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Brief Communication

Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis

Kun-Ho Song1, Mineo Hayasaki2, Kyu-Woan Cho1, Sang-Eun Lee1, Duck-Hwan Kim1
The Korean Journal of Parasitology 2002;40(4):195-198.
Published online: December 30, 2002

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Corresponding author (dhkim@cnu.ac.kr)
• Received: October 24, 2002   • Accepted: November 27, 2002

Copyright © 2002 by The Korean Society for Parasitology

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  • This study was performed to investigate whethere there is cross-reactivity between Dirofilaria immitis and three intestinal nematodes of dogs. In ELISA, D. immitis-infected dog sera obtained at the 4th molting stage (9-11 weeks) and microfilaremic stage (25-30 weeks) were shown to be highly reactive with crude extract of T. canis. In immunoblotting, some antigenic fractions, 44, 57, 88, 100 kDa of crude extract of T. canis, were found to be positive reaction with sera of dogs infected with D. immitis. However, little or no cross-reaction were observed between sera of D. immitis-infected dogs and crude extract antigen of T. vulpis or A. caninum. These result suggest that there are partial cross reaction between sera of D. immitis-infected dogs and the antigen of T. canis.
  • Key words: Dirofilaria immitis, cross-reactivity, ELISA, immunoblotting, Toxocara canis
Employing the indirect hemagglutination assay (Hayasaki, 1981) and Dot-ELISA (Matsumura et al., 1984), the authors found no cross-reactivity was observed between Dirofilaria immitis and intestinal nematodes including T. canis. On the other hand, Ott et al. (1985) reported the presence of immunologically distinguishable species-specific non-cross reactive antigens and the presence of cross-reactive antigens in antigenic extracts obtained from adult D. immitis and T. canis. Additionally, Konno et al. (1995) reported that D. immitis possessed a partial antigenic cross reactivity to T. canis, revealed by using immunoblotting. The above consideration indicate that more strict analysis is required for the study of antigenic cross-reactivity between D. immitis and other parasites. This study was performed to examine on cross-reactivity between D. immitis and three intestinal nematodes of dogs.
Five healthy mongrel juvenile dogs, approximately three months of age, were housed in mosquito-proof runs and treated with piperazine (100 mg/kg, PO) for intestinal parasites prior to experimentation. Three dogs were experimentally infected with D. immitis by the procedures described by Hayasaki (1982). Briefly, mosquitoes, Aedes togoi, collected during the larval stage from their natural spawning areas were reared under laboratory conditions and inoculated by feeding on a dog with about 200 circulating microfilarial (Mf) counts per 20 µl of blood. Two dogs were used as the negative control. Infective larvae (L3) of D. immitis were recovered from the proboscis of the infected mosquitoes 10 to 14 days after blood feeding and suspended in saline during microscopic observation. Three dogs were subcutaneously injected in the inguinal region with 228 (dog No. 1), 278 (dog No. 2) and 248 (dog No. 3) infective larvae (L3). The blood was collected from the cephalic vein once a week, and the sera were stored at -70℃ until analysis. The crude extract antigens of T. canis, T. vulpis and A. caninum were prepared as previously described (Hayasaki, 1982). Briefly, the crude antigens used in this study were extracted from adult worms of T. canis, A. caninum and T. vulpis by phosphate buffered saline (PBS, pH 7.2, 0.1 M). These worms were homogenized and sonicated by tissue homogenizer (15 min, 4℃) and ultrasonicator (50 watt, 15 min, 4℃), respectively, and then allowed to incubate overnight at 4℃. After centrifugation at 18,000 g, the supernatant as antigen was collected and kept at -70℃. The protein concentration of the antigens was determined using the methods of Lowry et al. (1951). ELISA was performed by method described by Grieve et al. (1981). SDS-PAGE and immunoblotting was performed as described by Gonno and Hayasaki (1995). In this study, by ELISA, D. immitis-infected sera obtained at the 4th molting stage (9-11 weeks after infection) and microfilaremic stage (25-30 weeks after infection) were strongly reacted with crude extract of T. canis. However, little or no cross-reactivities were found between D. immitis-infected sera and crude extracts of T. vulpis or A. caninum.
Immunoblotting analysis are more effective application of immunologic diagnosis (Kaneko et al., 1990; Hayasaki et al., 1994). In this study, 44, 57, 88 and 100 kDa, which were protein bands of crude extract of T. canis, were shown to be strongly reactive with D. immitis-infected sera. Based on these findings, we suggest that D. immitis share a common antigen with T. canis. This cross-reactivity may be dependent on many antigenic epitopes existing in somatic components of the parasites because previous report indicate that D. immitis consists of many protein components and its antigenicity is very complex, and also these two parasites may be originated from a same progenitor and still possess partially similar antigenicity. Therefore, certain interspecies antigenic epitopes may be isolated from one species which may be useful in eliciting a highly specific immune response in a host toward cross-reactive epitopes in different species (Hayasaki et al., 1994).
Further studies are needed to elucidate the immunological implications antigenic cross-reactivity between D. immitis and intestinal nematodes including T. canis.
  • 1. Grieve RB, Johnson MM, Jacobson RH. Enzyme-linked immunosorbent assay for measurement of antibody responses to Dirofilaria immitis in experimentally infected dogs. Am J Vet Res 1981;42:66-69.
  • 2. Hayasaki M. Indirect hemmaglutination test for diagnosis of canine filariasis. Nippon Juigaku Zasshi 1981;43:21-26.
  • 3. Hayasaki M. Reaginic and hemmaglutinating antibody production in dogs infected with Dirofilaria immitis. Nippon Juigaku Zasshi 1982;44:63-70.
  • 4. Hayasaki M, Nakamura F, Konno K. Immunoblotting analysis of somatic components of Dirofilaria immitis. J Vet Med Sci 1994;56:1181-1183.
  • 5. Kaneko H, Hayasaki M, Ohishi I. Antigenic identification of excretory-secretory products of adult Dirofilaria immitis. Nippon Juigaku Zasshi 1990;52:995-1000.
  • 6. Konno K, Hayasaki M. Antigenic cross reactivity among Dirofilaria immitis and the four intestinal parasite-species in the dog. Jpn J Parasitol 1995;44:161-164.
  • 7. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275.
  • 8. Matsumura K, Kazuta Y, Endo R, Tanaka K. Detection of circulating toxocaral antigens in dogs by sandwich enzyme-immunoassay. Immunology 1984;51:609-613.
  • 9. Ott RA, Staples M, Weekley M, Maggio ET. Demonstration of both immunogenically unique and common antigenic determinants in Dirofilaria immitis and Toxocara canis using monoclonal antibodies. Vet Immunol Immunopathol 1985;10:147-153.
Fig. 1
Antibody levels of D. immitis-infected dog serum (No. 1) against crude extract antigens of three intestinal nematodes, measured by ELISA. Asterisk represents the range of optical density of control group.
kjp-40-195-g001.jpg
Fig. 2
Antibody levels of D. immitis-infected dog serum (No. 2) against crude extract antigens of three intestinal nematodes, measured by ELISA. Asterisk represents the range of optical density of control group.
kjp-40-195-g002.jpg
Fig. 3
Antibody levels of D. immitis-infected dog serum (No. 1) against crude extract antigens of three intestinal nematodes, measured by ELISA. Asterisk represents the range of optical density of control group.
kjp-40-195-g003.jpg
Fig. 4
Protein fractions of adult T. canis were separated by SDS-PAGE stained with Coomassie brilliant blue R-250 (A), and antigenic cross reactions between T. canis antigens and sera from dogs experimentally infected with D. immitis by immunoblotting (B). Lane 1-3: uninfected serum, lane 4-6: sera collected at the period of prepatent infection, lane 7-9: sera collected at the period of patent infection (lane 1, 4, 7: dog 1, land 2, 5, 8: dog 2, lane 3, 6, 9: dog 3).
kjp-40-195-g004.jpg

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Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis
Korean J Parasitol. 2002;40(4):195-198.   Published online December 30, 2002
DOI: https://doi.org/10.3347/kjp.2002.40.4.195
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Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis
Korean J Parasitol. 2002;40(4):195-198.   Published online December 30, 2002
DOI: https://doi.org/10.3347/kjp.2002.40.4.195
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Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis
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Fig. 1 Antibody levels of D. immitis-infected dog serum (No. 1) against crude extract antigens of three intestinal nematodes, measured by ELISA. Asterisk represents the range of optical density of control group.
Fig. 2 Antibody levels of D. immitis-infected dog serum (No. 2) against crude extract antigens of three intestinal nematodes, measured by ELISA. Asterisk represents the range of optical density of control group.
Fig. 3 Antibody levels of D. immitis-infected dog serum (No. 1) against crude extract antigens of three intestinal nematodes, measured by ELISA. Asterisk represents the range of optical density of control group.
Fig. 4 Protein fractions of adult T. canis were separated by SDS-PAGE stained with Coomassie brilliant blue R-250 (A), and antigenic cross reactions between T. canis antigens and sera from dogs experimentally infected with D. immitis by immunoblotting (B). Lane 1-3: uninfected serum, lane 4-6: sera collected at the period of prepatent infection, lane 7-9: sera collected at the period of patent infection (lane 1, 4, 7: dog 1, land 2, 5, 8: dog 2, lane 3, 6, 9: dog 3).

Fig. 1

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Fig. 3

Fig. 4

Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis
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