Bong Kyun Park

Differential Detection of Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus by Duplex RT-PCR

Transmissible gastroenteritis (TGE) and porcine epidemic diarrhea (PED) are highly contagious enteric diseases of piglets. The clinical signs of these diseases are very similar and include watery, yellowish diarrhea. Thus, the effective differential detection of TGE virus and PED virus is required. In the present study, a duplex reverse transcription-polymerase chain reaction (RT-PCR) was established for the differential detection of TGE and PED viruses. The primers were designed for the S gene of each virus. RNA was extracted from the intestines and stool samples that were collected from the swine with diarrhea. The RT-PCR test could detect both TGE and PED viruses with 2 TCID50/200 μl. Among 90 clinical samples, 7 TGE viruses and 2 PED viruses were detected by the duplex RT-PCR. This duplex RT-PCR may be a useful diagnostic method for the rapid, specific, and sensitive differential detection of TGE and PED viruses using clinical samples.

Detection of Porcine Circovirus Type 2 in Feces of Pigs with or without Enteric Disease by Polymerase Chain Reaction

To establish the sensitive polymerase chain reaction(PCR) method and detect porcine circovirus type 2 (PCV2) from intestines and feces of commercial swine herds with or without enteric disease, intestinal samples from 68 pigs and 29 fecal samples from commercial swine farms were collected. A primer set, forward primer 5′-GAAGAATGGAAGAAGCGG-3′ and reverse primer 5′-CTCACAGCAGTAGACAGGT-3′, could detect the virus at a concentration as low as 2 infectious virions per milliliter under controlled conditions using PK-15 cell-adapted PCV2. The genomic nucleotide sequences of open reading frame 1 (ORF1) PCR products from fecal samples were found to have complete homology with other PCV2s deposited in the GenBank database. Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) as the other enteric pathogens were also investigated by performing duplex reverse transcription-PCR (RT-PCR). Among 63 pigs with clinical enteric disease, 18 PCV2s (14 from intestines and 4 from feces), 7 TGEVs from intestines, and 18 PEDVs (14 from intestines and 4 from feces) were detected by PCR and the duplex RT-PCR. In 34 pigs (14 from intestines and 20 from feces) without clinical enteric disease, only PCV2 was detected in 19 pigs (3 from intestines and 16 from feces). Both PEDV and PCV2 were found in 6 pigs with clinical enteric disease. Among 15 PCV2 samples that were PCR-positive, 4 were culture-positive at passage level 3 in PK-15 cells. These results reveal that PCV2 is shed through the feces of pigs without clinical enteric disease, which suggests the potentiality of the fecal–oral transmission of PCV2 in feces.

Evaluation of an Indirect Fluorescent IgM Antibody Test for the Detection of Pigs with Recent Infection of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused major economic losses to swine producers in recent years due to severe reproductive failure in pregnant sows and respiratory disease in young pigs. Antibodies to PRRSV in swine sera can be tested by various serologic methods including immunoperoxidase monolayer assay, 12 indirect fluorescent antibody (IFA) test, 14 enzymelinked immunosorbent assay, and serum neutralization (SN) test 3,15 However, none of the tests can adequately determine the time of infection. Development of a serologic test that could estimate the infection time or identify a recent infection would be valuable to swine farmers when introducing pigs from a PRRSV-infected farm. Demonstration of specific IgM antibodies has been a useful marker for recent infection to different viral infections. Virusspecific IgM antibody has been detected regularly in primary infection with herpes simplex virus, rubella virus, cytomegalovirus, hepatitis A and B viruses and pseudorabies viTUS. For example, IgM antibody to pseudorabies virus in pigs was first detected in serum 6 days postinoculation (PI), reached the highest titers 10 days PI, and was detectable up to 35 days PI. The objectives of this study were to evaluate an IFA test for the detection of IgM antibody specific to PRRSV and to compare the detection of IgM antibody and the presence of virus in sera from pigs with or without known days PI. PRRSV MN-1b was used for the IFA test, and the MN-H and MN-W isolates were used for animal inoculation. A permissive clone (MARC-145) of the African green monkey kidney (MA-104) cell line was used for virus propagation, virus isolation, and serology. The MARC-145 cells were maintained in Eagle’s minimum essential medium supplemented with 3% fetal bovine serum, 0.15% sodium bicarbonate, and antibiotics. Sera used were collected at known intervals from 3-weekold pigs following experimental infection with PRRSV MNH. Sera from sows infected experimentally with PRRSV MN-W or MN-H were also used. Field serum samples were collected from pigs of known age in 2 different farms with endemic PRRSV infection, and the samples were transported on the same day to the laboratory for virus isolation and serology. Virus isolation from sera was performed using 24-well microplates by inoculating 0.05 ml of the undiluted sera to 1 ml of MARC-145 cell suspensions (2 x 10 cell/ml). Infected monolayers were observed daily for cytopathic changes

Isolation of H3N2 swine influenza virus in South Korea

Swine influenza is a significant respiratory disease causing occasional reproductive problems in nïve swine herds. Although different subtypes of swine influenza virus (SIV) have been implicated in clinical outbreaks of swine influenza in Asian countries, no virus isolation has been made to identify SIV of subtypes other than the H1N1 subtype in the Korean swine population. In December 1998, an outbreak of acute respiratory disease was identified in a commercial swine farm located in the Kyunggi province of South Korea. A causative agent, which agglutinated rooster red blood cells, was detected from the lungs of 3 piglets from the index herd and was determined to be type A influenza virus using a commercial influenza virus typing kit. Hemagglutination activity (HA) of the isolates was completely inhibited by a swine antiserum against a recent US H3N2 SIV isolate (A/Sw/IA/41305/1998) but not by H1N1 swine antiserum (A/Sw/IA/1979). Reverse transcription–polymerase chain reaction (RT-PCR) revealed all 3 isolates were H3 SIV subtypes. Sequence analysis of hemagglutinin gene PCR products supported the belief that the Korean H3 SIV isolates were genetically similar to the known mammalian H3 influenza viruses. This is the first report on a clinical outbreak of swine influenza caused by the H3N2 virus in Korea.

Evaluation of the effects of nursery depopulation on the persistence of porcine reproductive and respiratory syndrome virus and the productivity of 34 farms

Nursery depopulation has been described as an effective strategy for improving the performance of weaned pigs. In order to assess whether the strategy was effective under a wide range of conditions, a study was carried out on 34 farms in the USA. Four groups with different depopulation protocols were designed on the basis of the location of the depopulated facility (on site vs off site) and the period for which the nursery remained empty (seven days vs 14 days). The changes in average daily liveweight gain, percentage mortality, feed efficiency and treatment cost per pig produced were assessed 12 months before and after nursery depopulation. The ability to eliminate porcine reproductive and respiratory syndrome (PRRs) virus was examined by indirect fluorescent antibody testing of the nursery pigs. Significant improvements (P<0.0001) were detected in both average daily gain and percentage mortality after depopulation when the differences within an individual group were analysed, but no significant differences (P>O.14) were observed between the study groups. Serological testing indicated that antibodies to PRRS virus were still present in 14 of the 34 farms after depopulation.

Indirect fluorescent IgM antibody response of pigs infected with porcine reproductive and respiratory syndrome syndrome virus

IgG and IgM antibody responses were examined by an indirect fluorescent antibody method in pigs following inoculation with different porcine reproductive and respiratory syndrome virus (PRRSV) isolates or a vaccine virus. Viremia was also examined in the pigs. The IgG antibody was first detected between 9 and 14 days post inoculation (PI) and maintained high titers for at least 7 weeks PI. No change in IgG antibody titers was observed when the pigs were reinoculated with PRRSV 35 days PI. IgM antibody was detected between 5 and 28 days PI in the pigs. Reinoculation at 35 days PI caused a short term rise of IgM antibody. Virus was isolated from sera collected between 2 and 21 days PI. The IgM antibody was detected regularly in sera collected during viremia and up to 1-2 weeks after the viremic periods. These results suggest that pigs with detectable IgM antibody are probably pigs with recent infection and that routine testing of IgM antibody in purchased breeding pigs from seropositive farms may be useful in identification of pigs with recent infection.

Genetic Characterization of Canine Rotavirus Isolated from a Puppy in Korea and Experimental Reproduction of Disease

Canine rotavirus was isolated from feces of a Korean Jindo dog with mild diarrhea, and the isolate was genetically characterized. Rotaviral antigen was detected in the feces using a commercial rotavirus antigen detection kit and cytopathic effects were observed in a cell line inoculated with the feces. The virus isolate (GC/KS05) was identified as subtype G3P[3] using reverse transcription polymerase chain reaction (RT-PCR). The strain displayed 98% and 90% identity with the VP7 genes of a canine rotavirus isolate (RV52/96) from Italy and the simian rotavirus strain (RRV) respectively. However, the GC/KS05 isolate exhibited only 83% and 82% identity, respectively, with the G3 serotype canine strains, RV198/95 and K9. Phylogenetic analysis of the VP7 and VP4 genes of GC/KS05 strain led to the classification of VP7 in a different cluster than other canine rotavirus VP7 genes, and VP4 within the cluster of canine rotavirus VP4 genes. The Korean isolate was thus more closely related to the RV52/96 isolate than the other isolates for which sequence data is available. Detailed analysis of the VP7 region revealed 6 amino acid variations between the new isolate and RV52/96. After 5 passages in cell culture, the GC/KS05 strain remained pathogenic for young pups, in which inoculation resulted in diarrhea and virus shedding in the feces.

Gouleako and Herbert Viruses in Pigs, Republic of Korea, 2013

Several viruses in the family Bunyaviridae are pathogenic to animals and cause vector-borne zoonoses. In 2013, investigation of cause of death of 9 pigs on 1 farm in the Republic of Korea found infection with Gouleako and Herbert viruses. Subsequent investigation revealed high prevalence of these viruses among pigs throughout the country.