[Preliminary application of PCR-based assay for the detection of Neospora caninum in bovine aborted fetus].
OBJECTIVE
To establish a PCR diagnostic method based on Nc-5 gene of Neospora caninum, for being used to detect Neospora in brain tissues of bovine aborted fetus.


METHODS
Specific primers were designed and synthesized based on the reported Nc-5 gene of N. caninum (GenBank Accession No. AY459289). Using genomic DNA from N.caninum as templates, Nc-5 gene was amplified by PCR. The PCR product was cloned into pMD18-T vector, transformed into Escherichia coli JM109 and then sequenced. To evaluate the specificity of the PCR, genomic DNA of Theileria annulata, Babesia bovis, Toxoplasma gondii, Leishmania donovani and standard strain of N. caninum were used as a template in the PCR. For determining the detection limit of amplification procedure, PCR was run on a dilution series of genomic DNA from N. caninum (1.562 5-200 ng/ml). Brain tissue samples of 32 aborted fetuses were detected by PCR-based assay, and 23 blood samples from mothers were tested by ELISA.


RESULTS
The amplified DNA fragment (350 bp) had a high identity of 98% with the Nc-5 gene sequence of N. caninum (GenBank Accession No. AY459289). The PCR was specific for N. caninum and allowed the detection of 3.125 pg DNA of the parasite, while no amplification occurred with the other four species of protozoa. PCR-based assay and ELISA showed a positive rate of 18.8% (6/32) and 17.4% (4/23) of the samples tested, respectively. Moreover, all the 4 antibody positive samples showed PCR positive. There is no significant difference between the two assays (P > 0.05).


CONCLUSIONS
PCR diagnostic method is promising in detecting Neospora infection in brain tissues of aborted bovine.
Wang
Chun-Ren
CR
College of Animal Science and Technology, Heilongjiang August-First Land Reclamation University, Daqing 163319, China. chunrenwang@yahoo.com.cn
Zhai
Yan-Qing
YQ
Zhao
Xing-Chun
XC
Tan
Qiu-Ju
QJ
Chen
Jia
J
Chen
Ai-Hua
AH
Wang
Yu
Y
D037881:Aborted Fetus / Q000469:parasitology; D000034:Abortion, Veterinary / Q000469:parasitology*; D000818:Animals; D001921:Brain / Q000469:parasitology; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000469:parasitology*; D016054:DNA, Protozoan / Q000235:genetics; D005260:Female; D008969:Molecular Sequence Data; D018688:Neospora / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy
D016054:DNA, Protozoan