Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA

Abstract

A pair of synthetic oligonucleotide primers, designed from the gene encoding a 32-kDa intraerythrocytic piroplasm surface protein of Theileria sergenti, were used to amplify parasite DNA from the blood of T. sergenti-infected cattle by means of the polymerase chain reaction (PCR). PCR-amplified DNA was examined by electrophoresis and by dot blot or microplate hybridization using a parasite-specific cDNA probe. PCR was specific for T. sergenti, since no amplification was detected with DNA from Anaplasma centrale, Babesia ovata, uninfected erythrocytes, and leukocytes. This method was sensitive enough to detect about 4.5 parasites per I.al of blood with a 10-p.l sample volume. Moreover, of 66 specimens from grazing cattle, 40 were microscopically positive, whereas PCR revealed that 54 samples were positive. Therefore, PCR provides a useful diagnostic tool for detecting T. sergenti-infected cattle, and it is significantly more sensitive than the current methods.

Detection of Theileria annulata in blood samples of carrier cattle by PCR

“…With the availability of sequenced parasite genes and PCR, it is possible to detect parasites within samples of blood (4,8,17,24). In this study, we developed a PCR assay for improved detection of T. annulata in blood samples from carrier cattle.…”

“…3A). The advantage of microplate hybridization is that the PCR-amplified product can be identified in an enzymelinked immunosorbent assay format (24). However, our PCR samples required precipitation before hybridization to obtain comparable results.…”

“…Microplate hybridization. Hybridization on microtiter plates was performed as previously described (24). After heat denaturation at 95ЊC for 5 min, 10 l of PCR-amplified product was ethanol precipitated and subsequently diluted with 90 l of a mixture of 10 mM sodium phosphate, 10 mM EDTA, and 1.5 M NaCl (pH 7.0) in Maxisorp microplate wells (Nunc, Roskilde, Denmark).…”

“…The A 490 was determined with a microplate colorimetric reader (Bio-Rad, Richmond, Calif.). Samples with an optical density greater than 0.1 were considered positive (24).…”

“…Therefore, animals with a negative serological test can still infect ticks. The advent of PCR (18) has allowed the development of sensitive diagnostic assays for Theileria parva (4) and Theileria sergenti (24).…”