Evaluation of a Real-Time PCR Test for the Detection and Discrimination of Theileria Species in the African Buffalo (Syncerus caffer)

Abstract

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected.Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

“…While real-time assays that detect T. orientalis have been developed, most have focused on species-level detection (not discrimination of genotypes) (34), discrimination of different Theileria spp. (43,44), or are only semiquantitative (28). The UIC multiplex qPCR assay developed here was both sensitive and specific for T. orientalis detection compared to cPCR and reliably identified the clinically relevant Ikeda and Chitose genotypes.…”

The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes

“…(buffalo) ( Allsopp et al., 1993 ). Subsequent studies have found this Theileria genotype to be well conserved and widespread in buffalo populations in both Uganda and South Africa, frequently in co-infections with T. parva ( Chaisi et al., 2013; Mans et al., 2011a; Mans et al., 2015; Oura et al., 2011a,b; Pienaar et al., 2011b; Pienaar et al. 2014 ).…”

Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

“…Serological tests have been developed to detect circulatory antibodies against the parasites, but these generally have poor sensitivity and specificity due to involving cross-reactions or nonspecific immune responses (Passos et al , 1998), and only detect previous exposure as opposed to current infection. Conventional PCR methods are useful in the detection of particular haemoprotozoan species for which the reagents and conditions have been developed, but can have limitations in the detection of other species and lack scalability (Bilgic et al , 2013; Chaisi et al , 2013; Gubbels et al , 1999; Kundave et al , 2018). In contrast, the deep amplicon sequencing method is potentially providing more reliable and accurate in the automated high throughput analysis of all haemoprotozoan species.…”

“…Determination of sequence variations in the hyper-variable 18S rDNA cistron can discriminate between haemoprotozoan parasites (Gubbels et al , 1999) and overcome limitations of traditional gross parasitological methods for the diagnosis of haemoprotozoa at species level (Agudelo et al , 2013; Haanshuus et al , 2013; Lee et al , 2015; Lefterova et al , 2015; Mens et al , 2006; Rougemont et al , 2004; Steenkeste et al , 2009). Various PCR methods (reverse line blot (RLB)-PCR, quantitative (q)PCR, multiple PCR) have been described to amplify the 18S region for sequence determination, but these are low throughput, hence relatively expensive, and can be error-prone (Bilgic et al , 2013; Chaisi et al , 2013; Gubbels et al , 1999; Kundave et al , 2018). These methods depend on the use of species-specific primers and probes, hence can only identify the tested species.…”