The role of thresholds in the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae

Internotional Journd/or Parasitology Vol. 14, No. 4, pp. 423-428, 1984. Printed in Great Britain.

0

OOZO-7519/84/$3.00+0.00 Pergamon Press Ltd. I984 Australian Society for Parasitology

THE ROLE OF THRESHOLDS IN THE RESPONSE OF LAMBS TO VACCINATION WITH IRRADIATED TRZCHOSTRONGYLUS COL. UBRZFORMZS LARVAE R. G. WINDON,

J. K. DINEEN,

P. GREGG,*

CSIRO Division of Animal Health, McMaster Laboratory, and -j-CSIRO Division of Mathematics and Statistics,

D. A.

Gmmms-f

and A. D.

DONALD

Private Bag No. 1, P.O. Glebe, N.S.W. 2037, Australia P.O. Box 218, Lindfield, N.S.W. 2070, Australia

(Received 12 September 1983) Abstract-WINDON R. G., DINEEN J. K., GREGG P., GRIFFITHSD. A. and DONALDA. D. 1984. The role of thresholds in the response of lambs to vaccination with irradiated Trichostrongylus colubr$ormis larvae. InternationalJournalfor Parusitofogy 14: 423-428. A piecewise logarithmic model fitted to worm counts of ewe lambs vaccinated and challenged in pens with a range of doses of irradiated and normal Trichostrongylus colubriformis larvae respectively, indicated that the threshold for response to both vaccine (V, = 4400) and challenge dose is exceeded by 5000 larvae. Whereas response was vaccine dose dependent, it was independent of challenge dose. Ram lambs vaccinated at low dose levels were as resistant against challenge as ewe lambs, but by contrast, failed to show increased protection after vaccination with high doses of irradiated larvae. Serum titre of antiworm complement-fixing antibodies at the time of challenge also indicated that ram lambs were less responsive immunologically than ewe lambs following vaccination at the higher dose levels. A field study showed that response to vaccination was only apparent after transfer of the sheep to heavily contaminated pastures, suggesting that previous exposure of the vaccinated animals to the low dose of infective larvae available on a lightly contaminated pasture constituted a challenge which was below the threshold.

INDEX KEY WORDS: Thresholds; vaccination; challenge; complement-fixing antibodies; pen study; field study.

T. colubriformis; sheep; regression

model;

lambs, for, in addition to immediately depressing production parameters, long-lasting deleterious effects may extend into the animal’s mature life. A vaccination programme therefore, should at least aim to lower the immunological threshold to a level whereby the pathogenic effects of the residual worm burden are substantially reduced and considerable economic benefits achieved (Dineen, 1978). It is important to concentrate efforts to protect susceptible categories of sheep, for example the young, lactating and nutritionally stressed. There have been several reports concerning regulation of Trichostrongylus colubriformis in lambs exposed to continuous infection with larvae. The studies of Gibson & Parfitt (1972, 1973) and Chiejina & Sewell (1974a, b) have demonstrated that regulation is achieved only after a period of increasing worm burdens. This regulation first operates by inhibiting establishment of incoming larvae and later acts to cause expulsion of adult worms. More recently, Waller & Thomas (1981) demonstrated a similar phenomenon for naturally acquired field infections in young lambs. These workers suggested that such mechanisms are under threshold control,

INTRODUCTION IN EARLIER

publications it was suggested that the immunological response of the host plays a crucial role during evolution of the host-parasite relationship and by natural selection is involved in establishing a dynamic equilibrium between host and parasite to ensure survival of both (Dineen, 1963; Dineen, 1978). Furthermore it was suggested that regulation of the equilibrium occurs at that level of antigenic information which causes immunological recognition of the parasitic biomass and may be subject to genetically-determined variation between individuals in an outbred host population. Above the critical level, or threshold, immune mechanisms are expected to be triggered and act to control the parasite burden, while below the threshold a certain number of parasites remain undetected by the host. The pathogenic effects of low worm burdens can cause substantial loss of liveweight gain and wool growth (Steel, Symons & Jones, 1980). In this respect trichostrongylosis is especially important in young *Present address: Department of Microbiology, New England University, Armidale, N.S.W. 2350, Australia. 423

424

R. G. WINDON, J. K. DINEEN, P. GREGG, D. A. GRIFFITHS and A. D. DONALD

as resistance to larval establishment occurred earlier with higher levels of larval intake. In the present studies an irradiated T. colubriformis larval vaccine was used to investigate threshold response to vaccination and challenge in young and mature sheep. Experiment 1 assessed the role of different vaccination and challenge levels on the subsequent response of ewe lambs, while Expt. 2 examined differences between male and female lambs to increasing vaccination doses. In a third experiment mature sheep were vaccinated in the field and exposed to low and then high levels of larval contamination on pasture. MATERIALS

Sheep. Random-bred

AND METHODS

Merino ram, wether and ewe lambs were reared in pens under worm-free conditions from birth and weaned at 11 weeks of age. Parasitological techniques. Third stage larvae were cultured from the faeces of sheep carrying pure T. colubriformis infections. Larvae used for vaccination were exposed to 500 Gy of y-radiation from a 38.46 TBq cobalt60 source at a rate of 3.24 Gy min-1. Gregg, Dineen & Griffiths (1976) have described the methods used for irradiating larvae and dosing sheep. Faecal worm-egg counts were performed by the modified McMaster technique (Whitlock, 1948). During field challenge the proportion of eggs attributable to each species of gastrointestinal nematode was obtained by differentiation of larvae after culture of faecal samples. Donald, Morley, Wailer, Axelsen & Donnelly (1978) described the methods used for digestion of abomasal mucosa, sampling and worm counting. Complement fixation test. Complement fixation tests (CFT) were carried out using the technique described by Windon & Dineen (1981). A crude extract of T. colubriformis third stage larvae (TcL3) prepared by the method of Stewart (1950) was used as antigen. Experimental design. In Expt. 1, groups of 12 ewe lambs were vaccinated at 12 and 16 weeks of age with either 5000, 20,000 or 80,000 irradiated T. cofubriformis larvae. All animals, including 12 unvaccinated ewe lambs, were treated with anthelmintic (320 mg levamisole; Nilverm, ICI Australia Ltd.) at 20 weeks of age. One week later, half of each group was challenged with 5000 normal infective larvae and the remainder challenged with 80,000 normal larvae. All animals were killed for worm counts 4 weeks after challenge. In Expt. 2, groups consisting of five to seven ram and seven ewe lambs were vaccinated with either 10,000, 20,000, 40,000, 80,000 or 160,000 irradiated T. colubriformis larvae at 8 and 12 weeks of age. At 16 weeks vaccinated and unvaccinated control rams and ewes were treated with anthelmintic (320 mg levamisole) and a week later challenged with 20,000 normal larvae. The animals were killed for worm counts 4 weeks after challenge. In Expt. 3, 15-month-old Merino ewes and wethers raised and maintained under worm-free conditions were injected subcutaneously with 10 mg kg-l Disophenol (Ancylol, Cyanamid Australia Pty Ltd) 1 week before pasturing to prevent Haemonchus contortus infections. Eight of these animals were vaccinated twice with 20,000 irradiated T. colubriformis larvae 14 days apart and a further eight animals served as unvaccinated controls. On the same day as the first vaccination all the sheep were put

I.I.P. VOL. 14. 1984

out to graze a paddock which had relatively light larval contamination, and 6 weeks later they were transferred to highly contaminated pastures. Faecal egg counts and differential larval cultures were carried out at fortnightly intervals. Four vaccinated and four unvaccinated sheep were killed for worm counts 9 weeks after transfer and the remaining animals were killed IO weeks later.

RESULTS

Worm count as a function of vaccination challenge dose (Expts. 1 and 2)

and

A preliminary analysis of the data suggested that vaccine dose was an important determinant of the response (decreased worm count relative to controls) but that challenge dose was unimportant, except as a determinant of the worm count (WC) in controls. For this reason the following regression model was fitted to the combined data of Expts. 1 and 2:,v
ci KY) = Ci-b( V-V,),

I’ > Vo

log 10 WC, WC > 0 where

y=

cj=

log,o25,

WC=0

expected

log

ith challenge

v= zz

logarithmically

lo

WC for controls

at the

dose scaled vaccine dose

log2 (vaccine dose/2500) so that doses of 5, 10, 20, 40, 80 and 160 x 103 irradiated larvae are scaled as 1 to 6 respectively

v,=

scaled threshold vaccine dose for response. As there was considerable heterogeneity, group variances within each challenge and vaccine dose regime were used to determine a weight function for the data in the fit of the model. As shown previously (Windon & Dineen, 1981) and confirmed in the current studies, ram lambs are significantly less responsive than ewe lambs. For this reason the data for ram lambs were not included in the analysis, and as the ewe lambs vaccinated with 80,000 irradiated larvae and challenged with 5000 normal larvae showed an aberrant response, this group was also excluded from analysis. Observed group logarithmic mean worm counts and expected values based on the model fitted to the combined data of Expts. 1 and 2 and fitted separately to each of the three challenge levels are given in Table 1. The differences between challenge levels were not significant (~2 = 0.5, 3dfl and an overall lack of fit test confirmed that the model provided a satisfactory description of the data.

Antiworm complement-fixing Although serum samples intervals during vaccination

antibodies in serum were obtained at regular and challenge, attention

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TABLE

Expt. No.

1 -OBSERVED

Challenge dose

1

1

2

*Aberrant

Threshold

5000

80,000

20,000

group-data

AND FITTED

Vaccine dose

response

to vaccination

WORM COUNTS

Logarithmically scaled vaccine dose

Control 5000 10,000 20,000 40,000 80,000* 160,000 _

Control 5000 10,000 20,000 40,000 80,000 160,000

1 2 3 4 5 6

Control 5c00 10,000 20,000 40,000 80,000 160,000

AND ESTIMATED

PROTECTION

425

AS A FUNCTION

OF VACCINE

DOSE

Fit of piecewise linear model to combined data from three challenge levels

Fit of model to each challenge level separately

Observed log WC Mean (s.E.)

log WC

log WC

3.35 (0.05) 3.28 (0.08) _

3.35 3.30

2.73 (0.21) _

2.70 _

3.21 (0.06)

2.10

4.54 (0.02) 4.52 (0.05)

4.54 4.49

3.73 (0.29)

3.89 _

3.28 (0.25) _

3.29

4.15 (0.02)

4.15

3.76 3.82 2.78 2.83 3.02

not included

T. colubriformis

with

(0.16) (0.07) (0.25) (0.33) (0.30)

% protection (95% confidence limits)

11 55 78 89 94 97

( 0, 36) (43, 65) (70,83) (83,93) (90,97) (94.99)

ll( 55 78 89 94 97

0,36) (43,65) (70,83) (83,93) (90, 97) (94, 99)

3.35 3.28 2.73 2.18 4.54 4.51 3.84 3.16

% protection

16 55 76 87 93 96

7 57 80 91 96 98

4.15 11( 0,36) 55 (43,65) 78 (70,83) 89 (83,93) 94 (90.97) 97 (94.99)

3.80 3.50 3.20 2.90 2.60

3.80 3.52 3.24 2.95 2.67

15 56 77 88 94 97

in analysis.

was focused on antibody titres at the time of challenge. Retrospective analysis of earlier data generated in studies on the responses of high- and low-responder lambs to vaccination and challenge with T. colubr~$ormis (Windon & Dineen, 1981) showed a high correlation (r > 0.8) between serum antibody titre at this time and WC of individual animals after challenge. The relationship between logarithmic reduction of WCs of vaccinated ewe lamb groups from the mean count for unvaccinated controls and tirre of serum complement-fixing antiworm antibodies at challenge in Expt. 1 is summarized in Table 2. With the exception of the aberrant group (vaccinated with 80,000 larvae and TABLE

~-MEAN

SERUM

AT

TITRES

CHALLENGE

FROM UNVACCINATED

Vaccine dose

OF

AND

challenged with 5000 larvae) high level of serum antibody at challenge was associated with substantial logarithmic reduction in WC. Figure 1 shows that logarithmic reduction in WCs of both ram- and ewe-lambs of Expt. 2 also correlated with serum antibody at challenge. Field vaccination and challenge (Expt. 3) Faecal egg counts of Trichostrongylus spp. showed no difference between vaccinated and unvaccinated groups during the period of grazing when larval pasture contamination was low. However, after transfer to highly contaminated pastures, egg counts of the unvaccinated sheep rose markedly while those

COMPLEMENT-FIXING LOG10

CONTROLS

REDUCTION OF EWE

ANTIBODIES IN

WORM

LAMBS--EXPT.

IN

COUNTS

](?I =

Challenge dose

Log2 antibody titre at challenge (k s.E.)

5000

5000 80,000

2.2 t 0.8 2.3 f 0.6

0.07 2 0.08 0.02 k 0.02

20,000

5000 80,000

6.1 f 0.8 5.4 f 1.2

0.59 * 0.20 0.80 -t 0.29

80,000

5000* 80,000

5.9 -c 0.8 6.8 f 0.5

0.14 2 0.02 1.25 ? 0.50

*Aberrant

group-see

text

Reduction

6)

log,0

ki’c

(2

in S.E.)

I.J.P. VOL. 14. 1984

R. G. WINDON, J. K. DINEEN, P. GREGG, D. A. GRIFFITHS~~~ A. D. DONALD

426

Egg counts cattitbutabie to-e

OLf 3 Log2

c

I

4 antibody

‘,i%; i(

10 12

low contamnatton

1

5 6 7 titre at challenge

6 5

8 5

FIG. 2.

Trichostrongylus

during

FIG. 1. Relationship between serum antibody titres at challenge and logarithmic reduction in worm counts of ~malelarnbs,O----a lambs from Expt. 2 (a---

female lambs). of the vaccinates remained low (see Fig. 2). Worm counts carried out 9 weeks after transfer showed that intestinal Trichostrongylus spp. accounted for most of the contamination, and confirmed that vaccinated animals had lower worm burdens than unvaccinated controls (p = 0.025). At the time of second slaughter vaccinated animals again had lower intestinal Tric~ostrongyius spp, burdens than controls @= O-095), and in addition also had lower counts of Ostertagia spp. (p = 0448). DISCUSSION

Worm burdens of animals exposed to natural field infections are characterised by marked betweenanimal variance. This heterogeneity in the responsiveness of animals has been attributed to differences in the antigenic relationship between individuals of an

periods

spp. faecal egg counts

l ) and unvaccinated (O-

vaccinated (* -

4 0

attributable to Egg CountS -----+I htgh contaminatnn

+

of

low then high tamination.

larval

Killed March Vaccinated Unvaccinated

Killed June Vaccinated Unvaccinated

Abomasal: 0

0

2180

2000

1700

1430

4660

10,080

630

760

3650

4230

4990

32,380

7450

29,540

12

75

100

240

Intestinal:

Trichostrongylus spp. Nematodirus spp.

*Ail animais

con-

outbred host population and the parasite, which in turn affects the threshold for immunological responsiveness (Dineen, 1963), and is also a feature of the reaction of animals to vaccination and challenge in the pen. The responsiveness or immunological threshold of animals is also affected by age, sex, physiological and nutritional status and previous antigenic experience, but regardless of mechanistic detail, a major component (ca. 60%) of variance is determined by genetic constitution of host as shown by selection of high- and low-responder lines of sheep to vaccination and challenge with T. co~abrt~ormis (Dineen & Windon, 1980; Windon & Dineen, 1981). Although it would be expected that response against challenge infection would be a function of both vaccine and challenge dose, in the present pen studies (Expts. 1 and 2) vaccine dose played the major role. Vaccine doses of 5000,2O,COOand 80,000 irradiated larvae and challenge doses of 5000 and 80,000 normal larvae were used in Expt. 1 and vaccine doses ranging from 10,000 to 160,000 larvae and a challenge dose of 20,000 larvae were used in

TABLE ~-DIFFERENTIAL WORM COUNTS OF MATURE SHEEP AFTER FIELD INFECTION KILLED 3 AND 5 MONTHSAFTER VACCINATION (FOUR ANIMALS PER GROUP) .._

Haemonchus contort&f Ostertogiu spp. $z Tri~host~ong~lus axei

pasture

of

0)

injected with 10 mg kg-I SC. Disophenol in December. *All H. contortus counted were at the early L4 stage. *The percentage of adult Ostertagiu spp. from sheep killed in June were 15.3 and 33.7 for vaccinated and unvaccinated groups respectively.

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427

Threshold response to vaccination with ‘K cohbriformis

Expt. 2. The fit of the model to the combined data implies that for the regime used in these experiments, the threshold for response of most lambs to both vaccine and challenge dose is exceeded by 5000 larvae, and, whereas the response is vaccine dose dependent above the threshold, it is challenge dose independent. The threshoid dose of vaccine larvae (V,), strictly relevant only to the age, sex and breed of lambs and the regime used in the present study, may be estimated from the linear regression model. This extrapolation suggests that the treshold vaccine dose is about 4400 irradiated larvae (95% CLs 2700 and 7200). Both vaccine and challenge doses exceeded their respective thresholds for most animals in the pen studies. However, the field study showed that response to vaccination was only apparent after transfer of the animals to heavily contaminated pastures (see Fig. 2), suggesting that the previous exposure of the vaccinated sheep to the few infective larvae available on the lightly contaminated pasture constituted a chailenge which was below the relevant threshold. The response (percent protection-see Table 1) of the ewes vaccinated with 80,000 irradiated larvae and challenged with 5000 normal larvae (Expt. 1) was inconsistent with those of all other ewe groups and WCs of this aberrant group were not used for the purpose of fitting the model. Failure of these lambs to give the expected response is also reflected in the relationship between antiworm complement-fixing antibodies in their sera at the time of challenge and subsequent level of protection (logarithmic reduction of WC-see Table 3). Although the lambs in the aberrant group responded normally to vaccination, in so far as titre of CF antibodies in serum are considered to reflect rather than mediate the relevant (protective) immunological reaction, these lambs failed to show the commensurate level of protection. Two explanations immediately present themselves. If the challenge threshold, like the vaccination threshold, is close to 5000 larvae, then this dose may not be recognised by some individuals even though these may have been vaccinated (cf. the field challenge-Fig. 2). Although less likely, we cannot exclude the possibility that a deficiency in the immunologically non-specific effector component of parasite immunity (Dineen, Gregg, Windon, Donald & Kelly, 1977) may have limited the expression of resistance in this otherwise immunologically responsive group. The model has only been fitted to the WCs of ewe lambs although ram lambs were used in Expt, 2. These animals were as responsive as ewe lambs to vaccination with 10,000 and 20,000 larvae, but failed to show dose-dependent levels of protection (logarithmic reduction of WCs) at the higher vaccine doses (see Fig. 3). Figure 1 shows that for given titres of antibody at challenge there was no significant difference in the level of resistance expressed by ram

019 ‘:

c”‘$5 IO Vaccine

io dose

Lo sb 160 x10b3

FIG. 3. Logarithmic reduction in worm counts from and female (0 - - 2 0) lambs for male (@ -0) increasing vaccination IeveIs. The fitted model for combined female data is also shown (-e-e-). The threshold dose for vaccination (Vo) is 4400.

and ewe lambs at lower titres, whereas resistance was not expressed as vigorously in ram lambs with higher levels of serum antibody, suggesting some incompetence of the non-specific effector mechanism. However, this suggestion alone does not adequately explain ihe difference between ram and ewe lambs as antibody titres (Fig. 4) ciearly show that, in addition, ewe lambs were more immunologically reactive at the higher dose levels. Thus although the difference in responsiveness of ram and ewe lambs, as previously reported by Windon & Dineen (1981), may in part be

I 2,““c”,\

5

I

10 Vaccine

I 20

J f+o

60

160

dose x10e3

Frc. 4. Mean serum antibody titres at challenge in male (O-

0) and female (0 - - - - 0) lambs for increasing vaccination levels.

428

R. G. WINDON, J. K. DINEEN, P. GREG(2, D. A. GRIFFITHS~~~ A. D. DONALD

due to non-specific components of resistance, coincidence of antibody titre and reduction in worm count over the full range of vaccine doses (cf. Figs. 3 and 4) suggests that the specific immunological components of resistance (Dineen et al., 1977) also play a role. The results of the present study suggest that the efficacy of vaccination may plateau in both sexes but earlier and at lower levels in ram than ewe lambs at high vaccine doses. Conceptually, response to vaccination could even decline due to pathogenicity of the irradiated larvae at very high doses. A plateau model was constructed but discarded because fit to the ewe lamb WCs was not improved by consideration of this factor and insufficient data were available for more critical appraisal. Acknowledgements-We wish to Elizabeth thank Townsend, Athol Luker and Stephen Ferguson for their excellent technical assistance, Cyril Samways of the School of Chemical Engineering, University of New South Wales, for irradiation of T. colubrlformis infective larvae, and Robert Dobson and Elaine Smith for statistical analyses. This work was carried out while R.G.W. was undertaking a Ph.D. degree at the School of Wool and Pastoral Science, University of New South Wales.

REFERENCES CHIEJINA S. N. & SEWELL M. M. H.

1974a. Experimental infections with Trichostrongylus colubriformis (Giles, 1892) Loos, 1905 in lambs: worm burden, growth rate and host resistance resulting from prolonged escalating infections. Parasitology 69: 301-314. CHIEJINA S. N. & SEWELL M. M. H. 1974b. Worm burdens, acquired resistance and live weight gains in lambs during prolonged daily infections with Trichostrongylus colubriformis (Giles, 1892) Loos, 1905. Parasitology 69: 315-327. DINEEN J. K. 1963. Immunological aspects of parasitism. Nature (London) 197: 268-269. DINEEN J. K. 1978. The nature and role of immunological control in gastrointestinal helminthiasis. In: The Epidemiology and Control of Gastrointestinal Parasites of Sheep in Australia (Edited by DONALD A. D., SOU~HCOTT W. H. & DINEEN J. K.), pp. 121-135. Division of Animal Health, CSIRO, Australia.

I.J.P. VOL. 14. 1984

J. K., GREGG P., WINDON R. G., DONALD A. D. & KELLY J. D. 1977. The role of immunologically specific and non-specific components of resistance in crossprotection to intestinal nematodes. International Journal for Parasitology 7: 21 l-215. DINEEN J. K. & WINDON R. G. 1980. The effect of sire selection on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journalfor Parasitology 10: 189-196. DONALD A. D., MORLEY F. H. W., WALLER P. J., AXELSEN A. & DONNELLY J. R. 1978. Availability to grazing sheep of gastrointestinal nematode infection arising from summer contamination of pastures. Australian Journal of Agricultural Research 29: 189-204. GIBSON T. E. & PARFITT J. W. 1972. The effect of age on the development by sheep of resistance to Trichostrongylus colubriformis. Research in Veterinary Science 13: 529-535. GIBSON T. E. & PARFITT J. W. 1973. The development of resistance to Trichostrongylus colubriformis by lambs under conditions of continuous infection. Research in Veterinary Science 15:220-223. GREGG P., DINEEN J. K. & GRIFFITHS D. A. 1976. The effect of y-radiation on the development of infective larvae of Trichostrongylus colubrlformis in guinea pigs and sheep. Veterinary Parasitology 2: 363-375. STEEL J. W.. SYMONSL. E. A. & JONES W. 0. 1980. Effects of level of larval intake on the productivity and physiological and metabolic responses of lambs infected with Trichostrongylus colubriformis. Australian Journal of Agricultural Research 31: 821-838. STEWART D. F. 1950. Studies on resistance of sheep to infestation with Haemonchus contortus and Trichostrongylus spp. and on the immunological reactions of sheep exposed to infestation. I. The preparation of antigens for the complement fixation test and the reactivity of the biochemical fractions of H. contortus. Australian Journal of Agricultural Research 1: 285-300. WALLER P. J. & THOMAS R. J. 1981. The natural regulation of Trichostrongylus spp. populations in young grazing sheep. Veterinary Parasitology 9: 47-55. WHITLOCK H. V. 1948. Some modifications of the McMaster helminth egg-counting technique apparatus. Journal of the Council for Scientific and Industrial Research 21: 177-180. WINDON R. G. & DINEEN J. K. 1981. The effect of selection of both sire and dam on the response of Fl generation lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology 11: 1 l-18.

DINEEN