis of adult female Ostertagia spp. worm burdens showed a significant difference (P < 0.05) between lambs slaughtered immediately and those held for two weeks for both principals and tracers. However, a direct comparison of female Ostertagia burdens, including developing forms as well as adults, is made difficult by the fact that lambs slaughtered immediately off pasture contain a proportion of developing stages, some of which are destined to remain as inhibited fourth stage larvae. To overcome this, the number of inhibited larvae was calculated for each of these lambs, based on the actual percentage inhibition recorded in the corresponding lamb removed from the same plot at the same time but slaughtered after two weeks. The estimated number of inhibited larvae was subtracted from the total immatures and the remainder was multiplied by the proportion of adult females present. This figure was added to the actual number of adult females present to give a ‘derived’ total female burden consisting of immature females assumed destined to develop to adults, and mature females, for lambs slaughtered immediately off pasture (see Fig. I). Analysis of this data showed there was no significant difference between the derived female worm burdens of lambs slaughtered immediately and the actual female Ostertagia infections in lambs slaughtered after 2 weeks for both principal and tracer lambs. Female worm counts were similar to total Ostertagia worm burdens, showing a progressive increase to September, followed by a sharp and significant (P < 0.05) fall in October and November, for both principals and tracers. Likewise, the number of Ostertagia females recorded in principals and tracers were not significantly different for each individual month. However, there were differences in numbers of Type I and Type 4 females between lamb types, slaughter groups and times. From August to October the numbers of Type 4 in principals were significantly less (P CC 0.05) than in tracers. Although there were more Type 1 females in principals than tracers in September, October and November these differences were not significant. Comparisons made between lambs slaughtered immediately and those slaughtered after two weeks showed no significant difference in
I .J.P.
VOL.
8.
1978
Morphology
of Ostertagia circumcincta in grazing sheep
Figure
I. Principals
2 records the percentage adult female of the total adult burden. There were no significant differences between lambs slaughtered immediately and after 2 weeks for either the principals or tracers. Results therefore were pooled for each lamb type. Between August and November inclusive, the proportion of females always exceeded the 95% confidence limits about a sex ratio of one in principal lambs but never in tracers. In both principal and tracer lambs, female Ostertagia showed marked reductions in length and width between June and August, followed by slight inOstertagia
9
Illri, 6
7
A
367
B
4
17
IO
6
12
I_:
2.Trocers
creases
10000
in autumn
(see Fig. 3).
r
i
6000
E
s *
12 -
20
E
la 1 Mean
adult
6000 4000
female
length
. -
Principal
*--o
Tracer
lambs lambs
IO
52 33
rl-l
:
39
“I 9
\
‘\
\
46&
ce
; o:,
‘1 6-
7-
,
FIG. 1. Female Ostertugia spp. Worm burdens mean worm counts of lambs slaughtered immediately off pasture (A) and those slaughtered after 2 weeks (B), and the mean percentage Type 4 adults (numerals above bars). i:3 Immature females assumed destined to develop to adults (see text); n Type 4 adults; n Type 1 adults.
the number of Type 4 females whereas there was a significant increase (P < 0.05) in Type 1 numbers in the latter groups for both principals and tracers.
?L..;~
\
Nov
I July
June
06,-
(b)
Meanadult
.
E 06 071
\
\
0.5 -
\
\
JUW
July
female
e-e
Principal
w--a
Tracer
\
I NOV
width lambs lambs
o-_--
\ \
04
I OCt
I Sept
I August
-0
=-?--*,*
August
sept
I OCI
I NW
FIG. 3. 0. circumcincta infection in lambs.
A striking feature is the larger size of the relatively small number of parasites recovered in June, which were significantly larger than those found in any other month. DISCUSSION
I June
I
I
I
I
July
I
Aug
Sept
OCl
NW
Slaughter
month
FIG. 2. Percentage adult female of total adult Ostertagiu worm burden. e-0 Principal lambs; O---O tracer lambs; ---95% confidence limits about 50% of females.
The continuously grazed principal lambs and the tracers were obtained from the same flock, were similar in age, and grazed the same pastures, differing only in the duration of exposure to infection. They showed a similarity in the seasonal rise and fall in the size of the Ostertagia populations, and in the numbers of developing and adult females recovered each month, which suggests that no accumulation of these parasites occurred in the principal lambs. The autumn decline in numbers could be attributed in
368
P. J. WALLERand R. J. THOMAS
both groups to increasing age of the lambs, but the rise and fall in worm population corresponds closely to the rise and fall in pasture larval availability, which will be reported in detail elsewhere and appears to be simply a reflection of larval intake. These findings indicate that turnover was operating in the principal lambs, similar to that suggested by Michel (1970) for experimental infections with 0. ostertagi in calves. He showed turnover to be rapid, the size of the adult worm population being proportional to the rate of larval intake and the worm loss directly related to the number present. In this work the similarity in worm numbers between the principal lambs and the tracers suggest that turnover was operating at approximately monthly intervals. If this is so then in any one month the populations in both groups of lambs should have been present for the same length of time, and developed from the same pool of infective larvae. From July to September the numbers of Type 4 females in the principal lambs remained relatively constant at below 500 worms but the numbers of Type 1 increased progressively (Fig. 1). Signjfi~antly more Type 4 females were recovered from tracers than principals during these months, with proportionately fewer Type 1. These differences, despite the similarity among principals and tracers in total adult female numbers, provides strong evidence that host induced responses suppressing flap development were operating in the principal lambs. Further evidence is provided by comparing the numbers of each flap type between lambs slaughtered immediately and those slaughtered after 2 weeks. Total numbers of Ostertagia females between the two slaughter groups were not significantly different (Fig. l), showing that no worm loss occurred over the holding period. Type 4 female numbers also showed no significant difference, but there was a significant increase in the number of Type I females over two weeks in both principals and tracers. This clearly indicates that the majority of developing stages present in the lambs when removed from the pasture become Type 1 adult females rather than Type 4. It may be concluded that this is due to a host-induced effect resulting from previous exposure to infection, and since it occurs in tracers as well as principals this exposure period need be no more than 4 weeks to produce some effect. Michet et al. (1972b) demonstrated this change in flap pattern in 0. ostertagi in experilnenta1 challenge infections and suggested that it results from a deterioration in the parasite environment consequent on previous experience of infection, which arrests Rap development without arresting development of the worm as a whole. The similarity between Michel’s results and those reported here suggest that, as in 0. osteutagi, vulva] flap types identified in 0. circumciircta are not genetic morphs but are stages in the development of flap, and that Type 1, the earliest stage, is most susceptible to arrest. Further-
I.J.P. VOL. 8. 1978
more, it demonstrates that the effect produced experimentally in 0. ostertagi occurs during the course of natural seasonal infection in 0. circumcintta. While host-induced responses appear to have a major effect on flap development, approximately 100-200 of females had fully developed flaps even in November. Michel et al. (1976) showed that the tendency to complete or incomplete development of the flap in 0. ostertagi could be inherited, possibly an inherited variation in susceptibility to the factors arresting development. A similar explanation would account for the residue of Type 4 females in the principal lambs. A similar seasonal change in the frequency of different flap types has been reported in H. contartus (1-e Jambre & Ractliffe, 1971) for Types A and B of the linguiform complex. However, these authors attribute the flap change in H. contortus to a change in the relative fitness of two genetically distinct types. They consider that the dominant allele of Type A overwinters more successfully while the larval stage of the recessive Type B a!iele survives better during the summer, and suggest that a gradual shift in the prevalent morph type occurs over a series of generations from spring to autumn. This explanation cannot be applied to the flap change in 0. ciwumcincta. Lambs in this study grazed on clean pasture where no overwintered larvae were present, and all larvae at least until August were derived from the ewe egg output in spring (Boag & Thomas, 1971) and represent only a single generation. In addition the relatively constant proportions of Type 4 and Type I flaps in the tracers, exposed to the same larval population, confirms that no major change in the available infective larvae occurred over this period, i.e. preferential survival of one type showing better climatic adaptation did not occur. Since the explanation proposed by Le Jambre & Ractliffe (1971) does not hold for 0. ri~~33mri33cta. the question arises whether on alternative interpretation of the H. ~offt5~tus data can be made. Although breeding experiments with different morph types (Le Jambre, 1977) suggest a genetic basis controlling the expression of A and B types, none of the published material by Lc Jambre and co-workers excludes the possibility that at least part of expression of phenotypic characters is modified by host age, experience of infection or density dependence. Daskalov (1972) presents evidence that cuticular inflations near or over the vulva1 flap in H. ro~t5rt13s~ which are the basis of classification into Types A and B, are nonheritable characters, and claims that their appearance is associated with increasing worm age and reproductive activity. This interpretation is open to question however, since if the actual worm counts rather than percentages of flap types are examined host selection seems apparent. Additionally, DaskaIov reports slower maturation rates and a lower percentage of females with cuticular in~ations in
I.J.P. VOL. 8.
1978
Morphology
of Ostertagia circumcincta in grazing sheep
infected goats, while virtually no worms with cuticular inflations developed from a superimposed larval dose given to a lamb with a pre-existing H. ~on~o~~~fsburden. These results are better explained as host effects, the development of cuticular inflations being modified by an abnormal or resistant host. Such a modification is likely to be in the direction of suppressing development and this is consistent with Michel’s hypothesis for 0. ostertagi and the present work with 0. circumcincta. Of the other characters studied, the percentage of adult worms in principal lambs slaughtered after July exceeds the 95 % confidence limits for equal sex distribution. This is in contrast to the levels of adult females in the tracers which were within these limits on all slaughter occasions. The percentage of males in the principal lambs fell rapidly in October and November, when high levels of larval inhibition were recorded, and the correlation of inhibition with the development of fewer males in previously exposed hosts is in agreement with work by Fox (1976) on Obeliscoides cuniculi.
This unequa! sex distribution could be due to an innate difference in the rate of development or turnover of the sexes, but appears more likely to be due to a greater susceptibility of male worms to the effects of host control mechanisms. Although not statistically significant, size variations were recorded in worms from both principals and tracers from July to November. In tracers, an initial decline in size was followed by a terminal rise and this correlates inversely with the rise and fall in parasite numbers and in the rate of infection, and thus size appears to be influenced by a density dependent factor. A similar initial fall occurred in the principal Iambs, the terminal rise was less marked, and the smallest worms were recovered in October when adult worm populations had fallen below those in August and September, so that in these lambs host responses also appeared to play a part in limiting worm size. These results agree with those reported by 0. ostertagi by Michel et al. (1971), who found worm length to be affected by both population size and developing host resistance associated with increasing duration of exposure to infection. It is clear that in the field a number of factors influence the morphologica development of 0. civcumcincfa, and while their effects may be cumulative, different morphological features vary in their sensitivity to individual factors. In particular vulva1 flap development appears to be highly sensitive to host-induced effects and will indicate a host response to prior infection which is not apparent from the worm burden levels. It may therefore prove useful as a tool in the investigation of the development of host resistance. Ack~ow~e~ge~~nTs-The authors are indebted to Mrs. J. Snowden, Mrs. C. Briertey and Mr. J. Wightman for technical assistance and to the Agricultural Research
369
Council for financial support. P. J. Waller was the holder of a C.S.K.R.O. Overseas Divisional Studentship whilst this work was being carried out. REFERENCES Bono B. & THOMASR. J. 1971. Epidemiological studies on gastrointestinal nematode parasites of sheep. Infection patterns on clean and autumn-contaminated pasture. Research in Veterinary Science 12: 132-139. DAS K. M. & WHITLOCKJ. H. 1960. Subspeciation in Haenmchus contortus (Rudlophi J 803) Nemata, Tr~~hostrongy~oidea. Cornell Vererinarian 50: 182-197. DASKAL~VP. R; 1971. ~ae~~~c~us contorrus : genetically determined polymorphism in females. Experimental Parasitology 29: 351-366. DASKALOVP. B. 1972. ~ae~unc~as contortus: factors determining the polymorphism of linguiform females. Experimental Parasitology 32: 364-368. DENHAM D. A. 1969. The development of Ostertugia circumcincta in lambs. Journal of Heimintho[ogy 43: 299-310. Fox J. C. 1976. Inhibited development of Obe/iscoides cuniculi in rabbits: the effects of active and passive immunization and resumption of larval development. Veterinary Paras~to~og~~1: 209-220. LE JAMBREL. F. 1977. Geneties of vutvar morph types in ffaemonchus contortus: Huenmtchus contorfus cayugensis from the Finger Lakes region of New York. I~~ernali~na~J~l~r~u~f#r Parasitology 7: 9-14. LE JAIWBREL. F. & RACTLIFFEL. H. 1971. Seasonal change in a balanced polymorphism in Haew;onchus contortus populations. Purasito~ogy 62: 151-155. LE JAMBREL. F. & RACTLIFFEL. H. 1976. Response of Hae~onchus rontortus cayugensis to a change in the ratio of smooth to hnguiform. ~ffru~~~u/~~~73: 213222. LE JAMBREL F. & WHITLOCK J H. I968 Seasonal fluctuation in linguiform morphs of ~ffe~~~izc~lrs contortus cayugensis. Journal of Parasitology 54: 827-830. MCKENNA P B. 1971~. Morphological evidence of subspeciation in Huemonchus conrarrus from New Zealand sheep: the vulvai flap formula. Nenl Zealand Journaf of~gric~~~iu$a~Research 14: 902-914. MCKENNA P B. 19716. The effect of season on the constancy of the vulva1 flap formula of Haenzonchus ronfortus.Nenj Zealand Journal of~gr~cl~~tl~ralResearch 14: 91.5-922. MI~HEL J. F. 1970. The regulation of populations of Ostertagia ostertqgi in calves. Parasitology 61: 43% 447. MICHELJ. F LANCASTERM. 3. & HONG C. 1971. Hostinduced effects on the size and body proportions of Osfert@-i osterfagi (Stiles, 1893) Ransom, 1907, a nematode parasite of cattle. Journal qf Parasitology 57: 1185-1189. MICHELJ. F,, LANCASTERM. B. & HONG C. 1972a. The development and symmetry of the vulva2 flap of Ostertagia Ostertqgi. Internatiunai Journal for Parasito/ogy 2: 297-304. MICHELJ. F., LANCASTERM. B. & HONG C. 1972b. Hostinduced effects on the vulva1 Rap of Ostertagia ostertagi International Jourml for Parasitobgy 2: 305-3 17. MICHELJ. F., LANCASTERM. B. & Hor~c C. 1976. The effect of genetic factors on the vulva1 flap of Ostertagia os1ertugi. ~nternario~ai Journal for Parasifol~gy 6: 83-86
370
P. J. WALLER and R. J. THOMAS
O’SULLIVAN B. M. & DONALD A. D. 1970. A field study of nematode parasite populations in the lactating ewe. Pnuasitology 61: 301-3 15.
TALIAPERRO W. H. reactions in the and immune rats muris. Journal of
I.J.P. VOL. 8. 1978 & SARLES M. P. 1939. The cellular skin, lungs and intestine of normal after infection with Nippostrongylus lnfkctious Diseases 64: 157- 192.
Great
Britain.
HOST-INDUCED EFFECTS ON THE MORPHOLOGY OF OSTERTAGIA CIRCUMCINCTA IN GRAZING SHEEP P. J. WALLER* and R. J. THOMAS? *CSIRO Division of Animal Health, McMaster Laboratory, Private Bag No. 1, P.O. Glebe, N.S.W. 2037, Australia and TDepartment of Agriculture, The University of Newcastle-upon-Tyne, England (Received 23 November
1977)
AbSfIW&--WALLER P. J. and THOMAS R. J. 1978.Host-induced effects on the morphology of Ostevrugia circumcincta in grazing sheep. Internutianal Journal for Parasitology 8: 365-370. Morphological changes are described in adult Ostertugia circumcincfa recovered from lambs slaughtered monthly
from June to November. Comparisons were made between continuously grazed principal lambs exposed to infection for increasingly long periods, and tracer lambs exposed for only four weeks. Lambs were either slaughtered immediately off pasture or held 2 weeks under worm free conditions before slaughter. Although no resistance to establishment of infection occurred, shown by comparable worm burdens of principals and tracers, a rapid parasite population turnover was observed in the principal lambs which ceased once the lambs were removed from the infected pasture. Significantly fewer adult female Ostertqqia with fully developed vulva1 flaps were recorded in principals compared to tracers. Such differences are considered to be primarily host induced responses which rapidly come into play, evidenced by the entire populations of developing female worms in tracers slaughtered immediately off pasture showing arrested vulva1 flap development after they had completed development in those tracers held for a further two weeks. A progressive reduction in the proportion of adult male worms also occurred in the principal lambs. Population density was shown to have an inverse correlation with worm size. By analogy, it is suggested that seasonal change in the presence of cuticular inflations in Huemonchus contortus females with linguiform vulva1 flaps may be primarily host induced, rather than a change in the relative fitness of discrete genotypes. INDEX KEY WORDS: sex-ratio; length, width.
Morphology;
Ostertagiu
INTRODUCTION INTRASPECIFIC variability in morphological characters of trichostrongylid nematodes has received considerable attention since Taliaferro & Sarles (1939) reported that adult worms were larger in fully susceptible hosts than in animals which had developed resistance following initial infection. The source of this variation appears to be genetic polymorphism in the parasite and/or host-induced effects on parasite development. The relative importance of these two sources may vary with the species concerned.
Among the nematodes of ruminants a number of morphological types of Haernonchus contortus has been described based on the form of the vulva1 flap (Das & Whitlock, 1960). The three main morph types namely linguiform, knobbed and smooth are distinct genotypes (Daskalov, 1971; Le Jambre & Ractliffe, 1971) and the frequency of these forms does not show seasonal variation in any one environment (Le Jambre & Whitlock, 1968; McKenna, 1971~). However within the linguiform complex, types A and B show seasonal fluctation (Le Jambre
circumcincta
host induced effects; vulva1 flap;
& Whitlock, 1968; Le Jambre & Ractliffe, 1971, 1976; McKenna, 19716). Le Jambre & Ractliffe (1971) consider these types to be genetic variants which exhibit different relative fitness to the free living and host environment resulting in a shift in the prevalent morph type from spring to autumn based on the premise that several parasite generations occur during this time. This change in frequency in vulva1 flap types is analagous to that reported by Michel and co-workers for the cattle nematode, Ostertagia ostertagi. However, in this nematode, variations in the vulva1 flap have been shown to be developmental stages (Michel, Lancaster & Hong, 1972~) and an increase in the frequency of reduced flaps is primarily due to host induced effects associated with resistance (Michel, Lancaster & Hong, 19726), with genetic factors of the parasite playing only a minor role (Michel, Lancaster & Hong, 1976). All observations on 0. ostertagi are based on experimental infections, not on naturally acquired worm populations and no information is available on 0. circumcincta in sheep apart from a description of common flap types by Denham (1969). This paper describes observations made during an epidemio365
366
P. J.
WALLFR
and R.J.
logical study of parasitism in North-East England, in which 0. circumcincta was the dominant species. The design of the experiment enables comparisons to be made between Ostertagia populations in tracer lambs and lambs continuously exposed to larval pickup, i.e. between susceptible lambs and potentially resistant lambs, through the course of seasonal exposure to natural infection. This made it possible to identify changes in morphology, associated with previous experience and intensity of infection under field conditions, and to relate these changes to manifestations of resistance. MATERIALS
AND METHODS
Four one acre plots of previously ungrazed pasture were each stocked with six ewes and twin lambs in April 1973. The lambs were weaned on 23 July by removing the ewes. Each month from June to November two continuously grazed ‘principal’ lambs per plot were randomly selected for slaughter. In addition, from July to November two worm-free ‘tracer’ lambs of the same breed and age as principals were added to each plot and allowed to graze for four weeks before removal on the same day as principals. Of the two principals and tracers per plot removed each month, one was slaughtered immediately and the other after 2 weeks grazing on wormfre: pasture to allow time for development of immature forms to adults. Abomasal worm counts were performed by the methods described by O’Sullivan and Donald (1970). Species differentiation showed over 90% of the Ostertagia population were 0. circunxincta. From the abomasal contents of each lamb, 100 adult Ostertagia females were classified according to vulva1 flap structure into four types: Type l-vulva1 flap entirdly absent; Type 2-rudimentary flao: Tvoe 3-small. knob like flap; Type &fully f&me&‘pr&nent flap. From each lamb taken off one plot, a further 100 adult female Ostertagia were drawn under a camera lucida and measurements of length, and width immediately anterior to the genital orifice were made using an opisometer. RESULTS Analysis of total Ostertagia spp. worm counts showed no significant difference between lambs slaughtered immediately and those slaughtered after 2 weeks for either principal or tracer lambs at each slaughter month. These results are therefore pooled for each lamb type and are shown in Table 1, together with the percentage inhibition estimated from those lambs held 2 weeks before slaughter. The pattern of a rising worm burden to September, followed by a sharp and significant decline (P < O-05) in October and November associated with the onset of accelerating inhibition, is common to both principal and tracer lambs. In addition the numbers of Ostertagia present in principals and tracers were not significantly different for each individual month. Examining the adult female Ostertagia morphology it was found that of the four classes of vulva1 flap, Types 2 and 3 were very infrequent and
TABLE
I.J.P. VOL. 8. 1978
THOMAS
I-TOTAL Ostertagia WORM BURDENS CENTAGE LARVALINHIBITION
Slaughter month June July August September October November
Principal lambs 0, /0 Total Inhibition 0 658 3 4258 5 6399 14,396 4 6362 38 4221 78
AND
PER-
Tracer lambs _ % Total Inhibition 8660 4 2 12,525 3 12,751 9488 48 4427 84 _____.
never accounted for more than 5 y0 of the adult females. These two types have therefore been excluded from further consideration. Analysis of adult female Ostertagia spp. worm burdens showed a significant difference (P < 0.05) between lambs slaughtered immediately and those held for two weeks for both principals and tracers. However, a direct comparison of female Ostertagia burdens, including developing forms as well as adults, is made difficult by the fact that lambs slaughtered immediately off pasture contain a proportion of developing stages, some of which are destined to remain as inhibited fourth stage larvae. To overcome this, the number of inhibited larvae was calculated for each of these lambs, based on the actual percentage inhibition recorded in the corresponding lamb removed from the same plot at the same time but slaughtered after two weeks. The estimated number of inhibited larvae was subtracted from the total immatures and the remainder was multiplied by the proportion of adult females present. This figure was added to the actual number of adult females present to give a ‘derived’ total female burden consisting of immature females assumed destined to develop to adults, and mature females, for lambs slaughtered immediately off pasture (see Fig. I). Analysis of this data showed there was no significant difference between the derived female worm burdens of lambs slaughtered immediately and the actual female Ostertagia infections in lambs slaughtered after 2 weeks for both principal and tracer lambs. Female worm counts were similar to total Ostertagia worm burdens, showing a progressive increase to September, followed by a sharp and significant (P < 0.05) fall in October and November, for both principals and tracers. Likewise, the number of Ostertagia females recorded in principals and tracers were not significantly different for each individual month. However, there were differences in numbers of Type I and Type 4 females between lamb types, slaughter groups and times. From August to October the numbers of Type 4 in principals were significantly less (P CC 0.05) than in tracers. Although there were more Type 1 females in principals than tracers in September, October and November these differences were not significant. Comparisons made between lambs slaughtered immediately and those slaughtered after two weeks showed no significant difference in
I .J.P.
VOL.
8.
1978
Morphology
of Ostertagia circumcincta in grazing sheep
Figure
I. Principals
2 records the percentage adult female of the total adult burden. There were no significant differences between lambs slaughtered immediately and after 2 weeks for either the principals or tracers. Results therefore were pooled for each lamb type. Between August and November inclusive, the proportion of females always exceeded the 95% confidence limits about a sex ratio of one in principal lambs but never in tracers. In both principal and tracer lambs, female Ostertagia showed marked reductions in length and width between June and August, followed by slight inOstertagia
9
Illri, 6
7
A
367
B
4
17
IO
6
12
I_:
2.Trocers
creases
10000
in autumn
(see Fig. 3).
r
i
6000
E
s *
12 -
20
E
la 1 Mean
adult
6000 4000
female
length
. -
Principal
*--o
Tracer
lambs lambs
IO
52 33
rl-l
:
39
“I 9
\
‘\
\
46&
ce
; o:,
‘1 6-
7-
,
FIG. 1. Female Ostertugia spp. Worm burdens mean worm counts of lambs slaughtered immediately off pasture (A) and those slaughtered after 2 weeks (B), and the mean percentage Type 4 adults (numerals above bars). i:3 Immature females assumed destined to develop to adults (see text); n Type 4 adults; n Type 1 adults.
the number of Type 4 females whereas there was a significant increase (P < 0.05) in Type 1 numbers in the latter groups for both principals and tracers.
?L..;~
\
Nov
I July
June
06,-
(b)
Meanadult
.
E 06 071
\
\
0.5 -
\
\
JUW
July
female
e-e
Principal
w--a
Tracer
\
I NOV
width lambs lambs
o-_--
\ \
04
I OCt
I Sept
I August
-0
=-?--*,*
August
sept
I OCI
I NW
FIG. 3. 0. circumcincta infection in lambs.
A striking feature is the larger size of the relatively small number of parasites recovered in June, which were significantly larger than those found in any other month. DISCUSSION
I June
I
I
I
I
July
I
Aug
Sept
OCl
NW
Slaughter
month
FIG. 2. Percentage adult female of total adult Ostertagiu worm burden. e-0 Principal lambs; O---O tracer lambs; ---95% confidence limits about 50% of females.
The continuously grazed principal lambs and the tracers were obtained from the same flock, were similar in age, and grazed the same pastures, differing only in the duration of exposure to infection. They showed a similarity in the seasonal rise and fall in the size of the Ostertagia populations, and in the numbers of developing and adult females recovered each month, which suggests that no accumulation of these parasites occurred in the principal lambs. The autumn decline in numbers could be attributed in
368
P. J. WALLERand R. J. THOMAS
both groups to increasing age of the lambs, but the rise and fall in worm population corresponds closely to the rise and fall in pasture larval availability, which will be reported in detail elsewhere and appears to be simply a reflection of larval intake. These findings indicate that turnover was operating in the principal lambs, similar to that suggested by Michel (1970) for experimental infections with 0. ostertagi in calves. He showed turnover to be rapid, the size of the adult worm population being proportional to the rate of larval intake and the worm loss directly related to the number present. In this work the similarity in worm numbers between the principal lambs and the tracers suggest that turnover was operating at approximately monthly intervals. If this is so then in any one month the populations in both groups of lambs should have been present for the same length of time, and developed from the same pool of infective larvae. From July to September the numbers of Type 4 females in the principal lambs remained relatively constant at below 500 worms but the numbers of Type 1 increased progressively (Fig. 1). Signjfi~antly more Type 4 females were recovered from tracers than principals during these months, with proportionately fewer Type 1. These differences, despite the similarity among principals and tracers in total adult female numbers, provides strong evidence that host induced responses suppressing flap development were operating in the principal lambs. Further evidence is provided by comparing the numbers of each flap type between lambs slaughtered immediately and those slaughtered after 2 weeks. Total numbers of Ostertagia females between the two slaughter groups were not significantly different (Fig. l), showing that no worm loss occurred over the holding period. Type 4 female numbers also showed no significant difference, but there was a significant increase in the number of Type I females over two weeks in both principals and tracers. This clearly indicates that the majority of developing stages present in the lambs when removed from the pasture become Type 1 adult females rather than Type 4. It may be concluded that this is due to a host-induced effect resulting from previous exposure to infection, and since it occurs in tracers as well as principals this exposure period need be no more than 4 weeks to produce some effect. Michet et al. (1972b) demonstrated this change in flap pattern in 0. ostertagi in experilnenta1 challenge infections and suggested that it results from a deterioration in the parasite environment consequent on previous experience of infection, which arrests Rap development without arresting development of the worm as a whole. The similarity between Michel’s results and those reported here suggest that, as in 0. osteutagi, vulva] flap types identified in 0. circumciircta are not genetic morphs but are stages in the development of flap, and that Type 1, the earliest stage, is most susceptible to arrest. Further-
I.J.P. VOL. 8. 1978
more, it demonstrates that the effect produced experimentally in 0. ostertagi occurs during the course of natural seasonal infection in 0. circumcintta. While host-induced responses appear to have a major effect on flap development, approximately 100-200 of females had fully developed flaps even in November. Michel et al. (1976) showed that the tendency to complete or incomplete development of the flap in 0. ostertagi could be inherited, possibly an inherited variation in susceptibility to the factors arresting development. A similar explanation would account for the residue of Type 4 females in the principal lambs. A similar seasonal change in the frequency of different flap types has been reported in H. contartus (1-e Jambre & Ractliffe, 1971) for Types A and B of the linguiform complex. However, these authors attribute the flap change in H. contortus to a change in the relative fitness of two genetically distinct types. They consider that the dominant allele of Type A overwinters more successfully while the larval stage of the recessive Type B a!iele survives better during the summer, and suggest that a gradual shift in the prevalent morph type occurs over a series of generations from spring to autumn. This explanation cannot be applied to the flap change in 0. ciwumcincta. Lambs in this study grazed on clean pasture where no overwintered larvae were present, and all larvae at least until August were derived from the ewe egg output in spring (Boag & Thomas, 1971) and represent only a single generation. In addition the relatively constant proportions of Type 4 and Type I flaps in the tracers, exposed to the same larval population, confirms that no major change in the available infective larvae occurred over this period, i.e. preferential survival of one type showing better climatic adaptation did not occur. Since the explanation proposed by Le Jambre & Ractliffe (1971) does not hold for 0. ri~~33mri33cta. the question arises whether on alternative interpretation of the H. ~offt5~tus data can be made. Although breeding experiments with different morph types (Le Jambre, 1977) suggest a genetic basis controlling the expression of A and B types, none of the published material by Lc Jambre and co-workers excludes the possibility that at least part of expression of phenotypic characters is modified by host age, experience of infection or density dependence. Daskalov (1972) presents evidence that cuticular inflations near or over the vulva1 flap in H. ro~t5rt13s~ which are the basis of classification into Types A and B, are nonheritable characters, and claims that their appearance is associated with increasing worm age and reproductive activity. This interpretation is open to question however, since if the actual worm counts rather than percentages of flap types are examined host selection seems apparent. Additionally, DaskaIov reports slower maturation rates and a lower percentage of females with cuticular in~ations in
I.J.P. VOL. 8.
1978
Morphology
of Ostertagia circumcincta in grazing sheep
infected goats, while virtually no worms with cuticular inflations developed from a superimposed larval dose given to a lamb with a pre-existing H. ~on~o~~~fsburden. These results are better explained as host effects, the development of cuticular inflations being modified by an abnormal or resistant host. Such a modification is likely to be in the direction of suppressing development and this is consistent with Michel’s hypothesis for 0. ostertagi and the present work with 0. circumcincta. Of the other characters studied, the percentage of adult worms in principal lambs slaughtered after July exceeds the 95 % confidence limits for equal sex distribution. This is in contrast to the levels of adult females in the tracers which were within these limits on all slaughter occasions. The percentage of males in the principal lambs fell rapidly in October and November, when high levels of larval inhibition were recorded, and the correlation of inhibition with the development of fewer males in previously exposed hosts is in agreement with work by Fox (1976) on Obeliscoides cuniculi.
This unequa! sex distribution could be due to an innate difference in the rate of development or turnover of the sexes, but appears more likely to be due to a greater susceptibility of male worms to the effects of host control mechanisms. Although not statistically significant, size variations were recorded in worms from both principals and tracers from July to November. In tracers, an initial decline in size was followed by a terminal rise and this correlates inversely with the rise and fall in parasite numbers and in the rate of infection, and thus size appears to be influenced by a density dependent factor. A similar initial fall occurred in the principal Iambs, the terminal rise was less marked, and the smallest worms were recovered in October when adult worm populations had fallen below those in August and September, so that in these lambs host responses also appeared to play a part in limiting worm size. These results agree with those reported by 0. ostertagi by Michel et al. (1971), who found worm length to be affected by both population size and developing host resistance associated with increasing duration of exposure to infection. It is clear that in the field a number of factors influence the morphologica development of 0. civcumcincfa, and while their effects may be cumulative, different morphological features vary in their sensitivity to individual factors. In particular vulva1 flap development appears to be highly sensitive to host-induced effects and will indicate a host response to prior infection which is not apparent from the worm burden levels. It may therefore prove useful as a tool in the investigation of the development of host resistance. Ack~ow~e~ge~~nTs-The authors are indebted to Mrs. J. Snowden, Mrs. C. Briertey and Mr. J. Wightman for technical assistance and to the Agricultural Research
369
Council for financial support. P. J. Waller was the holder of a C.S.K.R.O. Overseas Divisional Studentship whilst this work was being carried out. REFERENCES Bono B. & THOMASR. J. 1971. Epidemiological studies on gastrointestinal nematode parasites of sheep. Infection patterns on clean and autumn-contaminated pasture. Research in Veterinary Science 12: 132-139. DAS K. M. & WHITLOCKJ. H. 1960. Subspeciation in Haenmchus contortus (Rudlophi J 803) Nemata, Tr~~hostrongy~oidea. Cornell Vererinarian 50: 182-197. DASKAL~VP. R; 1971. ~ae~~~c~us contorrus : genetically determined polymorphism in females. Experimental Parasitology 29: 351-366. DASKALOVP. B. 1972. ~ae~unc~as contortus: factors determining the polymorphism of linguiform females. Experimental Parasitology 32: 364-368. DENHAM D. A. 1969. The development of Ostertugia circumcincta in lambs. Journal of Heimintho[ogy 43: 299-310. Fox J. C. 1976. Inhibited development of Obe/iscoides cuniculi in rabbits: the effects of active and passive immunization and resumption of larval development. Veterinary Paras~to~og~~1: 209-220. LE JAMBREL. F. 1977. Geneties of vutvar morph types in ffaemonchus contortus: Huenmtchus contorfus cayugensis from the Finger Lakes region of New York. I~~ernali~na~J~l~r~u~f#r Parasitology 7: 9-14. LE JAIWBREL. F. & RACTLIFFEL. H. 1971. Seasonal change in a balanced polymorphism in Haew;onchus contortus populations. Purasito~ogy 62: 151-155. LE JAMBREL. F. & RACTLIFFEL. H. 1976. Response of Hae~onchus rontortus cayugensis to a change in the ratio of smooth to hnguiform. ~ffru~~~u/~~~73: 213222. LE JAMBREL F. & WHITLOCK J H. I968 Seasonal fluctuation in linguiform morphs of ~ffe~~~izc~lrs contortus cayugensis. Journal of Parasitology 54: 827-830. MCKENNA P B. 1971~. Morphological evidence of subspeciation in Huemonchus conrarrus from New Zealand sheep: the vulvai flap formula. Nenl Zealand Journaf of~gric~~~iu$a~Research 14: 902-914. MCKENNA P B. 19716. The effect of season on the constancy of the vulva1 flap formula of Haenzonchus ronfortus.Nenj Zealand Journal of~gr~cl~~tl~ralResearch 14: 91.5-922. MI~HEL J. F. 1970. The regulation of populations of Ostertagia ostertqgi in calves. Parasitology 61: 43% 447. MICHELJ. F LANCASTERM. 3. & HONG C. 1971. Hostinduced effects on the size and body proportions of Osfert@-i osterfagi (Stiles, 1893) Ransom, 1907, a nematode parasite of cattle. Journal qf Parasitology 57: 1185-1189. MICHELJ. F,, LANCASTERM. B. & HONG C. 1972a. The development and symmetry of the vulva2 flap of Ostertagia Ostertqgi. Internatiunai Journal for Parasito/ogy 2: 297-304. MICHELJ. F., LANCASTERM. B. & HONG C. 1972b. Hostinduced effects on the vulva1 Rap of Ostertagia ostertagi International Jourml for Parasitobgy 2: 305-3 17. MICHELJ. F., LANCASTERM. B. & Hor~c C. 1976. The effect of genetic factors on the vulva1 flap of Ostertagia os1ertugi. ~nternario~ai Journal for Parasifol~gy 6: 83-86
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O’SULLIVAN B. M. & DONALD A. D. 1970. A field study of nematode parasite populations in the lactating ewe. Pnuasitology 61: 301-3 15.
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I.J.P. VOL. 8. 1978 & SARLES M. P. 1939. The cellular skin, lungs and intestine of normal after infection with Nippostrongylus lnfkctious Diseases 64: 157- 192.