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Research Article
ScienceAsia 25 (1999) : 085-090 |doi: 10.2306/scienceasia1513-1874.1999.25.085
Detection of Bovine Hemoparasite Infection Using Multiplex Polymerase Chain Reaction
Chantra Tananyutthawongesea, Klaokwan Saengsombutb, Wasana Sukhumsirichata, Wanlaya Uthaisanga, Nopporn Sarataphanc and Kosum Chansiria*
ABSTRACT: A rapid and specific multiplex polymerase chain reaction has been developed for the diagnosis
of hemoparasitic infection of bovine blood by five of the most common protozoan hemoparasites. This
method relied on the detection of the presence of genomic DNA from the five different bovine
hemoparasites isolated from red blood cells. Infection was detected by a single multiplex PCR reaction
containing five pairs of oligonucleotide primers whereby each primer pair was specific for each parasite
species. These primer sets amplify 160, 257, 446, 689 and 1,100 bp fragments for Anaplasma marginale,
Trypanosoma evansi, Babesia bovis, Babesia bigemina and Theileria sp, respectively. The PCR products
derived from each parasite species were visualized in ethidium bromide-stained agarose gels, thus allowing
the rapid identification of any, or all, of the five bovine parasites, if present, in a single amplification
reaction. This multiplex PCR was sensitive with the ability to detect the presence of as little as 10-1 pg
of parasite DNA. The primers used in this multiplex PCR also showed highly specific amplification of
each respective parasite DNA without the presence of non-specific and non-target PCR products. This
multiplex PCR system was used to analyze 35 bovine blood samples for the presence of parasitic infection
and the results were compared with detection of hemoparasites by light microscopic examination after
Giemsa staining of thin film blood smears. A comparison of the two detection methods revealed that
86% of specimens showed concordant diagnoses with both techniques. However, 14% of the samples
examined showed single or multiple infection by multiplex PCR analysis, but such infection was not
detected by microscopy. The simplicity and rapidity of this specific multiplex PCR method make it
suitable for large-scale epidemiological studies and for follow-up of drug treatments.
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a Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand.
b Department of Biochemistry, Faculty of Science, Burapa University, Chonburi, Thailand.
c Parasitology Section, National Institute of Animal Health, Department of Livestock Development, Kaset Klang, Bangkhen, Bangkok 10900, Thailand.
* Corresponding author: Tel: 66-2-260 2950 ext 4605 Fax: 66-2-260 0125 E-mail: kosum@psm.swu.ac.th
Received 3 Mar 1999
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