Comparison of use of Vero cell line and suspension culture of murine macrophage to attenuation of virulence of Neospora caninum.

In this study the tachyzoite yields of Neospora caninum were compared in two cell lines: Vero (African Green Monkey Kidney) and suspension culture of murine macrophage (J774) cell lines. Then, N. caninum were continuously passaged in these cell lines for 3 months and the effect of host cells on virulence of tachyzoites was assessed by broiler chicken embryonated eggs. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs with different dilutions (0.5 × 10(4), 1.0 × 10(4), 1.5 × 10(4)) of tachtzoites isolated from these cell cultures. The mortality pattern and pathological changes of the dead embryos and hatched chickens were noted. Tissue samples of brain, liver and heart were examined by histopathological and detection of DNA of parasite by polymerase chain reaction (PCR). Also, consecutive sections of the tissues examined histologically were used for immunohistochemical (IHC) examination. Embryos inoculated with tachyzoites derived from Vero cell line (group V) showed a higher mortality rate (100%) than the embryos that received tachyzoites derived from J774 cell line (group J) (10% mortality rate). The results of this study indicated that the culture of N. caninum in J774 cell led to a marked increase in the number of tachyzoite yields and rapid attenuation in comparison to Vero, so the results were confirmed by IHC and PCR. This study is the first report of the significant effect of host cell on the attenuation of virulence of N. caninum tachyzoites. These findings could potentially provide a practical approach in the mass production of N. caninum tachyzoites, and also in producing live attenuated vaccine.

Development of a disperse dye immunoassay technique for detection of antibodies against Neospora caninum in cattle.

In this study a disperse dye immunoassay method was standardized and evaluated for detection of antibodies against Neospora caninum in cattle. Sera from 150 cattle with a recent history of abortion were collected and tested by commercial ELISA kit and a standardized in-house dye immunoassay system. The positivity rate for the sera used in this study was 34.6% for the disperse dye immunoassay (DDIA) compared to 32% obtained by ELISA kit. This study showed no significant difference between DDIA and ELISA. The results indicated that the DDIA provide an economic, simple, rapid and robust test for detection of N. caninum infection in cattle.

Immunohistochemical and polymerase chain reaction studies in Neospora caninum experimentally infected broiler chicken embryonated eggs.

The diagnostic characteristics of immunohistochemistry (IHC) and polymerase chain reaction (PCR) methods were studied in the tissues of broiler chicken embryos experimentally infected by Neospora caninum. An infection with N. caninum NC-1 isolate was conducted in 70 broiler chicken embryonated eggs randomly divided into seven equal groups. After 8 days of incubation, six groups were inoculated with 10, 10(2), 10(3), 10(4), 10(5), and 10(6) doses of tachyzoites/embryonated egg. The 7th group was considered as control. The mortality rate and pathological changes of the dead embryos and hatched chickens up to 60 days old were noticed. Consecutive sections to those used for histopathological examination including the liver, heart, brain, and chorioalantoic (CA) membrane were subjected to IHC. The intensity and distribution of the immunostaining was graded as highly to mildly positive. For PCR procedure, DNA was extracted from 50mg of the tissues and primer pair Np21/Np6 was used for amplification of the Nc-5 gene. The results of the immunosignaling ranged from variable degrees of mild to moderate staining as dark-brown to brown and coarsely to finely granular, mostly within the cytoplasm of infected cells such as the endothelial cells of blood vessels. The parasite aggregation was more predominant in the heart than other tissues. Immunoreactivity for N. caninum antigen was multifocally moderate positive in the heart, liver and CA of the 10(3) dose, and also heart, liver, brain and CA of the 10(4) dose. IHC showed mildly positive in the liver and heart of the chicken embryos infected with 10 and 10(2) tachyzoites, as well. The results of the PCR confirmed the existence of the parasite in all of the examined tissues from the 10(3) and 10(4) doses. In conclusion, the results indicate a good agreement between IHC and PCR in diagnosis of neospora antigen in the infected tissues.

Epididymis ligation: a minimally invasive technique for preparation of teaser rams.

OBJECTIVE: To describe a minimally invasive technique for preparation of teaser rams by needle-assisted ligation through the tail of the epididymis. STUDY DESIGN: Experimental study. ANIMALS: Mature rams (n=6), estrus-induced ewes (2). METHODS; After local anesthesia, epididymis ligation was achieved by restraining the testis distally within the scrotal sac and passing suture through a hypodermic needle inserted between tail of epididymis and distal pole of testis, caudomedial to craniolateral through the scrotum. The needle was removed leaving the suture in place and the testis pushed up dorsally, then the needle was reinserted through the original holes and the suture passed back through the needle, which was withdrawn. This resulted in the suture forming a complete loop around the epididymis. The suture ends were tied ligating the epididymis. Semen was evaluated pre- and postligation. Testes were removed after 30 days for gross and histologic examination. RESULTS: Epididymis ligation was accomplished without postoperative complications. Three weeks after the epididymis ligation, no motile and live spermatozoa were found in ejaculates. From 5 to 28 days after epididymis ligation, attraction to ewes and libido was unchanged and similar to 14 days before ligation. CONCLUSIONS: This novel minimally invasive technique is a simple, alternative method for preparation of teaser rams. CLINICAL RELEVANCE: This method is simply performed, without skin wounds, and minimal postoperative care is needed. The technique should be readily adaptable to other species.

Histopathological and clinical investigations in Neospora caninum experimentally infected broiler chicken embryonated eggs.

To date, despite the limited information about the role of birds in Neospora caninum infection, there are no reports regarding the role of broiler chickens. In the present study, an experimental infection with N. caninum NC-1 isolate was conducted in embryonated eggs from 70 Lohman broiler chickens randomly divided into seven equal groups. At 8 days of incubation, six groups were inoculated via chorioallantoic (CA) liquid with different dilutions (10, 10(2), 10(3), 10(4), 10(5), and 10(6)) of tachyzoites/embryonated egg. The 7th group was considered the control. The mortality rate, gross and histopathologic changes of tissues of the dead embryos and live hatched chickens up to 60 days old were studied and evaluated. There were no hatchings in groups with 10(5) and 10(6) tachyzoites. In groups with 10, 10(2), 10(3) and 10(4) tachyzoites there were 8, 7, 5 and 2 hatchings, respectively. From the surviving chickens only one 10(4) tachyzoites inoculated chicken showed clinical neurologic signs. In all groups the main gross lesions were hemorrhage associated with thickening of the CA membranes. Three chickens (one chicken with 10(4) tachyzoites and two with 10(5) tachyzoites) showed arthritis in the feet joints after two weeks of inoculation. Microscopic examination of the heart, liver and chorioallantoic membrane revealed acute neosporosis and, in some cases, granulomatous inflammation. Our findings implied that broiler chicken embryonated egg is a completely suitable animal model for biological studies of acute neosporosis studies and genetic susceptibility can be propounded in different chicken breeds. It seems 10(3) tachyzoites dilution is equal to LD50 in N. caninum infection in broiler chicken embryonated eggs and can be used in other experimental investigations.