Bovine spongiform encephalopathy surveillance in the Republic of Korea.

National surveillance for bovine spongiform encephalopathy (BSE) began in the Republic of Korea (ROK) in 1996. Surveillance programmes changed overtime to comply with the guidelines of the World Organisation for Animal Health (OIE). Bovine spongiform encephalopathy was designated as a notifiable disease in 1997. From July 2008, the BSE surveillance programme was intensified to test cattle in designated high-risk populations more effectively. New measures included the compulsory testing of all non-ambulatory cattle at abattoirs, and encouraging the testing of all dead cattle examined and recorded under the Mutual Aid Insurance Scheme (fallen stock). In addition, there was a vigorous search for animals suspected of being clinically infected. As a result, a total of 426,919 OIE points were achieved over a period of seven consecutive years to the end of October 2009. This enabled the submission of a successful application to the OIE in 2010 for recognition of the ROK's BSE disease status as being one of controlled risk, in accordance with Chapter 11.5. of the OIE Terrestrial Animal Health Code.

Monoclonal antibody-based immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fixed, paraffin-embedded intestinal tissues.

An immunohistochemistry technique was developed for the diagnosis of porcine epidemic diarrhea virus (PEDV). The technique was tested on formalin-fixed, paraffin-embedded intestinal tissues from piglets naturally infected with PEDV. Five different monoclonal antibodies (MAbs) were tested in this study. PEDV antigen was consistently detected in the PLP (4% paraformaldehyde, 100 mM L-lysine dihydrochloride, 10 mM sodium m-periodate in phosphate-buffered saline)-fixed PEDV-infected Vero cells or formalin-fixed, paraffin-embedded intestinal tissues from piglets naturally infected with PEDV. The C9-2-2 MAb gave the strongest reactivity and least background staining, detecting 10 of 10 infected pigs. The positive reaction was cytoplasmic. Positive enterocytes were distributed over the tip and along the sides of atrophied or fused villi in the jejunum and ileum. Positive-staining cells were not detected in the crypts. No staining was observed in cecum and colon. No positive cells were observed when the C9-2-2 MAb was reacted with the tissue sections from noninfected piglets or from transmissible gastroenteritus virus (TGEV)- and rotavirus-infected piglets. The selected anti-PEDV MAbs tested on formalin-fixed, paraffin-embedded tissue sections are useful for diagnosis when virus isolation is not available. This method would be of particular value in countries where both PEDV and TGEV are epizootic and would aid in differentiating between PEDV and TGEV infection.

Identification of the cross-reactive antigens between Marek's disease virus and pseudorabies virus by monoclonal antibodies.

Among the 33 monoclonal antibodies (MAbs) against pseudorabies virus (PRV) examined, three MAbs (24-17, 74-26, and 8) were found to react with cells infected with Marek's disease virus (MDV)-related viruses by immunofluorescence test. Two of the MAbs (24-17 and 74-26) reacted with the nuclei of cells infected with MDV serotype 1 (MDV1), MDV serotype 2 (MDV2), and herpesvirus of turkeys (HVT), whereas MAb 8 reacted with the cytoplasm of MDV2- and HVT-infected cells. However, none of the MAbs against MDV1, MDV2, and HVT that were examined reacted with PRV-infected cells. None of these three MAbs against PRV reactive with MDV-related viruses cross-reacted with the cells infected with other herpesviruses, such as herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, or human herpesvirus 6. Southern-blot hybridization under stringent or less-stringent conditions showed that no significant DNA homology was detected between PRV DNA and MDV DNA.

Expression of envelope protein (E2) of bovine viral diarrhea virus in insect cells.

The gene encoding the envelope glycoprotein (E2) of bovine viral diarrhea virus (BVDV) was expressed in a baculovirus. The expressed protein was detected on the surface of infected cells by immunofluorescence. Western blotting analysis showed the presence of the expressed protein of a similar molecular size to the E2 protein. The antigenicity of expressed protein were tested in guinea pigs and cattle. The immunized animals developed neutralizing antibodies against BVDV.

Rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction.

The diagnosis of porcine epidemic diarrhea virus (PEDV) infection in the laboratory is rather fastidious because of difficulties in virus propagation. The feasibility of virus propagation in vivo is also limited by the handling of a number of samples at the same time. In this study, the detection of PEDV by reverse transcription polymerase chain reaction (RT-PCR) is described. The RT-PCR could detect up to 10(4) TCID50/ml of PEDV and did not show any cross reaction with transmissible gastroenteritis virus or porcine rotavirus. Using this method, the detection of PEDV in experimentally inoculated piglets was possible as early as one day after inoculation. These results suggest that the RT-PCR could be applicable for a rapid diagnosis of PEDV infection.

Bovine herpes virus expressing envelope protein (E2) of bovine viral diarrhea virus as a vaccine candidate.

The gene encoding the envelope protein (E2) of bovine viral diarrhea virus (BVDV) was expressed under the thymidine kinase (TK) promoter of Korean bovine herpesvirus 1 (BHV-1) isolate. Thymidine kinase negative (TK-) BHV-1 recombinants expressing E2 of BVDV were constructed and the expression of E2 was identified by immunofluorescence and Western blotting. Compared to wild type BHV-1, the recombinant BHV-1 had a delayed cytopathogenic effect in cells. The immunogenicity of the recombinant BHV-1 was examined in guinea pigs and cattle. Although an increase in body temperature was detected for a few days, the inoculated cattle returned to normal temperature with the development of neutralizing antibodies to BVDV.

Detection of porcine epidemic diarrhea virus by immunohistochemistry with recombinant antibody produced in phages.

Several diagnostic methods including immunofluorescence, enzyme-linked immunosorbent assay, polymerase chain reaction and immunohistochemistry have been developed for the detection of porcine epidemic diarrhea virus (PEDV). An immunohistochemical method using a new recombinant antibody produced by a phage antibody system (PAS16) kit was investigated and compared with that using a monoclonal antibody for PEDV detection in PEDV-infected piglets. In both the immunohistochemical methods, PEDV antigens were detected in the cytoplasm of villous enterocytes and in the macrophages infiltrated in the lamina propria at 18 to 110 hr post inoculation. The positive signals with the recombinant PAS16 antibody were similar to those with the monoclonal antibody. This result suggests that the recombinant PAS16 antibody can be applicable for the rapid immunohistochemical diagnosis of PEDV infection.

Immunoprophylactic effect of chicken egg yolk immunoglobulin (Ig Y) against porcine epidemic diarrhea virus (PEDV) in piglets.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of neonatal diarrhea in piglets, which causes high mortality rates. In this study, the immunoprophylactic effects of chicken egg yolk immunoglobulin (Ig Y) against PEDV were investigated in neonatal pigs. Ig Y was found to reduce the mortality in piglets after challenge exposures. The field application of Ig Y also revealed significant differences in survival rates of piglets given Ig Y, as compared with placebo or control. The results in this study indicated that Ig Y against PEDV could be an alternative way of supplementing prophylactic measures like colostral antibodies from sows.

[Cloning and sequencing of p33 in a Korean isolate of Theileria sergenti].

The gene encoding the 33 kDa piroplasm surface protein of Theileria sergenti isolated in Korea was cloned and the nucleotide sequence was determined by dideoxy chain termination method. The cloned gene corresponds to 869 bp encoding an open reading frame 283 amino acids. Comparison of the sequence between Korean and Japanese isolates showed 99.4% homology rate in the nucleotide sequence and 98.9% homology rate in the amino acid sequence.

Immunohistochemical identification of porcine respiratory coronavirus antigen in the lung of conventional pigs.

Nine 3-week-old conventional pigs were inoculated intranasally with a Korean isolate of porcine respiratory coronavirus (PRCV). Three 3-week-old conventional pigs were kept as noninoculated controls. Three inoculated and one control pig were euthanatized at 5, 10, and 15 days postinoculation (DPI), respectively. All nine inoculated pigs developed respiratory disease. Interstitial pneumonia characterized by mononuclear inflammatory cell infiltration into alveolar septa were seen microscopically in all nine PRCV-infected pigs. Histopathologic changes were severe at DPI, modest at 10 DPI, and almost resolved at 15 DPI. PRCV antigen was not detected in bronchial and bronchiolar epithelium but was detected in interstitial macrophages in the lungs. This Korean isolate of PRCV appeared to replicate primarily within alveolar macrophages in the respiratory system of the pig.

Pathogenicity and vaccine efficacy of a thymidine kinase gene deleted infectious laryngotracheitis virus expressing the green fluorescent protein gene.

The deletion of the thymidine kinase (TK) gene of herpesviruses causes a reduction in their virulence. However, the effects of the TK gene in infectious laryngotracheitis virus (ILTV) have not been clearly elucidated. In the present study, we constructed a TK gene-deleted recombinant ILTV expressing the green fluorescent protein (GFP) gene as a marker. The GFP gene was inserted in place of the TK gene in both virulent and low virulence strains of ILTV. The GFP gene in the recombinants was expressed in chicken kidney cells, LMH cells and in the chorioallantoic membrane of embryonated chicken eggs. The recombinants produced cytopathic effects in chicken kidney cells and LMH cells and formed pocks in the chorioallantoic membrane of embryonated chicken eggs. The growth rate of the recombinant in chicken kidney cells was similar to that of wild type viruses. The recombinants showed a reduction of virulence compared to that of parental viruses and induced protection against virulent ILTV in specific pathogen free chickens. The recombinant expressing GFP gene may be a candidate for a genetically engineered vaccine and provide a means to study growth kinetics and mechanism of latent infection and reactivation of ILTV. In this study, we confirmed that the TK gene is directly related to virulence of ILTV. This is the first report to show the evidence that the TK gene is a major gene related to virulence of ILTV.

Expression of major piroplasm protein (p33) of Theileria sergenti (Korean isolate) and its immunogenicity in guinea pigs.

To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein band of p33. The antigenicity of expressed polypeptide was further examined through the inoculation of a guinea pig. The sera of guinea pigs immunized with p33 expressed cell lysate showed similar fluorescent antibody patterns and reacted with the same molecular weight protein of T. sergenti in immunoblotting analysis, thus indicating that this protein can be a promising candidate for a subunit vaccine in the future.

[Rapid detection of Theileria sergenti by polymerase chain reaction].

Four separate pairs of oligonucleotide primers within the coding region in a T. sergenti 33-kDa surface protein gene were selected to detect T. sergenti by PCR. The specificity of PCR-amplified DNA was examined by digestion with restriction enzyme and Southern blot hybridization using T. sergenti p33 DNA probe. PCR appears to be specific for T. sergenti, without detectable signals from uninfected erythrocytes, uninfected bovine leukocytes and other hemoparasites, including A. marginale and B. ovata. Although 46 of 71 specimens (64.8%) from grazing cattle were microscopically positive, PCR in this study showed that 64 specimens (88.7%) were positive. Therefore, PCR proves a useful diagnostic tool for detecting T. sergenti-infected cattle. In addition, it is also revealed that PCR was significantly more sensitive than traditional microscopic examination using Giemsa's stain.

Derivation of attenuated porcine epidemic diarrhea virus (PEDV) as vaccine candidate.

The field isolate of porcine epidemic diarrhea virus (PEDV) was serially passaged in Vero cells. The cell passaged PEDV, designated KPEDV-9, was tested for its pathogenicity in the neonatal pigs, immunogenicity and safety in the pregnant sows. The result indicated that KPEDV-9 at the 93rd passage revealed reduced pathogenicity in the neonatal pigs. Pregnant sows inoculated with the attenuated virus showed increased immune responses by ELISA. In addition, delivered piglets were protected from challenge of wild type PEDV. The safety test in pregnant sows indicated that all inoculated animals farrowed the average numbers of litters of piglets. The results of this study supported that the attenuated virus derived from serial passage could be applied as vaccine for protecting suckling piglets against PEDV infection.