In vitro Development of Cystoisospora suis in Swine Kidney Cell Line, SK-6.

Cystoisospora suis (syn. Isospora suis) is an apicomplexan parasite which causes an important piglet coccidiosis resulting in commercial losses worldwide. Successful coccidiosis control using chemoprevention and chemotherapy as the recommended approach, nonetheless, conduces the extensive use of anticoccidial drugs that may lead to the emergence of drug-resistant parasites. Consequently, it is necessary to continuously engage in drug discovery for alternative anticoccidials. To achieve the drug discovery aspect, an appropriate in vitro model for the endogenous development of the coccidium has to be established. Moreover, the in vitro model can reduce the cost and experimental animal use necessitated by in vivo systems. The aim of this study was to investigate the development of C. suis throughout its endogenous life cycle using swine kidney cell line, SK-6 for the in vitro system. The results showed that SK-6 cells seemed to be an excellent support system for the complete endogenous life cycle of C. suis. For ability to infect, sporozoites were able to penetrate into the host cells, first observed by 2 hours post inoculation. The intracellular penetrated sporozoites underwent asexual multiplications producing presumed type I meronts and merozoites. The meronts each formed 2-8 zoites by 3 days post infection (dpi) which were prominently seen on 3-5 dpi. Developing oocysts were initially observed on 5 dpi and went on sequential sexual development resulting in intracellular mature oocysts from 7-14 dpi. These became prominent from 9-12 dpi. Recycling of merozoites to form more meronts and merozoites also appeared by 10 dpi, indicated by merozoites burst from initially infected cells observed re-entering host cells. In conclusion, the presented in vitro system was able to support complete endogenous development of C. suis that was comparable to its in vivo life cycle. Furthermore, this provides an appropriate model for the study of a vast array of aspects relating to the coccidium, especially in applications with regard to the sexual development of the parasite.

RNA interference targeting nucleocapsid protein (C) inhibits classical swine fever virus replication in SK-6 cells.

The application of RNA interference (RNAi) strategy for controlling classical swine fever could become a promising alternative to the conventional eradication measures, as it was recently shown for foot-and-mouth disease (Chen et al., 2004), influenza (Ge et al., 2003), porcine reproductive and respiratory syndrome (He et al., 2007) and porcine transmissible gastroenteritis (Zhou et al., 2007). The use of synthetic siRNA which is corresponding to nucleotides 1130-1148 of the CSF virus strain Alfort, targeting the nucleocapsid protein (C) was investigated to show the inhibition of CSF virus replication. It could be shown that the virus titer of infected cells, which had been mock-transfected or transfected with control (non-silence) RNA were not affected. These data indicate that siRNA_253 is able to inhibit viral replication.

Are low-density granulocytes the major target cells of classical swine fever virus in the peripheral blood?

The target cells of classical swine fever (CSF) virus in the peripheral blood of pigs infected with recent field isolates from Germany were studied. Eight weaned pigs were inoculated oronasally with the CSF virus field isolate Visbek/Han 95 and three weaners were inoculated with the isolate Losten/Freese 98. All pigs showed severe clinical signs typical of CSF and died or had to be euthanized between 9 and 24 days post-infection (dpi). The first cells in the peripheral blood which became infected with CSF virus were mixed granulocytes (a combination of low- and high-density granulocytes). These cells yielded the highest infectivity for PK 15 cell cultures. On day 7 post-infection, the peripheral blood mononuclear cell (PBMC) fraction was virus positive, while the peripheral blood leucocyte (PBL), peripheral blood T lymphocyte (PBT) and high-density granulocyte fractions were either negative or their infectivity was lower than the infectivity of the PBMC fraction. These results indicate that PBMC contain more virus-positive cells than other fractions of leucocytes. These findings may also have diagnostic implications for the detection of CSF virus in blood samples. Because PBMC showed the highest infectivity in the early stages of CSF, it should be the sample of choice for CSF virus isolation.