Development and Evaluation of a Real-time PCR Assay for the Quantitative Detection of Theileria Annulata in Cattle

Overview

Journal Parasit Vectors

Publisher Biomed Central

Specialties Parasitology
Tropical Medicine

Date 2012 Aug 15

PMID 22889141

Citations 23

Authors

Amaia Ros-Garcia

Antoni Nicolas

Ana L Garcia-Perez

Ramon A Juste

Ana Hurtado

Affiliations

Soon will be listed here.

Abstract

Background: The tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered.

Results: We developed a real-time quantitative PCR (qPCR) assay for the detection and quantification of T. annulata that included an internal amplification control (IAC) to monitor for the presence of potential inhibitors. Specificity, sensitivity, precision, linear range and PCR efficiency were calculated and different methods for transformation of quantification cycle (Cq) values into quantities (Q) were evaluated. The assay was able to detect (100% probability) and quantify (linear response) 100 gene copies, and clinical sensitivity was set at 10 T. annulata per μl of blood. The assay was then validated on 141 bovine blood samples analyzed in parallel by a Luminex® suspension array, showing the utility of the qPCR assay developed here for the detection and quantification of the parasite in field conditions. Once validated it was used to monitor T. annulata parasitaemia throughout a year in 8 carrier animals from a farm in Minorca.

Conclusions: The developed qPCR assay offers a reliable and simple way to quantify T. annulata infection loads, which could prove crucial in studying the role of carrier animals as a source of the infection, or assessing the efficacy of treatment and control measures.

Citing Articles

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Multiplex ddPCR: A Promising Diagnostic Assay for Early Detection and Drug Monitoring in Bovine Theileriosis.

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Current and Future Molecular Diagnostics of Tick-Borne Diseases in Cattle.

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Epidemiology and molecular characterization of Theileria annulata in cattle from central Khyber Pakhtunkhwa, Pakistan.

Ullah R, Shams S, Khan M, Ayaz S, Akbar N, Din Q PLoS One. 2021; 16(9):e0249417.

PMID: 34529664 PMC: 8445462. DOI: 10.1371/journal.pone.0249417.

A Multi-copy Nucleic Acid-Based Diagnostic Test for Bovine Tropical Theileriosis.

Mohmad A, Saravanan B, Manjunathachar H, Chandra D, Ahmad S, Malla W Acta Parasitol. 2021; 67(1):504-510.

PMID: 34146240 DOI: 10.1007/s11686-021-00428-x.

References

Sivaganesan M, Seifring S, Varma M, Haugland R, Shanks O . A Bayesian method for calculating real-time quantitative PCR calibration curves using absolute plasmid DNA standards. BMC Bioinformatics. 2008; 9:120. PMC: 2292693. DOI: 10.1186/1471-2105-9-120. View

Georges K, Loria G, Riili S, Greco A, Caracappa S, Jongejan F . Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribution of ticks in Sicily. Vet Parasitol. 2001; 99(4):273-86. DOI: 10.1016/s0304-4017(01)00488-5. View

Habela M, Rol J, Anton J, Pena J, Corchero E, van Ham I . Epidemiology of Mediterranean theileriosis in Extremadura region, Spain. Parassitologia. 2000; 41 Suppl 1:47-51. View

Almeria S, Castella J, Ferrer D, Gutierrez J, Estrada-Pena A, Sparagano O . Reverse line blot hybridization used to identify hemoprotozoa in Minorcan cattle. Ann N Y Acad Sci. 2002; 969:78-82. DOI: 10.1111/j.1749-6632.2002.tb04354.x. View

Almeria S, Castella J, Ferrer D, Ortuno A, Estrada-Pena A, Gutierrez J . Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection. Vet Parasitol. 2001; 99(3):249-59. DOI: 10.1016/s0304-4017(01)00464-2. View

Bilgic H, Karagenc T, Shiels B, Tait A, Eren H, Weir W . Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals. Vet Parasitol. 2010; 174(3-4):341-7. DOI: 10.1016/j.vetpar.2010.08.025. View

Sivaganesan M, Haugland R, Chern E, Shanks O . Improved strategies and optimization of calibration models for real-time PCR absolute quantification. Water Res. 2010; 44(16):4726-35. DOI: 10.1016/j.watres.2010.07.066. View

Lund M, Nordentoft S, Pedersen K, Madsen M . Detection of Campylobacter spp. in chicken fecal samples by real-time PCR. J Clin Microbiol. 2004; 42(11):5125-32. PMC: 525216. DOI: 10.1128/JCM.42.11.5125-5132.2004. View

Gubbels J, De Vos A, van der Weide M, Viseras J, Schouls L, de Vries E . Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J Clin Microbiol. 1999; 37(6):1782-9. PMC: 84950. DOI: 10.1128/JCM.37.6.1782-1789.1999. View

10.

dOliveira C, van der Weide M, Habela M, Jacquiet P, Jongejan F . Detection of Theileria annulata in blood samples of carrier cattle by PCR. J Clin Microbiol. 1995; 33(10):2665-9. PMC: 228552. DOI: 10.1128/jcm.33.10.2665-2669.1995. View

11.

Sibeko K, Oosthuizen M, Collins N, Geysen D, Rambritch N, Latif A . Development and evaluation of a real-time polymerase chain reaction test for the detection of Theileria parva infections in Cape buffalo (Syncerus caffer) and cattle. Vet Parasitol. 2008; 155(1-2):37-48. DOI: 10.1016/j.vetpar.2008.03.033. View

12.

Kim C, Iseki H, Herbas M, Yokoyama N, Suzuki H, Xuan X . Development of TaqMan-based real-time PCR assays for diagnostic detection of Babesia bovis and Babesia bigemina. Am J Trop Med Hyg. 2007; 77(5):837-41. View

13.

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

14.

Nagore D, Garcia-Sanmartin J, Garcia-Perez A, Juste R, Hurtado A . Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain. Int J Parasitol. 2004; 34(9):1059-67. DOI: 10.1016/j.ijpara.2004.05.008. View

15.

Almeria S, Delgado-Neira Y, Adelantado C, Huguet M, Vinent J, Nicolas A . Mediterranean theileriosis and other tick transmitted piroplasmoses in cattle in Minorca (Balearic Islands, Spain): the effect of tick control on prevalence levels analyzed by reverse line blot (RLB) macroarrays. J Parasitol. 2008; 95(3):598-603. DOI: 10.1645/GE-1687.1. View

16.

Castella J, Estrada-Pena A, Almeria S, Ferrer D, Gutierrez J, Ortuno A . A survey of ticks (Acari: Ixodidae) on dairy cattle on the island of Menorca in Spain. Exp Appl Acarol. 2002; 25(10-11):899-908. DOI: 10.1023/a:1020482017140. View

17.

Gibello A, Blanco M, Moreno M, Cutuli M, Domenech A, Dominguez L . Development of a PCR assay for detection of Yersinia ruckeri in tissues of inoculated and naturally infected trout. Appl Environ Microbiol. 1999; 65(1):346-50. PMC: 91030. DOI: 10.1128/AEM.65.1.346-350.1999. View

18.

Bhoora R, Quan M, Franssen L, M Butler C, van der Kolk J, Guthrie A . Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa. Vet Parasitol. 2009; 168(3-4):201-11. DOI: 10.1016/j.vetpar.2009.11.011. View

19.

Hoorfar J, Malorny B, Abdulmawjood A, Cook N, Wagner M, Fach P . Practical considerations in design of internal amplification controls for diagnostic PCR assays. J Clin Microbiol. 2004; 42(5):1863-8. PMC: 404670. DOI: 10.1128/JCM.42.5.1863-1868.2004. View

20.

Ros-Garcia A, Juste R, Hurtado A . A highly sensitive DNA bead-based suspension array for the detection and species identification of bovine piroplasms. Int J Parasitol. 2012; 42(2):207-14. DOI: 10.1016/j.ijpara.2011.12.001. View