The in vitro culture of Toxoplasma gondii was evaluated using a JNUCK cell strain that was independently developed by our research team. The sensitivity of the JNUCK strain was compared with those of MDCK and vero cell strains, which were previously shown to be sensitive to T. gondii. Morphological observation and 3H-uracil absorption tests showed that JNUCK cell strain has the highest sensitivity, while the MDCK and vero cell strains showed lower sensitivities. The optimal culture conditions using the JNUCK cell strain were determined by inoculating T. gondii at multiplicity of infection (MOI) 1, MOI 5, and MOI 25 and the numbers of T. gondii were measured 24, 48, and 72 hours after inoculation. The optimal medium composition was determined by adding different concentrations of FBS to DMEM medium for inoculation at MOI 5, and then calculating the numbers of tachyzoites inside the cells and in the medium. The numbers of tachyzoites inside the cell were highest for 10% FBS, while the numbers of tachyzoites in the medium peaked after addition of 30% FBS. These studies confirmed that the optimal culture condition of T. gondii using the JNUCK cell strain was achieved by inoculation at MOI 5, using medium containing 20% FBS and culturing for 72 hours.

Vulpes zerda is a fox that inhabits the desert regions of North Africa and Asia, whereas Vulpes pallida is a fox that inhabits the savannah zones of North Africa and the Sahara desert. Vulpes zerda, which is on the “Red List of Threatened Species” designated by the International Union for Conservation of Nature and Natural Resources (IUCN), belongs to the “Appendix II” group drawn up by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and hence its cross-border trade is prohibited. In contrast, Vulpes pallida is classified as being of “Least concern” (LC) by IUCN. However, distinguishing between Vulpes pallida and Vulpes zerda by shape alone is difficult. This study attempted to differentiate between the two species by examining the chromosomes of Vulpe szerda and Vulpes pallida that were raised in Korea. According to the examination, two of eight foxes believed to be Vulpes zerda were Vulpes pallida and one of six foxes believed to be Vulpes pallida was actually Vulpes zerda. Considering the ambiguity of making a formal distinction between Vulpes zerda and Vulpes pallida, a more scientific approach to differentiation will be required.

Several national control plans have been formulated and carried out to prevent domestic animals from various infectious diseases in Korea. Typical plans include the prevention of diseases by vaccination and the quarantine activities to reduce or cut off the occurrence and transmission. Although such great efforts to prevent diseases every year, difficulties have been experienced in controling transmittable diseases like foot-and-mouth disease and bird influenza. The present study was conducted to investigate the awareness, knowledge, difficulties and routine activities in controling infectious diseases on persons-in-charge of preventive measures in national live stock breeding facilities. A questionnaire survey composed of 81 questions in three categories was carried out on 30 subjects (12 veterinarians and 18 ordinary workers) serving in nine national breeding stock farms. According to the results of the survey analysis, the difficulties and concerns in carrying out preventive measures experienced by the veterinarians and the ordinary workers were similar. Shortage of budgets and manpower was the top ranked concern in performing preventive measures for both the veterinarians and the ordinary workers. Manual or guideline on controling infectious diseases for routine or emergency case were established in all of the facilities, however those were not executed practically in most cases. Instructional training on those manual and controling wild animal to prevent contacting breeding stock were not performed in most facilities surveyed.

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin–Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.

A cell line of bovine origin was immortalized to isolate foot-and-mouth disease virus (FMDV). The immortalization was performed by infection of bovine primary epithelial cells with a recombinant retrovirus that overexpressed the human telomerase (hTERT), after primary culture of fetal bovine kidney tissue and removal of fibroblasts. After cloning the immor- talized cell line into single cells, the cloned cell lines were named JNUBK-1, JNUBK-2, JNUBK-3 and JNUBK-4, according to their characteristics. To confirm the epithelial phenotype of the cell lines JNUBK-3 and JNUBK-4, which showed stable proliferation capability over 35 generations after immortalization, the expression of cytokeratin and fibronectin was measured. Finally, the FMDV titer in the JNUBK-3 and JNUBK-4 cell lines was measured and was 800∼2,000 times higher than that of the currently used cell line IRBS-2. In conclusion, more sensitive isolation and production of FMDV became possible through the use of the immortalized JNUBK-3 and JNUBK-4 cell lines.

Canine distemper virus (CDV) causes serious and often fatal disease in dogs. Currently, various cells or cell lines have been used to detect or produce CDV. In order to set up the conditions, we separated two different cell lines from Madin-Darby canine kidney (MDCK) and named as MDCK-F (fibroblast-like) and MDCK-E (epithelial-like) by Na2EDDA treatment. CDV seed virus was prepared using MDCK cells and inoculated into MDCK-F and MDCK-E including MDCK with various ranges of multiplicity of infection (MOI) to confirm the optimal amount of virus inoculation. The virus titer of TCID50/ml was calculated by inoculation of serially diluted virus into 96-well plate of MDCK cells. The titer and cytopathic effect (CPE) in MDCK-E were compared to those in MDCK-F. The titer of seed CDV was 1.24×106 TCID50/ml. Optimal MOI was about 0.1 for both MDCK-F and MDCK-E to obtain highest titers of 108 TCID50/ml and 5 × 108 TCID50/ml respectively. CPE in MDCK-E was shown 4 days after inoculation whether in MNCK-F 5 6 days after inoculation. We can obtain highest titer of 5 × 108 TCID50/ml with 0.1 MOI using MDCK-E. MDCK-E was more susceptible for CDV production than MDCK-F.