Optimization of In-House Indirect-ELISA & EITB Assays Employing Cysticercus cellulosae Antigens for Serological Detection and PCR Assays for Molecular Detection of Porcine Cysticercosis
-
Kaushik Satyaprakash
,
Wiqar Ahmed Khan
,
Sandeep Prabhakarro Chaudhari
,
Shilpshri Vasant Shinde
,
Sunil Waman Kolte
,
Nilesh Ramchandra Pansare
,
Amrut Vijay Likhite
Abstract
Background: Porcine cysticercosis, caused by metacestodes of Taenia solium is an important neglected zoonosis. We evaluated the presence of anti-cysticercal antibodies and T. solium specific DNA in pig sera and blood samples respectively collected from Maharashtra, India.
Methods: A total of three antigens (Scolex Antigen (SA), Membrane Body Antigen (MBA) and Excretory-Secretory Antigen (ESA)) were prepared from metacestodes of T. solium and employed in an in-house developed indirect-IgG ELISA for serological screening of 1000 porcine sera samples at Department of Veterinary Public Health, Nagpur Veterinary College, Maharashtra, India. The ELISA positive sera samples were subjected to EITB Assay for detection of immunodominant peptides. An effort has been made for molecular detection of porcine cysticercosis by PCR assay targeting large subunit rRNA gene of T. solium from blood samples of the corresponding ELISA-positive pigs.
Results: The overall seroprevalence of porcine cysticercosis employing SA, MBA and ESA was 12.6%, 8.7% and 12.5% respectively. The lower and medium molecular weight peptides were the most frequently recognised in EITB assay. The numbers of bands recognised in EITB assay were observed to be proportionate with the corresponding ELISA O.D. values. An amplification product of 286 bp was observed in 22.98% (20/87), 30.35% (30/99) and 17.14% (12/70) of the sero-positive samples against SA, ESA and MBA respectively.
Conclusion: EITB still remains the gold standard serodiagnosis test for cysticercosis. The inclusion of a greater number of positive samples and purification of antigens may improve the diagnostic efficacy of the tests.
1. Giri S, Parija SC. A review on diagnostic and preventive aspects of cystic echino-coccosis and human cysticercosis. Trop Parasitol. 2012; 2:99-108.
2. Garcia HH, Del Brutto OH. Taenia solium cysticercosis. Infect Dis Clin North Am. 2000;14(1):97-119.
3. Prasad KN, Prasad A, Gupta RK, et al. Neurocysticercosis in patients with active epilepsy from a pig farming community. Trans R Soc Trop Med Hyg. 2009;103:144-50.
4. Singh AK, Singh SK, Prasad KN, et al. Evaluation of ELISA, neck muscle, tongue and eyelid examinations for the diagnosis of swine cysticercosis in a highly endemic area of North India. Exp Parasitol. 2013;134:313-7.
5. Sciutto E, Hernandez M, Garcia G, et al. Diagnosis of porcine cysticercosis: A comparative study of serological tests for detection of circulating antibody and viable parasites. Vet Parasitol. 1998;78:185-94.
6. Boa ME, Kassuku AA, Willingham AL, et al. Distribution and density of cysticerci of Taenia solium by muscle groups and organs in naturally infected local finished pigs in Tanzania. Vet Parasitol. 2002;106(2):155-64.
7. Phiri IK, Dorny P, Gabriel S, et al. The prevalence of porcine cysticercosis in Eastern and Southern provinces of Zam-bia. Vet Parasitol. 2002;108(1):31-9.
8. Sako Y, Itoh S, Okamoto M, et al. Simple and reliable preparation of immunodiag-nostic antigens for Taenia solium cysticerco-sis. Parasitology. 2013;140:1589-94.
9. Gonzalez AE, Cama V, Gilman RH, et al. Prevalence and comparison of serologic assays, necropsy, and tongue examination for the diagnosis of porcine cysticercosis in Peru. Am J Trop Med Hyg. 1990;43:194-9.
10. Pinto PSA, Vaz AJ, Germano PML, et al. ELISA test for diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci. Rev Inst Med Trop Sao Paulo. 2000;42(2):71-9.
11. Cheng RWK, Ko RC. Cross-reactions between crude antigens of larva (Cysticercus cellulosae) and other helminthes of pigs. Vet Parasitol. 1991;39:161-70.
12. Dhanalakshmi H, Jagannath MS, D’Souza PE. Protein profile and serodiagnosis of Taenia solium bladder worm infections in pigs. Vet Arhiv. 2005;75(6):505-512.
13. Sahu PS, Parija SC, Narayan SK, et al. Evaluation of an IgG-ELISA strategy us-ing Taenia solium metacestode somatic and excretory-secretory antigens for diagnosis of neurocysticercosis revealing biological stage of the larvae. Acta Trop. 2009;110(1):38-45.
14. Sahu P, Parija SC. Antibody specific to 43 kDa excretory secretory antigenic peptide of Taenia solium metacestode as a potential diagnostic marker in human neurocysticer-cosis. Acta Trop. 2010;115(3):257-261.
15. Shukla N, Husain N, Jyotsna, et al. Com-parisons between scolex and membrane antigens of Cysticercus fasciolaris and Cysticer-cus cellulosae larvae for immunodiagnosis of neurocysticercosis. J Microbiol Immunol Infect. 2008;41(6):519-24.
16. Jardim EAGV, Linhares GFC, Torres FAG, et al. Specific discrimination between Taenia saginata and Taenia solium by one step PCR assay and duplex-PCR. Cienc Rural. 2006;36(1):166-72.
17. Sreedevi C, Hafeez M, Putcha AK, et al. PCR test for detecting Taenia solium cysti-cercosis in pig carcasses. Trop Anim Health Prod. 2012;44:95-9.
18. D’Souza PE, Hafeez M. Detection of Tae-nia solium Cysticercosis in pigs by ELISA with an excretory-secretory antigen. Vet Res Commun. 1999;23(5):293-8.
19. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265-75.
20. Shiguekawa KYM, Mineo JR, Moura LP, et al. ELISA and Western Blotting tests in the detection of IgG antibodies to Taenia solium metacestodes in serum samples in human neurocysticercosis. Trop Med Int Health. 2000;5(6);443-9.
21. Minozzo JC, Moura J, Almeida SM, et al. Crude antigen from Taenia crassiceps cysti-cercus used as heterologous antigen in ELISA and in EITB for neurocysticercosis diagnosis of patients from Paraná-Brazil. Braz Arch Biol Techn. 2008;51(6): 16784324.
22. Towbin H, Stachelin T, Gordon J. Elec-trophoretic transfer of proteins from pol-yacrylamide gel to nitrocellulose sheets: procedure and cone applications. Proc Natl Acad Sci USA. 1979;76(9):4350-4.
23. Laemmli UK. Cleavage of structural pro-teins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-5.
24. Martins TM, Pedro OC, Caldeira RA, et al. Detection of bovine babesiosis in Mozambique by a novel seminested hot-start PCR method. Vet Parasitol. 2008;153:225-30.
25. Hafeez M, Reddy CVS, Ramesh B, et al. Prevalence of porcine cysticercosis in South India. J Para Dis. 2004;28(2):118-20.
26. Mohan VR, Tharmalingam J, Muliyil J, et al. Prevalence of porcine cysticercosis in Vellore, South India. Trans R Soc Trop Med Hyg. 2013; 107(1):62-4.
27. Flisser A, Gonzalez D, Plancarte A, et al. Praziquantel treatment of porcine brain and muscle Taenia solium cysticiercosis. 2. Immunological and cytogenetic studies. Parasitol Res. 1990;76:640-2.
28. Soares KA, Silva MRM., Poleti MD, et al. Immunodiagnosis of cysticercosis in swine experimentally infected with Taenia solium eggs, using antigen of Cysticercus cellulosae scolex. Arq Bras Med Vet Zoo. 2006;58(1):15-20.
29. Lisiane VE, Maia AAM, Pianetti G, et al. Immunological evaluation of a 26-kDa an-tigen from Taenia solium larvae for specific immunodiagnosis of human neurocysti-cercosis. Parasitol Res. 1999;85(2):98-102.
30. Neto FI., Filho GP, Araújo RN, et al. Im-munodiagnosis of human neurocysticer-cosis by using semipurified scolex antigens from Taenia solium cysticerci. Rev Soc Bras Med Tro.2007;40(2):163-9.
31. Ko RCC, Ng TF. Evaluation of excreto-ry/secretory products of larval Taenia solium as diagnostic antigens for porcine and human cysticercosis. J Helminthol. 1998;72(2):147-54.
32. Brandt JRA, Geerts S, Deken R, et al. A monoclonal antibody-based ELISA for the detection of circulating excretory-secretory antigens in Taenia saginata cysti-cercosis. Int J Parasitol. 1992;22(4):471-7.
33. Santamaria E, Plancarte A, Aluja AS. The experimental infection of pigs with differ-ent numbers of Taenia solium eggs: immune response and efficiency of establishment. J Parasitol. 2002;88(1):69-73.
34. Molinari J, García-mendoza E, Garza YD, et al. Discrimination between active and inactive neurocysticercosis by metacestode excretory/secretory antigens of Taenia soli-um in an enzyme-linked immunosorbent assay. Am J Trop Med Hyg. 2002;66(6):777-81.
35. Garcia HH, Gilman RH, Gonzalez AE, et al. Hyperendemic human and porcine Tae-nia solium infection in Peru. Am J Trop Med Hyg. 2003;68(3):268-75.
36. Gavidia CM, Verastegui MR, Garcia HH, et al. Relationship between serum antibod-ies and Taenia solium larvae burden in pigs raised in field conditions. PLOS Neg Trop Dis. 2013;7(5):e2192.
37. Gonzalez AE, Lopez-Urbina T, Tsang BY, et al. Short report: secondary transmission in porcine cysticercosis: description and their potential implications for control sus-tainability. Am J Trop Med Hyg. 2005;73(3):501-3.
38. Sciutto E, Chavarria A, Fragoso G, et al. The immune response in Taenia solium cys-ticercosis: protection and injury. Parasite Immunol. 2007;29(12):621-636.
39. Flisser A. Risk factors and control measures for taeniosis/cysticercosis. In: Craig P, Pawlowski Z, editors. Cestode zoonoses: echinococcosis and cysticerco-sis, an emergent and global problem. NATO Science Series, vol. 341. Amster-dam: IOS Press; 2002. p. 335-42.
40. Aluja AS, Villalobos ANM, Plancarte A, et al. Experimental Taenia solium cysticercosis in pigs: characteristics of the infection and antibody response. Vet Parasitol. 1996; 61(1-2):49-59.
41. Cho SY, Kang SY, Kim SI. Analysis of antigen specificity using monoclonal and polyclonal antibodies to Cysticercus cellulosae by enzyme-linked immunotransfer blot technique. Kisaengchunghak Chapchi. 1987;25(2):159-167.
42. Ramahefarisoa RM, Rakotondrazaka M, Jambou R, et al. Comparison of ELISA and PCR assays for the diagnosis of por-cine cysticercosis. Vet Parasitol. 2010; 173(3-4):336-9.
2. Garcia HH, Del Brutto OH. Taenia solium cysticercosis. Infect Dis Clin North Am. 2000;14(1):97-119.
3. Prasad KN, Prasad A, Gupta RK, et al. Neurocysticercosis in patients with active epilepsy from a pig farming community. Trans R Soc Trop Med Hyg. 2009;103:144-50.
4. Singh AK, Singh SK, Prasad KN, et al. Evaluation of ELISA, neck muscle, tongue and eyelid examinations for the diagnosis of swine cysticercosis in a highly endemic area of North India. Exp Parasitol. 2013;134:313-7.
5. Sciutto E, Hernandez M, Garcia G, et al. Diagnosis of porcine cysticercosis: A comparative study of serological tests for detection of circulating antibody and viable parasites. Vet Parasitol. 1998;78:185-94.
6. Boa ME, Kassuku AA, Willingham AL, et al. Distribution and density of cysticerci of Taenia solium by muscle groups and organs in naturally infected local finished pigs in Tanzania. Vet Parasitol. 2002;106(2):155-64.
7. Phiri IK, Dorny P, Gabriel S, et al. The prevalence of porcine cysticercosis in Eastern and Southern provinces of Zam-bia. Vet Parasitol. 2002;108(1):31-9.
8. Sako Y, Itoh S, Okamoto M, et al. Simple and reliable preparation of immunodiag-nostic antigens for Taenia solium cysticerco-sis. Parasitology. 2013;140:1589-94.
9. Gonzalez AE, Cama V, Gilman RH, et al. Prevalence and comparison of serologic assays, necropsy, and tongue examination for the diagnosis of porcine cysticercosis in Peru. Am J Trop Med Hyg. 1990;43:194-9.
10. Pinto PSA, Vaz AJ, Germano PML, et al. ELISA test for diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci. Rev Inst Med Trop Sao Paulo. 2000;42(2):71-9.
11. Cheng RWK, Ko RC. Cross-reactions between crude antigens of larva (Cysticercus cellulosae) and other helminthes of pigs. Vet Parasitol. 1991;39:161-70.
12. Dhanalakshmi H, Jagannath MS, D’Souza PE. Protein profile and serodiagnosis of Taenia solium bladder worm infections in pigs. Vet Arhiv. 2005;75(6):505-512.
13. Sahu PS, Parija SC, Narayan SK, et al. Evaluation of an IgG-ELISA strategy us-ing Taenia solium metacestode somatic and excretory-secretory antigens for diagnosis of neurocysticercosis revealing biological stage of the larvae. Acta Trop. 2009;110(1):38-45.
14. Sahu P, Parija SC. Antibody specific to 43 kDa excretory secretory antigenic peptide of Taenia solium metacestode as a potential diagnostic marker in human neurocysticer-cosis. Acta Trop. 2010;115(3):257-261.
15. Shukla N, Husain N, Jyotsna, et al. Com-parisons between scolex and membrane antigens of Cysticercus fasciolaris and Cysticer-cus cellulosae larvae for immunodiagnosis of neurocysticercosis. J Microbiol Immunol Infect. 2008;41(6):519-24.
16. Jardim EAGV, Linhares GFC, Torres FAG, et al. Specific discrimination between Taenia saginata and Taenia solium by one step PCR assay and duplex-PCR. Cienc Rural. 2006;36(1):166-72.
17. Sreedevi C, Hafeez M, Putcha AK, et al. PCR test for detecting Taenia solium cysti-cercosis in pig carcasses. Trop Anim Health Prod. 2012;44:95-9.
18. D’Souza PE, Hafeez M. Detection of Tae-nia solium Cysticercosis in pigs by ELISA with an excretory-secretory antigen. Vet Res Commun. 1999;23(5):293-8.
19. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265-75.
20. Shiguekawa KYM, Mineo JR, Moura LP, et al. ELISA and Western Blotting tests in the detection of IgG antibodies to Taenia solium metacestodes in serum samples in human neurocysticercosis. Trop Med Int Health. 2000;5(6);443-9.
21. Minozzo JC, Moura J, Almeida SM, et al. Crude antigen from Taenia crassiceps cysti-cercus used as heterologous antigen in ELISA and in EITB for neurocysticercosis diagnosis of patients from Paraná-Brazil. Braz Arch Biol Techn. 2008;51(6): 16784324.
22. Towbin H, Stachelin T, Gordon J. Elec-trophoretic transfer of proteins from pol-yacrylamide gel to nitrocellulose sheets: procedure and cone applications. Proc Natl Acad Sci USA. 1979;76(9):4350-4.
23. Laemmli UK. Cleavage of structural pro-teins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-5.
24. Martins TM, Pedro OC, Caldeira RA, et al. Detection of bovine babesiosis in Mozambique by a novel seminested hot-start PCR method. Vet Parasitol. 2008;153:225-30.
25. Hafeez M, Reddy CVS, Ramesh B, et al. Prevalence of porcine cysticercosis in South India. J Para Dis. 2004;28(2):118-20.
26. Mohan VR, Tharmalingam J, Muliyil J, et al. Prevalence of porcine cysticercosis in Vellore, South India. Trans R Soc Trop Med Hyg. 2013; 107(1):62-4.
27. Flisser A, Gonzalez D, Plancarte A, et al. Praziquantel treatment of porcine brain and muscle Taenia solium cysticiercosis. 2. Immunological and cytogenetic studies. Parasitol Res. 1990;76:640-2.
28. Soares KA, Silva MRM., Poleti MD, et al. Immunodiagnosis of cysticercosis in swine experimentally infected with Taenia solium eggs, using antigen of Cysticercus cellulosae scolex. Arq Bras Med Vet Zoo. 2006;58(1):15-20.
29. Lisiane VE, Maia AAM, Pianetti G, et al. Immunological evaluation of a 26-kDa an-tigen from Taenia solium larvae for specific immunodiagnosis of human neurocysti-cercosis. Parasitol Res. 1999;85(2):98-102.
30. Neto FI., Filho GP, Araújo RN, et al. Im-munodiagnosis of human neurocysticer-cosis by using semipurified scolex antigens from Taenia solium cysticerci. Rev Soc Bras Med Tro.2007;40(2):163-9.
31. Ko RCC, Ng TF. Evaluation of excreto-ry/secretory products of larval Taenia solium as diagnostic antigens for porcine and human cysticercosis. J Helminthol. 1998;72(2):147-54.
32. Brandt JRA, Geerts S, Deken R, et al. A monoclonal antibody-based ELISA for the detection of circulating excretory-secretory antigens in Taenia saginata cysti-cercosis. Int J Parasitol. 1992;22(4):471-7.
33. Santamaria E, Plancarte A, Aluja AS. The experimental infection of pigs with differ-ent numbers of Taenia solium eggs: immune response and efficiency of establishment. J Parasitol. 2002;88(1):69-73.
34. Molinari J, García-mendoza E, Garza YD, et al. Discrimination between active and inactive neurocysticercosis by metacestode excretory/secretory antigens of Taenia soli-um in an enzyme-linked immunosorbent assay. Am J Trop Med Hyg. 2002;66(6):777-81.
35. Garcia HH, Gilman RH, Gonzalez AE, et al. Hyperendemic human and porcine Tae-nia solium infection in Peru. Am J Trop Med Hyg. 2003;68(3):268-75.
36. Gavidia CM, Verastegui MR, Garcia HH, et al. Relationship between serum antibod-ies and Taenia solium larvae burden in pigs raised in field conditions. PLOS Neg Trop Dis. 2013;7(5):e2192.
37. Gonzalez AE, Lopez-Urbina T, Tsang BY, et al. Short report: secondary transmission in porcine cysticercosis: description and their potential implications for control sus-tainability. Am J Trop Med Hyg. 2005;73(3):501-3.
38. Sciutto E, Chavarria A, Fragoso G, et al. The immune response in Taenia solium cys-ticercosis: protection and injury. Parasite Immunol. 2007;29(12):621-636.
39. Flisser A. Risk factors and control measures for taeniosis/cysticercosis. In: Craig P, Pawlowski Z, editors. Cestode zoonoses: echinococcosis and cysticerco-sis, an emergent and global problem. NATO Science Series, vol. 341. Amster-dam: IOS Press; 2002. p. 335-42.
40. Aluja AS, Villalobos ANM, Plancarte A, et al. Experimental Taenia solium cysticercosis in pigs: characteristics of the infection and antibody response. Vet Parasitol. 1996; 61(1-2):49-59.
41. Cho SY, Kang SY, Kim SI. Analysis of antigen specificity using monoclonal and polyclonal antibodies to Cysticercus cellulosae by enzyme-linked immunotransfer blot technique. Kisaengchunghak Chapchi. 1987;25(2):159-167.
42. Ramahefarisoa RM, Rakotondrazaka M, Jambou R, et al. Comparison of ELISA and PCR assays for the diagnosis of por-cine cysticercosis. Vet Parasitol. 2010; 173(3-4):336-9.
| Files | ||
| Issue | Vol 18 No 1 (2023) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v18i1.12376 | |
| Keywords | ||
| Cysticercosis Enzyme-linked Immuno-sorbent assay Seroprevalence | ||
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How to Cite
1.
Satyaprakash K, Khan W, Chaudhari S, Shinde S, Kolte S, Pansare N, Likhite A. Optimization of In-House Indirect-ELISA & EITB Assays Employing Cysticercus cellulosae Antigens for Serological Detection and PCR Assays for Molecular Detection of Porcine Cysticercosis. Iran J Parasitol. 2023;18(1):19-29.
