Discrimination between six species of Theileria using oligonucleotide probes which detect small subunit ribosomal RNA sequences

Abstract

SUMMARYThe complete small subunit ribosomal RNA (srRNA) gene of Theileria parva was cloned and sequenced. Two primers were designed which permitted the specific amplification of part of the Theileria srRNA gene from Theileria-infected cell line samples which were predominantly (> 95%) bovine DNA. The sequence of the central (variable) region of the srRNA genes of T. annulata, T. taurotragi, T. mutans and two unidentified parasites referred to as Theileria sp. (buffalo) and Theileria sp. (Marula) were obtained. An alignment of the sequences was generated from which 6 oligonucleotide probes, corresponding to species-specific regions, were designed. These probes were demonstrated to provide unequivocal identification of each of the 6 species either by direct detection of parasite srRNA or by hybridization to amplified parasite srRNA genes. The probes were not able to distinguish buffalo-derived T. parva, the causal agent of Corridor disease, from cattle-derived T. parva, the causal agent of East Coast fever.

MixedTheileriainfections in free-ranging buffalo herds: implications for diagnosingTheileria parvainfections in Cape buffalo (Syncerus caffer)

“…This might explain the observation that free-ranging buffalo tested previously were 100% positive on IFAT for T. parva (Allsopp et al 1993). In addition, its close resemblance to T. parva in the 18S rRNA raised the question whether this was a variant strain of T. parva (Conrad et al 1987;Allsopp et al 1993). Subsequently, this parasite was detected in buffalo from the KNP that showed that T. sp.…”

“…For this a 1101 bp fragment of the 18S rRNA gene for T. parva and T. sp. (buffalo) were amplified, respectively, using Theileria genus-specific primers 989 and 990 (Allsopp et al 1993), from cloned and sequenced products for T. parva and T. sp. (buffalo).…”

Diversity in the 18S SSU rRNA V4 hyper-variable region ofTheileriaspp. in Cape buffalo (Syncerus caffer) and cattle from southern Africa

“…A 1101 bp fragment of the 18S rRNA gene was amplified using Theileria genus-specific primers designated as 989 and 990 by Allsopp et al (1993). Reaction conditions included the use of 25 μl GoTaq ® Green Ready reaction mix (Promega), 2·5 μl of DNA template and 10pmol of each primer up to a total volume of 50 μl.…”

“…(buffalo) and T. sp. (bougasvlei) known to occur in buffalo and thought to be apathogenic (Allsopp et al 1993;Zweygarth et al 2008). Of these, T. parva was found in buffalo and cattle, while T. taurotragi was limited to cattle.…”

“…Sequencing of the 18S ribosomal RNA (rRNA) gene showed, however, that T. sp. (buffalo) was a distinct entity from T. parva (Allsopp et al 1993). Subsequently, the systematics of the Piroplasmida has been extensively studied by analysis of the 18S rRNA gene (Allsopp et al 1994;Chae et al 1999;Chansiri et al 1999;Criado-Fornelio et al 2004;Criado et al 2006;Reichard et al 2005;Altay et al 2007;Bhoora et al 2009).…”