Original Article

Development and Evaluation of a Loop-mediated Isothermal Amplification Assay for Rapid Detection of Theileria annulata Targeting the Cytochrome B Gene


Background: Theileria annulata is an economically important cattle disease in North Africa that occurs in subtropical and tropical areas. Accurate and rapid, molecular diagnosis of tropical theileriosis is an important issue that allows early treatment and, prevents transmission. We developed and validated a Theileria annulata specific LAMP assay targeting the cytochrome b multicopy gene, in order to increase the DNA detection sensitivity.

Methods: The methodology was used to evaluate the occurrences of T. annulata in 88 field samples collected in Northern Tunisia during 2013-2014. The specificity and sensitivity of the LAMP assays were compared to conventional cytochrome b PCR and routine microscopy commonly used on naturally infected cattle blood samples.

Results: The PCR assay showed a sensitivity of 70% and specificity around 75%. Our LAMP assay showed a suitable sensitivity 78.7% and specificity 87.5%, with, however, positive (98.4%) and negative (29.1%) predictive values.

Conclusion: The LAMP assay is a simple and convenient diagnostic tool for tropical theileriosis. Moreover, LAMP does not require experienced staff and specialized equipment for sampling procedures and it is practical outside laboratories and can be used for field diagnosis.

Robinson PM. Theileriosis annulata and its transmission-a review. Trop Anim Health Prod. 1982; 14(1):3-12.

Darghouth MA, Bouattour A, Kilan M. Tropical theileriosis in Tunisia: epidemiology and control. Parassitologia. 1999; 41 Suppl 1:33-6.

Gharbi M, Sassi L, Dorchies P, Darghouth MA. Infection of calves with Theileria annulata in Tunisia: Economic analysis and evaluation of the potential benefit of vaccination. Vet Parasitol. 2006; 137(3-4):231-41.

Jeong W, Kweon CH, Kim JM, Jang H, Paik SG. Serological investigation of Theileria sergenti using latex agglutination test in South Korea. J Parasitol. 2005; 91(1):164-9.

Burridge MJ, Kimber CD, Young AS. Use of the indirect fluorescent antibody technique in serologic studies of Theileria lawrencei infections in cattle. Am J Vet Res. 1973;34(7):897-900.

Kachani M, Spooner RL, Rae P, Bell-Sakyi L, Brown CG. Stage-specific responses following infection with Theileria annulata as evaluated using ELISA. Parasitol Res. 1992; 78(1):43-7.

Renneker S, Kullmann B, Gerber S, Dobschanski J, Bakheit MA, Geysen D et al. Development of a competitive ELISA for detection of Theileria annulata infection. Transbound Emerg Dis. 2008; 55(5-6):249-56.

Gubbels JM, de Vos AP, van der Weide M, Viseras J, Schouls LM, de Vries E, Jongejan F. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J Clin Microbiol. 1999; 37(6):1782-9.

Schnittger L, Yin H, Qi B, Gubbels MJ, Beyer D, Niemann S, Jongejan F, Ahmed JS. Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting. Parasitol Res. 2004; 92(3):189-96.

d'Oliveira C, van der Weide M, Habela MA, Jacquiet P, Jongejan F. Detection of Theileria annulata in blood samples of carrier cattle by PCR. J Clin Microbiol. 1995; 33(10):2665-9.

Shayan P, Biermann R, Schein E, Gerdes J, Ahmed JS. Detection and differentiation of Theileria annulata and Theileria parva using macroschizont-derived DNA probes. Ann N Y Acad Sci. 1998; 849:88-95.

Kirvar E, Ilhan T, Katzer F et al. Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences. Parasitology. 2000; 120 ( Pt 3):245-54.

Habibi G. Phylogenetic Analysis of Theileria annulata Infected Cell Line S15 Iran Vaccine Strain. Iran J Parasitol. 2012; 7(2):73-81.

Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000; 28(12):E63.

Salih DA, Liu Z, Bakheit MA, Ali AM, El Hussein AM, Unger H, Viljoen G, Seitzer U, Ahmed JS. Development and evaluation of a loop-mediated isothermal amplification method for diagnosis of tropical theileriosis. Transbound Emerg Dis. 2008; 55(5-6):238-43.

Liu Z, Hou J, Bakheit MA, Salih DA, Luo J, Yin H, Ahmed JS, Seitzer U. Development of loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of ovine theileriosis in China. Parasitol Res. 2008; 103(6):1407-12.

Thekisoe OM, Rambritch NE, Nakao R, Bazie RS, Mbati P, Namangala B, Malele I, Skilton RA et al. Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes. Int J Parasitol. 2010; 40(1):55-61.

Wang LX, He L, Fang R, Song QQ, Tu P, Jenkins A, Zhou YQ, Zhao JL. Loop-mediated isothermal amplification (LAMP) assay for detection of Theileria sergenti infection targeting the p33 gene. Vet Parasitol. 2010; 171(1-2):159-62.

Liu A, Guan G, Du P, Liu Z, Gou H, Liu J et al. Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences. Exp Parasitol. 2012; 131(1):125-9.

Liu A, Guan G, Du P, Gou H, Zhang J, Liu Z, Ma M et al. Rapid identification and differentiation of Theileria sergenti and Theileria sinensis using a loop-mediated isothermal amplification (LAMP) assay. Vet Parasitol. 2013; 191(1-2):15-22.

Salem GH, Liu X, Johnsrude JD, Dame JB, Roman Reddy G. Development and evaluation of an extra chromosomal DNA-based PCR test for diagnosing bovine babesiosis. Mol Cell Probes. 1999; 13(2):107-13.

Moretti A, Mangili V, Salvatori R, Maresca C, Scoccia E, Torina A, Moretta I et al. Prevalence and diagnosis of Babesia and Theileria infections in horses in Italy: a preliminary study. Vet J. 2010; 184(3):346-50.

Rodríguez-Cortés A, Ojeda A, Francino O, López-Fuertes L, Timón M, Alberola J. Leishmania infection: laboratory diagnosing in the absence of a "gold standard". Am J Trop Med Hyg. 2010; 82(2):251-6.

Shahnawaz S, Ali M, Aslam MA, Fatima R, Chaudhry ZI, Hassan MU, Ali M, Iqbal F. A study on the prevalence of a tick-transmitted pathogen, Theileria annulata, and hematological profile of cattle from Southern Punjab (Pakistan). Parasitol Res. 2011; 109(4):1155-60.

Bilgic HB, Karagenç T, Shiels B, Tait A, Eren H, Weir W. Evaluation of cytochrome b as a sensitive target for PCR based detec-tion of T. annulata carrier animals'. Vet Par-asitol. 2010; 174(3-4):341-7.

26- Voet JG, Voet D. BAMBED Changes Publisher: Farewell, ASBMB; welcome, John Wiley. Biochem Mol Biol Educ.2006; 34(6): 401.

Kuru T, Janusz N, Gadisa E, Gedamu L, Aseffa A. Leishmania aethiopica: development of specific and sensitive PCR diagnostic test. Exp Parasitol. 2001; 128(4):391-5.

Weir W, Karagenc T, Gharbi M, Simuunza M, Aypak, S, Aysul, N, Darghouth, MA, et al. Population diversity and multiplicity of infection in Theileria annulata. Int J Parasitol. 2001; 41(2): 193-203

Ndao M. Diagnosis of parasitic diseases: old and new approaches. Interdiscip Perspect Infect Dis. 2009;2009:278246.

Amirabadi AR, Shahhosseiny MH, Yousefi JV, Ghahri M. LOOP mediated isothermal AMPlification (LAMP) in diagnosis of ne-orocryptococcosis.Afr J Biotechnol. 2012; 11: 3986-3992.

Chaouch M, Fathallah-Mili A, Driss M, Lahmadi R, Ayari C, Guizani I, Benabder-razak S. Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis. Acta Trop. 2013;125(3):357-65.

Reithinger R, Espinoza JC, Courtenay O, Davies CR. Evaluation of PCR as a diagnostic mass-screening tool to detect Leishmania (Viannia) spp. in domestic dogs (Canis familiaris). J Clin Microbiol. 2003; 41(4):1486-93.

Al-Soud WA, Rådström P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol. 2001; 39(2):485-93.

Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, Sugimoto C, Igarashi I. Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol. 2003; 41(12):5517-24.

Adams ER, Schoone GJ, Ageed AF, Safi SE, Schallig HD. Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples. Am J Trop Med Hyg. 2010; 82(4):591-6.

IssueVol 13 No 2 (2018) QRcode
SectionOriginal Article(s)
Theileria annulata LAMP Cytochrome b Tunisia Diagnosis

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How to Cite
CHAOUCH M, MHADHBI M, LIMAM S, DARGHOUTH MA, BENABDERRAZAK S. Development and Evaluation of a Loop-mediated Isothermal Amplification Assay for Rapid Detection of Theileria annulata Targeting the Cytochrome B Gene. Iran J Parasitol. 2018;13(2):225-234.