• 대한수의학회지
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• 제29권3호
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• pp.343-359
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• 1989

To study the effects of ibaraki virus on preimplantation mouse embryos collected from prepubertal ICR and BALB/cByJ mice (30~40days old) by superovulation, zona pellucidaintact(ZPI) or free(ZPF) embryos(n=774) of 4- to 8-cell and morulae were exposed to $10^{5.8}$ $TCID_{50}$ of the virus up to 96 hours. The embryos were examined morphologically by observing the degeneration and hatching rates, and virologically and immunologically by determining the presence of infection with the virus, in addition, the effect of washing the embryos to remove virus possibly attached to was also investigated. The ZPI 4- to 8-cell embryos and morulae exposed to the virus showed considerably higher degeneration rate than those not exposed, for 96, and for 72 to 96 hours, respectively(p<0.01). The ZPF 4- to 8-cell embryos and morulae exposed to the virus showed considerably higher degeneration rates than those not exposed, throughout the whole culture hours in vitro (p<0.01). The ZPI 4- to 8-cell embryos and morulae not exposed to the virus showed considerably higher rates of hatched blastocyst than those exposed (p<0.01). The virus infection rates of the ZPF 4- to 8-cell embryos and morulae were significantly higher than those of the ZPI embryos according to cell culture system. The viral antigen was detected exclusively on the zona pellucida of ZPI embryos, while the antigen was evenly distributed in the blastomeres of ZPF embryos by the immunofluorescent assay. In the ZPI embryos exposed to ibaraki virus, the virus was detected in the two times-washing groups, but not in the ten times-washing groups. The results indicated that zona pellucida of murine embryos would provide an effective protection and that ten times-washing of the ZPI embryos previously exposed to the virus was effective to remove virus from the embryos.

• 대한수의학회지
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• 제37권4호
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• pp.793-807
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• 1997

We tried to develop detection system of porcine reproductive and respiratory syndrome virus(PRRSV) by in situ hybridization(ISH) in the piglets experimentally infected with KPRRS-2, the Korean isolate(12 piglets) or Mn-1b, the American isolate(4 piglets), and in the natural infection suspected 6 piglets. Twelve 30-days-old piglets(two pigs per each inoculated group) were inoculated by nasal instillation of KPRRS-2 virus(total dose $10^{4.5}TCID_{50}$), Six piglets(one pig per each group) were induced contact infection with inoculated piglets, during the experiment, and two piglets were used as control. Inoculated or contacted piglets were euthanized at 1, 3, 5, 7, 14 and 21 days postinoculation(DPI). The respiratory signs such as coughing and nasal discharge were observed on day 3 DPI, and ear cyanosis were on day 5 DPI, including contacted piglets. Through the necropsy, purple discolorization of dorsal part of lung, and hypertrophy of local lymph nodes were observed. The histopathological lesions of lung were interstitial pneumonia characterized by type 2 pneumocyte hyperplasia. We prepared the probe for ISH by RNA isolation from KPRRS-2, RT-PCR, and biotin labeling. We performed the ISH within only 1~2 hours using $Microprobe^{TM}$ capillary action system. As the results, the strong red specific positive signals, means PRRSV distribution, was mainly observed in the cytoplasm of alveolar macrophages. And also signals were detected in some type 2 pneumocytes and bronchiolar epithelium of lung, myocardium, liver, kidney, tonsil, spleen, gastrointestinal mucosa, testis and lymph nodes.

• 대한수의학회지
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• 제34권3호
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• pp.583-591
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• 1994

This study was carried out to investigate the pathogenesis of canine herpesvirus(CHV) infection in dogs. The 17 puppies, one day old, delivered from CHV seronegative 3 dams were divided into two groups. The 13 puppies were inoculated intranasally with 1ml of CHV-KK inoculum($5{\times}10^{5.6}TCID_{50}/ml$) and 4 puppies were served as control. And then the puppies were sacrificed at 2, 4, 6 and 7 days after the treatment, and sampled nasal mucosa, trigeminal nerve, trigeminal ganglion, bone marrow, eye, brain and other major organs for the immunohistochemical examination. Distribution of CHV antigens was limited in cytoplasms and nuclei of necrotic nasal epthelia at 2 days after infection. At 4 days after infection, CHV antigens were detected in vascular walls and peripheral nerves of nasal lamina propria, reticuloendothelial cells of spleen, interstitium of kidney, leptomeningeal vascular walls and alveolar walls, At 6 and 7 days after infection, CHV antigens were detected in all of the necrotic area. CHV antigens were also detected in vascular endothelial cells of various organs and in blood leukocytes from 4 days after infection. Among the six puppies in which necrotic lesions of central nervous system were observed, CHV antigens were detected in trigeminal ganglion, trigeminal nerve and ventroposteriomedial nucleus of four puppies and in spinal trigeminal nucleus of three puppies. These results indicate that the generalized focal necrosis of all organs including brain and eyes in canine herpesvirus infection were resulted from generalized vasculitis with leukocyte-associated viremia, and also the hemonecrotizing meningoencephalitis was resulted from spreading of CHV via blood and nerve trunk.

• 한국어병학회지
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• 제5권1호
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• pp.1-7
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• 1992

CHSE-214 무지개송어 세포주는 10% fetal bovine serum과 2mM-glutamine이 첨가된 Eagle's 기본배지에서 성장하였다. CHSE-214 세포주의 최적온도는 $20^{\circ}C$였다. IPN바이러스는 CHSE-214 세포주에서 복제되었으며, 세포변성효과를 나타내었다. 또한 IPN 바이러스의 감염시간에 따른 감염증상을 역위상차 현미경으로 관찰하였다. 감염 6-12시간 후 세포는 정상세포와 비슷하였다. 감염 18-24시간 후 세포는 일부가 정상 세포보다 더 둥근형태로 되었고, 일부세포는 세포배양용 플라스크 바닥에서 떨어져 부유상태로 되었다. 감염 30시간 후 세포는 더더욱 비정상적인 형태로 되었다. 감염 48~68시간 후 감염된 세포는 파열되었고, 아주 높은 세포 병변 효과를 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.873-879
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• 2005

Red sea bream iridovirus (RSIV) obtained from infected rock bream was propagated by Bluegill fry-2 (BF-2) cell culture. The virus titer was determined as $10^{5.5}\;TCID_{50}/ml$ on confluent BF-2 cell monolayers. The integrin binding site of ORF 055L of infectious spleen and kidney necrosis virus (ISKNV) was selected for the construction of a primer to obtain the RSIV ORF 055L gene. The genes were amplified using RSIV gene lyzate by PCR. The homologies of the ORF 055L sequence of RSIV with ISKNV and rock bream iridovirus (RBIV) were approximately $96\%$ and $100\%$, respectively. DNA vaccine was constructed by cloning the ORF 055L of RSN into pcDNA 3.1 (+), containing a cytomegalovirus (CMV) promoter. For antibody production, pcDNA-055 DNA vaccine was injected to BALB/c mice. The production of antibodies against pcDNA-055 DNA vaccine was confirmed by the Western blot analysis. The antibodies produced by the pcDNA-055 DNA vaccine showed efficacy to neutralize the RSIV in the neutralization test in BF-2 cell culture.

• Parasites, Hosts and Diseases
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• 제51권2호
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• pp.247-253
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• 2013

Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was $10^9TCID_{50}/ml$. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-${\gamma}$ and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.

• 한국동물위생학회지
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• 제19권1호
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• pp.1-29
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• 1996

This study was conducted to elucidate the pathogenesis of Aujeszky's disease virus(ADV) by histopathologic examination. The first Korean ADV Isolate, which was isolated from piglets with clinical signs of Aujeszky's disease in Yangsan(YS) county, Kyungnam province, was inoculated into 32 days old piglets with a dose of $10^{5.9}$$TCID_{50}/ml$ through intranasal or intramuscular route. These piglets were sacrificed at intervals of every 24hrs for 8 days. The virulence of YS strain was determined by the observation of clinical signs, gross findings, and histopathologic changes in tissues. The virus recovery test was performed from brain, spleen, lung and tonsil in cell culture. The pathogenesis of YS strain was determined by the observation of histopathologlc lesions in CNS and neuronal tracts. The major clinical signs were fever, anorexia, dyspnea, constipation, tremor, ataxia, circling movement, hindleg paralysis and salivation. The clinical signs were more severe in piglets of the group inoculated intranasally than those of the intramuscularly inoculated gorup. Lymphocytopenia was detected on day 5 to day 6 postinoculation (PI). The ADV was recovered from the tissue homogenates of tonsil, lung, spleen and cerebrum in cell culture. The highest virus titer was detected from tonsil between day 6 and day 7 PI. Reddish sublobar consolidation foci were scattered in the apical and cardiac lobes of lung. Although yellowish necrotic foci were detected in tonsil and liver, hemorrhagic lesions were mainly observed in heart, kidney and lymph nodes. Histopathologically, degeneration and necrosis of nerve cells, nonsuppurative meningoe-ncephalitis, nodular gliosis and perivascular cuffings were observed in CNS. Multifocal fibronecrotic foci were observed in lung, liver, lymph nodes and spleen. The major pathologic changes were detected in the midbrain, pons and medulla oblongata. Eosinophilic intranuclear inclusion bodies were mainly observed in epithelia and /or macrophages of tonsil, liver, lung, spleen and submandibular lymph nodes, and neurons of brain, respectively. Observation of viral particles at various stages of replication were possible from the endothelial cells of the alveolar capillaries and tonsillar crypt epithelia by transmission electron microscope.

• 한국동물위생학회지
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• 제36권3호
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• pp.157-162
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• 2013

A good level of biosecurity practice is important for efficient porcine reproductive and respiratory syndrome (PRRS) control. In the current study, disinfecting ability of ozone against PRRS virus (PRRSV) was evaluated in comparison with ultraviolet (UV) and an organic acid-based disinfectant to assess the possible use of ozone for disinfecting farm vehicles, equipments, and materials to reduce the risk of new virus introduction. For in vitro evaluation, the levels of infectious virus and viral RNA were determined on the swabs collected from the floor surface of each room treated with either ozone, UV or the disinfectant up to 30 min after contamination with 100 mL of VR2332 ($10^5\;TCID_{50}/mL$). For in vivo evaluation, 3, 4-week old, PRRS-free pigs were housed into those rooms right after the last swab collection. Then the pigs in each room were injected intramuscularly with the corresponding swab samples collected at the last time point and pooled per each room. Although ozone, UV, and the disinfectant significantly reduced the levels of PRRSV RNA contamination, ozone was most effective in removing the viral RNA. In addition, the virus collected after at least 10 min exposure to ozone failed to replicate in pigs while the virus collected after treatment with UV and the disinfectant for 30 min still replicated in pigs. Based on the results, it was concluded that ozone is more effective in inactivating PRRSV as compared with UV and the organic acid-based disinfectant.

• 한국수의병리학회지
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• 제2권2호
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• pp.117-125
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• 1998

Pathological studies were performed on the five piglets experimentally infected with Aujeszky's disease virus(pseudorabies), NYJ isolate, isolated from the naturally infected pigs in Korea: two piglets were inoculated intramuscularly, two piglets intranasally, and one piglet subcutaneously at the dose of 1$m\ell$ per animal with the 105.5 $TCID_50$/0.1ml titer. Clinical signs included dyspnea, high fever(＞$41^{\circ}C$), anorexia, vomiting, diarrhea or constipation, ataxia, circling movement, posterior paralysis, intermittent convulsion, and coma followed by death although some variations by age and inoculated routes were observed. Gross features included multiple necrotic foci in the liver, congestion and hemorrhage in the lymph nodes and spleen, petechial hemorrhage in the kidney, hemorrhagic pneumonia, marked meningeal congestion, severe sub meningeal hemorrhage in the spinal cord, excessive cerebrospinal fluid retention, and muscular necrosis at the inoculated area. Microscopically, non suppurative meningoencephalitis with gliosis and perivascular cuffing in CNS, ganglioneuritis, necrohemorrhagic splenitis, necrotic hepatitis, tonsillitis and rhinitis, hemorrhagic or interstitial pneumonia, and non-suppurative myositis in the injected area were observed. Eosinophilic intranuclear inclusion bodies were found in a variety of tissues the including the liver, kidney, adrenal gland, spleen, lymph nodes, tonsil, and lung. Ultrastructurally, virus particles were confirmed in nucleus and cytoplasms of pneumocytes around the necrotic areas.

• 대한수의학회지
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• 제40권1호
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• pp.42-48
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• 2000

Akabane disease is transmitted through mosquitoes in cattle, sheep and goats. It shows congenital abnormalities including encephalomyetitis, hydranencephaly, neurogenic arthrogryposis, and deformed neonatal calves. Akabane viruses, 93FMX and K-9 strain, were isolated from fetal matrix of aborted cow and blood of healthy cow, respectively. S gene sequences of 93FMX and K-9 showed 100% homology with that of OBE-1 strain isolated in Japan. Based upon our sequencing data, we synthesized specific primers for PCR diagnosis. Using these primers, we were able to amplify the S gene of Akabane virus not only from the culture fluid of Vero cells but also from the brain tissue of suckling mouse inoculated with, Akabane virus. These PCR products were confirmed by Southern blot hybridization. Not only the sensitivity of PCR test was high enough to detect the viruses of $10^{1.0}TCID_{50}/ml$, but also the time for diagnosis was significantly shorter than that of the virus isolation by tissue culture method. This method was also effective for the detection of Akabane virus in the cerebrum of fetus. RT-PCR method may be used for a useful diagnostic test of the clinical cases of Akabane disease.