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Evaluation of Schistosoma bovis adult worm extract for vaccination of calves
Preventive Veterinary Medicine, 16 ( 1993 ) 7 7 - 8 4
77
Elsevier Science Publishers B.V., A m s t e r d a m
Evaluation of Schistosoma bovis adult worm extract for vaccination of calves I.E. Aradaib a, B. Abbas ~, H.O.
Bushara ~and M.G. Taylord
~Department of Veterinary Pathology, School of Veterina~3,Medicine, Unirersio'of Calfornia, Davis, CA 95616, USA bDepartment of Medicine, Pharmacologyand Toxicology, Faculty of Veterinao, Science, University of Khartoum, Khartoum, Sudan ~Department of Veterinary Pathology, Faculty of I'eterinaryScience, Universityof Khartoum, Khartoum, Sudan ~Department of Medical Hehninthology, London School of Hygiene and Tropical Medicine, University of London, London, UK (Accepted 24 November 1992 )
ABSTRACT Aradaib, I.E., Abbas, B., Bushara, H.O. and Taylor, M.G., 1993. Evaluation of Schistosoma boris adult work extract for vaccination of calves. Prey. Vet. Med., 16: 77-84. Six calves were immunized with adult worm extract of Schistosoma boris emulsified in Freund's adjuvant. The immune response was monitored by agar-gel immunodiffusion. Precipitin lines were observed when sera from immunized calves were tested against adult worm antigen, but no lines were observed with sera from control calves. The immunized calves together with six control calves were each challenged with 20 000 cercariae of Schistosoma boris administered percutaneously to the shaved tail. There was no significant difference between the immunized and the control groups as judged by fecal and tissue egg counts, worm recovery and hematological parameters ( including packed cell volume and hemoglobin concentration). This indicates failure of S. boris adull worm extract to induce resistance against S. boris challenge. Hence, lhe antibody response detected in the AGID test seemed lo have no association with protection.
INTRODUCTION
Bovine schistosomiasis, caused by Schistosoma bovis, is one of the major veterinary problems in many African and Mediterranean countries, including the Sudan (McCauly et al., 1984; Aradaib and Abbas, 1985 ). The losses occur principally in animals 6-30 months of age and are mainly due to mortality, retarded growth rate, liver contamination and poor subsequent reproductive performance (McCauly et al., 1984). In general, the prevalence of bovine Correspondence to. I.E. Aradaib~ D e p a r t m e n t of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0 1 6 7 - 5 8 7 7 / 9 3 / $ 0 6 . 0 0
78
I.E. ARADAIB ET AL
schistosomiasis increases in those areas where development in agriculture and industry depends on the construction of new dams and irrigation canals which provide suitable habitats for the snail intermediate host, Bulinus spp. However, the control of contaminated water a n d / o r destruction of the snail intermediate host is impractical and not economical. In spite of the development of effective and relatively safe drugs, prevention of rapid re-infection has remained a problem which requires repeated drug applications over a long period of time. Partial protection develops in cattle following exposure to S. boris (Hussien, 1968). This indicates that vaccination could be a feasible and effective method for limiting the extent of pathology and level of transmission (Villella et al., 1961; Hussien and Bushara, 1976; Taylor and Bickle, 1988). Significant levels of immunity to infection can be induced in laboratory animals by using irradiated schistosomular vaccine (James et al., 1981; Damian et al., 1984). Under field conditions in the endemic area of the White Nile province, a single intramuscular injection of 10 000 cercariae ofS. bovis irradiated at 3 kilo-rad induced 60% reduction in worm recovery and 80% reduction in fecal egg counts. Improved survival and body weight in calves were recorded. Protection lasted at least 40 weeks (Majid et al., 1980). The efficacy of the vaccine was 70% ( McCauly et al., 1984). The administration of various preparations of dead schistosome or non-schistosome origin as anti-schistosome vaccine has been tried. Unfortunately, experimental immunization in laboratory animals with dead antigen has been largely unsuccessful (Murrell et al., 1975; Maddison et al., 1978). However, successful immunization was achieved by Hillyer ( 1979 ), who reported a significant reduction in S. bovis worm burdens of mice vaccinated with a crude antigenic preparation from Fasciola gigantica injected with Freund's complete adjuvant. There was also some degree of success by vaccinating mice against S. mansoni using repeated intraperitoneal injections of cercarial antigen in alum (Balloul et al., 1987). However, no work has yet been carried out in cattle. In the present investigation, zebu calves were injected with schistosome antigen prepared from adult worms of S. bovis in an attempt to stimulate resistance to challenge with this trematode parasite. MATERIALS AND METHODS
Experimental animals Twelve 6- to 8-months-old zebu calves were purchased from an area known to be free from schistosomiasis and each animal was subjected to parasitological and clinical examinations. All the animals were healthy and free from trematode infection. They were then divided randomly (by lottery) into two groups. Six calves (Group 1 ) were immunized with 0.5 ml adult worm anti-
SCHISTOSOMA BOVISADULT WORM EXTRACT FOR CALF VACCINATION
79
gen (AWA) emulsified with an equal volume of Freund's complete adjuvant (Difco Laboratories, USA). Administration was by two intrasmuscular injections of approximately 0.2 ml each in the hind limbs and the rest was subcutaneously injected in at least three sites in the back. Two weeks after the first immunization, the calves were re-injected with the same initial dose emulsified in an equal volume of Freund's incomplete adjuvant (Difco Laboratory, USA). The antigen was administered at Weeks 0, 2, 4, 6, 8 and 12 (where 0 represents the first immunization). The i m m u n e response was monitored by agar-gel diffusion test until a sufficiently strong response had been obtained. Six additional calves (Group 2 ) were kept as non-immunized controls. Six months after the first immunization all the calves were challenged with 20 000 cercariae of S. bovis administered percutaneously to the shaved tail. During the course of the experiment, the animals were maintained together indoors and were fed a ration of concentrate and hay with water ad libitum.
Parasites The cercariae were obtained from 50-70 Bulinus africanus snails collected from Elmoglad in Western Sudan. After screening to exclude already parasitized snails, they were infected with miracidia obtained from experimentally infected goats. When necessary, cercariae were collected, using a light source, from shedding snails for 4-6 h.
Antigen preparation Two experimentally infected calves were slaughtered and perfused for collection of adult schistosomes. The recovered worms were washed thoroughly in physiological saline. The worms then were placed in a glass homogenizer together with an equal volume of phosphate buffered saline (pH 7.2) and sonicated with intermittent cooling in ice until no distinct worms were left (usually 3-5 min). The homogenate was then centrifuged at 12 000 rpm for 1 h in a cold centrifuge. The supernatant was taken off immediately using a Pasteur pipette and stored frozen at - 20 ° C. The protein content of the antigen was 20 mg m l - 1.
Blood samples Blood samples were collected from the jugular vein for hematology and serology. Sera were divided into 0.5 ml aliquots and stored until used. Packed cell volume (PCV) was determined in a microhematocrit centrifuge, and hemoglobin concentration (Hb) by the cyanmethemoglobin method.
80
I.E. AR~DAIB E I AL
Egg counts After challenge, weekly fecal samples were collected from the rectum and the fecal egg counts were calculated as described by Pitchford and Visser ( 1975 ). Samples from the liver and the intestines were collected at the time of necropsy and were kept in suitable polythene bags at - 2 0 ° C until used. The tissue egg determination was made according to the digestion method of Cheever ( 1968 ).
Perfusion After slaughter, perfusion of calves for worm recovery and tissue egg counts was done as described by Bushara et al. (1978). The worms were preserved in Rudabush solution and counted individually.
Agar-gel immunodiffusion (AGID) AGID was carried out basically as described by Ouchterlony ( 1958 ). Briefly, 15 ml of 1.5% Noble agar in barbitone buffer (2,76 g barbitone, 15.44 g barbitone sodium, 0.1 g sodium azide and 1 1 distilled water), pH 8.2, was poured into a leveled petri dish. A pattern consisting of one central and six peripheral wells was cut in the agar. Each well was filled twice with 25/tl of reactant fluid. The antigen was used at a concentration of 10 mg m l - ~.
Statistical analysis Geometric means were calculated for parasitological parameters (fecal egg counts, tissue egg counts and worm recoveries), and arithmetic means were calculated for the hematological parameters (PCV and Hb). The differences between means were tested by comparison against 95% confidence intervals (CI) (Schwabe et al., 1977). RESULTS
Serology Precipitin lines were first detected in sera from three calves in the vaccinated group by the 4th week post-vaccination and in sera of all vaccinated calves by the 5th week after the first vaccination, and all vaccinated animals remained test-positive throughout the experimental period. No precipitin lines were formed with sera from the control calves.
SCHISTOSOMA BOVIS ADULT WORM EXTRACT FOR CALF VACCINATION
81
Hematology Haemoglobin concentrations (Hb) (gm dl- ~) and PCV (%) were similar in the two groups. All the animals showed a progressive reduction in their PCV and Hb concentrations compared to their initial values (Figs. 1 and 2 ). 32"
30 i 28
26"
24"
22-
20-
1 ~
2
3
4
5
6 Weel(s
acc (Mean ±SD) ]
7
8
post
infection
9
10
11
12
13
14
15
Cont (Mean ± SD)
Fig. 1. Packed cell volumes of calves vaccinated with S. bovis worm extracts and in non-vaccinated controls. 11
10
o
g
7"
6
.... 1
r~
2
3
4
Cont (Mean ± SD) J Vacc. (Mean ± SD)
5
6 Weeks
7
8
9
post
infection
10
11
12
, .... i .... , ]3 14 15
I
Fig. 2. Hemoglobin concentration in calves vaccinated with S. boris worm extracts and in nonvaccinated controls.
82
I.E. A R A D A I B E l AL.
Clinical observations All animals in both groups developed mucoid and then hemorrhagic diarrhoea, loss of appetite, emaciation, roughness of the skin and pale mucous membranes from around the 6th week post-challenge (which coincided with time of oviposition). The severity of these signs increased between the 7th and the 9th week when fecal egg counts were highest. The animals became dull and depressed by the time of slaughter.
Parasitological findings (Fig. 3) Fecal egg counts Eggs first appeared in the feces at Week 6 post-challenge and all the animals were shedding by Week 7. There was no significant difference between the
4oo! 350
~ 30o "
q
i
!
2s0 1 N ~
200
i
8 ~
150
J
--
~
100
50
-~-0
~
1
~ 2
~ 3
4
5 Weeks
.
. ~
.
95% k
post
- T--
~
l
•
7
8
9
10
T-11
1 12
Infection
.
vac --
6
95% L --
95% U
~
-
cl
95% U
Fig. 3. Fecal egg counts of calves vaccinated with S. boris w o r m extracts and the controls.
83
SCHISTOSOMA BOVIS ADULT WORM EXTRACT FOR CALF VACCINATION
TABLE 1 Tissue egg count (eggs per gram of tissue ) by type of tissue Tissue
Liver Small intestines Large intestines
Vaccinated
Controls
Mean
95% CI
Mean
95% CI
259 2160 1439
185, 362 1298, 3593 988, 2096
175 1504 1042
128,240 1087, 2065 747, 1453
means of the vaccinated calves and the controls. Tissue egg counts (Table 1 ) were highest in the small intestines. However, there was no significant difference in means of tissue egg counts between the vaccinated and the control calves. There was no statistically significant difference in worm recovery between the vaccinated calves ( m e a n = 2 3 0 5 , 95% C I = 1295, 4083) and the controls ( m e a n = 1682, 95% C I = 8 9 6 , 3156).
DISCUSSION
Our results showed no significant difference between the calves vaccinated with adult worm antigen and their controls in fecal and tissue egg counts and worm recovery. The pathogenicity of S. bovis is mainly due to oviposition, which results in hemorrhagic diarrhoea with consequent progressive reduction in PCV and Hb concentrations in all animals (Figs. 1 and 2). This indicates failure of antigen prepared from adult schistosomes of S. bovis to stimulate resistance to challenge with this trematode parasite. We recognize that we had small sample size, but the responses of the groups were so similar that we do not believe that lack of significance was due merely to poor statistical power. In general, experimental immunization with dead schistosome antigens has been unsuccessful (Smithers and Doenhoff, 1983 ). The main reason for this failure has been attributed to insufficient relative antigen, i.e. antigenic competition from contaminating material in the preparation or stimulation of irrelevant antibody (Murrell et al., 1975; Maddison et al., 1978). The antibodies detected in this study seemed to have no association with protection. In laboratory animals (rats and mice), a DNA-recombinant vaccine in the presence of alum or complete Freund's adjuvant resulted in up to 70% protection as judged by worm recovery (Balloul et al., 1987 ). This study suggests that the recent advances in cell immunology and molecular biology such as DNA recombinant techniques should be used to develop vaccines for controlling schistosomal infection in cattle.
84
I.E. ARADAIB EF -kL
REFERENCES Aradaib, I.E. and Abbas, B., 1985. A retrospective study of diseases diagnosed at the U niversit,x of Khartoum Veterinary Medical Teaching Hospital. Sudan J. Vet. Sci. Anita. Husb., 2: 5566. Balloul, J.M., Grzych, J.M., Piercec, R.J. and Capron, A., 1987. A purified 28 000 dalton prorein from Schislr)soma mansoni adull worms protects rat and mice against experimental schistosomiasis. J. Immunol., 138: 3448-3453. Bushara, H.O., Hussien, M.F., Saad, A.M., Taylor, M.G., Dargie, J.D., Marshal, T.E. and Nelson, G.S., 1978. Immunization of calves against &'hislosoma ho~,i,r using irradiated cercariae or schistosomula orS. boris. Parasilology, 77: 303-310. Cheerer, A.W., 1968. Conditions affecting the accuracy of potassium hydroxide digestion techniques for counling &'hislosoma mansoni eggs in tissues. Bull. W.H.O., 39:328-331. Damian, R.T., Robert, M.T., Powell, M,R., Clark, J.D., Lewis, F.A. and Stirewalt, M.A., 1984. Schistosoma mansoni egg granuloma size reduction in challenged baboon after vaccination with cryopreserved schistosomula. Helminthol. Abstr., 51-54. Hillyer, G.V., 1979. &'histosoma mansoni: reduced worm burden in mice immunized with isolated 1,asciola hepalica anligen. Exp. Parasitol., 48: 287-295. Hussien, M.F., 1968. The pathology of natural and experimental bovine Schistosomiasis. Trans. R. Soc. Trop. Med. Hyg., 62: 9-12. Hussien, M.F. and Bushara, H.O., 1976. The investigation on the development of an irradiated vaccine for animal schistosomiasis. In: Nuclear Techniques in Animal Production and Health. Inlernational Atomic Energy Agency, Austria, pp. 421-431. James, E.R., Labine, M. and Sher, A,, 1981. Mechanism of protective immunit3, against ,S~'hi.> losoma mansoni infection in mice vaccinated with irradiated cercariae. Cell. lmmunol., 65: 75-83. Maddison, S.E., Slemenad, S.B., Chandler, F.M. and Kagan, I.G., 1978. Studies on putative adull worm-derived vaccine and adjuvant for protection against ,fchisto.so,ta ma~,~o~i. J. Parasitol., 64: 986-993. Majid~ A.A., Bushara, H.O., Saad, A.M., Hussien, M.F,~ Taylor~ M.G., Dargic, ,I.D., Marshal, T.F. de C. and Nelson, G.S., 1980. Field testing of an irradiated ,gchisto.s'(,na ho~'i.s vaccine. Am. J. Trop. Med. Hyg., 29: 452-455. McCauley, E.H., Majid, A.A. and Taycb, A., 1984. Economic evaluation of thc production impact of bovine schistosomiasis and vaccination in the Sudan. Prey. Vet. Med., 6: 735-754. Murrell, K.D., Dean, D.A. and Stafferd, E.E., 1975. Resistance to infection to ,Sk'hi,s'~o.5o~,~a ma~.~'oni after immunization with worm extracts or live ccrcariae. Am. J. Hyg. Trop. Med., 24: 955. Ouchlerlony, O., 1958. Diffusion in gel: Mclhod for immunological anabsis. Prog. Allergy, 5: 1-78.
Pitchford, R.J. and Visscr, P.S., 1975. A simple and rapid technique for quantitative estimation of helminth eggs in human and animal excreta with special reference to scbislosomc spp. Trans. R. Soc. Trop. Med. Hyg., 69:318-322. Schwabe, C.W., Riemann, H.P. and Franti, C.E., 1977. Epidemiology in Veterinary Practice. Lea and Febiger, Philadelphia, PA, 303 pp. Smithers, S.R. and Doenhoff, M.J., 1983. Schistosomiasis. In: S. Cohen and K.S. Warren (Editors), Immunology of Parasitic Infection. 2nd edn. Blackwell Scientific. Oxford, pp. 527607. Taylor, M.G. and Bickle. Q.D., 1988. Irradialed schistosome vaccine. Parasitol. Today, 5: 132134. Villella, J.B., Somberg, H.J. and Gould, S.E., 1961. Immunization to Schist~s~,,~la ma~.~oni in mice inoculated with irradialed cercariae. Science, N. 1/ 143:1073-1074.
77
Elsevier Science Publishers B.V., A m s t e r d a m
Evaluation of Schistosoma bovis adult worm extract for vaccination of calves I.E. Aradaib a, B. Abbas ~, H.O.
Bushara ~and M.G. Taylord
~Department of Veterinary Pathology, School of Veterina~3,Medicine, Unirersio'of Calfornia, Davis, CA 95616, USA bDepartment of Medicine, Pharmacologyand Toxicology, Faculty of Veterinao, Science, University of Khartoum, Khartoum, Sudan ~Department of Veterinary Pathology, Faculty of I'eterinaryScience, Universityof Khartoum, Khartoum, Sudan ~Department of Medical Hehninthology, London School of Hygiene and Tropical Medicine, University of London, London, UK (Accepted 24 November 1992 )
ABSTRACT Aradaib, I.E., Abbas, B., Bushara, H.O. and Taylor, M.G., 1993. Evaluation of Schistosoma boris adult work extract for vaccination of calves. Prey. Vet. Med., 16: 77-84. Six calves were immunized with adult worm extract of Schistosoma boris emulsified in Freund's adjuvant. The immune response was monitored by agar-gel immunodiffusion. Precipitin lines were observed when sera from immunized calves were tested against adult worm antigen, but no lines were observed with sera from control calves. The immunized calves together with six control calves were each challenged with 20 000 cercariae of Schistosoma boris administered percutaneously to the shaved tail. There was no significant difference between the immunized and the control groups as judged by fecal and tissue egg counts, worm recovery and hematological parameters ( including packed cell volume and hemoglobin concentration). This indicates failure of S. boris adull worm extract to induce resistance against S. boris challenge. Hence, lhe antibody response detected in the AGID test seemed lo have no association with protection.
INTRODUCTION
Bovine schistosomiasis, caused by Schistosoma bovis, is one of the major veterinary problems in many African and Mediterranean countries, including the Sudan (McCauly et al., 1984; Aradaib and Abbas, 1985 ). The losses occur principally in animals 6-30 months of age and are mainly due to mortality, retarded growth rate, liver contamination and poor subsequent reproductive performance (McCauly et al., 1984). In general, the prevalence of bovine Correspondence to. I.E. Aradaib~ D e p a r t m e n t of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0 1 6 7 - 5 8 7 7 / 9 3 / $ 0 6 . 0 0
78
I.E. ARADAIB ET AL
schistosomiasis increases in those areas where development in agriculture and industry depends on the construction of new dams and irrigation canals which provide suitable habitats for the snail intermediate host, Bulinus spp. However, the control of contaminated water a n d / o r destruction of the snail intermediate host is impractical and not economical. In spite of the development of effective and relatively safe drugs, prevention of rapid re-infection has remained a problem which requires repeated drug applications over a long period of time. Partial protection develops in cattle following exposure to S. boris (Hussien, 1968). This indicates that vaccination could be a feasible and effective method for limiting the extent of pathology and level of transmission (Villella et al., 1961; Hussien and Bushara, 1976; Taylor and Bickle, 1988). Significant levels of immunity to infection can be induced in laboratory animals by using irradiated schistosomular vaccine (James et al., 1981; Damian et al., 1984). Under field conditions in the endemic area of the White Nile province, a single intramuscular injection of 10 000 cercariae ofS. bovis irradiated at 3 kilo-rad induced 60% reduction in worm recovery and 80% reduction in fecal egg counts. Improved survival and body weight in calves were recorded. Protection lasted at least 40 weeks (Majid et al., 1980). The efficacy of the vaccine was 70% ( McCauly et al., 1984). The administration of various preparations of dead schistosome or non-schistosome origin as anti-schistosome vaccine has been tried. Unfortunately, experimental immunization in laboratory animals with dead antigen has been largely unsuccessful (Murrell et al., 1975; Maddison et al., 1978). However, successful immunization was achieved by Hillyer ( 1979 ), who reported a significant reduction in S. bovis worm burdens of mice vaccinated with a crude antigenic preparation from Fasciola gigantica injected with Freund's complete adjuvant. There was also some degree of success by vaccinating mice against S. mansoni using repeated intraperitoneal injections of cercarial antigen in alum (Balloul et al., 1987). However, no work has yet been carried out in cattle. In the present investigation, zebu calves were injected with schistosome antigen prepared from adult worms of S. bovis in an attempt to stimulate resistance to challenge with this trematode parasite. MATERIALS AND METHODS
Experimental animals Twelve 6- to 8-months-old zebu calves were purchased from an area known to be free from schistosomiasis and each animal was subjected to parasitological and clinical examinations. All the animals were healthy and free from trematode infection. They were then divided randomly (by lottery) into two groups. Six calves (Group 1 ) were immunized with 0.5 ml adult worm anti-
SCHISTOSOMA BOVISADULT WORM EXTRACT FOR CALF VACCINATION
79
gen (AWA) emulsified with an equal volume of Freund's complete adjuvant (Difco Laboratories, USA). Administration was by two intrasmuscular injections of approximately 0.2 ml each in the hind limbs and the rest was subcutaneously injected in at least three sites in the back. Two weeks after the first immunization, the calves were re-injected with the same initial dose emulsified in an equal volume of Freund's incomplete adjuvant (Difco Laboratory, USA). The antigen was administered at Weeks 0, 2, 4, 6, 8 and 12 (where 0 represents the first immunization). The i m m u n e response was monitored by agar-gel diffusion test until a sufficiently strong response had been obtained. Six additional calves (Group 2 ) were kept as non-immunized controls. Six months after the first immunization all the calves were challenged with 20 000 cercariae of S. bovis administered percutaneously to the shaved tail. During the course of the experiment, the animals were maintained together indoors and were fed a ration of concentrate and hay with water ad libitum.
Parasites The cercariae were obtained from 50-70 Bulinus africanus snails collected from Elmoglad in Western Sudan. After screening to exclude already parasitized snails, they were infected with miracidia obtained from experimentally infected goats. When necessary, cercariae were collected, using a light source, from shedding snails for 4-6 h.
Antigen preparation Two experimentally infected calves were slaughtered and perfused for collection of adult schistosomes. The recovered worms were washed thoroughly in physiological saline. The worms then were placed in a glass homogenizer together with an equal volume of phosphate buffered saline (pH 7.2) and sonicated with intermittent cooling in ice until no distinct worms were left (usually 3-5 min). The homogenate was then centrifuged at 12 000 rpm for 1 h in a cold centrifuge. The supernatant was taken off immediately using a Pasteur pipette and stored frozen at - 20 ° C. The protein content of the antigen was 20 mg m l - 1.
Blood samples Blood samples were collected from the jugular vein for hematology and serology. Sera were divided into 0.5 ml aliquots and stored until used. Packed cell volume (PCV) was determined in a microhematocrit centrifuge, and hemoglobin concentration (Hb) by the cyanmethemoglobin method.
80
I.E. AR~DAIB E I AL
Egg counts After challenge, weekly fecal samples were collected from the rectum and the fecal egg counts were calculated as described by Pitchford and Visser ( 1975 ). Samples from the liver and the intestines were collected at the time of necropsy and were kept in suitable polythene bags at - 2 0 ° C until used. The tissue egg determination was made according to the digestion method of Cheever ( 1968 ).
Perfusion After slaughter, perfusion of calves for worm recovery and tissue egg counts was done as described by Bushara et al. (1978). The worms were preserved in Rudabush solution and counted individually.
Agar-gel immunodiffusion (AGID) AGID was carried out basically as described by Ouchterlony ( 1958 ). Briefly, 15 ml of 1.5% Noble agar in barbitone buffer (2,76 g barbitone, 15.44 g barbitone sodium, 0.1 g sodium azide and 1 1 distilled water), pH 8.2, was poured into a leveled petri dish. A pattern consisting of one central and six peripheral wells was cut in the agar. Each well was filled twice with 25/tl of reactant fluid. The antigen was used at a concentration of 10 mg m l - ~.
Statistical analysis Geometric means were calculated for parasitological parameters (fecal egg counts, tissue egg counts and worm recoveries), and arithmetic means were calculated for the hematological parameters (PCV and Hb). The differences between means were tested by comparison against 95% confidence intervals (CI) (Schwabe et al., 1977). RESULTS
Serology Precipitin lines were first detected in sera from three calves in the vaccinated group by the 4th week post-vaccination and in sera of all vaccinated calves by the 5th week after the first vaccination, and all vaccinated animals remained test-positive throughout the experimental period. No precipitin lines were formed with sera from the control calves.
SCHISTOSOMA BOVIS ADULT WORM EXTRACT FOR CALF VACCINATION
81
Hematology Haemoglobin concentrations (Hb) (gm dl- ~) and PCV (%) were similar in the two groups. All the animals showed a progressive reduction in their PCV and Hb concentrations compared to their initial values (Figs. 1 and 2 ). 32"
30 i 28
26"
24"
22-
20-
1 ~
2
3
4
5
6 Weel(s
acc (Mean ±SD) ]
7
8
post
infection
9
10
11
12
13
14
15
Cont (Mean ± SD)
Fig. 1. Packed cell volumes of calves vaccinated with S. bovis worm extracts and in non-vaccinated controls. 11
10
o
g
7"
6
.... 1
r~
2
3
4
Cont (Mean ± SD) J Vacc. (Mean ± SD)
5
6 Weeks
7
8
9
post
infection
10
11
12
, .... i .... , ]3 14 15
I
Fig. 2. Hemoglobin concentration in calves vaccinated with S. boris worm extracts and in nonvaccinated controls.
82
I.E. A R A D A I B E l AL.
Clinical observations All animals in both groups developed mucoid and then hemorrhagic diarrhoea, loss of appetite, emaciation, roughness of the skin and pale mucous membranes from around the 6th week post-challenge (which coincided with time of oviposition). The severity of these signs increased between the 7th and the 9th week when fecal egg counts were highest. The animals became dull and depressed by the time of slaughter.
Parasitological findings (Fig. 3) Fecal egg counts Eggs first appeared in the feces at Week 6 post-challenge and all the animals were shedding by Week 7. There was no significant difference between the
4oo! 350
~ 30o "
q
i
!
2s0 1 N ~
200
i
8 ~
150
J
--
~
100
50
-~-0
~
1
~ 2
~ 3
4
5 Weeks
.
. ~
.
95% k
post
- T--
~
l
•
7
8
9
10
T-11
1 12
Infection
.
vac --
6
95% L --
95% U
~
-
cl
95% U
Fig. 3. Fecal egg counts of calves vaccinated with S. boris w o r m extracts and the controls.
83
SCHISTOSOMA BOVIS ADULT WORM EXTRACT FOR CALF VACCINATION
TABLE 1 Tissue egg count (eggs per gram of tissue ) by type of tissue Tissue
Liver Small intestines Large intestines
Vaccinated
Controls
Mean
95% CI
Mean
95% CI
259 2160 1439
185, 362 1298, 3593 988, 2096
175 1504 1042
128,240 1087, 2065 747, 1453
means of the vaccinated calves and the controls. Tissue egg counts (Table 1 ) were highest in the small intestines. However, there was no significant difference in means of tissue egg counts between the vaccinated and the control calves. There was no statistically significant difference in worm recovery between the vaccinated calves ( m e a n = 2 3 0 5 , 95% C I = 1295, 4083) and the controls ( m e a n = 1682, 95% C I = 8 9 6 , 3156).
DISCUSSION
Our results showed no significant difference between the calves vaccinated with adult worm antigen and their controls in fecal and tissue egg counts and worm recovery. The pathogenicity of S. bovis is mainly due to oviposition, which results in hemorrhagic diarrhoea with consequent progressive reduction in PCV and Hb concentrations in all animals (Figs. 1 and 2). This indicates failure of antigen prepared from adult schistosomes of S. bovis to stimulate resistance to challenge with this trematode parasite. We recognize that we had small sample size, but the responses of the groups were so similar that we do not believe that lack of significance was due merely to poor statistical power. In general, experimental immunization with dead schistosome antigens has been unsuccessful (Smithers and Doenhoff, 1983 ). The main reason for this failure has been attributed to insufficient relative antigen, i.e. antigenic competition from contaminating material in the preparation or stimulation of irrelevant antibody (Murrell et al., 1975; Maddison et al., 1978). The antibodies detected in this study seemed to have no association with protection. In laboratory animals (rats and mice), a DNA-recombinant vaccine in the presence of alum or complete Freund's adjuvant resulted in up to 70% protection as judged by worm recovery (Balloul et al., 1987 ). This study suggests that the recent advances in cell immunology and molecular biology such as DNA recombinant techniques should be used to develop vaccines for controlling schistosomal infection in cattle.
84
I.E. ARADAIB EF -kL
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