Neospora caninum was isolated in Vero cell culture from the brain of a stillborn calf. This isolate (designated NC-LivB1) is the first to be obtained from cattle in the United Kingdom and was confirmed as N. caninum by immunofluorescence with specific antibodies and by internal transcribed spacer 1 (ITS1) sequence analysis. Differences were found between NC-LivB1, other bovine isolates and canine isolates of N. caninum and closely related protozoal parasites, using random amplified polymorphic DNA polymerase chain reaction (RAPD - PCR) techniques.
"In isolation procedures reported elsewhere, the period between introduction of suspicious material to cell culture and first detection of N. caninum in cultures varied between 5 days (McInnes et al., 2006) and 9.5 weeks (Pastusiak et al., 2005). According to Davison et al. (1999b) the earliest observation of parasites in culture was after more than 1 month, and it was found to be directly associated with the number and viability of parasites used for the initial infection. The nPCR applied in the present study was used to confirm N. caninum infection, both in the brain tissues of seropositive aborted fetuses and in cell cultures. "
[Show abstract][Hide abstract] ABSTRACT: First Israeli Neospora caninum isolates were obtained from brain tissues of aborted fetuses (NcIs491 and NcIs580) from dairy farms endemic for neosporosis and maintaining cattle on zero grazing. Tissues from different parts of the fetus brains were used to infect Vero cells. Tachyzoites of N. caninum were first observed in cultures from days 30 and 32 after infection. To confirm the identity of the isolated parasites, DNA extracts from brains and cultures were tested by PCR with specific primers based on the Nc5 gene. Specific fragments were amplified by PCR from infected cultures of both fetuses on day 25. Susceptible seronegative gerbils (Meriones tristrami) were inoculated intraperitoneally with 10(3) to 10(5) tenfold dilutions of subculture tachyzoites. The inoculated gerbils developed specific antibodies to N. caninum, with end-point serum dilution of 1:4096 in the IFA assay, whereas no neurological signs or deaths were seen during 4 months of observation.
"Since the first isolation of N. caninum by Dubey et al. (1988b) a number of isolates have been characterised from bovine as well as canine sources. Successful isolation of N. caninum is however, difficult due to low numbers and/or viability of parasites and observation of parasites in primary culture can often take more than a month (Davison et al., 1999; Otter et al., 1995). This paper describes the first successful isolation and in vitro culture of N. caninum from a skin lesion of a naturally infected dog in Australia and the characterisation of the isolate by indirect immunowww .elsevier.com/locate/vetpar "
[Show abstract][Hide abstract] ABSTRACT: Neospora caninum was isolated and established in vitro from the skin lesion of a naturally infected dog. The identity of the parasite was evaluated by immunofluorescent antibody test (IFAT), microscopy, Western blotting and polymerase chain reaction (PCR). N. caninum DNA was detected in the whole blood, serum, skin lesion, rectal scrapings and faeces of the infected dog utilising a nested PCR targeting the Nc-5 gene of N. caninum. Antigenic and genetic characterisation of the isolate, designated WA-K9, at a number of loci including the Nc-5 gene, heat shock protein 70 (HSP-70) gene, alpha-tubulin and beta-tubulin genes revealed no variation between this isolate and two N. caninum isolates from different geographic areas. Clinical aspects of this case, which included cutaneous and neurological disease, are also discussed.
"One reason for this difficulty in recovering viable N. caninum is because most N. caninum in aborted bovine fetuses are dead, and the sensitivity of the cell culture to isolate N. caninum from autolysed bovine fetuses is probably low (Conrad et al., 1993; Dubey and Lindsay, 1996). Therefore , there are only a few isolates of N. caninum from bovine fetuses available worldwide, and little is known of their antigenic variability (Conrad et al., 1993; Yamane et al., 1997; Stenlund et al., 1997; Davison et al., 1999; Kim et al., 2000; McAllister et al., 2000). In the present paper, we describe isolation of the first strain of N. caninum from a dairy cattle fetus in Portugal by bioassays in immunosuppressed mice co-infected with mouse sarcoma cells. "
[Show abstract][Hide abstract] ABSTRACT: Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.
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