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Precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae
Veterinary Parasitology, 43 (1992) 243-247 Elsevier Science Publishers B.V., Amsterdam
243
Precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae C.A.O. Adeyefa Department of Veterinary Medicine, Universityof lbadan, Ibadan, Nigeria (Accepted 16 January 1992)
ABSTRACT Adeyefa, C.A.O., 1992. Precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae. Vet. Parasitol., 43: 243-247. The precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae was investigated. IgG(T) from the sera of horses naturally infected with S. vulgaris adults and arterial larvae recognised the presence of two antigenic components of the mitogenic fractions. The results obtained seem to confirm that these antigens are immunogenicin stimulating the production of increased levels of IgG(T) in infected animals, and showed that the procedures could be used as immunological tools in the diagnosis ofS. vulgaris infection.
INTRODUCTION
The fourth and fifth stage larvae of Strongylus vulgaris migrate extensively in the mesenteric arterial system of infected horses (Drudge et al., 1966; Amborski et al., 1974; Ogbourne and Duncan, 1977). This results in large increases in the serum protein concentration, particularly the fl-globulin fraction, as seen in experimental infection with S. vulgaris (Drudge et al., 1966; Round, 1970; Amborski et al., 1974). Patton et al. (1978), Bailey et al. (1984a) and Adeyefa (1989a) also reported increases in the total serum protein concentration in naturally infected horses. The mitogen produced by the migrating arterial larvae has been incriminated in the elevation of the fl-globulin fraction of serum protein, particularly IgG (T) (Bailey et al., 1982, 1984a, b; Adeyefa et al., 1985; Adeyefa, 1989b). The characterisation of this mitogen by gel filtration and ion-exchange chromatography showed that it was contained in the third and fourth fractions of the crude larval extract separated on Sephadex G-200, and in Fraction 4C of the fourth fraction separated on Correspondence to: C.A.O. Adeyefa, Department of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
244
C.A.O. ADEYEFA
DEAE-Sephadex A-25 (Adeyefa, 1983; Bailey et al., 1984b; Adeyefa et al., 1985). The purpose of the present study was to determine the immunoprecipitation activities of this mitogen against I g G ( T ) from the sera of horses naturally infected with S. vulgaris. MATERIALS AND METHODS
Fourth and fifth stage larvae ofS. vulgaris were collected from the anterior mesenteric arteries of horses slaughtered at Potters and Sons, Bishop Sutton, UK. Extracts were prepared from them and fractionated as previously described (Bailey et al., 1984b; Adeyefa et al., 1985). Briefly, the somatic extracts were fractionated by upward flow chromatography on Sephadex G-200 in 0.15% phosphate-buffered saline and the fractions concentrated by vacu u m dialysis. DEAE-Sephadex A-25 ion-exchange chromatography was performed with a stepwise gradient of 0.01 M (pH 8.6) to 0.75 M (pH 7.4) Tris-HC1 buffer to further purify the mitogenic fraction. The protein concentration of the crude arterial larval extract, as well as the fractions, was approximately 25 mg ml-~. The somatic extract eluate from Sephadex G-200 was pooled into five different fractions, F I - F 5 . Fraction F4 (the mitogenic fraction) was further eluted into five fractions, 4A-4E, on DEAE-Sephadex A-25. Fraction 4C was the mitogenic fraction and was eluted with 0.3m to 0.35m Tris-HC1 (pH 7.4). Blood was collected into plain universal bottles from 40 horses from which arterial larvae were collected. These horses were also naturally moderately or heavily infected with adult S. vulgaris and other helminth parasites in their small intestines, colon and caecum. The blood samples were allowed to clot overnight and sera were harvested the following day and stored in aliquots of 2 ml at - 2 0 ° C. The fractions of the larval antigen and the horse sera were then brought to Nigeria where the rest of the study was conducted. Twenty millilitres of the sera from the 40 horses were pooled and fractionated by DEAE ion-exchange chromatography with 0.01 M phosphate buffer (pH 8.0 ). The IgG (T)-rich fraction was concentrated by vacuum dialysis and stored at - 2 0 °C until required for analyses. Control sera were obtained from unborn foetuses removed from the uteri of the slaughtered horses. Antisera to the various fractions were raised in rabbits, as described by Wynne et al. ( 1981 ). Immunodiffusion and immunoelectrophoresis were performed as previously reported (Adeyefa, 1983; Adeyefa et al., 1985). The mitogenic fractions F4 from gel filtration and 4A-4D from ion-exchange chromatography were reacted against the IgG (T)-rich fraction of horse sera and the antisera to the mitogenic fractions. In immunodiffusion, the central well contained the IgG(T)-rich fraction and the peripheral wells contained Fractions 4A, 4B, 4C and 4D, while in immunoelectrophoresis the wells contained Frac-
PRECIPITIN RESPONSE OF M1TOGEN PRODUCED BY S I'ULGARIS
245
tions F4 and 4C, and the trough contained the IgG (T)-rich fraction. The slides were washed and stained with Ponceaus as previously described (Adeyefa et al., 1985). RESULTS
Precipitin lines developed within 36 h in the immunodiffusion slides of IgG (T) against Fractions 4A-4D. The reaction revealed the presence of two proteins in these fractions (Fig. 1 ). The presence of lines of complete identity indicated a common protein in Fractions 4A-4D, while the spur of partial identity would indicate an additional protein in Fraction 4C recognised by IgG (T). In immunoelectrophoresis of IgG (T) against Fraction F4, the precipitin lines indicate the presence of one protein (Fig. 2 ), while that of IgG (T) against Fraction 4C revealed the presence of two proteins which migrated at different speeds (Fig. 3). Similar results were obtained when the two fractions were reacted against the antisera raised in rabbits. This supports the finding that IgG (T) is the principal component offl-globulin fractions, which is elevated in S. vulgaris infection. No detectable precipitin lines were observed with the foetal control serum. 4B
z+A 0
4c
~
0
40
Fig. 1. Line drawing of precipitin lines developed by mitogenic Fractions 4A-4D in immunodiffusion with IgG (T).
IgG(T) Cathode
Anode
Fig. 2. Line drawing of precipitin lines developed by Fraction F4 in immunoelectrophoresis with IgG(Y).
Anode
~4C
IgO(T) Cathode
Fig. 3. Line drawing ofprecipitin line developed by Fraction 4C in immunoelectrophoresis with IgG(T).
246
CA.O. ADEYEFA
DISCUSSION
The results of these studies indicate that the IgG (T) from the sera of horses with naturally acquired S. vulgaris infection recognised the proteins in Fractions F4 and 4C from S. vulgaris arterial larval mitogen as antigens. The antigen-antibody reactions gave the precipitin lines observed in the immunodiffusion and immunoelectrophoresis slides. It has been reported earlier that the extracts of arterial larvae of S. vulgaris produce a mitogen (Bailey et al., 1982, 1984a, b; Adeyefa et al., 1985 ) and that this mitogen stimulated polyclonal proliferation of equine peripheral blood lymphocytes (Bailey et al., 1982, 1984a,b; Adeyefa, 1983, 1989b; Adeyefa et al., 1985), as well as lymphocytes from other lymphoid organs (Bailey et al., 1984a,b). The large increases in total serum protein concentration in natural and experimental S. vulgaris infection reported by many workers (Drudge et al., 1966; Round, 1970; Amborski et al., 1974; Ogbourne and Duncan, 1977; Patton et al., 1978: Bailey et al., 1984a,b; Adeyefa, 1989a) have been shown to be responsible for the high degree of acquired immunity to S. vulgaris infection in young horses. This immunity has been attributed to increases in//-globulins, particularly IgG(T), by these workers. It has also been shown that the increase in IgG(T) level is probably due to the mitogen produced by the arterial larvae during their extensive migration in the mesenteric arterial system of infected horses (Bailey et al., 1982, 1984a,b; Adeyefa et al., 1985; Adeyefa, 1989b). The results of the present study would seem to confirm that the proteins present in the mitogenic fractions F4 and 4C are antigenic stimulants for the production of IgG (T) in the serum of horses naturally or experimentally infected with S.
vulgaris. The above results could be used as an immunological tool in the diagnosis of S. vulgaris infection in horses. They could also serve as fruitful models in the production of effective immunising agents against S. vulgaris infection from the mitogen. However, further studies would be necessary to elucidate the in vivo serological activities of this mitogen since polyctonal activation of host lymphocytes in vivo has been postulated to be associated with the immunosuppression often observed in parasitic infections in field situations. ACKNOWLEDGEMENTS
The author is grateful to Dr. Mick Bailey for the antisera used in this study, and to the staff of Potters and Sons for permission and assistance in collecting arterial larvae and blood from slaughtered horses. I am also grateful to Professor Mark Nwagwu for permission to use his laboratory facilities to fractibnate the serum and to Miss Adetilure for technical assistance.
PRECIPITIN RESPONSE OF MITOGEN PRODUCED BY S. VULGARIS
247
REFERENCES Adeyefa, C.A,O., 1983. Studies on the mitogenic factor produced by the arterial larval Strongylus vulgaris in the horse. MSc Thesis, University College of Wales. Adeyefa, C.A.O., 1989a. The effects of anthelmintic treatment and improved management on the haematological values of horses with naturally acquired helminthiasis. Trop. Vet., 7:177184. Adeyefa, C.A.O., 1989b. The response of equine peripheral blood lymphocytes to Strongylus vulgaris arterial larval mitogen. Trop. Vet., 7: 58-64. Adeyefa, C.A.O., Bailey, M., Lloyd, S. and Soulsby, E.J.L., 1985. Larval mitogen fractionation. Trop. Vet., 3: 66-69. Amborski, G.F., Bello, T.R. and Torbet, E.J., 1974. Host response to experimentally induced infections of Strongylus vulgaris in parasite free and naturally infected ponies. Am. J. Vet. Res., 35: 1181-1188. Bailey, M., Lloyd, S. and Soulsby, E.J.L., 1982. Non-specific immunostimulatory effect of antigens of Strongylus vulgaris. Proceedings of the British Society for Parasitology. Abstract in Parasitology 85: 34. Bailey, M., Kent, T., Martin, S.C., Lloyd, S. and Soulsby, E.J.L., 1984a. Haematological and biochemical values in horses naturally infected with Strongylus vulgaris. Vet. Rec., 115:144147. Bailey, M., Lloyd, S., Martin, S.C. and Soulsby, E.J.L., 1984b. In-vitro induction of lymphocyte responsiveness by Strongylus vulgaris-derived mitogen. Z. Parasitenkd., 70: 229-242. Drudge, J.H., Lyons, E.T. and Szanto, J., 1966. Pathogenesis of migrating stages of helminths with special reference to Strongylus vulgaris. In: E.J.L. Soulsby (Editor), Biology of Parasites, Emphasis on Veterinary Parasites. Academic Press, New York/London, pp. 199-214. Ogbourne, C.P. and Duncan, J.L., 1977. Strongylus vulgaris in the horse, its biology and veterinary importance. Commonw. Inst. Helminthol. Misc. PUN., 4: 1-40. Patton, S., Mock, R.E., Drudge, J.H. and Morgan, D., 1978. Increase in immunoglobulin concentration in ponies as a response to experimental infection with the nematode Strongylus vulgaris. Am. J. Vet. Res., 39: 19-23. Round, M.C., 1970. The development of strongyles in horses and the associated serum protein changes. In: J.T. Bryans and M. Gerber (Editors), Equine Infectious Diseases II. Proceedings of the 2nd International Conference on Equine Infectious Diseases, 1979, at Paris. Karger, Basel, pp. 290-303,323-325. Wynne, E., Sloombe, J.O.D. and Wilkie, B.H., 1981. Antigenic analyses of tissues and excretory and secretory products from Strongylus vulgaris. Can. J. Comp. Med., 45: 259-265.
243
Precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae C.A.O. Adeyefa Department of Veterinary Medicine, Universityof lbadan, Ibadan, Nigeria (Accepted 16 January 1992)
ABSTRACT Adeyefa, C.A.O., 1992. Precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae. Vet. Parasitol., 43: 243-247. The precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae was investigated. IgG(T) from the sera of horses naturally infected with S. vulgaris adults and arterial larvae recognised the presence of two antigenic components of the mitogenic fractions. The results obtained seem to confirm that these antigens are immunogenicin stimulating the production of increased levels of IgG(T) in infected animals, and showed that the procedures could be used as immunological tools in the diagnosis ofS. vulgaris infection.
INTRODUCTION
The fourth and fifth stage larvae of Strongylus vulgaris migrate extensively in the mesenteric arterial system of infected horses (Drudge et al., 1966; Amborski et al., 1974; Ogbourne and Duncan, 1977). This results in large increases in the serum protein concentration, particularly the fl-globulin fraction, as seen in experimental infection with S. vulgaris (Drudge et al., 1966; Round, 1970; Amborski et al., 1974). Patton et al. (1978), Bailey et al. (1984a) and Adeyefa (1989a) also reported increases in the total serum protein concentration in naturally infected horses. The mitogen produced by the migrating arterial larvae has been incriminated in the elevation of the fl-globulin fraction of serum protein, particularly IgG (T) (Bailey et al., 1982, 1984a, b; Adeyefa et al., 1985; Adeyefa, 1989b). The characterisation of this mitogen by gel filtration and ion-exchange chromatography showed that it was contained in the third and fourth fractions of the crude larval extract separated on Sephadex G-200, and in Fraction 4C of the fourth fraction separated on Correspondence to: C.A.O. Adeyefa, Department of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
244
C.A.O. ADEYEFA
DEAE-Sephadex A-25 (Adeyefa, 1983; Bailey et al., 1984b; Adeyefa et al., 1985). The purpose of the present study was to determine the immunoprecipitation activities of this mitogen against I g G ( T ) from the sera of horses naturally infected with S. vulgaris. MATERIALS AND METHODS
Fourth and fifth stage larvae ofS. vulgaris were collected from the anterior mesenteric arteries of horses slaughtered at Potters and Sons, Bishop Sutton, UK. Extracts were prepared from them and fractionated as previously described (Bailey et al., 1984b; Adeyefa et al., 1985). Briefly, the somatic extracts were fractionated by upward flow chromatography on Sephadex G-200 in 0.15% phosphate-buffered saline and the fractions concentrated by vacu u m dialysis. DEAE-Sephadex A-25 ion-exchange chromatography was performed with a stepwise gradient of 0.01 M (pH 8.6) to 0.75 M (pH 7.4) Tris-HC1 buffer to further purify the mitogenic fraction. The protein concentration of the crude arterial larval extract, as well as the fractions, was approximately 25 mg ml-~. The somatic extract eluate from Sephadex G-200 was pooled into five different fractions, F I - F 5 . Fraction F4 (the mitogenic fraction) was further eluted into five fractions, 4A-4E, on DEAE-Sephadex A-25. Fraction 4C was the mitogenic fraction and was eluted with 0.3m to 0.35m Tris-HC1 (pH 7.4). Blood was collected into plain universal bottles from 40 horses from which arterial larvae were collected. These horses were also naturally moderately or heavily infected with adult S. vulgaris and other helminth parasites in their small intestines, colon and caecum. The blood samples were allowed to clot overnight and sera were harvested the following day and stored in aliquots of 2 ml at - 2 0 ° C. The fractions of the larval antigen and the horse sera were then brought to Nigeria where the rest of the study was conducted. Twenty millilitres of the sera from the 40 horses were pooled and fractionated by DEAE ion-exchange chromatography with 0.01 M phosphate buffer (pH 8.0 ). The IgG (T)-rich fraction was concentrated by vacuum dialysis and stored at - 2 0 °C until required for analyses. Control sera were obtained from unborn foetuses removed from the uteri of the slaughtered horses. Antisera to the various fractions were raised in rabbits, as described by Wynne et al. ( 1981 ). Immunodiffusion and immunoelectrophoresis were performed as previously reported (Adeyefa, 1983; Adeyefa et al., 1985). The mitogenic fractions F4 from gel filtration and 4A-4D from ion-exchange chromatography were reacted against the IgG (T)-rich fraction of horse sera and the antisera to the mitogenic fractions. In immunodiffusion, the central well contained the IgG(T)-rich fraction and the peripheral wells contained Fractions 4A, 4B, 4C and 4D, while in immunoelectrophoresis the wells contained Frac-
PRECIPITIN RESPONSE OF M1TOGEN PRODUCED BY S I'ULGARIS
245
tions F4 and 4C, and the trough contained the IgG (T)-rich fraction. The slides were washed and stained with Ponceaus as previously described (Adeyefa et al., 1985). RESULTS
Precipitin lines developed within 36 h in the immunodiffusion slides of IgG (T) against Fractions 4A-4D. The reaction revealed the presence of two proteins in these fractions (Fig. 1 ). The presence of lines of complete identity indicated a common protein in Fractions 4A-4D, while the spur of partial identity would indicate an additional protein in Fraction 4C recognised by IgG (T). In immunoelectrophoresis of IgG (T) against Fraction F4, the precipitin lines indicate the presence of one protein (Fig. 2 ), while that of IgG (T) against Fraction 4C revealed the presence of two proteins which migrated at different speeds (Fig. 3). Similar results were obtained when the two fractions were reacted against the antisera raised in rabbits. This supports the finding that IgG (T) is the principal component offl-globulin fractions, which is elevated in S. vulgaris infection. No detectable precipitin lines were observed with the foetal control serum. 4B
z+A 0
4c
~
0
40
Fig. 1. Line drawing of precipitin lines developed by mitogenic Fractions 4A-4D in immunodiffusion with IgG (T).
IgG(T) Cathode
Anode
Fig. 2. Line drawing of precipitin lines developed by Fraction F4 in immunoelectrophoresis with IgG(Y).
Anode
~4C
IgO(T) Cathode
Fig. 3. Line drawing ofprecipitin line developed by Fraction 4C in immunoelectrophoresis with IgG(T).
246
CA.O. ADEYEFA
DISCUSSION
The results of these studies indicate that the IgG (T) from the sera of horses with naturally acquired S. vulgaris infection recognised the proteins in Fractions F4 and 4C from S. vulgaris arterial larval mitogen as antigens. The antigen-antibody reactions gave the precipitin lines observed in the immunodiffusion and immunoelectrophoresis slides. It has been reported earlier that the extracts of arterial larvae of S. vulgaris produce a mitogen (Bailey et al., 1982, 1984a, b; Adeyefa et al., 1985 ) and that this mitogen stimulated polyclonal proliferation of equine peripheral blood lymphocytes (Bailey et al., 1982, 1984a,b; Adeyefa, 1983, 1989b; Adeyefa et al., 1985), as well as lymphocytes from other lymphoid organs (Bailey et al., 1984a,b). The large increases in total serum protein concentration in natural and experimental S. vulgaris infection reported by many workers (Drudge et al., 1966; Round, 1970; Amborski et al., 1974; Ogbourne and Duncan, 1977; Patton et al., 1978: Bailey et al., 1984a,b; Adeyefa, 1989a) have been shown to be responsible for the high degree of acquired immunity to S. vulgaris infection in young horses. This immunity has been attributed to increases in//-globulins, particularly IgG(T), by these workers. It has also been shown that the increase in IgG(T) level is probably due to the mitogen produced by the arterial larvae during their extensive migration in the mesenteric arterial system of infected horses (Bailey et al., 1982, 1984a,b; Adeyefa et al., 1985; Adeyefa, 1989b). The results of the present study would seem to confirm that the proteins present in the mitogenic fractions F4 and 4C are antigenic stimulants for the production of IgG (T) in the serum of horses naturally or experimentally infected with S.
vulgaris. The above results could be used as an immunological tool in the diagnosis of S. vulgaris infection in horses. They could also serve as fruitful models in the production of effective immunising agents against S. vulgaris infection from the mitogen. However, further studies would be necessary to elucidate the in vivo serological activities of this mitogen since polyctonal activation of host lymphocytes in vivo has been postulated to be associated with the immunosuppression often observed in parasitic infections in field situations. ACKNOWLEDGEMENTS
The author is grateful to Dr. Mick Bailey for the antisera used in this study, and to the staff of Potters and Sons for permission and assistance in collecting arterial larvae and blood from slaughtered horses. I am also grateful to Professor Mark Nwagwu for permission to use his laboratory facilities to fractibnate the serum and to Miss Adetilure for technical assistance.
PRECIPITIN RESPONSE OF MITOGEN PRODUCED BY S. VULGARIS
247
REFERENCES Adeyefa, C.A,O., 1983. Studies on the mitogenic factor produced by the arterial larval Strongylus vulgaris in the horse. MSc Thesis, University College of Wales. Adeyefa, C.A.O., 1989a. The effects of anthelmintic treatment and improved management on the haematological values of horses with naturally acquired helminthiasis. Trop. Vet., 7:177184. Adeyefa, C.A.O., 1989b. The response of equine peripheral blood lymphocytes to Strongylus vulgaris arterial larval mitogen. Trop. Vet., 7: 58-64. Adeyefa, C.A.O., Bailey, M., Lloyd, S. and Soulsby, E.J.L., 1985. Larval mitogen fractionation. Trop. Vet., 3: 66-69. Amborski, G.F., Bello, T.R. and Torbet, E.J., 1974. Host response to experimentally induced infections of Strongylus vulgaris in parasite free and naturally infected ponies. Am. J. Vet. Res., 35: 1181-1188. Bailey, M., Lloyd, S. and Soulsby, E.J.L., 1982. Non-specific immunostimulatory effect of antigens of Strongylus vulgaris. Proceedings of the British Society for Parasitology. Abstract in Parasitology 85: 34. Bailey, M., Kent, T., Martin, S.C., Lloyd, S. and Soulsby, E.J.L., 1984a. Haematological and biochemical values in horses naturally infected with Strongylus vulgaris. Vet. Rec., 115:144147. Bailey, M., Lloyd, S., Martin, S.C. and Soulsby, E.J.L., 1984b. In-vitro induction of lymphocyte responsiveness by Strongylus vulgaris-derived mitogen. Z. Parasitenkd., 70: 229-242. Drudge, J.H., Lyons, E.T. and Szanto, J., 1966. Pathogenesis of migrating stages of helminths with special reference to Strongylus vulgaris. In: E.J.L. Soulsby (Editor), Biology of Parasites, Emphasis on Veterinary Parasites. Academic Press, New York/London, pp. 199-214. Ogbourne, C.P. and Duncan, J.L., 1977. Strongylus vulgaris in the horse, its biology and veterinary importance. Commonw. Inst. Helminthol. Misc. PUN., 4: 1-40. Patton, S., Mock, R.E., Drudge, J.H. and Morgan, D., 1978. Increase in immunoglobulin concentration in ponies as a response to experimental infection with the nematode Strongylus vulgaris. Am. J. Vet. Res., 39: 19-23. Round, M.C., 1970. The development of strongyles in horses and the associated serum protein changes. In: J.T. Bryans and M. Gerber (Editors), Equine Infectious Diseases II. Proceedings of the 2nd International Conference on Equine Infectious Diseases, 1979, at Paris. Karger, Basel, pp. 290-303,323-325. Wynne, E., Sloombe, J.O.D. and Wilkie, B.H., 1981. Antigenic analyses of tissues and excretory and secretory products from Strongylus vulgaris. Can. J. Comp. Med., 45: 259-265.