Cross-Genera PCR Amplification of DNA from Apicomplexan Parasites
AbstractBackground: The discovery of an unexpected genetic sequence raised doubts about the specificity of a primer pair targeting Babesia spp. and Theileria spp. This study aimed to check the specificity of this primer pair.Methods: Conventional end-point PCR and real-time PCR protocols using primers targeting the 18S rRNA gene V4 hypervariable region of Babesia spp. and Theileria spp. were tested for potential cross-genera amplification using DNA from a palette of parasitic protists and pathogenic bacteria as a template. These investigations took place at the Ludwig Maximilian University of Munich (Germany) in 2010 as part of the EDEN project.Results: Successful amplification was obtained with DNA from five apicomplexan genera: Babesia, Theileria, Hepatozoon, Toxoplasma, and Hammondia. No amplicons were obtained when DNA from Leishmania infantum or bacteria within the genera Borrelia, Leptospira or Anaplasma was used as a template.Conclusion: This cross-genera amplification ability is useful for the quick exclusion of many parasite species from PCR negative diagnostic samples. Accurate species identification from PCR positive samples requires genetic sequencing of the amplicon.
Aktas M, Altay K, Dumanli N (2005) De-velopment of a polymerase chain re-ac¬tion method for diagnosis of Babe-sia ovis infection in sheep and goats. Vet Parasitol. 133: 277–281.
Bourdoiseau G (2006) Canine babesiosis in France. Vet Par. 138: 118–125.
Chaudhry ZI, Suleman M, Younus M, Aslim A (2010) Molecular Detection of Babe-sia bigemina and Babesia bovis in Cross¬bred Carrier Cattle Through PCR. Pa¬kistan J Zool. 42(2): 201–204.
Esmaeilnejad B, Tavassoli M, Asri-Rezaei S, Dalir-Naghadeh B, Mardani K, Go¬labi M, Arjmand J, Kazemnia A, Jalil¬zadeh G (2015) Determination of prev¬alence and risk factors of infection with Babesia ovis in small ruminants from West Azerbaijan Province, Iran by pol-ymerase chain reaction. J Arthropod Borne Dis. 9(2): 246–252.
García ATC (2006) Piroplasma infection in dogs in northern Spain. Vet Par. 138: 97–102.
Gorenflot A, Moubri K, Precigout E, Car-cy B, Schetters TP (1998) Human babesi¬osis. Ann Trop Med Parasitol. 92: 489–501.
Njiiri NE, Bronsvoort BMDC, Collins NE, Steyn HC, Troskie M, Vorster I, Thumbi SM, Sibeko KP, Jennings A, van Wyk IC, Mbole-Kariuki M, Kiara H, Poole EJ, Hanotte O, Coetzer K, Oosthuizen MC, Woolhouse M, Toye P (2015) The epidemiology of tick-borne haemo-par¬asites as determined by the reverse line blot hybridization assay in an intensive¬ly studied cohort of calves in western Kenya. Vet Par¬asitol. 210(1–2): 69–76.
Singh H, Haque JM, Singh NK, Rath SS (2013) PCR based detection of sub-clinical bovine babesiosis in Punjab. Indian J Anim Res. 47(6): 543–546.
Vannier E, Krause PJ (2012) Human Babesi-osis. N Engl J Med. 366: 2397–2407.
Zhou X, Xia S, Huang JL, Tambo E, Zhuge HX, Zhou XN (2014) Human babesio¬sis, an emerging tick-borne dis¬ease in the People’s Republic of Chi¬na. Para¬sit Vectors. 7: 509.
de Mendonça PG (2008) The EDEN pro¬ject. Parasitol Res. 103(Suppl.1): 158–159.
Gubbels JM, de Vos AP, Van Der Weide M, Viseras J, Schouls LM, de Vries E, Jongejan F (1999) Simultaneous de¬tection of bovine Theileria and Babe¬sia species by reverse line blot hy¬bridization. J Clin Microbiol. 37(6): 1782–1789.
Georges K, Loria GR, Riili S, Greco A, Caracappa S, Jongejan F, Sparagano O (2001) Detection of Haemopara-sites in cattle by reverse line blot hybrid¬i¬sa¬tion with a note on the distribution of ticks in Sicily. Vet Parasitol. 99(4): 273–286.
Tonetti N, Berggoetz M, Rühle C, Preto¬rius AM, Gern L (2009) Ticks and tick-borne pathogens from wildlife in the Free State Province, South Africa. J Wildl Dis. 45(2): 437–446.
Cox FEG (2002) Systematics of the par¬a¬sitic Protozoa. Trends Parasitol. 18 (3): 108.
Cox FEG (2011) Taxonomy and Classifi¬ca¬tion of Human Parasitic Protozoa and Helminths. In: Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (Eds) Manual of Clinical Microbiology. 10th edition. ASM Press, Washington DC, pp. 2041–2046.
Allsopp MTEP, Allsopp BA (2006) Mo-lec¬ular sequence evidence for the re-classification of some Babesia spe-cies. Ann NY Acad Sci. 1081: 509–517.
Uilenberg G (2006) Babesia - A histori-cal overview. Vet Parasitol. 138: 3–10.