Cloning, nucleotide sequencing and bioinformatics study of NcSRS2 gene, an immunogen from Iranian isolate of Neospora caninum

Neosporosis is caused by an obligate intracellular parasitic protozoa Neospora caninum which infect variety of hosts. NcSRS2 is an immuno-dominant antigen of N. caninum which is considered as one of the most promising targets for a recombinant or DNA vaccine against neosporosis. As no study has been carried out to identify the molecular structure of N. caninum in Iran, as first step, we prepared a scheme to identify this gene in this parasite in Iran. Tachyzoite total RNA was extracted and cDNA was synthesized and NcSRS2 gene was amplified using cDNA as template. Then the PCR product was cloned into pTZ57R/T vector and transformed into E. coli [DH5alpha strain]. Finally, the recombinant plasmid was extracted from transformed E. coli and sequenced. Bioinformatics analysis also carried out. The PCR product of NcSRS2 gene was sequenced and recorded in GenBank. The deduced amino acid sequence of NcSRS2 in current study was compared with other N. caninum NcSRS2 and showed some identities and differences. NcSRS2 gene of N. caninum successfully cloned in pTZ57R/T. Recombinant plasmid was confirmed by sequencing, colony PCR and enzymatic digestion. It is ready to express recombinant protein for further studies

Molecular and pathological study of bovine aborted fetuses and placenta from Neospora caninum infected dairy cattle

The objective of the study was to evaluate the presence of Neospora caninum organisms in the brain of aborted fetuses and placentas of full-term calves born of seropositive cows. During 2006-2007, 12 brains of aborted calves from Neospora seropositive cattle and 7 placentas from seropositive dams giving birth to full-term calves, from four dairy cattle farms located around Tehran province, Iran were examined by Nested-PCR and histopathology techniques. The Nested-PCR demonstrated that all of 12 aborted fetal brain samples and 5 of 7 placentas were infected by N. caninum. Mild to severe placentitis was observed in 5 placentas. Severe hyperemia and perivascular and perineuronal edema revealed in all fetal brain. In 3 out of 12 brains, scattered foci of hemorrhages, neuropilar necrosis and gliosis were present. In addition, nonpurulent encephalitis with severe lymphohistiocytic perivascular cuffing in one case and a small tissue cyst like Neospora caninum cyst in other calf were observed. Our results confirmed the molecular and histopathologic findings of other studies about Neospora caninum infection and it seems to support the hypothesis that Neospora infection is associated with bovine abortion in Iran