Veterinary Parasitology, 35 (1990) 175 178 Elsevier Science Publishers B.V., A m s t e r d a m - - P r i n t e d in T h e Netherlands
175
Short Communication
A n t i g e n s a n d A n t i b o d i e s in the A s c i t i c Fluid of Mice I n f e c t e d w i t h Fasciola gigantica S. Y O S H I H A R A and K. S U Z U K I
Poultry Disease Laboratory, National Institute of Animal Health, 4909-58 Kurachi, Seki-shi, Gifu 501-32 (Japan) (Accepted for publication 24 July 1989)
ABSTRACT Yoshihara, S. and Suzuki, K., 1990. Antigens and antibodies in the ascitic fluid of mice infected with Fasciola gigantiea. Vet. Parasitol., 35: 175-178. Fluke antigens were detected in the ascitic fluid of mice infected with Fasciola gigantica. The precipitating antibodies reactive against the antigens from metacercariae were detected as early as 2 weeks after the infection, a n d those against the adult a n d immature worms appeared 3 and 4 weeks later.
INTRODUCTION
Medwar and Voller (1975) studied schistosomiasis in mice and detected circulating soluble antigens and antibody. Yokogawa et al. (1976) found Paragonimus antigens and IgG and IgE in pleural exudates of patients infected with
Paragonimus miyazakii. Kruyt and van der Steen (1971) studied fascioliasis in mice and observed blood-stained fluids in the abdominal cavity 9 days after infection with Fasciola hepatica. In addition, Dawes (1962) speculated that immature flukes might leave the liver of mice and re-enter the abdominal cavity, producing a bloodstained exudate. These findings suggest that antigens from immature worms and antibody may have existed in the exudate. The present work was conducted to demonstrate the presence of Fasciola antigens and precipitating antibodies in the exudate (ascitic fluid) of mice infected with F. gigantica. MATERIALS AND METHODS
The cercariae emerged from Lymnaea ollula experimentally infected with F. gigantica in a polyethylene bag containing water and encysted on the wall of 0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
176
S. YOSH1HARA AND K. SUZUKI
the container. The metacercariae (Mc) were stored in a moist chamber at 4 ° C. Each of three male rabbits, averaging 2.5 kg weight, was inoculated orally with 30 Mc. Forty days later, they received another 50 Mc and were killed 21 days later to recover their sera. The pooled serum was designated Fasciola-infected rabbit serum and was used to detect antigens in the ascitic fluid. Forty male C F W mice weighing ~ 25 g were each given 3 Mc. Groups of three mice were killed weekly for 7 weeks, their pooled abdominal fluids centrifuged at 700 × g for 7 min at 4 °C and the supernatant portion used in the present study. The antigens used were the extracts of adult flukes from infected rats (Wistar strain ), 14-day-old immature flukes from mice and Mc. Extraction was carried out according to the technique of Oldham (1983). The fourth antigen consisted of excretory/secretory products ( E S P ) of young worms. Incubation of the flukes was performed by a modification of Technique A described by Rajasekariah and Howell (1978). NaC1 solution (0.15 M) in a Carrel flask was used as medium in the present study. The agar gel precipitation test (AGT) was carried out using 1% agarose dissolved in 0.15 M NaC1 solution on a microscope slide. The wells in gel were filled with reactants, the slides kept in a moist chamber at 4 °C for 4 days and stained with amido black. Ascitic fluids from mice infected with Schistosoma japonicum and from non-infected animals were used as controls. The abdominal fluid collected 4 weeks after inoculation of mice with Mc was precipitated with a 33 or 50% saturated solution of ammonium sulphate. After centrifugation, the supernatant portion and also the precipitates, dissolved in 0.15 M saline solution, were dialysed for 24 h against 0.15 M phosphate-buffered saline solution. Each of the portions was concentrated by carboxymethyl cellulose sodium salt at 4 ° C and tested for antigenic activity. RESULTS Macroscopically, the ascitic fluid was present in mice killed 2 weeks after inoculation with the Mc, b u t had disappeared at 7 weeks. About one-third of the mice died within 7 weeks of infection. The serological relationships between antigens in fluid collected from 2 weeks after inoculation and antigens from various developmental stages of the flukes are shown in Fig. 1. Antigen in the fluid reacted with the Fasciola-infected rabbit serum, forming a line of precipitate. This line coalesced with a line formed by antigens from adult and immature worms. The line also fused with a line which was observed between E S P and the rabbit serum, but crossed a line produced by Mc (Fig. 2 ). The antibodies in the ascitic fluid reacting with antigen from Mc were also detected, as indicated by the line between Wells 1 and 5 (Fig. 1) or 2 and 3 (Fig. 2 ). Antibodies against antigen from adult and immature worms and E S P were present in the ascitic fluids collected 3 weeks after infection.
MICEINFECTEDWITHFASCIOLA GIGANTICA
177 f
j
A
,I i
H~j
~
Fig. 1. Agar gel precipitation test using ascitic fluid collected from Fasciola-infected mice 2 weeks after inoculation (1), extracts from adult worms (2), immature worms (3) and Mc (5), and excretory/secretory products of immature worms (4) with Fasciola-infected rabbit serum (6).
Fig. 2. Agar gel precipitation test using the ascitic fluid collected from Fasciola-infected mice: excretory/secretory products of immature worms (1), 2 weeks after innoculation (2) and extract from Mc (3) with Fasciola-infected rabbit serum (4).
Fig. 3. Agar gel precipitation test using the ascitic fluid collected from Fasciola-infected mice 4 weeks after inoculation {1), 33% ammonium sulphate supernatant fluid (2), 33% ammonium sulphate precipitate (3), 50% ammonium sulphate supernatant fluid (4) and 50% ammonium sulphate precipitate fluid (5) with Fasciola-infected rabbit serum (6).
178
S. YOSHIHARAANDK. SUZUKI
The fluids from control mice formed no lines of precipitate with any of the reactants. The antigens were detected in both the precipitate and supernatant portions of the fluid treated with ammonium sulphate (Fig. 3 ). DISCUSSION
This study demonstrated the presence of Fasciola antigen in the ascitic fluid of mice infected with F. gigantica and suggested that the migratory living immature worms in the liver may release antigenic substance (s) into the bloodstained ascitic fluid during the early acute infection. When the ascitic fluids were treated with 33 or 50% saturated solutions of ammonium sulphate, antigens were distributed in both the precipitate and the supernatant portions. From this result, it is suggested that antigens in ascitic fluid may be associated with the globulin and albumin fraction of mouse serum. The precipitating antibodies reacting with antigens from various developmental stages of the flukes were also detected in the fluid. Kojima (1983) investigated the pleural exudates of a patient with paragonimiasis and suggested that complexes of antigens, antibodies and eosinophile-chemotactic factor of complement (ECF-C) from activated complement due to reaction of IgG and antigen, might also bear a part in the mechanism of eosinophils in the infection. Further experimentation would be thus required to demonstrate the pathogenic role of ascitic fluid with Fasciola antigens and antibodies in early acute fascioliasis in the mouse. ACKNOWLEDGEMENTS
The authors wish to thank Dr. H. Hata, Chiba University, School of Medicine, for the supply of ascitic fluid from mice infected with S. japonicum.
REFERENCES Dawes, B., 1962. On the growth and maturation of Fasciola hepatica. J. Helminthol., 36: 11-38. Kojima, S., 1983. Eosinophils in parasitic infections. Taisha, 20:41-48 (in Japanese). Kruyt, W. and van der Steen, 1971. Fasciola hepatica: Screening of chemical compounds on immature stage in the mouse. 1. Pathology and method of investigation. Exp. Parasitol., 30: 375384. Medwar, M.A. and Voller, A., 1975. Circulating soluble antigens and antibody in schistosomiasis. Br. Med. J., 1: 435-436. Oldham, G., 1983. Protection against Fasciola hepatica in rats with adult fluke antigen in Freund's adjuvant: influence of antigen batch, antigen dose and number of sensitising injections. Res. Vet. Sci., 34: 240-244. Rajasekariah, G.R. and Howell, M.J., 1978. Acquired immunity to the trematode Fasciola hepatica in rats. Aust. J. Exp. Biol. Med. Sci., 56: 747-756. Yokogawa, M., Kojima, S., Araki, K., Tomioka, H. and Yoshida, S., 1976. Immunoglobulin E: Raised levels in sera and pleural exudates with paragonimiasis. Am. J. Trop. Med. Hyg., 25: 581 586.
175
Short Communication
A n t i g e n s a n d A n t i b o d i e s in the A s c i t i c Fluid of Mice I n f e c t e d w i t h Fasciola gigantica S. Y O S H I H A R A and K. S U Z U K I
Poultry Disease Laboratory, National Institute of Animal Health, 4909-58 Kurachi, Seki-shi, Gifu 501-32 (Japan) (Accepted for publication 24 July 1989)
ABSTRACT Yoshihara, S. and Suzuki, K., 1990. Antigens and antibodies in the ascitic fluid of mice infected with Fasciola gigantiea. Vet. Parasitol., 35: 175-178. Fluke antigens were detected in the ascitic fluid of mice infected with Fasciola gigantica. The precipitating antibodies reactive against the antigens from metacercariae were detected as early as 2 weeks after the infection, a n d those against the adult a n d immature worms appeared 3 and 4 weeks later.
INTRODUCTION
Medwar and Voller (1975) studied schistosomiasis in mice and detected circulating soluble antigens and antibody. Yokogawa et al. (1976) found Paragonimus antigens and IgG and IgE in pleural exudates of patients infected with
Paragonimus miyazakii. Kruyt and van der Steen (1971) studied fascioliasis in mice and observed blood-stained fluids in the abdominal cavity 9 days after infection with Fasciola hepatica. In addition, Dawes (1962) speculated that immature flukes might leave the liver of mice and re-enter the abdominal cavity, producing a bloodstained exudate. These findings suggest that antigens from immature worms and antibody may have existed in the exudate. The present work was conducted to demonstrate the presence of Fasciola antigens and precipitating antibodies in the exudate (ascitic fluid) of mice infected with F. gigantica. MATERIALS AND METHODS
The cercariae emerged from Lymnaea ollula experimentally infected with F. gigantica in a polyethylene bag containing water and encysted on the wall of 0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
176
S. YOSH1HARA AND K. SUZUKI
the container. The metacercariae (Mc) were stored in a moist chamber at 4 ° C. Each of three male rabbits, averaging 2.5 kg weight, was inoculated orally with 30 Mc. Forty days later, they received another 50 Mc and were killed 21 days later to recover their sera. The pooled serum was designated Fasciola-infected rabbit serum and was used to detect antigens in the ascitic fluid. Forty male C F W mice weighing ~ 25 g were each given 3 Mc. Groups of three mice were killed weekly for 7 weeks, their pooled abdominal fluids centrifuged at 700 × g for 7 min at 4 °C and the supernatant portion used in the present study. The antigens used were the extracts of adult flukes from infected rats (Wistar strain ), 14-day-old immature flukes from mice and Mc. Extraction was carried out according to the technique of Oldham (1983). The fourth antigen consisted of excretory/secretory products ( E S P ) of young worms. Incubation of the flukes was performed by a modification of Technique A described by Rajasekariah and Howell (1978). NaC1 solution (0.15 M) in a Carrel flask was used as medium in the present study. The agar gel precipitation test (AGT) was carried out using 1% agarose dissolved in 0.15 M NaC1 solution on a microscope slide. The wells in gel were filled with reactants, the slides kept in a moist chamber at 4 °C for 4 days and stained with amido black. Ascitic fluids from mice infected with Schistosoma japonicum and from non-infected animals were used as controls. The abdominal fluid collected 4 weeks after inoculation of mice with Mc was precipitated with a 33 or 50% saturated solution of ammonium sulphate. After centrifugation, the supernatant portion and also the precipitates, dissolved in 0.15 M saline solution, were dialysed for 24 h against 0.15 M phosphate-buffered saline solution. Each of the portions was concentrated by carboxymethyl cellulose sodium salt at 4 ° C and tested for antigenic activity. RESULTS Macroscopically, the ascitic fluid was present in mice killed 2 weeks after inoculation with the Mc, b u t had disappeared at 7 weeks. About one-third of the mice died within 7 weeks of infection. The serological relationships between antigens in fluid collected from 2 weeks after inoculation and antigens from various developmental stages of the flukes are shown in Fig. 1. Antigen in the fluid reacted with the Fasciola-infected rabbit serum, forming a line of precipitate. This line coalesced with a line formed by antigens from adult and immature worms. The line also fused with a line which was observed between E S P and the rabbit serum, but crossed a line produced by Mc (Fig. 2 ). The antibodies in the ascitic fluid reacting with antigen from Mc were also detected, as indicated by the line between Wells 1 and 5 (Fig. 1) or 2 and 3 (Fig. 2 ). Antibodies against antigen from adult and immature worms and E S P were present in the ascitic fluids collected 3 weeks after infection.
MICEINFECTEDWITHFASCIOLA GIGANTICA
177 f
j
A
,I i
H~j
~
Fig. 1. Agar gel precipitation test using ascitic fluid collected from Fasciola-infected mice 2 weeks after inoculation (1), extracts from adult worms (2), immature worms (3) and Mc (5), and excretory/secretory products of immature worms (4) with Fasciola-infected rabbit serum (6).
Fig. 2. Agar gel precipitation test using the ascitic fluid collected from Fasciola-infected mice: excretory/secretory products of immature worms (1), 2 weeks after innoculation (2) and extract from Mc (3) with Fasciola-infected rabbit serum (4).
Fig. 3. Agar gel precipitation test using the ascitic fluid collected from Fasciola-infected mice 4 weeks after inoculation {1), 33% ammonium sulphate supernatant fluid (2), 33% ammonium sulphate precipitate (3), 50% ammonium sulphate supernatant fluid (4) and 50% ammonium sulphate precipitate fluid (5) with Fasciola-infected rabbit serum (6).
178
S. YOSHIHARAANDK. SUZUKI
The fluids from control mice formed no lines of precipitate with any of the reactants. The antigens were detected in both the precipitate and supernatant portions of the fluid treated with ammonium sulphate (Fig. 3 ). DISCUSSION
This study demonstrated the presence of Fasciola antigen in the ascitic fluid of mice infected with F. gigantica and suggested that the migratory living immature worms in the liver may release antigenic substance (s) into the bloodstained ascitic fluid during the early acute infection. When the ascitic fluids were treated with 33 or 50% saturated solutions of ammonium sulphate, antigens were distributed in both the precipitate and the supernatant portions. From this result, it is suggested that antigens in ascitic fluid may be associated with the globulin and albumin fraction of mouse serum. The precipitating antibodies reacting with antigens from various developmental stages of the flukes were also detected in the fluid. Kojima (1983) investigated the pleural exudates of a patient with paragonimiasis and suggested that complexes of antigens, antibodies and eosinophile-chemotactic factor of complement (ECF-C) from activated complement due to reaction of IgG and antigen, might also bear a part in the mechanism of eosinophils in the infection. Further experimentation would be thus required to demonstrate the pathogenic role of ascitic fluid with Fasciola antigens and antibodies in early acute fascioliasis in the mouse. ACKNOWLEDGEMENTS
The authors wish to thank Dr. H. Hata, Chiba University, School of Medicine, for the supply of ascitic fluid from mice infected with S. japonicum.
REFERENCES Dawes, B., 1962. On the growth and maturation of Fasciola hepatica. J. Helminthol., 36: 11-38. Kojima, S., 1983. Eosinophils in parasitic infections. Taisha, 20:41-48 (in Japanese). Kruyt, W. and van der Steen, 1971. Fasciola hepatica: Screening of chemical compounds on immature stage in the mouse. 1. Pathology and method of investigation. Exp. Parasitol., 30: 375384. Medwar, M.A. and Voller, A., 1975. Circulating soluble antigens and antibody in schistosomiasis. Br. Med. J., 1: 435-436. Oldham, G., 1983. Protection against Fasciola hepatica in rats with adult fluke antigen in Freund's adjuvant: influence of antigen batch, antigen dose and number of sensitising injections. Res. Vet. Sci., 34: 240-244. Rajasekariah, G.R. and Howell, M.J., 1978. Acquired immunity to the trematode Fasciola hepatica in rats. Aust. J. Exp. Biol. Med. Sci., 56: 747-756. Yokogawa, M., Kojima, S., Araki, K., Tomioka, H. and Yoshida, S., 1976. Immunoglobulin E: Raised levels in sera and pleural exudates with paragonimiasis. Am. J. Trop. Med. Hyg., 25: 581 586.
