Molecular identification technique of Trypanosoma evansi by Multiplex Polymerase Chain Reaction

Dyah H. Sawitri, April H. Wardhana, H. Wibowo, M. Sadikin, Fitrine Ekawasti

Abstract

Trypanosoma evansi is a Hemoflagella parasite that infects cattle and is known as the agents of Surra. Several other trypanosome species infects mammals: T. equiperdum, T. b. rhodesiense, T. b. gambiense, T. vivax, T. congolense, T.theileri. Some of these species is quite difficult to be distinguished morphologically with T. evansi through conventional techniques (thin blood smear). Molecular technique by polymerase chain reaction (PCR) is reported to have the ability to identify, characterize and diagnose trypanosomes accurately. However, a single PCR used is relatively expensive because it takes at least two or more pairs of primers to determine T. evansi. The purpose of this study is to develop T. evansi species identification techniques by multiplex PCR/mPCR (the three pairs of primer in one reaction) that takes the relatively fast and inexpensive. A total of 31 isolates T.evansi were obtained from Bblitvet Culture Collection (BCC) and the Department of Parasitology BBLitvet used in this study. Isolates represent isolates from endemic areas and Surra outbrake isolated from 1988-2014. DNA extraction performed on each sample, including Bang 87 isolates which has been purified as a positive control. Primers used are specific for T. evansi, the ITS-1, Ro Tat 1.2 VSG and ESAG 6/7. Before running mPCR, each primer is optimized by using a single PCR. The results showed that the three primers can be combined in a single reaction with mPCR technique and amplify each DNA fragment target perfectly, so identified 31 isolates as T. evansi. This technique can be applied in the field with a lower cost and faster time.

Key Words: Trypanosoma evansi, Identification, Multiplex PCR

Keywords

Trypanosoma evansi; Identification; Multiplex PCR

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References

Ahmed, Heba A, Picozzi K, Welburn SC, Macleod ET. 2013. A Comparative Evaluation of PCR- Based Methods for species- specific determination of African animal Trypanosomes in Ugandan cattle. Parasites & Vectors 6:316-7.

Bajyana SE, Hamers R. 1988. A Card Agglutination Test (CATT) for veterinary use based on an early VAT RoTat 1/2 of Trypanosoma evansi. Ann Soc Belg Med Trop. 68:233-240.

Batra, Arun S, Krupanidhi S, Tuteja U. 2013. A sensitive & specific Multiplex PCR assay for simultaneous detection of Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei & Brucella species. Indian J Med Res. 138: 111–116

Bitter W, Gerrits H, Kieft R, Borst P. 1998. The role transferrin-reseptor variation in the host range of Trypanosoma brucei. Nature. 391:499-503.

Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT. 1988. Deletion Screening of the Duchenne Muscular Dystrophy Locus via Multiplex DNA Amplification. Nucl Acids Res. 16:11141-11156.

Claes, Filip, Radwanska M, Urakawa T, Majiwa PAO, Goddeeris B, Büscher P. 2004. Kinetoplastid biology and disease diagnostic tool for the detection of Trypanosoma evansi infections. Kinetoplastid Biol Dis. 6:1-6. doi: 10.1186/1475-9292-3-3.

Davila AM, Herrera HM, Schlebinger T, Souza SS, Traub-Cseko YM. 2003. Using PCR for unraveling the cryptic epizootiology of livestock Trypanosomosis in the Pantanal. Braz Vet Parasitol. 117:1-13.

De Sá ARN, Steindel M, Demeu LMK, Lückemeyer DD, Grisard EC, Neto QADL, de Araújo SM, Toledo MJDO, Gomes ML. 2013. Cytochrome oxidase subunit 2 gene allows simultaneous detection and typing of Trypanosoma reangeli and Trypanosoma cruzi. Parasites & Vectors. 6:363. doi: 10.1186/1756-3305-6-363.

Desquesnes M, Dávila AMR. 2002. Applications of PCR-based tools for detection and identification of animal Trypanosomes: A Review and Perspectives. Vet Parasitol. 109:213-231. doi: 10.1016/S0304-4017(02)00270-4.

Desquesnes M, McLaughlin G, Zoungrana A, Davila AMR. 2001. Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA. Int J Parasitol. 31:610-614.

[Ditjennak] Direktorat Jenderal Peternakan. 2012. Pedoman Pengendalian Dan Pemberantasan Penyakit Trypanosomiasis (Surra). Jakarta (Indones): Direktorat Kesehatan Hewan, Direktorat Jenderal Peternakan dan Kesehatan Hewan, Kementerian Pertanian.

Ekawasti F, Sawitri DH, Wardhana AH. 2014. Perbandingan metode penyimpanan darah vektor Surra (Lalat Haematophagus) untuk analisis Multiplex Polimerase Chain Reaction. Pamungkas D, Widiawati Y, Noor SM, Purwantari ND, Widiastuti R, Brahmantiyo B, Herawati T, Kusumaningsih A, Handiwirawan E, Puastuti W, editors. Prosiding Seminar Nasional Peternakan dan Veteriner. Bogor (Indones): Pusat Penelitian dan Pengembangan Peternakan. p. 209-217.

Fernandez D, Gonzalez-Baradat B, Eleizalde M, Gonzalez-Marcano E, Perrone T, Mendoza M. 2009. Trypanosoma evansi: A comparison of PCR and parasitological diagnostic tests in experimentally infected mice. Trypanosoma Exp Parasitol. 121:1-7.

Gibson W. 2009. Species-specific probes for the identification of the African Tsetse-Transmitted Trypanosomes. Parasitol. 136:1501-1507.

Hernandez P, Martin-Parras L, Martinez-Robles ML, Schvartz-Man JB. 1993. Conserved features in the mode of replication of eukaryotic ribosomal RNA genes. EMBO J. 12:1475-1485.

Herrera HM, Dávila MR, Norek A, Abreu UG, Souza SS, D’Andrea PS, Jansen AM. 2004. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet Parasitol. 125:263-275. doi:10.1016/j.vetpar.2004.07.013.

Herrera HM, Aquino LPCT, Menezes RF, Marques LC, Moraes MAV, Werther K, Machado RZ. 2001. Trypanosoma evansi experimental infection in the South American coati (Nasua nasua): Clinical, parasitological and humoral immune response. Vet Parasitol. 102:209-216. doi: 10.1016/S0304-4017(01)00532-5.

Hoare CA. 1972. The Trypanosomes of mammals. Oxford (UK): Blackwell Scientific Publications.

Holland WC, My LN, Dung TV, Thanh NG, Tam PT, Vercruysse J, Goddeeris BM. 2001. The influence of Trypanosoma evansi infection on the immunoresponsive- ness of experimentally infected water buffaloes. Vet Parasitol. 102:225-234.

Isobe T, Holmes EC, Rudenko G. 2003. the transferrin receptor genes of Trypanosoma equiperdum are less diverse in their transferrin binding site than those of the broad-host range Trypanosoma brucei. J Mol Evol. 56:377-386. doi: 10.1007/s00239-002-2408-z.

Jittapalapong, Sathaporn, Pinyopanuwat N, Inpankaew T, Sangvaranond A, Phasuk C, Chimnoi W, Kengradomkij C, Kamyingkird K, Sarataphan N, Desquesnes M and Arunvipas P. 2009. Prevalence of Trypanosoma evansi infection causing abortion in dairy cows in central Thailand. Kasetsart J Nat Sci. 43:53-57.

Kabiri M, Steverding D. 2001. Identification of a developmentally regulated iron superoxide dismutase of Trypanosoma brucei. Biochem J. 260:173-177.

Konnai, Satoru, Hirohisa Mekata, Claro N Mingala, Nancy S Abes, Charito a Gutierrez, Jesus Rommel V Herrera, Alan P Dargantes. 2009. Development and application of a quantitative real-time PCR for the diagnosis of Surra in water buffaloes. Infect Genet Evol. 9:449-452. doi: 10.1016/j.meegid.2009.01.006.

Markoulatos P, Siafakas N, Moncany M. 2002. Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal. 16:47-51.

Masake RA, Njuguna JT, Brown CC. 2002. The application of PCR-ELISA to the detection of Trypanosoma brucei and T. vivax infections in livestock. Vet Parasitol. 105:179-189.

Mekata, Hirohisa, Konnai S, Witola WH, Inoue N, Onuma M, Ohashi K. 2009. Molecular detection of Trypanosomes in cattle in South America and genetic diversity of Trypanosoma evansi based on expression-site-associated gene 6. Infect Genet Evol: J Mol Epidemiol Evol Genet Infect Dis. 9:1301-1305. doi: 10.1016/j.meegid.2009.07.009.

Mulumbu JM. 2006. The importance of trypanosoma congolense strain diversity in the epidemiology of bovine trypanosomiasis (Thesis). Faculteit Diergeneeskunde, Universiteit Gent.

Njiokou F, Simo G, Nkinin SW, Laveissi C. 2004. Infection rate of Trypanosoma brucei S.L., T. vivax, T. congolense ‘Forest Type’, and T. simiae in small wild vertebrates in South Cameroon. 92:139-146. doi: 10.1016/j.actatropica.2004.04.011.

Njiru ZK, Constantine CC, Masiga DK, Reid SA, Thompson RCA, Gibson WC. 2006. Characterization of Trypanosoma evansi type B §. Infect Genet Evol. 6:292-300. doi: 10.1016/j.meegid.2005.08.002.

Njiru NK, Constantine CC, Guya S, Crowther J, Kiragu JM, Thompson RC, Dávila AM. 2005. The use of ITS1 rDNA PCR in detecting pathogenic African Trypanosomes. Parasitol Res. 95:186-192.

Njiru ZK, Constantine CC, Ndung’u JM, Robertson I, Okaye S, Thompson RCA, Reid SA. 2004. Detection of Trypanosoma evansi in camels using PCR and CATT/T. evansi tests in Kenya. Vet Parasitol. 124:187-199. doi:10.1016/j.vetpar.2004.06.029.

[OIE] Office International des Epizooties Terrestrial Manual, 2012. Chapter 2.1.17. Trypanosoma evansi infection (surra).

Payne RC, Sukanto IP, Bazeley K, Jones TW. 1993. The effect of Trypanosoma evansi infection on the oestrous cycle of Friesian Holstein heifers. Vet Parasitol. 51:1-11. doi: 10.1016/0304-4017(93)90190-X.

Payne RC, Sukanto IP, Partoutomo S, Jones TW. 1994. Efficacy of Cymelarsan treatment of suramin resistant Trypanosoma evansi in cattle. Trop Anim Health Prod. 26:92-94.

Powers TO, Todd TC, Burnell AM, Murray PCB, Fleming CC, Szalanki AL, Adams BA, Harris TS. 1997. The internal transcribed spacer region as a taxonomic marker for nematodes. J Nematol. 229:441-450.

Pruvot M, Kamyingkird K, Desquesnes M, Sarataphan N. 2010. Veterinary parasitology a comparison of six primer sets for detection of Trypanosoma evansi by Polymerase Chain Reaction in Rodents and Thai Livestock. Vet Parasitol. 171:185-193. doi: 10.1016/j.vetpar.2010.04.001.

Reid SA. 2002. Trypanosoma evansi control and containment in Australasia. Trends Parasitol. 18:219-224. doi: 10.1016/S1471-4922(02)02250-X.

Salim B, Bakheit MA, Kamau J, Nakamura I, Sugimoto C. 2011. Molecular epidemiology of camel Trypanosomiasis based on ITS1 rDNA and RoTat 1 . 2 VSG gene in the Sudan. Parasites Vectors. 4. BioMed Central. Ltd: 31. doi: 10.1186/1756-3305-4-31.

Salmon D, Geuskens M, Hanocq F, Hanocq-Quertier J, Nolan D, Ruben L, Pays E. 1994. A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei. Cell. 78:75-86.

Sawitri DH. 2016. Studi virulensi T. evansi isolat Indonesia dengan penentuan marka molekuler DNA mikrosatelit dan analisis profil sitokin pada mencit (Thesis). [Jakarta (Indones)]: Faculty of Medicine, Universitas Indonesia.

Schell D, Borowy NK, Overath P. 1991. Transferrin in a growth factor for the bloodstream form of Trypanosoma brucei. Parasitol Res. 30:558-560.

Shahzad W, Munir R, Khan MS, Ahmad MD, Ijaz M, Ahmad A, Iqbal M. 2010. Prevalence and molecular diagnosis of Trypanosoma evansi in Nili-Ravi buffalo (Bubalus bubalis) in different districts of Punjab (Pakistan). Trop Anim Health Prod. 42:1597-1599. doi: 10.1007/s11250-010-9616-0.

Sharma P, Juyal PD, Singla LD, Chachra D, Pawar H. 2012. Comparative evaluation of Real Time PCR assay with conventional parasitological techniques for diagnosis of Trypanosoma evansi in cattle and buffaloes. Vet Parasitol. 190:375-382. doi: 10.1016/j.vetpar.2012. 07.005.

Steverding D, Stierhof YD, Fuchs H, Tauber R, Overath P. 1995. Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei. J Cell Biol. 131:1173-1782.

Sukanto IP, Solihat L, Politedy F, Dachlan M, Wardhana AH, Satria E. 2000. Peran Diptera Hematofagus sebagai vektor Trypanosoma evansi. Haryanto B, Darminto, Hastiono S, Sutama I-K, Partoutomo S, Subandriyo, Sinurat AP, Darmono, Supar, Butar SOB, editors. Prosiding Seminar Nasional Teknologi Peternakan dan Veteriner. Bogor (Indones): Pusat Penelitian dan Pengembangan Peternakan. p. 481-487.

Uilenberg G. 1998. A field guide for the diagnosis, treatment and prevention of African animal Trypanosomes. In FAO Corporate Document Repository. Agriculture Costumer Production. https://www.fao.org/docrep/006 /x0413e/x0413e02.htm.

Urakawa T, Verloo D, Moens L, Büscher P, Majiwa PAO. 2001. Trypanosoma evansi: Cloning and expression in Spodoptera frugiperda [correction of Fugiperda] insect cells of the diagnostic antigen RoTat1.2. Experiment Parasitol. 99:181-189. doi: 10.1006/expr.2001.4670.

Verloo D, Magnus E, Büscher P. 2001. General expression of RoTat 1. 2 variable antigen type in Trypanosoma evansi isolates from different origin. Vet Parasitol. 97:183-189.

Welburn SC, Picozzi K, Fèvre EM, Coleman PG, Odiit M, Carrington M, Maudlin I. 2001. Identification of human-infective Trypanosomes in Animal reservoir of sleeping sickness in Uganda by means of Serum-Resistance-Associated (SRA) gene. The Lancet. 358:2017-2019.

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