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Western blot analysis of the humoral response of dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866)
Veterinary Parasitology 106 (2002) 83–87
Short communication
Western blot analysis of the humoral response of dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866) M.C. Cury a , M.P. Guimarães b , W.S. Lima b , R.W.A. Vitor b,∗ a
Laboratory of Parasitology, Federal University of Uberlˆandia, Uberlˆandia, Minas Gerais, Brazil b Department of Parasitology, Federal University of Minas Gerais, ICB-UFMG, P.O. Box 486, CEP 31270-901 Belo Horizonte, Minas Gerais, Brazil Received 7 August 2001; received in revised form 15 January 2002; accepted 23 January 2002
Abstract Seven cross-bred dogs were inoculated with Angiostrongylus vasorum and serum samples were analyzed using the enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). ELISA detected specific antibodies anti-A. vasorum, from 14 to 28 days after inoculation (DAI) and persisted throughout the experiment. Using WB, the main antigens detected had molecular weight of approximately 115, 102, 86, 76, 69, 56, 41, 32, 28, 20–22 and 10 kDa. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Angiostrongylus vasorum; Dog; Western blot analysis
1. Introduction Canine angiostrongylosis is a disease caused by Angiostrongylus vasorum (Baillet, 1866), a nematode from the Protostrongylidea family, whose adult form is found in the right ventricle and pulmonary artery (and its branches) in domestic dogs and wild carnivores. The definite host infected by accidentally ingesting aquatic or terrestrial molluscs contaminated with third-stage larvae. It has been reported in many countries, but appears to be particularly frequent in dogs in the France, England and Ireland (Cury and Lima, 1996). ∗ Corresponding author. Tel.: +55-31-3499-2853; fax: +55-31-3499-2970. E-mail address: [email protected] (R.W.A. Vitor).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 3 1 - 6
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M.C. Cury et al. / Veterinary Parasitology 106 (2002) 83–87
Several clinical signs have been associated with the infection, including signs of pulmonary and cardiac diseases (Patteson et al., 1993; Cury and Lima, 1996). Diagnosis is based on the detection of first-stage larvae in feces or in postmortem examination by the finding of lesions or presence of adult parasites in the heart and lung. Serological diagnosis is rarely used, with a few experimental results published using complement fixation and agglutination in latex (Guilhon et al., 1971; Mishra and Benex, 1972). Cury et al. (1996) demonstrated that antibodies to A. vasorum antigens are stimulated by experimental infection of dogs and that this response is reflected in generally increasing enzyme-linked immunosorbent assay (ELISA) values. At present, no attention has been paid to the characterization of the individual antigens in A. vasorum adult worms which elicit immune response in the definitive host. The purpose of this study was to analyze the kinetics of anti-A. vasorum antibodies in the serum and to characterize the targets of canine humoral immune response by the Western blot (WB) technique. 2. Materials and methods Seven cross-bred dogs were infected with a varying number of infective larvae of A. vasorum, contained in the tissue of an intermediate host, an aquatic mollusc (Biomphalaria glabrata) maintained in the laboratory and experimentally contaminated with the first-stage larvae of this nematode. The dogs were randomly divided into two groups: group A with four animals (dogs 7, 9, 12 and 13) and group B with three animals (dogs 16, 18 and 20). The animals were inoculated by oral route with 50 larvae/kg of body weight (group A) and 100 larvae/kg of body weight (group B). Two animals (dogs 1 and 2) were not inoculated and served as controls. Fecal samples were collected daily from 25 days after inoculation (DAI). Five milliliters of blood were collected from the jugular vein of each animal to determine antibody levels up to 5 months after infection. ELISA was carried out as described by Cury et al. (1996) in order to determine the IgG antibodies. WB analyses were performed as described by Graeff-Teixeira et al. (1997) with modifications. Briefly, female worms were frozen and homogenized in a mortar while immersed in liquid nitrogen. The worm sample was homogenized in a sample buffer with 1 mM phenylmethylsulphonyl fluoride (PMSF) and 1 mM N-p-tosyl-l-lysine-chloromethyl ketone (TLCK) and centrifuged at 15,000 × g for 2 min at 4 ◦ C. Electrophoretic separation of this soluble fraction was performed in a 10% polyacrylamide gel under reduction conditions in the Bio-Rad® Mini Protean II system (three females per minigel). After separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose membranes for 20 h at 30 V and 40 mA, followed by 1 h at 90 mA. Detection of proteins was carried out by an immunoenzymatic method on 5 mm strips using 4-chloro-1-naphthol as substrate. In order to minimize interference of antibodies of Toxocara canis, frequently found in dogs, sera were previously absorbed with T. canis adult worm antigen. The pre-absorption experiments were performed according to Conway (1993), by mixing and incubating each serum for 1 h with 20 g/ml PBS-soluble adult T. canis extract.
M.C. Cury et al. / Veterinary Parasitology 106 (2002) 83–87
85
Table 1 Specific antibody response detected by ELISA in dogs inoculated with A. vasorum Dog
1 2 7 9 12 13 16 18 20
DAI 0
14
28
56
112
156
0.006 0.167 0.009 0.008 0.053 0.012 0.048 0.047 0.020
0.046 ND 1.065 0.028 ND ND ND 0.180 0.008
0.127 0.066 0.707 0.153 0.318 0.201 0.338 0.073 0.041
0.090 0.052 1.571 0.333 0.905 0.194 0.674 0.293 0.329
0.048 0.030 1.488 1.194 1.255 0.167 1.270 0.233 0.840
0.082 0.057 1.169 1.606 2.502 0.724 2.643 0.113 0.884
Optical density at 490 nm. ND: not done.
3. Results A. vasorum larvae appeared in the feces of the inoculated dogs 28–108 DAI and were recovered until the end of the experiment. All of the inoculated animals developed IgG antibodies to A. vasorum based on the ELISA (Table 1). IgG was detected in the serum in two of the seven dogs on 14 DAI (dogs 7 and 18), in four dogs on 28 DAI (dogs 9, 12, 13
Fig. 1. WB of IgG responses of dogs to adult female worm antigen of A. vasorum. Individual lanes illustrate the reactivity of serum collected at various time intervals (days) post-infection. (a) Non-absorbed serum; (b) serum absorbed with T. canis antigen. The apparent molecular weights (kDa) of protein standards are given on the left.
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M.C. Cury et al. / Veterinary Parasitology 106 (2002) 83–87
and 16) and in one dog on 56 DAI (dog 20). A strong antibody response on 156 DAI was observed for dogs 7, 9, 12 and 16. Antibodies from all of the A. vasorum inoculated dogs reacted with a large number of antigens from 14 DAI until the end of the observation period (Fig. 1), while sera from non-inoculated dogs showed little or no reactivity (data not shown). The pattern of recognition was not uniform for the dogs. Antigenic components were recognized from 14 DAI by the dogs 7, 13 and 18 and from 28 DAI by the dogs 9, 12, 16 and 20. The principal antigens recognized by dog IgG had molecular weights of 115, 102, 86, 76, 69, 56, 41, 32, 28, 20–22, 12 and 10 kDa. Antigens of 69, 56, 20–22, 12 and 10 kDa were weakly and sporadically recognized. An antigen of 41 kDa was recognized from 14 to 112 DAI in dogs 7 and 18 and from 28 to 156 DAI in dogs 12 and 16. Serum anti-A. vasorum IgG of all inoculated dogs recognized a 28 kDa antigen from 14 to 156 DAI, with the exception of dog 9, that recognized this antigen from 112 DAI. Antigens with molecular weight of 115 and 102 kDa were strongly recognized by all of the experimentally infected dogs, on 14 DAI for dogs 7, 13 and 20; 28 DAI for dogs 9, 12 and 16; and 112 DAI for the dog 18. The application of sera pre-absorption did not significantly reduce the detection of antigens of A. vasorum for all seven dogs. Representative results of pre-absorption experiments of dog 20 are shown (Fig. 1).
4. Discussion The results of this study showed that antibodies for antigens of A. vasorum are stimulated by the experimental infection and this humoral response is reflected in increasing values of ELISA and by the reactivity to specific antigens by WB. Both the tests presented results with no apparent relationship with the pre-patent or patent period. Up to now, there has been no work about the recognition of antigens of A. vasorum adult worms. While comparing the results of ELISA to those of WB, it was observed that dogs 7, 12 and 16, whose values in ELISA were high, also showed a strong recognition of antigens of the parasite, especially the 115, 102 and 28 kDa antigens. On the other hand, in the animals with low values for ELISA (dogs 9, 13, 18 and 20), less intensity of recognition of antigens by WB was observed although they also recognized the antigens of 115, 102 and 28 kDa. These results indicate that these antigens may be the most immunogenic proteins in infections with A. vasorum. This study could show that in dogs experimentally infected with A. vasorum, ELISA and WB may present homogeneous results on the detection of specific antibodies, independent of the test. However, they differ regarding intensity, with a greater sensitivity occurring in WB. The specificity of WB using antigen of the adult worm of the parasite may be demonstrated as the pre-absorption of sera with antigen of T. canis had only a minimum effect in the reduction of the intensity of the recognition of antigens of A. vasorum. Graeff-Teixeira et al. (1997) observed sera with false-positive results by ELISA while evaluating the human infection produced by A. costaricencis. These authors observed that the reaction was less intense when the sera were pre-absorbed with Ascaris suum. Both ELISA and WB did not present differences with regard to the inoculum, as some dogs infected with double the number of larvae presented less intensity of reading and of recognition of proteins when compared to those which received a smaller inoculum.
M.C. Cury et al. / Veterinary Parasitology 106 (2002) 83–87
87
As ELISA can be used as a reference to obtain results in the cases of canine angiostrongylosis (Cury et al., 1996), WB can also be an auxiliary technique for diagnosing this disease with the advantage of recognizing antigens specific to A. vasorum, even when ELISA values were low. It should also be remarked that the use of crude antigenic extract reduces the specificity, making it necessary to elaborate fractionated antigens as surface and excretion–secretion antigens to improve test ability in detecting antibodies for A. vasorum in infected dogs.
Acknowledgements We thank Rosalida Estevam Nazar Lopes for technical assistance. References Conway, D.J., 1993. Immunodiagnosis of Strongyloides stercoralis infection: a method for increasing of the specificity of the indirect ELISA. Trans. R. Soc. Trop. Med. Hyg. 87, 173–176. Cury, M.C., Lima, W.S., 1996. Aspectos cl´ınicos de cães infectados experimentalmente com Angiostrongylus vasorum (Baillet, 1866) Kamensky, 1905. Arq. Bras. Med. Vet. Zootecnol. 48 (1), 27–34. Cury, M.C., Lima, S.K., Vitor, R.W.A., 1996. Enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Angiostrongylus vasorum (Baillet, 1866) infection in dogs. Rev. Méd. Vét. 147, 525–530. Graeff-Teixeira, C., Agostini, A., Camilo-Coura, L., Ferreira-da-Cruz, M.F., 1997. Seroepidemiology of abdominal angiostrogyliasis: the standardization of an immunoenzymatic assay and prevalence of antibodies in two localities in southern Brazil. Trop. Med. Int. Health 2 (3), 254–260. Guilhon, J., Benex, J., Misha, G.S., 1971. Premiers essais de diagnostic immunologique de l’angiostrongylose canine à Angiostrongylus vasorum. Bull. Soc. Pathol. Exot. 64 (2), 220–228. Mishra, G.S., Benex, J., 1972. Imunological specificity of Angiostrongylus cantonensis and A. vasorum (Nematoda). Indian J. Anim. Sci. 42 (8), 614–617. Patteson, M.W., Gibbs, C., Wotton, P.R., Day, M.J., 1993. Angiostrongylus vasorum infection in seven dogs. Vet. Rec. 4, 565–570.
Short communication
Western blot analysis of the humoral response of dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866) M.C. Cury a , M.P. Guimarães b , W.S. Lima b , R.W.A. Vitor b,∗ a
Laboratory of Parasitology, Federal University of Uberlˆandia, Uberlˆandia, Minas Gerais, Brazil b Department of Parasitology, Federal University of Minas Gerais, ICB-UFMG, P.O. Box 486, CEP 31270-901 Belo Horizonte, Minas Gerais, Brazil Received 7 August 2001; received in revised form 15 January 2002; accepted 23 January 2002
Abstract Seven cross-bred dogs were inoculated with Angiostrongylus vasorum and serum samples were analyzed using the enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). ELISA detected specific antibodies anti-A. vasorum, from 14 to 28 days after inoculation (DAI) and persisted throughout the experiment. Using WB, the main antigens detected had molecular weight of approximately 115, 102, 86, 76, 69, 56, 41, 32, 28, 20–22 and 10 kDa. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Angiostrongylus vasorum; Dog; Western blot analysis
1. Introduction Canine angiostrongylosis is a disease caused by Angiostrongylus vasorum (Baillet, 1866), a nematode from the Protostrongylidea family, whose adult form is found in the right ventricle and pulmonary artery (and its branches) in domestic dogs and wild carnivores. The definite host infected by accidentally ingesting aquatic or terrestrial molluscs contaminated with third-stage larvae. It has been reported in many countries, but appears to be particularly frequent in dogs in the France, England and Ireland (Cury and Lima, 1996). ∗ Corresponding author. Tel.: +55-31-3499-2853; fax: +55-31-3499-2970. E-mail address: [email protected] (R.W.A. Vitor).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 3 1 - 6
84
M.C. Cury et al. / Veterinary Parasitology 106 (2002) 83–87
Several clinical signs have been associated with the infection, including signs of pulmonary and cardiac diseases (Patteson et al., 1993; Cury and Lima, 1996). Diagnosis is based on the detection of first-stage larvae in feces or in postmortem examination by the finding of lesions or presence of adult parasites in the heart and lung. Serological diagnosis is rarely used, with a few experimental results published using complement fixation and agglutination in latex (Guilhon et al., 1971; Mishra and Benex, 1972). Cury et al. (1996) demonstrated that antibodies to A. vasorum antigens are stimulated by experimental infection of dogs and that this response is reflected in generally increasing enzyme-linked immunosorbent assay (ELISA) values. At present, no attention has been paid to the characterization of the individual antigens in A. vasorum adult worms which elicit immune response in the definitive host. The purpose of this study was to analyze the kinetics of anti-A. vasorum antibodies in the serum and to characterize the targets of canine humoral immune response by the Western blot (WB) technique. 2. Materials and methods Seven cross-bred dogs were infected with a varying number of infective larvae of A. vasorum, contained in the tissue of an intermediate host, an aquatic mollusc (Biomphalaria glabrata) maintained in the laboratory and experimentally contaminated with the first-stage larvae of this nematode. The dogs were randomly divided into two groups: group A with four animals (dogs 7, 9, 12 and 13) and group B with three animals (dogs 16, 18 and 20). The animals were inoculated by oral route with 50 larvae/kg of body weight (group A) and 100 larvae/kg of body weight (group B). Two animals (dogs 1 and 2) were not inoculated and served as controls. Fecal samples were collected daily from 25 days after inoculation (DAI). Five milliliters of blood were collected from the jugular vein of each animal to determine antibody levels up to 5 months after infection. ELISA was carried out as described by Cury et al. (1996) in order to determine the IgG antibodies. WB analyses were performed as described by Graeff-Teixeira et al. (1997) with modifications. Briefly, female worms were frozen and homogenized in a mortar while immersed in liquid nitrogen. The worm sample was homogenized in a sample buffer with 1 mM phenylmethylsulphonyl fluoride (PMSF) and 1 mM N-p-tosyl-l-lysine-chloromethyl ketone (TLCK) and centrifuged at 15,000 × g for 2 min at 4 ◦ C. Electrophoretic separation of this soluble fraction was performed in a 10% polyacrylamide gel under reduction conditions in the Bio-Rad® Mini Protean II system (three females per minigel). After separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose membranes for 20 h at 30 V and 40 mA, followed by 1 h at 90 mA. Detection of proteins was carried out by an immunoenzymatic method on 5 mm strips using 4-chloro-1-naphthol as substrate. In order to minimize interference of antibodies of Toxocara canis, frequently found in dogs, sera were previously absorbed with T. canis adult worm antigen. The pre-absorption experiments were performed according to Conway (1993), by mixing and incubating each serum for 1 h with 20 g/ml PBS-soluble adult T. canis extract.
M.C. Cury et al. / Veterinary Parasitology 106 (2002) 83–87
85
Table 1 Specific antibody response detected by ELISA in dogs inoculated with A. vasorum Dog
1 2 7 9 12 13 16 18 20
DAI 0
14
28
56
112
156
0.006 0.167 0.009 0.008 0.053 0.012 0.048 0.047 0.020
0.046 ND 1.065 0.028 ND ND ND 0.180 0.008
0.127 0.066 0.707 0.153 0.318 0.201 0.338 0.073 0.041
0.090 0.052 1.571 0.333 0.905 0.194 0.674 0.293 0.329
0.048 0.030 1.488 1.194 1.255 0.167 1.270 0.233 0.840
0.082 0.057 1.169 1.606 2.502 0.724 2.643 0.113 0.884
Optical density at 490 nm. ND: not done.
3. Results A. vasorum larvae appeared in the feces of the inoculated dogs 28–108 DAI and were recovered until the end of the experiment. All of the inoculated animals developed IgG antibodies to A. vasorum based on the ELISA (Table 1). IgG was detected in the serum in two of the seven dogs on 14 DAI (dogs 7 and 18), in four dogs on 28 DAI (dogs 9, 12, 13
Fig. 1. WB of IgG responses of dogs to adult female worm antigen of A. vasorum. Individual lanes illustrate the reactivity of serum collected at various time intervals (days) post-infection. (a) Non-absorbed serum; (b) serum absorbed with T. canis antigen. The apparent molecular weights (kDa) of protein standards are given on the left.
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M.C. Cury et al. / Veterinary Parasitology 106 (2002) 83–87
and 16) and in one dog on 56 DAI (dog 20). A strong antibody response on 156 DAI was observed for dogs 7, 9, 12 and 16. Antibodies from all of the A. vasorum inoculated dogs reacted with a large number of antigens from 14 DAI until the end of the observation period (Fig. 1), while sera from non-inoculated dogs showed little or no reactivity (data not shown). The pattern of recognition was not uniform for the dogs. Antigenic components were recognized from 14 DAI by the dogs 7, 13 and 18 and from 28 DAI by the dogs 9, 12, 16 and 20. The principal antigens recognized by dog IgG had molecular weights of 115, 102, 86, 76, 69, 56, 41, 32, 28, 20–22, 12 and 10 kDa. Antigens of 69, 56, 20–22, 12 and 10 kDa were weakly and sporadically recognized. An antigen of 41 kDa was recognized from 14 to 112 DAI in dogs 7 and 18 and from 28 to 156 DAI in dogs 12 and 16. Serum anti-A. vasorum IgG of all inoculated dogs recognized a 28 kDa antigen from 14 to 156 DAI, with the exception of dog 9, that recognized this antigen from 112 DAI. Antigens with molecular weight of 115 and 102 kDa were strongly recognized by all of the experimentally infected dogs, on 14 DAI for dogs 7, 13 and 20; 28 DAI for dogs 9, 12 and 16; and 112 DAI for the dog 18. The application of sera pre-absorption did not significantly reduce the detection of antigens of A. vasorum for all seven dogs. Representative results of pre-absorption experiments of dog 20 are shown (Fig. 1).
4. Discussion The results of this study showed that antibodies for antigens of A. vasorum are stimulated by the experimental infection and this humoral response is reflected in increasing values of ELISA and by the reactivity to specific antigens by WB. Both the tests presented results with no apparent relationship with the pre-patent or patent period. Up to now, there has been no work about the recognition of antigens of A. vasorum adult worms. While comparing the results of ELISA to those of WB, it was observed that dogs 7, 12 and 16, whose values in ELISA were high, also showed a strong recognition of antigens of the parasite, especially the 115, 102 and 28 kDa antigens. On the other hand, in the animals with low values for ELISA (dogs 9, 13, 18 and 20), less intensity of recognition of antigens by WB was observed although they also recognized the antigens of 115, 102 and 28 kDa. These results indicate that these antigens may be the most immunogenic proteins in infections with A. vasorum. This study could show that in dogs experimentally infected with A. vasorum, ELISA and WB may present homogeneous results on the detection of specific antibodies, independent of the test. However, they differ regarding intensity, with a greater sensitivity occurring in WB. The specificity of WB using antigen of the adult worm of the parasite may be demonstrated as the pre-absorption of sera with antigen of T. canis had only a minimum effect in the reduction of the intensity of the recognition of antigens of A. vasorum. Graeff-Teixeira et al. (1997) observed sera with false-positive results by ELISA while evaluating the human infection produced by A. costaricencis. These authors observed that the reaction was less intense when the sera were pre-absorbed with Ascaris suum. Both ELISA and WB did not present differences with regard to the inoculum, as some dogs infected with double the number of larvae presented less intensity of reading and of recognition of proteins when compared to those which received a smaller inoculum.
M.C. Cury et al. / Veterinary Parasitology 106 (2002) 83–87
87
As ELISA can be used as a reference to obtain results in the cases of canine angiostrongylosis (Cury et al., 1996), WB can also be an auxiliary technique for diagnosing this disease with the advantage of recognizing antigens specific to A. vasorum, even when ELISA values were low. It should also be remarked that the use of crude antigenic extract reduces the specificity, making it necessary to elaborate fractionated antigens as surface and excretion–secretion antigens to improve test ability in detecting antibodies for A. vasorum in infected dogs.
Acknowledgements We thank Rosalida Estevam Nazar Lopes for technical assistance. References Conway, D.J., 1993. Immunodiagnosis of Strongyloides stercoralis infection: a method for increasing of the specificity of the indirect ELISA. Trans. R. Soc. Trop. Med. Hyg. 87, 173–176. Cury, M.C., Lima, W.S., 1996. Aspectos cl´ınicos de cães infectados experimentalmente com Angiostrongylus vasorum (Baillet, 1866) Kamensky, 1905. Arq. Bras. Med. Vet. Zootecnol. 48 (1), 27–34. Cury, M.C., Lima, S.K., Vitor, R.W.A., 1996. Enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Angiostrongylus vasorum (Baillet, 1866) infection in dogs. Rev. Méd. Vét. 147, 525–530. Graeff-Teixeira, C., Agostini, A., Camilo-Coura, L., Ferreira-da-Cruz, M.F., 1997. Seroepidemiology of abdominal angiostrogyliasis: the standardization of an immunoenzymatic assay and prevalence of antibodies in two localities in southern Brazil. Trop. Med. Int. Health 2 (3), 254–260. Guilhon, J., Benex, J., Misha, G.S., 1971. Premiers essais de diagnostic immunologique de l’angiostrongylose canine à Angiostrongylus vasorum. Bull. Soc. Pathol. Exot. 64 (2), 220–228. Mishra, G.S., Benex, J., 1972. Imunological specificity of Angiostrongylus cantonensis and A. vasorum (Nematoda). Indian J. Anim. Sci. 42 (8), 614–617. Patteson, M.W., Gibbs, C., Wotton, P.R., Day, M.J., 1993. Angiostrongylus vasorum infection in seven dogs. Vet. Rec. 4, 565–570.