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.

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.