Complement fixing antibody response of calves to Dictyocaulus viviparus

J.

COMPo PATH. 1963. VOL.

73.

COMPLEMENT FIXING ANTIBODY RESPONSE OF CALVES TO DICTYOCAULUS VIVIPARUS IV. A COMPARISON OF ANTIGENS By

R. L.

CORNWELL

*

Department qf Veterinary Preventive Medicine, University of Liverpool INTRODUCTION

The natural and experimental infection of calves with Dictyocaulus viviparus produces a strong active immunity and complement fixing (C.F.) antibodies are detectable in the blood serum (Jarrett, Jennings, McIntyre, Mulligan and Urquhart, 1957; Weber, 1958; Cornwell and Michel, 1960). The pattern of C.F. response using heated whole worm antigen was found by Jarrett, et al. (1957) to consist of an initial rise in titre at 30 to 35 days, reaching a peak at about 80 days and then falling to a low level. Reinfection produced a secondary response in 7 days, reaching a peak in 21 to 28 days. These results were confirmed by Cornwell and Michel (1960), who extended their observations to include natural infections. Weber (1958), using a "low temperature" antigen, obtained an earlier and more prolonged response. Although serum from hyperimmunised calves containing a high level of C.F. antibodies conferred a passive immunity on susceptible calves (Jarrett, Jennings, McIntyre, Mulligan and Urquhart, 1955), it was demonstrated by Michel and Cornwell (1959) that the level of C.F. antibody was not directly related to the immune status of the calf. The ability of injections of dead worm material to stimulate C.F. antibodies without the production of a strong immunity was demonstrated by Jarrett, Jennings, McIntyre, Mulligan and Urquhart, (1960), and Cornwell (I 960c) produced a secondary response in a previously infected calf by the injection of dead worms. Jarrett et al. (1960) in their experiments with irradiated larvae found a negligible C.F. response following a single dose of larvae. Cornwell (1960 a and b, 1961) found that a second dose of irradiated larvae stimulated a variable response in the majority of calves and that challenge evoked a secondary response. Two injections of 4th stage larvae were without effect in producing a C.F. response, though a moderate degree of imnwnity resulted (Cornwell, 1962), whilst normal infections, successfully treated at the 14 to 17 day stage with diethylcarbamazine, resulted in a greatly reduced C.F. response (Cornwell, 1963). Since this published work refers to the use of adult worm antigen only, a comparison of antigens prepared from different stages of the parasite and the biochemical fractions of adults was made along with an assessment of the degree of cross-reaction with other parasites. The results are reported here. METHODS

Preparation rif antigens. Heated antigens were prepared by the method of Stewart (1950) from the following stages of D. viviparus: mixed adults, *Present address Pfizer Ltd., Sandwich, Kent.

C. F. ANTIGENS OF

Dictyocaulus viviparus

adult males, adult females, immature adults, third stage larvae, eggs and first stage larvae. Similar heated antigens were prepared from Haemonchus contortus adults, H. contortus third stage larvae, mixed intestinal spp. (calf), mixed intestinal spp. (goat), Ascaris lumbricoides, Metastrongylus apri, Fasciola hepatica and Moniezia expansa. Saline extracts of D. viviparus were prepared as follows: One gramme of adult worm material was chopped up finely in 3 ml. distilled water and the resulting coarse suspension broken down finely with an ultrasonic homogeniser in a small bottle surrounded by ice. The fine suspension was made up to 25 ml., rendered isotonic, and centrifuged at 1500 r.p.m. for 10 minutes. The supernatant was bottled and stored at 4°C. Saline extracts were also prepared by the method of Weber (1958), who subjected a ground-up suspension of worms to quick-freezing at -40°C in an alcohol-dry ice bath, and stored the antigen at - 38°C. Saline suspensions of 3rd stage larvae of H. contortus or D. viviparus were extracted overnight at 4°c' after grinding in an agate mortar with the addition of powdered glass. Saline suspensions of the residues of several parasites after extraction with ether or alcohol were re-ground and kept at 4°C overnight. The suspension was then centrifuged and the supernatant recovered. Alcohol soluble and ether soluble lipid extracts of D. viviparus, M. apri and A. lumbricoides were prepared by the method of Jennings (1949). Protein fractions and polysaccharide fractions of D. viviparus were prepared by the methods of Campbell (1937). An alcohol/acetone extract of F. hepatica was prepared by the method of Spuhler, Moosbrugger and Meyer (1958). Antigens from the "metabolic products" of adult worms and exsheathed 3rd stage larvae of D. viviparus were obtained by the method of Soulsby, Sommerville and Stewart (1959) and were concentrated by dialysis against polyethylene glycol. Complement fixation tests. C.F. tests were carried out as previously described (Cornwell and Michel, 1960a). Antigens were titrated in doubling dilutions against known positive and negative D. viviparus sera, using three 50 per cent. units of complement, and also without serum, using one unit of complement. For full details see Cornwell (1960c). Mter titration, antigens were compared in parallel C.F. tests with the same series of sera. Apart from the two sources acknowledged, the sera used in these experiments were selected from samples collected during experimental work on D. viviparus and stored at -20°C (Cornwell 1960 a and b, 1961). Positive sera for antigen titration consisted of pooled samples collected from calves 2 to 4 weeks after re-infection. Negative sera were collected from calves reared free from lungworms. RESULTS

Comparison of D. viviparus Antigens Potent antigens were obtained from all the stages of the parasite (Table I). The low-temperature saline extract of adult worms (Weber, 1958) had a much lower potency than a similar extract prepared at room temperature with the aid of an ultrasonic disintegrator. A potent antigen was prepared from metabolic products

299

R. L. CORNWELL TABLE

I

TITRATION OF ANTIGENS PREPARED FROM D. VIVIPARUS AGAINST KNOWN POSITIVE AND NEGATIVE CALF ANTISERA I

Antigen

---

I

Adults mixed Females Heated antigens

Males Eggs & LI

Serum

Pos.

Neg. Pos. Neg. Pos. Neg. Pos.

Neg. Immatures POI. Neg.

---~~

Extracts

Low temp. Pos. saline Neg. adult (Weber) UltraPos. sonic saline Neg. adult Metabolic Pos. prod. Neg. adult. Metabolic Po•. prod. L3 Neg. SalineL3 Pos. 4'C Neg. Protein

Polysaccharide Lipid (alcohol) Biochem. Lipid (ether) fraction Saline adult. extract \AlcohoI.res. Saline extract Ether residue

-Pos.Neg. Po. Neg. Pos. Neg. Pos. Neg. Pos. Neg. Pos. Neg.

Antigen dilutions

Remmks

III

112

114

+++ +++ +++ +++ +++ +++

+++ +++ +++ +++ +++

+++ +++ +++ +++ +++

+ +++ +++ ++ +++

++ +++ +++ +++ +++ +++ ++ Zone +++ +++ +++ + -- Slight PIC --- Marked PIC A/C at ++ ++ ++ + +++ +++ +++ + - - 1/4 -+++ ++ + Not active Slightly +++ +++ +++ +++ +++ +++ +++ AIC Marked +++ +++ +++ +++ +++ ++ A/C +++ +++ +++ ++ ++ Slight +++ +++ +++ ++ PIC -

-

-+++ +++

+++ +++ +++ -

118

+++ +++ +++ +++ +++ +++ ++ + -

A/C = anticomplementary. PIC = procomplementary. + + + = no haemoly.is. + + - 30% haemolysis. + - - 100% haemoly.i•.

-

1116

+++ +++ +++ +++ ++ -

1132

1164

11128

+++ ++ + +++ +++ +++ + +++ +++ ++ ++ --

--Slight A/C

" "

"

70% haemolysis.

of adult worms, but a similar antigen from 3rd stage larvae was inactive and pro-complementary. Of the biochemical fractions of adult worms the alcohol-soluble lipid fraction was highly potent, the protein fraction was of low potency and the polysaccharide fraction did not react in the test. Ether extracts of adult worms proved markedly anti-complementary, possibly due to some residual ether. Saline extracts of the residues of worms after lipid extraction reacted in the test, presumably due to their protein content. Using the heated adult whole worm antigen as a standard, comparisons were made with the other antigens by testing them in parallel against the same series of lungworm sera. Fig. I shows the results of a comparison between the heated antigens of mixed adults, females, males, immatures, and eggs with 1st stage larvae. The general pattern of antibody response to each antigen with serum 164 was the same, but differences in the titre of the serum were evident

300

Dictyocaulus viviparus

C. F. ANTIGENS OF

Fig.

I.

160 SERUM 164 ILl

40

ex:

tt-

10

o

5

WEEKS

15

10

H<" AJE"P ANTIGENS P VIVIPABUIi

_ _ ADU"LTS MIXED

~

_

FEMALES

tr-----to I MMA TUBES

MALES

I)---(J

EGGS AN 0 L.I.

Comparison between the titres produced by heated antigens from different stages of D. viviparus against a series of sera from calf 164. This calf was infected with 3,200 larvae at week 0 and reinfected with 40,000 larvae at week 12. Fig.

2.

28 SERUM 245

320

80

o

5

15 WEEKS

20

25

30

Comparison between the titres produced by heated adult worms and a saline extract of 3rd stage larvae of D. viviparus against a series of sera from calf 245. This calf was dosed with 1,000 larvae at weeks 0 and 4 and challenged with 12,500 larvae at week 26.

with the different antigens. The highest titres were detected by the mixed adults and the females, whilst the males reacted to a much lower titre. This would indicate the importance of the mature female worms in the stimulation of this particular antibody response. A comparison is shown in Fig. 2 between the adult worm antigen and the yd stage larval antigen, another calf serum (245) being used.

301

R. L. CORNWELL

Fig. 3.

320 SERUM

806

.w a:

80

l-

I-

ADULT Q.lllll1PAB!.I5 0--0.0 HEATEO EXTRACT

20

--........

LOW TEMR PROTEIN

o-,a

5

o

5

WEEKS

LIPID

15

10

Comparison of the titres developed to different antigens from adult D. viviparus in a series of sera from calf 806. This calf had been vaccinated previously and challenged with 5,000 larvae at week 4.

Fig. 4.

640

160

w a: l-

I-

o

5

10

WEEKS

15

20

Comparison of whole worm antigen and lipid-free residue of D. viviparus in a series of sera from calf 150. This calf was vaccinated at weeks 0 and 4 and challenged with 7,000 larvae at week 12.5.

C. F. ANTIGENS OF

Dictyocaulus viviparus

The larval antigen gave no titre until late in the primary infection, but after challenge a similar sharp rise in antibodies was detected by both antigens. A comparison of the heated and unheated antigens with the lipid and protein fractions again revealed no difference in general pattern of response and the only obvious difference in titre occurred with the lipid fraction which gave a titre about half that of the other' three (Fig. 3). The antigen from the residue after ether extraction behaved in a manner similar to the protein extract in that little difference was apparent between the titres of sera tested with this antigen and with the heated antigen (Fig. 4). The antigen from metabolic products of adult worms gave a similar pattern to the heated adult worm antigen, but with a slightly later onset and a lower titre (Fig. 5). Fig. 5. 1~80

SERUM

298

320

w a:

80

~

....

D. VII!IPARU5

20

5 0

5

15

10

20

25

WEEKS

,Comparison of titres to heated antigen and metabolic products in a composite series of sera from calves vaccinated at weeks 0 and 4 and challenged with 5,000 larvae at week 13.

Cross Reactions with Antigens from other Parasites Antigens from other nematodes reacted with D. viviparus positive sera but those from F. hepatica and M. expansa did not (Tables 2 and 3)· The antibody response of a series of sera collected from calves infected with lungworms against heated D. viviparus compared with heated antigens from H. contortusJ intestinal worms of goat origin, M. apri and A. lumbricoides; and the lipid-free residues of M. apri and A. lumbricoides are shown in Figs. 6 to g. The comparison reveals the same pattern of response in all cases, with a higher titre and more rapid onset with the D. viviparus antigen. The lipid-free antigens,

R. L. CORNWELL TABLE

2

TITRATION OF HEATED ANTIGENS FROM OTHER HELMINTHS AGAINST D. VIVIPARUS POSITIVE AND NEGATIVE ANTISERA FROM CALVES

Antigen

Serum

--

IX;-I

112

114

I

Antigen dilutions .

118

Ascaris

Pos. Neg.

+++

+++

+++

M. apri

Pos.

H. contortus adults

Neg.

H. contortus L3

Pos. Neg.

+++ +++ +++

+++ +++ +++

Mixed worms Goat intestine

Pos. Neg.

+++ +++ +++ + +++ -

+++ +++ ++ ++

+++ -

++

Mixed wormS Calf intestine

Pos.

F. hepatica

Pos.

+++ -

+++ -

M. expansa

Neg. Pos.

Neg. Neg.

Pos.

Neg.

-

+++ + -

-

-

+++ +++ -

-

-

-

-

+++ ++ -

++ -

+ + +++ -

-

-

AIC - anticomplementary. PIC - procomplementary. + + + - no haemolysis. + + = 30% haemolysis. + - - 100% haemolysis.

1116

-

I

I 1132

'1164

11128

Remarks

+++ ++ ++ -

++

++ + -

Slight PIC

-

-

-

+ -

+++

++

-

-

-

--

-

-

-

-

+ -

Not

-

active

-

"

70% haemolysis.

TABLE 3 TITRATION OF ANTIGENS CONSISTING OF THE BIOCHEMICAL FRACTIONS OF OTHER HELMINTHS AGAINST POSITIVE AND NEGATIVE SERA FROM CALVES INFECTED WITH D. VIVIPARUS

Antigen

I Serum

Pos. F. hepatica Alcoholl acetone Neg. Ascaris

Pos. Neg.

Lipid-alcohol

Ascaris

Pos. Neg.

M.apri Lipid-ether

Pos. Neg.

M. apri Lipid-alcohol

Pos.

Lipid-ether

Neg.

Pos. Residue-alcohol Neg.

Ascaris

Ascaris Residue-ether

Pos.

Neg.

Pos. M. apri Residue-alcohol Neg. M. apri

Residue-ether

Pos.

Neg.

Antigen dilution

--'1-1-'~1--11-4-1--11-8-1~ - ; ; ; ; -

1164

I 11128

--------- --- -----------' +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ - +++ +++ +++ -+++ ++ +++ +++ +++ + -+++ +++ +++ + - +++ +++ +++ ++ - +++ +++ ++ +++ +++ I

AIC - anticomplementary. PIC - procomplementary. + + + - no haemolysis. + + - 30% haemolysis. + - - 100% haemolysis.

-

70% haemolysis.

Remarks

Not

active

Marked AIC

Marked AIC

" Slight PIC

..

C. F. ANTIGENS OF

Dictyocaulus viviparus

although still detecting a response, did so to a lower titre than the heated antigens from the same worms. Samples of D. viviparus positive sera previously incubated for 30 minutes at 37°C with a 1/10 dilution of a crude saline suspension of Ascaris, were then subjected to the C.F. test against heated D. viviparus antigen. The result shown in Table 4 indicates that antibodies to the lungworm antigen can be partially absorbed by Ascaris. Fig. 6.

320 SERUM

330 80 w

HEATED

C(

D. VIVIPARUS

~

~

20 H. CONTORTUS

5

o

15 10 WEEKS Comparison of titres to heated adult D. viviparus and heated adult H. contortus

5

in a series of sera from calf 330. This calf was vaccinated at weeks 0 and 6 and turned on to infected pasture at week II. Fig. 7.

32 SERUM 73

w

80 HEATEO

C(

D. VIVIPARUS

~ ~

20

INTESTINAL WORMS [GOAT1

5

o

5

10

15

WEEKS

Comparison of titres to heated antigens from D. viviparus and mixed intestinal nematodes from a goat, in a series of sera from calf 73. This calf was vaccinated at weeks 0 and 4 and turned out to infected pasture at week 8.

R. L. CORNWELL

Fig. 8. 640 SERUM

HEATED

300

p----<>--q_ .. --. ',

, , ,,

,

'tl

'1>---<1\ ,

5

o

't/

LIPID-FREE M:APRI

5

15

10

25

20

40

30

WEEKS

Comparison oftitres to heated D. viviparus antigen, heated and lipid-free M. apri antigens in a series of sera from calf 300. This calf was dosed with vaccine at weeks 0 and 6 and turned out to infected pasture at week I I •

Fig. g. HEATED O.VIVIPAAUS

320

SERUM

240 8

,,

, ,,

,, ,,, , ,, ,,

LIPID-FREE ASCARIS

,0----<1;

,, 0

5

10

15

:20'

25

30

WEEKS Comparison of titres developed to heated D. viviparus and Ascaris antigens and lipid-free Ascaris antigens in a series of sera from calf 240. This calf was vaccinated at weeks 0 and 6 and turned on to infected pasture at week I I.

306

C. F. ANTIGENS OF

Dictyocaulus viviparus

TABLE 4 C. F. TITRES, USING HEATED D. VIVIPARUS ANTIGEN, OF LUNGWORM POSITIVE SERA ABSORBED WITH ASCARIS, COMPARED WITH UNABSORBED SERA

Date qf sample Calf 207

Unabsorbed sera

Absorbed with Ascaris

2.6.60 23.6.60 30.6.60 7.7. 60 15.7.60 21.7.60

10 640 640 480 480 320

0 240 160 80 80 60

DISCUSSION

A large number of sera from experiments on lungworm infection have been tested in the C.F. test using a heated whole worm antigen. Early results (Michel and Cornwell, 1959; Cornwell and Michel, 1960) agreed substantially with those of jarrett,jennings, McIntyre, Mulligan, Thomas and Urquhart, (1959). Weber (1958) used an antigen prepared by a low temperature (-40°C) method and obtained different results which he attributed to the smaller doses of larvae used for infection. As it seemed more likely that the different method of antigen preparation was the explanation the experiments described in this paper were conducted to test this point and to search for an antigen which might give a better indication of the immune status of the host than the heated adult worms. The results show that a similar pattern of response was obtained with antigens from all stages of the parasite, although some differences occurred in the titre. The antigen prepared by the method of Weber (1958) did not differ from the heated antigen or from an extract made at 4°C in parallel tests against the same sera. It was, however, less potent in the antigen titration, though it was completely free from the small amount of anticomplementary activity shown by the other antigens. These results suggest that all stages of the parasite contain similar somatic antigens which are capable of detecting C.F. antibodies in lungworm sera. The adult worm antigen and the metabolic products of adult worms are very similar in their capacity to fix complement in the presence of positive sera. The results obtained with the biochemical fractions show that the protein fraction, whilst not as potent an antigen in the titration as the lipid fraction, gave results in serial tests approximating closely to those given by the adult worm extracts, whereas the lipid fraction detected antibodies to a much lower titre. The polysaccharide fraction did not fix complement in the presence of positive sera l

R. L. CORNWELL

suggesting that the active antigen may be a combination of protein and lipid. Stewart (1950), working with H. contortus and Trichostrongylus spp. in sheep, found that satisfactory antigens for the C.F. test could be prepared from adults, 3rd stage larvae of Trichostrongylus spp. and from 3rd stage larvae of Haemonchus, but that adult Haemonchus, especially if "aged", were less satisfactory. Jennings (1949) prepared biochemical fractions of H. contortus and found that only the lipid fraction reacted with natural sheep antisera. This fraction was of low specificity, however, and cross reactions occurred between H. contortus sera and the lipid fractions of several nematodes and M. expansa, but not F. hepatica, Paramphistomum cervi or vertebrate tissues. Stewart (1950) prepared antisera in rabbits to each of the saline suspensions of lipid-free residues of H. contortus, Nematodirus spp., M. expansa, A. lumbricoides and F. hepatica. He found a greater degree of specificity was shown by the lipid-free material from these helminths than by the lipid itself. However, absolute specificity was shown only by F. hepatica. In the results reported here, the degree of specificity of A. lumbricoides and M. apri when tested against natural lungworm sera, was increased by the removal of lipid material. Antibodies were still detected, however, by these lipid-free antigens. CONCLUSIONS

A comparison of different antigens of Dictyocaulus viviparus in the complement-fixation test was made. Potent antigens were prepared from all the stages of the parasite and metabolic products of adults. Lipid and protein fractions of adult worms, but not the polysaccharide fraction, also reacted. All the antigens were compared with the heated adult antigen in tests with a series of sera, and the same general pattern of response was obtained, with some differences in titre. Cross-reactions were demonstrated between heated antigens from other nematodes and lungworm positive sera, but not with M. expansa or F. hepatica. In serial samples of sera, titres to these heated antigens were lower than those to the lungworm antigen. Lipid-free fractions of three species of nematodes tested gave titres lower than the heated antigens from the same worms. ACKNOWLEDGMENTS

Thanks are due to Professor E. G. White for his encouragement and advice, and to Miss Jean Berry for technical assistance. Mr. J. F. Michel of Weybridge kindly supplied samples of serum 164 and quantities of worm material. Mr. A. Mackenzie of Compton supplied samples of serum 245, and Col. D. J. Anthony of Marsh & Baxter Ltd. sent pig lungs from which specimens of M. apri were recovered. I am indebted to the Agricultural Research Council for financial assistance to carry out this work.

308

c.

F. ANTIGENS OF

Dictyocaulus viviparus

REFERENCES

Campbell, D. H. (1937). J. Parasit., 23,248. Cornwell, R. L. (1960a). J. camp. Path., 70, 494; (1960b). Ibid., 499; (1960c). M.V.Sc. Thesis; University of Liverpool; (1961). J. camp. Path., 71, 19 I; (1962). Ibid., 72, 181; (1963). Res. vet. Sci., In press. Cornwell, R. L., and Michel, J. F. (1960). J. camp. Path., 70, 482. Jennings, A. C. (1949). Aust. J. sci. Res., 2,408. Jarrett, W. F. H., Jennings, F. W., McIntyre, W. 1. M., Mulligan, W., and Urquhart, G. M. (1955). Vet. Rec., 67, 291; (1957). Ibid., 69, 1329; (1960). Immunology, 3, 135. Jarrett, W. F. H., Jennings, F. W., McIntyre, W. 1. M., Mulligan, W., Thomas, B. A. C., and Urquhart, G. M. (1959). Ibid., 2,252. Michel, J. F., and Cornwell, R. L. (1959). Vet. Rec., 71, 9I2. Soulsby, E. J. L., Sommerville, R. 1., and Stewart, J. F. (I959). Nature, Land., 183, 153. Spuhler, V., Moosbrugger, G. S., and Meyer, K. (I958). Schwei;:;. Arch. Tierheilk., 100, 610. Stewart, D. F. (1950). Aust. J. agric. Res., 1, 427. Weber, T. B. (I958). Amer. J. vet. Res., 19, 338. [Received for publication, March I 5th, I963]