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Oesophagostomum radiatum: Successful vaccination of calves with high molecular weight antigens
0020-7519/X9$3.00 fO.00 Pergamon 0 1989 Austra/ian Society for Parorrrology
lnte,nanonalJoloumalforPa,as~rology Vol.19,No. 3,pp. 271-274.1989. Printed in Great Brnain.
Press plc
OESOPHAGOSTOhfUA4
RADIA TUA4: SUCCESSFUL VACCINATION OF CALVES WITH HIGH MOLECULAR WEIGHT ANTIGENS I. J. EAST,* D. A. BERRIE
CSIRO,
Division
of Tropical
Animal
and C. J. FITZGERALD
Production, Long Pocket Laboratories, Queensland 4068, Australia
Private Bag No. 3, P.O., Indooroopilly,
(Received 21 July 1988; accepted 27 November 1988) Abstract--EAST I. J., BERRIED. A. and FITZGERALDC. J. 1989. Oesophagostomum radiatum: successful vaccination of calves with high molecular weight antigens. International Journalfor Parasitology 19: 271274. Extracts of adult Oesophagostomum radiatum were resolved into four fractions by gel filtration chromatography on a Superose 12 column. ELISA assays on these four fractions showed that the antibodies produced by naturally infected calves predominantly reacted with the void volume fraction (VV). Three trials were conducted in which calves were vaccinated with the VV fraction of adult extract. When compared to untreated controls, vaccination with the VV of adult extract significantly (PC 0.05) reduced worm establishment and faecal egg output after challenge infection relative to untreated controls. INDEX
KEY WORDS:
Oesophagostomum
radiatum ; cattle; vaccination;
INTRODUCTION CALVES infected
with Oesophagostomum radiatum, a pathogenic parasite of the gastro-intestinal tract, acquire a strong resistance to reinfection which is manifested against the late fourth stage larvae (L4) (Roberts, Elek & Keith, 1962). This acquired immunity can be stimulated artificially by immunization with worm components. Keith & Bremner (1973) successfully vaccinated calves with homogenates of both L4 and adult 0. radiatum. The soluble components of L4. grown in vitro, also contain potent protective
antigens (East, Berrie & Fitzgerald, 1988). However, Herlich, Douvres & Romanowski (1973) were unable to protect cattle from challenge infections with soluble extracts of either adults or larvae of 0. radiatum subfractions derived by gel filtration. Both homogenates and soluble
extracts
are hetero-
geneous mixtures of complex components, and the production of efficacious vaccines will almost certainly rely on identification of defined protective antigens. Little work has been done on fractionation of 0. radiatum components. In this paper, the successful vaccination of calves with the high molecular weight fraction of 0. radiatum adult worms obtained by gel filtration chromatography is reported. MATERIALS AND METHODS Worms. Adult 0. rudiatum were collected from culture calves as described by Keith & Bremner (1973). Soluble extracts were prepared by homogenization in 50 rn~ imidazole. 150mM NaCl, 5 mM Na:EDTA, pH 7.4 (INE)
*Author
to whom correspondence
should be addressed. 271
high molecular
weight antigens.
using a ground glass homogenizer, followed by centrifugation (50,000 g, 30 min, 4°C) and filtration through a 0.22 pm filter (Flow Labs, Rickmansworth, England). Protein concentrations were determined by the method of Bradford (1976). Chromatography. Two hundred microlitre samples of extract were applied to a Superose- 12 column (1 X 30 cm) (Pharmacia, Uppsala, Sweden) equilibrated and run in INE buffer. The column was controlled through a Pharmacia fast system, protein liquid chromatography eluted at 0.5 ml min’ and 0.5 ml fractions were collected. Calves. Friesian bull calves were reared worm-free in concrete pens as previously described (Roberts CI nl., 1962) and first vaccinated at 4 months of age. Vaccination. The vaccine fraction was emulsified with an equal volume of Freund’s Complete Adjuvant (Commonwealth Serum Laboratories, Parkville, Australia). Each calf received 50 pg of protein per subcutaneous injection in a single site in the neck. Control calves received an injection containing INE buffer emulsified with the same adjuvant. The injection was repeated 28 days later and 56 days after the first injection the test calves received a final subcutaneous booster of 50 pg of antigen in INE without adjuvant. Control calves received INE buffer. On days 63, 64 and 65 after the first injection, each calf was infected with 3000 0. radiufum infective larvae per OS. Faecal egg counts were made on days Y9, IO4 and 106 by the method of Roberts & O’Sullivan (1950) and reported as eggs per gram of faeces. The calves were necropsied on days 110, 111 and 112 and the worm burden determined (Roberts el al., 1962). The trial was repeated three times with groups of four, six and five calves per treatment, respectively. StafisGcs. Worm burdens are reported as arithmetic means f standard error. Faecal egg counts are the arithmetic mean + standard error of three determinations for all animals within each group. Worm burdens and faecal egg counts were square root transformed (East & Bourne, 1988) and the transformed data assessed by analysis of variance.
I. J. EAST, D. A. BERME and C. J. FITZGERALD
272
ELISA. The antigenicity of the various column fractions was determined using an enzyme-linked immunosorbent assay (ELISA). Microtitre plates were coated with aliquots of the column fractions adjusted to 100 pg ml-’ of protein. Serum was obtained from three cattle which had each received five infections with 0. radiutum. The relative proportions of antibody in these sera, specific for each column fraction, were determined by the method of East el al. (1988). Results are expressed as the ratio of each test absorbance to the mean absorbance of five known negative sera. The antibody titre of each test vaccine calf was measured by ELISA on the day of the first injection and 7 days after each of the secondary and tertiary injections. The plates were coated with the VV fraction, and each serum was diluted one in 850. The detailed method is described by East, Washington, Brindley, Monroy & Scott-Young (in press). Results again were expressed as the ratio of each test absorbance to the mean absorbance of five known negative sera. Electrophoresis. Samples were run on 10% sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) according to the method of Laemmli (1970). Gels were silver stained by the method of Morrissey (1981). Molecular weight standards were purchased from Pharmacia. RESULTS
SDS-PAGE showed that the components of the void volume fraction had molecular weights greater than 67,000 M,. More than 12 bands could be resolved in the fraction (Fig. 3). By the start of the challenge infection, all test calves had responded by
25r
0
1
2 Superose
3 Fraction
4
FIG. 2. The relative antigenicity of four Superose fractions determined using three hyperimmune anti-O.radiatum sera. Calf428 (~),calf436(111 ),calf489(\\).
The gel filtration elution profile of adult 0. r&uturn extract is shown in Fig. 1. The tubes corresponding to fractions as marked on Fig. 1 were pooled. Assay of the fractions with sera from immune cattle showed that most antigens reacting on ELISA resided in the void volume (VV) fraction (Fig. 2) and therefore this was the fraction used for vaccination. Fractions 3 and 4 were essentially non-reactive.
a
b
c
0.3
P $ 2
67
0.2
Z 5 0 b fn 9 0.1
0 I
,
I
0
5
I
10 Elution Volume
I
I
15 (ml)
20
FIG. 1. Elution profile of 0. radiatum adult extract chromatographed on Superose 12 (1 X 30 cm) equilibrated in INE (50 mM imidazole, 150 mM NaCl, 5 mM EDTA. pH 7.4) and run at 0.5 ml min-‘.
25----FIG. 3. SDS-polyacrylamide gel of 0. radiatum extracts. (a) Molecular standards; (b) adult extract; (c) adult VV fraction.
213
Vaccination of calves against 0. radiatum producing circulating antibodies which reacted with the void volume fraction (Fig. 4). The results of vaccination with 0. radiatum VV are shown in Table 1. In the three trials, the establishment of adult worms was reduced by 87, 70 and 39%, respectively, and the faecal egg count was reduced by 82,75 and 29%, respectively. The analysis of variance (Table 2) showed that both the overall average reduction in worm establishment (57%, P < 0.01) and faecal egg count (55%, P < 0.05) were significant.
However this difference may be associated with differences in dose of antigen or immunization schedule. Immunity acquired through repeated infection with 0. radiatum was directed mainly at high molecular weight components and this confirms the findings of East & Berrie (1986) who showed that most adult 0. radiatum antigens identified by Western immunoblotting were greater than 67,000 kl,. High molecular weight antigens have been used successfully to vaccinate against other gastro-intestinal nematodes including Nematospiroides dubius (Monroy & Dobson, 1986), Trichostrongylus coZubr@ormis (O’Donnell, Dineen, Rothwell & Marshall, 1985) and Haemonthus contortus (Neilson & Van de Walle, 1987). Sheep have also been successfully vaccinated with contortin (Munn, Greenwood & Coadwell, 1987), a protein of 60,000 M, derived from H. contortus which aggregates readily and would therefore, presumably, chromatograph on a gel filtration column at a much higher apparent molecular weight. This consistent success with high molecular weight
DISCUSSION Vaccination of young calves with high molecular weight fraction of an 0. radiatum extract caused a significant reduction in both the number of adult worms which established after challenge and the resultant egg count. This result represents a further step toward the isolation and identification of protective antigens suitable for incorporation into a commercial vaccine. The findings are in contrast to those of Herlich et al. (1973) who were unable to vaccinate successfully with fractions obtained by gel filtration. 30
b
a
C
A
25 0 A
20
,.--•
E
Fz
D
/
/
15 -
z w
‘1
;:
70
10 -
0 / x 5-
o-
.-. +
I!!0
4
8
+
L 12
4 Time
12
8
4
8
12
(weeks)
FIG. 4. ELISA titres of calves vaccinated with 0. radiatum adult VV. (a) First trial; (b) second trial; (c)third trial. +Indicates the average titre of control calves. Different symbols represent different animals within each experiment.
TABLE HIGH
l--WORM
BVRDENSANDFAEcALEGGcO"NTSOFCALVESVAcclNATEDWlTHTHE
MOLECULARWEIGHTFRACTION
OF
O.radiatum
ADULTEXTRACTANDOFCONTROL
CALVES
Trial number 1 2 3
Group
Calves/ treatment
Controls Vaccinates Controls Vaccinates Control Vaccinates *Values are group means +I S.E.M.
4 4 6 6 5 5
Worm burden 878.8 115.0 2805.0 858.0 3058.0 1886.0
f 386.7* f 45.8 AZ635.3 + 440.6 k 629.4 + 978.8
Faecal egg count, epg 76.7 13.4 344.0 85.4 322.8 230.2
f + f + k k
34.9 2.4 110.1 41.5 84.7 97.6
274
I. J. EAST, D. A. BERRIE and C. J. FITZGERALD
TABLE ~-ANALYSIS
OF VARIANCEFORVACCINATIONOF CATTLE AGAINST 0. radiatum WITHTHE HIGHMOLECULAR WEIGHTFRACTIONOF 0. radiutum
Worms
Trial Treatment interaction Error
Eggs
d.f.:
ssq.
F
S.Sq.
F
2 1 2 23
3793.3 2910.4 263.1 6631.9
6.58** 10.09**
470.7 251.0 53.1 816.X
6.63”” 7.07”
td.f., Degrees of freedom; S.Sq., sum of the squares: ratio; Students /-test: *P < 0.05, **P < 0.0 1.
p-
material is not surprising because immunogenicity of proteins increases with increasing molecular weight (Crumpton, 1974). Trials 1 and 3 were not statistically significant individually (both 0.05 < P < 0.1) because each contained one aberrant result. In trial 1, control calf no. 6 13 was totally resistant to infection; no worms or eggs were observed. Variation in susceptibility to infection with 0. radiatum exists in the general cattle population (Bremner, Keith &Winks, 1976) and calf 613 may represent one extreme of this variation in susceptibility. In trial 3, vaccinated calf no. 655 had a worm burden of 5550, the highest of all calves in the three trials. The exclusion of this one calf would reduce the group mean worm burden by 50% (from 1886 to 970) and increase the level of protection from 39 to 69%. The calf had only a minimal response to vaccination and only low levels of circulating antibody specific for 0. radiatum. Thus the problem with this calf was not that the vaccine was not protective but rather that the calf failed to elicit demonstrable antibody. The ability of one animal to affect drastically the result of a small trial highlights the need to use repeated trials and pooled results to ensure an accurate assessment of potential vaccines. Acknowledgements-The authors thank Mr T. Henderson and Mr P. Scheiwe for invaluable technical assistance.
REFERENCES BRADFORD M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyfical Biochemistry 12: 248-254. BREMNER K. C., KEITH R. K. & WINKS R. 1976. Age resistance of cattle to the nodular worm Oesophagostomum radiatum Research in Veterinary Science 2 0: 3 50351.
CRUMPTONM. J. 1974. Protein antigens: the molecular bases of antigenicity and immunogenicity. In: The Antigens, Vol. 2 (Edited by SELA M.), pp. l-78. Academic Press, New York. EAST I. J. & BERRIE D.A. 1986. Antigenic differences among the life cycle stages of Oesophagostomum radiaturn. Research in Veterinary Science 4 1: 129- 130. EAST I. J., BERRIE D. A. & FITZGERALD C. J. 1988. Oesophagostomum radiatum: successful vaccination of calves with an extract of in vitro cultured larvae. International Journalfor Parasitology 18: 125-127. East I. J. & BOURNE A. S. 1988. A comparison of worm burden and faecal egg count for measuring the efficacy of vaccination against Oesophagostomum radiatum. International Journalfor Parasitology 18: 863-864. EAST 1. J., WASHINGTON E. A., BRINDLEY P. J., MONROY F. G. & SCOTT-YOUNG N. (in press) Nematospiroides dubius: passive transfer of protective immunitv to mice with monoclonal antibodies.‘Experimental Parasitology. HERLICH H., DOUVRES F. W. & ROMANOWSKI R. D. 1973. Vaccination against Oesophagostomum radiatum by injecting killed worm extracts and in vitro grown larvae into cattle. Journal of Parasitology 59: 987-993. KEITH R. K. & BREMNER K. C. 1973. Immunization of calves against the nodular worm, Oesophagostomum radiatum. Research in Veterinary Science 15: 23-124. LAEMMLI U. K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227: 680-685. MONROY F. G. & DOBSON C. 1986. Protective antigens in fractions of adult Nematospiroides dubius. Australian Journal of Experimental Biology and Medical Science 64: 335-343. MORRISSEY J. H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analytical Biochemistry 117: 307310. MUNN E. A., GREENWOOD C. A. & COADWELL W. J. 1987. Vaccination of young lambs by means of a protein fraction extracted from adult Haemonchus contortus. Parasitology 94: 385-397. NEILSON J. T. M. & VAN DE WALLE M. J. 1987. Partial protection of lambs against Haemonchus contortus bv baccination with a fractionated preparation of the paral site. Veterinarv Parasitoloav 23: 21 l-221. O’DONNELL I. j,, DINEEN?. K., ROTHWELL T. L. W. & MARSHALL R. C. 1985. Attempts to probe the antigens and protective immunogens of Trichostrongylus colubriformis in immunoblots with sera from infected and hyperimmune sheep and high- and low-responder guinea pies. Internationaljournalfor Parasitology- 15: 12q-136. . ROBERTS F. H. S. & O’SULLIVAN P. J. 1950. Methods for egg counts and larval cultures for strongyles infesting the gastro-intestinal tract of cattle. Australian Journal of Agricultural Research 1: 99-l 02. ROBERTSF. H. S., ELEK P. &KEITH R. K. 1962. Studies on resistance in calves to experimental infections with the nodular worm, Oesophagostomum radiatum (Rudolphi, 1803) Railliet, 1898. Australian Journal of Agricultural Research 13: 551-573.
lnte,nanonalJoloumalforPa,as~rology Vol.19,No. 3,pp. 271-274.1989. Printed in Great Brnain.
Press plc
OESOPHAGOSTOhfUA4
RADIA TUA4: SUCCESSFUL VACCINATION OF CALVES WITH HIGH MOLECULAR WEIGHT ANTIGENS I. J. EAST,* D. A. BERRIE
CSIRO,
Division
of Tropical
Animal
and C. J. FITZGERALD
Production, Long Pocket Laboratories, Queensland 4068, Australia
Private Bag No. 3, P.O., Indooroopilly,
(Received 21 July 1988; accepted 27 November 1988) Abstract--EAST I. J., BERRIED. A. and FITZGERALDC. J. 1989. Oesophagostomum radiatum: successful vaccination of calves with high molecular weight antigens. International Journalfor Parasitology 19: 271274. Extracts of adult Oesophagostomum radiatum were resolved into four fractions by gel filtration chromatography on a Superose 12 column. ELISA assays on these four fractions showed that the antibodies produced by naturally infected calves predominantly reacted with the void volume fraction (VV). Three trials were conducted in which calves were vaccinated with the VV fraction of adult extract. When compared to untreated controls, vaccination with the VV of adult extract significantly (PC 0.05) reduced worm establishment and faecal egg output after challenge infection relative to untreated controls. INDEX
KEY WORDS:
Oesophagostomum
radiatum ; cattle; vaccination;
INTRODUCTION CALVES infected
with Oesophagostomum radiatum, a pathogenic parasite of the gastro-intestinal tract, acquire a strong resistance to reinfection which is manifested against the late fourth stage larvae (L4) (Roberts, Elek & Keith, 1962). This acquired immunity can be stimulated artificially by immunization with worm components. Keith & Bremner (1973) successfully vaccinated calves with homogenates of both L4 and adult 0. radiatum. The soluble components of L4. grown in vitro, also contain potent protective
antigens (East, Berrie & Fitzgerald, 1988). However, Herlich, Douvres & Romanowski (1973) were unable to protect cattle from challenge infections with soluble extracts of either adults or larvae of 0. radiatum subfractions derived by gel filtration. Both homogenates and soluble
extracts
are hetero-
geneous mixtures of complex components, and the production of efficacious vaccines will almost certainly rely on identification of defined protective antigens. Little work has been done on fractionation of 0. radiatum components. In this paper, the successful vaccination of calves with the high molecular weight fraction of 0. radiatum adult worms obtained by gel filtration chromatography is reported. MATERIALS AND METHODS Worms. Adult 0. rudiatum were collected from culture calves as described by Keith & Bremner (1973). Soluble extracts were prepared by homogenization in 50 rn~ imidazole. 150mM NaCl, 5 mM Na:EDTA, pH 7.4 (INE)
*Author
to whom correspondence
should be addressed. 271
high molecular
weight antigens.
using a ground glass homogenizer, followed by centrifugation (50,000 g, 30 min, 4°C) and filtration through a 0.22 pm filter (Flow Labs, Rickmansworth, England). Protein concentrations were determined by the method of Bradford (1976). Chromatography. Two hundred microlitre samples of extract were applied to a Superose- 12 column (1 X 30 cm) (Pharmacia, Uppsala, Sweden) equilibrated and run in INE buffer. The column was controlled through a Pharmacia fast system, protein liquid chromatography eluted at 0.5 ml min’ and 0.5 ml fractions were collected. Calves. Friesian bull calves were reared worm-free in concrete pens as previously described (Roberts CI nl., 1962) and first vaccinated at 4 months of age. Vaccination. The vaccine fraction was emulsified with an equal volume of Freund’s Complete Adjuvant (Commonwealth Serum Laboratories, Parkville, Australia). Each calf received 50 pg of protein per subcutaneous injection in a single site in the neck. Control calves received an injection containing INE buffer emulsified with the same adjuvant. The injection was repeated 28 days later and 56 days after the first injection the test calves received a final subcutaneous booster of 50 pg of antigen in INE without adjuvant. Control calves received INE buffer. On days 63, 64 and 65 after the first injection, each calf was infected with 3000 0. radiufum infective larvae per OS. Faecal egg counts were made on days Y9, IO4 and 106 by the method of Roberts & O’Sullivan (1950) and reported as eggs per gram of faeces. The calves were necropsied on days 110, 111 and 112 and the worm burden determined (Roberts el al., 1962). The trial was repeated three times with groups of four, six and five calves per treatment, respectively. StafisGcs. Worm burdens are reported as arithmetic means f standard error. Faecal egg counts are the arithmetic mean + standard error of three determinations for all animals within each group. Worm burdens and faecal egg counts were square root transformed (East & Bourne, 1988) and the transformed data assessed by analysis of variance.
I. J. EAST, D. A. BERME and C. J. FITZGERALD
272
ELISA. The antigenicity of the various column fractions was determined using an enzyme-linked immunosorbent assay (ELISA). Microtitre plates were coated with aliquots of the column fractions adjusted to 100 pg ml-’ of protein. Serum was obtained from three cattle which had each received five infections with 0. radiutum. The relative proportions of antibody in these sera, specific for each column fraction, were determined by the method of East el al. (1988). Results are expressed as the ratio of each test absorbance to the mean absorbance of five known negative sera. The antibody titre of each test vaccine calf was measured by ELISA on the day of the first injection and 7 days after each of the secondary and tertiary injections. The plates were coated with the VV fraction, and each serum was diluted one in 850. The detailed method is described by East, Washington, Brindley, Monroy & Scott-Young (in press). Results again were expressed as the ratio of each test absorbance to the mean absorbance of five known negative sera. Electrophoresis. Samples were run on 10% sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) according to the method of Laemmli (1970). Gels were silver stained by the method of Morrissey (1981). Molecular weight standards were purchased from Pharmacia. RESULTS
SDS-PAGE showed that the components of the void volume fraction had molecular weights greater than 67,000 M,. More than 12 bands could be resolved in the fraction (Fig. 3). By the start of the challenge infection, all test calves had responded by
25r
0
1
2 Superose
3 Fraction
4
FIG. 2. The relative antigenicity of four Superose fractions determined using three hyperimmune anti-O.radiatum sera. Calf428 (~),calf436(111 ),calf489(\\).
The gel filtration elution profile of adult 0. r&uturn extract is shown in Fig. 1. The tubes corresponding to fractions as marked on Fig. 1 were pooled. Assay of the fractions with sera from immune cattle showed that most antigens reacting on ELISA resided in the void volume (VV) fraction (Fig. 2) and therefore this was the fraction used for vaccination. Fractions 3 and 4 were essentially non-reactive.
a
b
c
0.3
P $ 2
67
0.2
Z 5 0 b fn 9 0.1
0 I
,
I
0
5
I
10 Elution Volume
I
I
15 (ml)
20
FIG. 1. Elution profile of 0. radiatum adult extract chromatographed on Superose 12 (1 X 30 cm) equilibrated in INE (50 mM imidazole, 150 mM NaCl, 5 mM EDTA. pH 7.4) and run at 0.5 ml min-‘.
25----FIG. 3. SDS-polyacrylamide gel of 0. radiatum extracts. (a) Molecular standards; (b) adult extract; (c) adult VV fraction.
213
Vaccination of calves against 0. radiatum producing circulating antibodies which reacted with the void volume fraction (Fig. 4). The results of vaccination with 0. radiatum VV are shown in Table 1. In the three trials, the establishment of adult worms was reduced by 87, 70 and 39%, respectively, and the faecal egg count was reduced by 82,75 and 29%, respectively. The analysis of variance (Table 2) showed that both the overall average reduction in worm establishment (57%, P < 0.01) and faecal egg count (55%, P < 0.05) were significant.
However this difference may be associated with differences in dose of antigen or immunization schedule. Immunity acquired through repeated infection with 0. radiatum was directed mainly at high molecular weight components and this confirms the findings of East & Berrie (1986) who showed that most adult 0. radiatum antigens identified by Western immunoblotting were greater than 67,000 kl,. High molecular weight antigens have been used successfully to vaccinate against other gastro-intestinal nematodes including Nematospiroides dubius (Monroy & Dobson, 1986), Trichostrongylus coZubr@ormis (O’Donnell, Dineen, Rothwell & Marshall, 1985) and Haemonthus contortus (Neilson & Van de Walle, 1987). Sheep have also been successfully vaccinated with contortin (Munn, Greenwood & Coadwell, 1987), a protein of 60,000 M, derived from H. contortus which aggregates readily and would therefore, presumably, chromatograph on a gel filtration column at a much higher apparent molecular weight. This consistent success with high molecular weight
DISCUSSION Vaccination of young calves with high molecular weight fraction of an 0. radiatum extract caused a significant reduction in both the number of adult worms which established after challenge and the resultant egg count. This result represents a further step toward the isolation and identification of protective antigens suitable for incorporation into a commercial vaccine. The findings are in contrast to those of Herlich et al. (1973) who were unable to vaccinate successfully with fractions obtained by gel filtration. 30
b
a
C
A
25 0 A
20
,.--•
E
Fz
D
/
/
15 -
z w
‘1
;:
70
10 -
0 / x 5-
o-
.-. +
I!!0
4
8
+
L 12
4 Time
12
8
4
8
12
(weeks)
FIG. 4. ELISA titres of calves vaccinated with 0. radiatum adult VV. (a) First trial; (b) second trial; (c)third trial. +Indicates the average titre of control calves. Different symbols represent different animals within each experiment.
TABLE HIGH
l--WORM
BVRDENSANDFAEcALEGGcO"NTSOFCALVESVAcclNATEDWlTHTHE
MOLECULARWEIGHTFRACTION
OF
O.radiatum
ADULTEXTRACTANDOFCONTROL
CALVES
Trial number 1 2 3
Group
Calves/ treatment
Controls Vaccinates Controls Vaccinates Control Vaccinates *Values are group means +I S.E.M.
4 4 6 6 5 5
Worm burden 878.8 115.0 2805.0 858.0 3058.0 1886.0
f 386.7* f 45.8 AZ635.3 + 440.6 k 629.4 + 978.8
Faecal egg count, epg 76.7 13.4 344.0 85.4 322.8 230.2
f + f + k k
34.9 2.4 110.1 41.5 84.7 97.6
274
I. J. EAST, D. A. BERRIE and C. J. FITZGERALD
TABLE ~-ANALYSIS
OF VARIANCEFORVACCINATIONOF CATTLE AGAINST 0. radiatum WITHTHE HIGHMOLECULAR WEIGHTFRACTIONOF 0. radiutum
Worms
Trial Treatment interaction Error
Eggs
d.f.:
ssq.
F
S.Sq.
F
2 1 2 23
3793.3 2910.4 263.1 6631.9
6.58** 10.09**
470.7 251.0 53.1 816.X
6.63”” 7.07”
td.f., Degrees of freedom; S.Sq., sum of the squares: ratio; Students /-test: *P < 0.05, **P < 0.0 1.
p-
material is not surprising because immunogenicity of proteins increases with increasing molecular weight (Crumpton, 1974). Trials 1 and 3 were not statistically significant individually (both 0.05 < P < 0.1) because each contained one aberrant result. In trial 1, control calf no. 6 13 was totally resistant to infection; no worms or eggs were observed. Variation in susceptibility to infection with 0. radiatum exists in the general cattle population (Bremner, Keith &Winks, 1976) and calf 613 may represent one extreme of this variation in susceptibility. In trial 3, vaccinated calf no. 655 had a worm burden of 5550, the highest of all calves in the three trials. The exclusion of this one calf would reduce the group mean worm burden by 50% (from 1886 to 970) and increase the level of protection from 39 to 69%. The calf had only a minimal response to vaccination and only low levels of circulating antibody specific for 0. radiatum. Thus the problem with this calf was not that the vaccine was not protective but rather that the calf failed to elicit demonstrable antibody. The ability of one animal to affect drastically the result of a small trial highlights the need to use repeated trials and pooled results to ensure an accurate assessment of potential vaccines. Acknowledgements-The authors thank Mr T. Henderson and Mr P. Scheiwe for invaluable technical assistance.
REFERENCES BRADFORD M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyfical Biochemistry 12: 248-254. BREMNER K. C., KEITH R. K. & WINKS R. 1976. Age resistance of cattle to the nodular worm Oesophagostomum radiatum Research in Veterinary Science 2 0: 3 50351.
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