Neospora caninum: quantification of DNA in the blood of naturally infected aborted and pregnant cows using real-time PCR.

This study quantified Neospora caninum DNA in the blood and brain of pregnant and aborted heifers by monitoring PCR product formation in real-time using SYBR Green I, a double-stranded DNA-binding dye. Primers were designed to amplify a 188 bp product specific to N. caninum from the Nc-5 gene fragment of N. caninum. Similarly, a 71 bp product was amplified from the 28S rRNA gene of bovine genomic DNA that served as a control. Agarose gel electrophoresis and analysis of the melting curve for PCR products showed that both primer pairs were specific to their targets. Standard curves were generated for both bovine and N. caninum genomic DNA, and were used to compute the relative concentration of parasite to bovine DNA in the test samples. The concentration of N. caninum DNA in 1 ng of bovine genomic DNA obtained from blood ranged between 0.097 ng at the 1st week of the observation and 0 ng at the 15th week in aborted cows. In pregnant cows, the values ranged between 0.080 ng at the 1st week and 0.155 ng at the 15th week of observation. There was a sustained decrease of DNA concentration in the aborted group after abortion and an increase in DNA concentration in the pregnant group. Comparison of parasite DNA in blood and brain of infected heifers showed a higher DNA concentration in brain than in blood. This study shows that N. caninum DNA can be quantified to obtain the relative concentration of parasite DNA to host genomic DNA in blood. This technique allows testing of a large number of samples at one time and can be done without the need for slaughter of tested animals.

Recognition patterns of Neospora caninum tachyzoite antigens by bovine IgG at different IFAT titres.

This study describes qualitative and quantitative antibody response in cows naturally infected with Neospora caninum. The study was carried out with 269 serum samples obtained from 24 cows over a period of 15 weeks. Prior to sample collection, the cows were tested with ELISA. The 269 samples were screened with IFAT and categorized into seven IFAT titre groups (< 1 : 80, 1 : 80, 1 : 200, 1 : 600, 1 : 1000, 1 : 2000, > 1 : 2000). The samples were finally analysed by Western blotting. Seven immunodominant antigens (approximately 18-, approximately 25-, approximately 33-, approximately 35-36-, approximately 45-46-, approximately 47-, approximately 60-62 kDa) and five minor antigens (approximately 25, approximately 51, approximately 64, approximately 77, approximately 116 kDa) were recognized by cow sera. The recognition of approximately 46 kDa antigen by cow sera was common to samples with IFAT titre 1 : 80 and above. Another common antigen was the approximately 18 kDa antigen, which was recognized by samples with IFAT titre 1 : 200 and above. The most remarkable observation was the presence of the 45-46 kDa, the 77 kDa, and absence of the 18 kDa antigenic bands in samples with IFAT titre 1 : 80. This observation was consistent even in the face of fluctuating antibody titre where serum antibody titres from an animal exceeded then failed to reach 1 : 80. Antibody fluctuation was observed across all cows (pregnant and aborted) with no discernible fluctuation pattern. However, the fluctuation in antibody titre observed appeared to be most remarkable in initially ELISA-negative pregnant cows, and to a lesser extent in ELISA-positive pregnant cows, and ELISA-positive aborted cows. Although there was fluctuation in antibody titre, the banding patterns of N. caninum tachyzoite antigens by cows within the same IFAT titre group remained similar.

The use of PCR to detect Neospora caninum DNA in the blood of naturally infected cows.

Twelve 2-year old heifers in their fifth month of gestation when pregnancy tested were used in this study. Six heifers aborted at approximately 4 months of gestation and had blood samples drawn less than 6 weeks after the abortions were identified. Blood samples were also drawn from three sero-positive pregnant and three sero-negative pregnant heifers. DNA was isolated from the samples and a 350 bp fragment of the Nc-5 gene was PCR amplified using primer pair Np21+ and Np6+. Also, the Internal Transcribed Spacer 1 (ITS1) was PCR amplified using Tim 3 and Tim 11 primer pair. The Nc-5 gene fragment was cloned, sequenced and the sequence BLAST-tested. Similarly, the ITS1 product was sequenced and BLAST-tested. The BLAST test results revealed that Neospora caninum DNA was present in these blood samples indicating that polymerase chain reaction can be used in the detection of N. caninum DNA in the blood of sero-positive cows.

Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand.

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates. METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent antibody test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice. RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3-7%. CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.