Specificity test

DNA from all parasite samples included in the panel was amplified with the universal 18S rRNA primers, generating amplification curve (Fig. 4A) and confirming the quality and amount of DNA loaded per reaction. The repetitive T.evansi DNA primers reacted only with T.evansi DNA, generating amplification curve. No amplification of the other blood protozoa with the repetitive T.evansi DNA primer at crossing point of 55 cycles (Fig. 4B).

Melting curve analysis of the real-time PCR amplified ten isolates of T.evansi originated from various hosts showed identical Tm (Fig. 5A), and DNA fragment 257 bp in size (Fig. 5B).

4. DISCUSSION AND CONCLUSION

The minimal limit of detectable purified T.evansi genomic DNA in this study was 0.00338 pg per reaction. It was higher sensitive than previously published PCR assays which the minimal level of detection at 0.5 to 1 pg per reaction of T.evansi DNA was reported by using the same primers in this study [28]; [22]. The minimal detection of real-time PCR was also higher sensitive than PCR-ELISA which could detect T.evansi DNA at the level as less as 0.01 pg per reaction by using the same primers in this study [6].

The minimal detection of the real-time PCR in this study for detection of T.evansi using DNA templates derived from chelex-100® preparation was 38 parasites per mL of blood. It was higher sensitive than conventional parasitological techniques which microscopic examination from Giemsa stained thin blood film has a sensitivity of 10⁵ Trypanosomes per mL of blood [16]. The diagnostic capability could be significantly improved by adopting simple, low-cost alternative, such as the hematocrit

centrifugation technique (HCT), which has a sensitive of 85 Trypanosomes per mL blood [17]. The mouse inoculation test (MI) is the most sensitive method for the isolation of T.evansi from infected animals. The MI test is able to detect T.evansi in bovine blood to a level of at least 25 parasites per mL of blood regardless of mouse strain used [18]. However, the real-time PCR assay on mouse blood in this study was higher in sensitivity than that shown recently [1]; for a real-time PCR assay for detection of T.brucei in human blood samples using Sybr Green I dye.

The DNA template derived from Chelex 100 resin preparation from blood sample on Whatman FTA cards. The detection limit of the assay was 100 Trypanosomes per mL blood. Furthermore, the assay in this study using whole blood shows proximately 2.5 times better sensitivity than using dried blood on Whatman FTA card.

The PCR product of purified T.evansi DNA analysed by agarose gel electrophoresis and stained with ethidium bromide could be seen as little as 0.5 pg per reaction [28]. This is equivalent to a genome content of 5 parasites, assuming that one Trypanosome parasite has a DNA content of 0.1 pg [4], but the amplicons of T.evansi amplified by the real-time PCR and analysed by agarose gel stained with SYBR gold could be seen as less as 0.00338 pg per reaction in this study.

Future studies should develop Real-time PCR assays for Trypanozoon (T.evansi, T.equiperdum, T.b.brucei, T.b.rhodesiense, T.b.gambiense) using detection by hydrolysis probes using ITS1 of rRNA primers [7]; and MORF2 primers [2].

Comparison of real-time PCR detection methods for Trypanozoon using ITS1 and MORF2 primers should be evaluated in term of diagnostic sensitivity in experimental parasite infected calves.

In conclusion, the real-time PCR assay is very sensitive and specificity assay that can be used to detect T.evansi in blood samples using T.evansi repetitive DNA primers and SYBR Green I fluorescent dye. The detection response was linear of purified T.evansi genomic DNA concentrations (r =-0.97) as well as parasite number (r =-0.96), and the assay was able to detect as little as 0.00338 pg of T.evansi genomic DNA and 39 parasites per mL of blood. The real-time PCR assay presents several important advantages over other methods of detection and quantification of T.evansi DNA. First, the real-time PCR is performed in a closed tube with no post-PCR manipulations, thereby reducing potential amplicon carryovers and post-PCR processing time. Second, the assay is quick; results can be confirmed within 1 h, thirdly, the assay response is sensitive and linear over a broad range of DNA concentrations.

ACNOWLEDGEMENTS

This research project number THA/11417-CRP D3.20.21 was supported under research contract No.11417/R3 of the International Atomic Energy Agency (IAEA) in the budget year 2004.

TABLE I. T.EVANSI ISOLATES ORIGINATED FROM VARIOUS NATURALLY INFECTED HOSTS WERE USED IN THIS STUDY

Host Year Place Genotype(2,19)

Sow 1991 Suphanburi 4/5

Cattle 1991 Petchaburi 3/5

Cattle 1991 Lopburi 3/6

Hog deer 1997 Samuthprakan 3/5

Sow 1997 Nakhonratchasima 5/6

Cattle 1999 Pichit 4/5

Sow 2000 Prachuabkirikan 4/5

Horse 2003 Bangkok 3/6

Elephant 2004 Lampang 3/6

Cattle 2005 Bangkok 3/6

TABLE II. SEQUENCES OF PRIMERS USED IN THIS STUDY

Gene and primer Sequence

Universal 18S rRNA (10)

Sense primer 5’-CGGCTACCACATCTAAGG-3’

Antisense primer 5’-TATACGCTATTGGAGCTGG-3’

Repetitive T.evansi DNA

Sense primer 5’-GCGCGGATTCTTTGCAGACGA-3’

Antisense primer 5’-TGCAGACACTGGAATGTTACT-3’

FIG. 1. Amplification curve of real-time PCR.

Amplified 10-fold serial dilutions (undiluted to 1:10,000,000) of purified T.evansi genomic DNA (16.9 ng, 1.69 ng, 0.169 ng, 1.69 pg, 0.169 pg, 16.9 fg,1.69 fg), (Sample no. 1-7,8:distilled water as negative control and 2-fold serial dilutions (undiluted to 1: 2,048) of T.evansi infected (5,000 to 2.44 parasites per mL) mouse blood (Sample no. 9-20) was performed with 2 µM each T.evansi repetitive DNA primer using Platinum SYBR ^RR Green qPCR SuperMix UDG supplemented with BSA.

[A: 50 cycles, B: 70 cycles]

FIG. 2. Melting curve analysis of the amplicon obtained from real-time PCR.

Purified T.evansi genomic DNA and T.evansi infected mouse blood at 50 cycles (A) and 70 cycles (B) showed melting temperature (Tm) and fluorescence.

FIG. 3. Comparison of the amplicons using different methods.

The 257 bp stained with ethidium bromide (A, B) and SYBR Gold stain (C). Identical 10 microliter of the amplicons obtained from triplicated real-time PCR (A: 50 cycles, B, C: 70 cycles) were separated on 1.5% agarose minigels by electrophoresis (Mupid EX) at 100 volts for 30 min., and the image was photographed using gel documentation (BIO RAD).

FIG. 4. Specificity test of real-time PCR.

Performed to amplify different purified T.evansi DNA concentration (14.8 ng to 2.43 pg) and other blood protozoan DNA with 18S rRNA primers (A) and 1 µM repetitive T.evansi DNA primers (B).

FIG. 5. Melting curve analysis of the amplicon obtained from real-time PCR.

Purified T.evansi genomic DNA and DNA obtained from 10 isolates of T.evansi infected mouse blood showed identical melting temperature without fluorescence from mouse, cow DNA templates, distilled water (A). Identical band(257 bp) of the amplicon (10 ul) was detected on 1.5% agarose gel stained with ethidium bromide (B).[M:100 bp DNA ladder, Lane 1:purified T.evansi DNA template(1.69 ng), Lane 2:

purified T.evansi DNA template(0.169 ng), Lane 3:purified T.evansi DNA template(0.0169 ng), Lane 4:mouse DNA, Lane 5:Cow DNA, Lane 6:distilled water, Lane 7-16:T.evansi DNA templates from mouse infected bloods originated from various hosts according to Table I).

REFERENCES

[1] BECKER, S., FRANCO, J.R., SIMARRO, P.P., STICH, A., ABEL, P.M., STEVERDING, D. Real-time PCR for detection of Trypanosoma brucei in human blood samples. Diag. Microbiol. Infect. Dis., 50: 193–199 (2004).

[2] BITEAU, N., BRINGAUD, F., GIBSON, W., TRUC, P., BALTZ, T.

Characterization of Trypanozoon isolates using a repeated coding sequence and microsatellite markers. Mol. Biochem. Parasitol., 105: 185–201 (2000).

[3] BOONYAWONG, T., CHANTARAPRATEEP, P. AND MUANGYAI, M.

Surra in horse. Thai. J. Vet. Med., 5: 665–672 (1975).

[4] BORST, P., VAN DER PLOEG, M., VAN HOEK, J.F.M., TOS, J., JAMES, J.

On the DNA content and ploidy of Trypanosomes. Mol. Biochem. Parasitol., 6:

13 (1982).

[5] CHAICHANAPUNPOL, I., SIRIRANGKAMANONT, T., TRONGWONGSA, L., SUVANWASI, P. Observations on Trypanosomiasis in a native bull.

Kasetsart Vet., 6: 1–9 (1985).

[6] CHANSIRI, K., KHUCHAREONTAWON, S., RATAPHAN, N. PCR-ELISA for diagnosis of Trypanosoma evansi in animals and vector. Mol. Cell. Probes.

16: 173–177 (2002).

[7] DESQUESNES, M., GERALD, M.L., ZOUNGRANA, A. DAVILA, A.M.R.

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 (2001)

[8] IJAZ, M., K., NUR-E-KAMAL, M. S., MOHAMAD, A. I., DAR, F. K.

Comparative studies on the sensitivity of polymerase chain reaction and microscopic examination for the detection of Trypanosoma evansi in experimentally infected mice. Comp. Immun. Microb. Inf. Dis., 21: 215–223 (1998).

[9] INDRAKAMHANG, P., MOHKAEW, K., ROONGUTHAI, P. Preliminary report on Trypanosoma sp. in native Sambar deer and imported Rusa deer raised in a deer farm in Thailand. In: Proc. 16th Ann. Livestock Conf., Dept. Livestock Development, Thailand, 3–4 MAY, pp. 55–67 (1996).

[10] JAUREGUI, L.H., HIGGINS, D., ZARLENGA, D., DUBEY, J.P., LUNNEY, J.K. Development of a real-time PCR assay for detection of Toxoplasma gondii in pig and mouse tissues. J. Clin. Microb., 39: 2065–2071 (2001).

[11] LANHAM, S.M., GODFREY, D.G. Isolation of salivarian Trypanosome from man and other mammals using DEAE-cellulose. Exp. Parasitol., 28: 521–534 (1980).

[12] LOEHR, K.F., PHOLPARK, S., SRIKITJAKARN, L. BETTERMAN, G., STAAK, C. Trypanosoma evansi infection in buffaloes in north-east Thailand.

1. Field investigation. Trop. Anim. Hlth. Prod., 17: 121–125 (1985).

[13] LOEHR, K.F., PHOLPARK, S., UPATUM, N., LEIDL, K., STAAK, C.

Trypanosoma evansi infection, a frequent cause of abortion in buffaloes. Trop.

Anim. Health Prod., 18: 103–108 (1986).

[14] MATTHIAS, E., MUANGYAI, M. Trypanosoma evansi infection in a swamp buffaloes calf. Thai. J. Vet. Med., 10: 47–54 (1980).

[15] OMAWA, S., RAO, J.R., BASAGOUDANAVAR, S.H., BUTCHAIAH, G.

Direct and sensitive detection of Trypanosoma evansi by polymerase chain reaction. Acta Vet. Hungary, 47: 351–359 (1999).

[16] PARIS J, MURRAY M., MCODIMBA F. A comparative evaluation of the parasitological techniques currently available for the diagnosis of African Trypanosomiasis in cattle. Acta Trop., 39: 307–316 (1982).

[17] REID, S.A, HUSEIN A., COPEMAN D.B. Evaluation and improvement of parasitological tests for Trypanosoma evansi infection. Vet. Parasitol., 102: 291–

297 (2001).

[18] REID, S.A., HUSEIN, A. Variation in the susceptibility of 6 strains of mice to infection with Trypanosoma evansi. RCPMI-Obihiro/OIE-Paris International Symposium on Strategies for research and control of surra Trypanosoma evansi infection, The research Center for Protozoan Molecular Immunology, Obihiro University, Japan. 19–22 (1998).

[19] RODTIAN, P., HIN-ON, W., UTHAIWAN, W., VITOORAKOOL, P., TRISANAROM, A., CHAIYASERT, S., KLAIHONG, S., SARATAPHAN, N., MUANGYAI, M. A case report: Trypanosoma evansi Infection in Timber Elephant at Lampang Province. Proc. 19th Ann. Livestock Conf., Dept.

Livestock Development, Thailand, 26–30 (2004).

[20] SARATAPHAN, N., BOONCHIT, S., SIRIWAN, C., INDRAKAMHAENG, P.

Genetic diversity of Trypanosoma evansi in Thailand based on a repeated DNA coding sequence marker. Proc. 19th Ann. Livestock Conf., Dept. Livestock Development, Thailand, 26–30 (2004)

[21] SUKHUMSIRICHART, W., KHUCHAREONTAWON, S., SARATAPHAN, N., VISESHAKUL, N., CHANSIRI, K. Application of PCR-based assay for diagnosis of Trypanosoma evansi in different animals and vector. J. Trop. Med.

Parasitol., 23:1–6 (2000).

[22] TANANYTTHAWONGESE, C., SAENGSOMBAT, K., SUKHUMSIRICHAT, W., UTHAISANG, W., SARATAPHAN, N., CHANSIRI, K. Detection of bovine haemoparasite infection using multiplex polymerase chain reaction.

Science Asia, 25:85–90 (1999).

[23] TEERAPRASERT, E., CHAICHANAPUNPOOL, I., TRONGWONGSA, L. A case report of Trypanosoma evansi infection in pigs. Proceedings of the 11th Ann. Livestock Conf. Dept. Livestock Development, Thailand, 12–14 December 1984, pp. 43–64 (1984).

[24] TRISANAROM, A., MARKMEE, S., PEDUGSORN, C. Trypanosoma evansi infection in Chiengmai dairy cattle. In: Proc. 6th Ann. Livestock Conf., Dept.

Livestock Development, Thailand, 18–19, pp. 1–12 (1987).

[25] TUNTASUVAN, D., MIMAPAN, S., SARATAPHAN, N., TRONGWONGSA, L., INTRARAKSA, R., LUCKINS, A.G. Detection of Trypanosoma evansi in brains of the naturally infected hog deer by streptavidine-biotin immunohistochemistry. Vet. Parasitol., 87: 223–230 (2000).

[26] TUNTASUVAN, D., SARATAPHAN, N., NISHIKAWA, H. CNS Trypanosomiasis in native cattle. Vet. Parasitol., 73: 357–363 (1997).

[27] WOO, P.T.K. Evaluation of the haematocrit centrifuge and other technique for field diagnosis of human Trypanosomiasis and filariasis. Can. J. Zool., 47: 921–

923 (1970).

[28] WUYTS, N., CHOKESAJJAWATEE, N., PANYIM, S. A simplified and highly sensitive detection of Trypanosoma evansi by DNA amplification. Southeast Asian J. Trop. Med. Public Health, 25: 266–271 (1994).

[29] WUYTS, N., CHOKESAJJAWATEE, N., SARATAPHAN, N., PANYIM, S.

PCR amplification of crude blood on microscope slides in the diagnosis of Trypanosoma evansi infection in dairy cattle. Annual Society of Belgium Medical Tropics, 75: 229–237 (1995).

APPLICATION OF DRIED BLOOD SAMPLE ON FTA® PAPER FOR