Short chain fatty acids facilitate protective immunity by macrophages and T cells during acute fowl adenovirus-4 infection.

Short chain fatty acids (SCFAs) are major gut metabolites that are involved in the regulation of dysfunction in immune responses, such as autoimmunity and cytokine storm. Numerous studies have reported a protective action of SCFAs against infectious diseases. This study investigated whether SCFAs have protective effect for immunity during fowl adenovirus-4 (FAdV-4) infection. We examined whether SCFA mixture (acetate, propionate, and butyrate) administration could protect against intramuscular challenge of a virulent viral strain. SCFA treatment promoted MHCII-expressing monocytes, the active form of T cells, and effector molecules in both peripheral and lymphoid tissues. It also boosted the production of immune molecules involved in pathogen elimination by intraepithelial lymphocytes and changed the intestinal microbial composition. We suggest that gut metabolites influence the gut microbial environment, and these changes stimulate macrophages and T cells to fight against the intramuscular challenge of FAdV-4.

Genetic and immunological characterization of commercial infectious bronchitis virus vaccines used in Korea.

The aim of the study was to investigate the genetic and immunogenic features of commercial vaccines against infectious bronchitis virus (IBV), which is a major contagious pathogen of poultry. Although numerous vaccines have been developed based on the genetic characteristics of field strains, the continual emergence of variants decreases vaccine efficacy and cross-protection. To address this issue, we compared the S1 gene sequences of three IBV vaccines commercially available in Korea with those of various field isolates. Phylogenetic analysis showed that the vaccine strains clustered into two different lineages. Comparison of commercial vaccines with their parental viruses showed that most of the genetic variability occurred around hypervariable regions (HVRs). Conversely, antigenic stimulation with commercial vaccines and regional IBV variants was not sufficient to alter major immune cell phenotypes. Our study suggests that vaccines should be selected carefully based on their genetic background because genetic variability can affect the antigenicity of vaccines and host immune responses.

Transcriptome analysis of primary chicken cells infected with infectious bronchitis virus strain K047-12 isolated in Korea.

Infectious bronchitis virus (IBV), an avian coronavirus, is highly contagious. Chickens with IBV infection develop acute pathogenesis in multiple organs, including the respiratory and urogenital tracts. Frequent recombination in the spike (S) glycoprotein gene has made vaccine strategies ineffective. To understand IBV pathogenesis, we analyzed the genetic distance between Korean IBV isolates and other coronaviruses, including SARS-CoV-2. To obtain comprehensive information about early immune responses such as innate cytokine production and associated immune regulation during IBV infection, we infected primary chicken embryonic kidney cells and performed transcriptome analysis. We observed that the functional pathways of innate immunity are regulated and confirmed expression of genes that coordinate early immune responses. Understanding the immune profile of the host cell may assist in vaccine development.

Role of inflammasome regulation on immune modulators.

Inflammatory responses are essential in eliminating harmful substrates from damaged tissue and inducing recovery. Several cytokines participate in and facilitate this response. Certain cytokines such as interleukin (IL)-1ß and IL-18 are initially produced in precursor form in response to toll-like receptor (TLR) ligands and undergo maturation by inflammasomes, which are cytosolic multi-protein complexes containing nucleotide-binding oligomerization domain (NOD)-containing protein 2-like receptors (NLRs). Immune modulators targeting inflammasomes have been investigated to control inflammatory diseases such as metabolic syndrome. However, most immune modulators possessing anti-inflammasome properties attenuate production of other cytokines, which are essential for host defense. In this review, we analyzed the effect of anti-inflammasome agents on the production of cytokines which are not regulated by inflammasome and involving in initial immune responses. As a result, the inflammasome inhibitors are put into three categories: non-effector, stimulator, or inhibitor of cytokine production. Even the stimulator of cytokine production ameliorated symptoms resulting from inflammasome activation in mouse models. Thus, we suggest ideal immune modulators targeting inflammasomes in order to enhance cytokine production while inhibiting cytokine maturation.

Triggers of NLRC4 and AIM2 inflammasomes induce porcine IL-1ß secretion.

Pigs are an important livestock and serve as a large animal model due to physiological and anatomical similarities with humans. Thus, components of the porcine immune system such as inflammasomes need to be characterized for disease control, vaccination, and translational research purposes. Previously, we and others elucidated porcine nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family Pyrin domain containing 3 (NLRP3) inflammasome activation. However, until now, porcine NLR family caspase recruitment domain (CARD)-containing 4 (NLRC4) and absent in melanoma 2 (AIM2) inflammasomes have been not well studied. In this study, we treated well defined NLRC4 and AIM2 inflammasome triggers to porcine peripheral blood mononuclear cells (PBMCs) and murine bone-marrow derived macrophages (BMDMs) and observed interleukin (IL)-1ß maturation as a readout of inflammasome activation. NLRC4 (flagellin) and AIM2 (dsDNA) triggers led to IL-1ß secretion in both porcine PBMCs and mice macrophages. In addition, porcine and mouse NLRC4 and AIM2 inflammasomes responded differently to NLRP3 inhibitors. Bacterial inflammasome triggers, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Escherichia coli, also induced IL-1ß secretion in porcine PBMCs. Taken together, we suggest that known triggers of NLRC4 and AIM2 inflammasomes in mice induce IL-1ß secretion in porcine PBMCs.

Genetic Diversity of Avian Paramyxovirus Type 6 Isolated from Wild Ducks in the Republic of Korea.

Eleven avian paramyxovirus type 6 (APMV-6) isolates from Eurasian Wigeon ( n=5; Anas penelope), Mallards ( n=2; Anas platyrhynchos), and unknown species of wild ducks ( n=4) from Korea were analyzed based on the nucleotide (nt) and deduced amino acid sequences of the fusion (F) gene. Fecal samples were collected in 2010-14. Genotypes were assigned based on phylogenetic analyses. Our results revealed that APMV-6 could be classified into at least two distinct genotypes, G1 and G2. The open reading frame (ORF) of the G1 genotype was 1,668 nt in length, and the putative F0 cleavage site sequence was 113PAPEPRL119. The G2 genotype viruses included five isolates from Eurasian wigeons and four isolates from unknown waterfowl species, together with two reference APMV-6 strains from the Red-necked Stint ( Calidris ruficollis) from Japan and an unknown duck from Italy. There was an N-truncated ORF (1,638 nt), due to an N-terminal truncation of 30 nt in the signal peptide region of the F gene, and the putative F0 cleavage site sequence was 103SIREPRL109. The genetic diversity and ecology of APMV-6 are discussed.

First identification and phylogenetic analysis of equine hepacivirus in Korea.

Non-primate hepacivirus (NPHV) corresponds a group of isolates recently characterized in horses and dogs that present similar genomic organization and are closely related to hepatitis C virus. Since canine hapacivirus, NPHV identified in dogs, was first discovered in dogs in the United States, equine hepacivirus (EqHV, NPHV identified in horses) has been identified in horses in several countries. However, no epidemiological studies have investigated EqHV in horses in Korea. In this study, a total of 74 (n=74) serum samples collected from horses in four regions of Korea were tested for EqHV RNA using nested RT-PCR. Overall, 14 samples were identified as positive (18.9%) and further analyzed according to gender, age, breed, and region. There were high positive rates in males, young horses, and Thoroughbreds; however, these rates differed regionally. Sequencing of the partial NS3 region of 12 samples and the polyprotein encoding regions of two samples positive for EqHV RNA revealed that the Korean EqHV isolates shared approximately 85.3-99.6% and 97.7-100% homology at the nucleotide and deduced amino acid level, respectively. Phylogenetic analysis revealed that the partial NS3 genes clustered with sequences previously reported as NPHV. Notably, sequences of EqHV detected in horses in the same region showed sequence divergence. The sequences of the polyprotein encoding region of two representative EqHVs shared 83.9% and 95.7% homology with each other at the nucleotide and deduced amino acid level, respectively. Comparison of the sequences of polyprotein encoding regions of Korean EqHV isolates and hepaciviruses from different hosts revealed that the NS3 and NS5B regions were most conserved among hepaciviruses. The results of the present study demonstrate that there is a high positive rate of EqHV in Korea and provide significant information regarding the geographical distribution and genetic variability of Korean EqHV isolates that will help improve global epidemiology of EqHV.

Identification and characterization of avian hepatitis E virus genotype 2 from chickens with hepatitis-splenomegaly syndrome in Korea.

A new avian hepatitis E virus (HEV) GI-B was identified in broiler breeders with hematomas, liver rupture, and splenomegaly, along with excessive abdominal fat, in Korea. Previously, genotype 1 had been identified in avian HEV strains in Korea. Complete sequence analyses revealed that the new avian HEV clustered in genotype 2, which has been identified in the USA and Spain; the GI-B isolate was closely related to the USA prototype avian HEV isolated from a chicken with hepatitis-splenomegaly syndrome. Although some HEV genotypes show a geographical distribution pattern, the discovery of genotype 2 in addition to genotype 1 in Korea suggests that the geographical grouping might be reconsidered. These findings have important implications for understanding the global epidemiology and spread of avian HEV.

Construction of an infectious cDNA clone of genotype 1 avian hepatitis E virus: characterization of its pathogenicity in broiler breeders and demonstration of its utility in studying the role of the hypervariable region in virus replication.

A full-length infectious cDNA clone of the genotype 1 Korean avian hepatitis E virus (avian HEV) (pT11-aHEV-K) was constructed and its infectivity and pathogenicity were investigated in leghorn male hepatoma (LMH) chicken cells and broiler breeders. We demonstrated that capped RNA transcripts from the pT11-aHEV-K clone were translation competent when transfected into LMH cells and infectious when injected intrahepatically into the livers of chickens. Gross and microscopic pathological lesions underpinned the avian HEV infection and helped characterize its pathogenicity in broiler breeder chickens. The avian HEV genome contains a hypervariable region (HVR) in ORF1. To demonstrate the utility of the avian HEV infectious clone, several mutants with various deletions in and beyond the known HVR were derived from the pT11-aHEV-K clone. The HVR-deletion mutants were replication competent in LMH cells, although the deletion mutants extending beyond the known HVR were non-viable. By using the pT11-aHEV-K infectious clone as the backbone, an avian HEV luciferase reporter replicon and HVR-deletion mutant replicons were also generated. The luciferase assay results of the reporter replicon and its mutants support the data obtained from the infectious clone and its derived mutants. To further determine the effect of HVR deletion on virus replication, the capped RNA transcripts from the wild-type pT11-aHEV-K clone and its mutants were injected intrahepatically into chickens. The HVR-deletion mutants that were translation competent in LMH cells displayed in chickens an attenuation phenotype of avian HEV infectivity, suggesting that the avian HEV HVR is important in modulating the virus infectivity and pathogenicity.

Genetic characterization of three novel chicken parvovirus strains based on analysis of their coding sequences.

Chicken parvovirus (ChPV) is one of the causative agents of viral enteritis. Recently, the genome of the ABU-P1 strain of ChPV was fully sequenced and determined to have a distinct genomic composition compared with that of vertebrate parvoviruses. However, no comparative sequence analysis of coding regions of ChPVs was possible because of the lack of other sequence information. In this study, we obtained the nucleotide sequences of all genomic coding regions of three ChPVs by polymerase chain reaction using 13 primer sets, and deduced the amino acid sequences from the nucleotide sequences. The non-structural protein 1 (NS1) gene of the three ChPVs showed 95.0 to 95.5% nucleotide sequence identity and 96.5 to 98.1% amino acid sequence identity to those of NS1 from the ABU-P1 strain, respectively, and even higher nucleotide and amino acid similarities to one another. The viral proteins (VP) gene was more divergent between the three ChPV Korean strains and ABU-P1, with 88.1 to 88.3% nucleotide identity and 93.0% amino acid identity. Analysis of the putative tertiary structure of the ChPV VP2 protein showed that variable regions with less than 80% nucleotide similarity between the three Korean strains and ABU-P1 occurred in large loops of the VP2 protein believed to be involved in antigenicity, pathogenicity, and tissue tropism in other parvoviruses. Based on our analysis of full-length coding sequences, we discovered greater variation in ChPV strains than reported previously, especially in partial regions of the VP2 protein.

Baculovirus expression of the avian paramyxovirus 2 HN gene for diagnostic applications.

Avian paramyxovirus 2 (APMV-2) infections are associated with respiratory diseases in poultry worldwide. The hemagglutination inhibition (HI) test is a useful tool for surveillance and monitoring of this virus. In this study, full-length hemagglutinin (HN) gene of APMV-2 was chemically synthesized based on its published sequence, cloned and expressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. The biological, antigenic and immunogenic properties of the expressed protein were evaluated to assess its ability to produce diagnostic reagents for HI testing. Recombinant APMV-2 HN protein showed two distinct bands with molecular masses of 64 and 75kDa, which showed hemagglutination (HA) and neuraminidase activities, respectively. The recombinant HN (rHN) protein extracted from infected cells produced high HA titers (2(13) per 25µL). HA activity of the protein was inhibited by APMV-2 antiserum, although there were weak cross reactions with other APMV serotype antisera. The rHN protein induced high titers of APMV-2-specific antibodies in immunized chickens based on the HI test. These results indicated that recombinant APMV-2 HN protein is a useful alternative to the APMV-2 antigen in HI assays.

Sequencing, phylogenetic analysis, and potential recombination events of infectious bronchitis viruses isolated in Korea.

The S2 glycoprotein and membrane (M) protein genes and S1 glycoprotein and nucleocapsid (N) genes of 11 Korean infectious bronchitis virus (IBV) isolates were amplified by RT-PCR, cloned, and sequenced. The resultant nucleotide sequences were compared with the published sequences for non-Korean IBV strains. Korean IBV isolates formed two independent subclusters within the phylogenetic tree based on S2 glycoprotein gene sequences. However, four and two different clusters were formed in the phylogenetic tree based on S1 glycoprotein and M gene sequences, respectively. In particular, Korean IBV K446-01 and K203-02 strains appeared to be the result of recombination between an indigenous Korean IBV strains and a vaccine strain (Massachusetts serotype) currently used in Korea. The recent IBV isolate, K026-10, formed a new subgroup that was closely related to traditional Korean IBV group in a phylogenetic tree based on the S1 and S2 genes, but it was grouped into the traditional Korean IBV cluster in a phylogenetic tree based on the M and N genes. Our data show that field IBVs in Korea are continuing to evolve and that vaccine strains might actually play a critical role in the appearance of new IBV strains via recombination in the field.

Serological prevalence, genetic identification, and characterization of the first strains of avian hepatitis E virus from chickens in Korea.

Avian hepatitis E virus (avian HEV) is associated with hepatitis-splenomegaly (HS) syndrome or big liver and spleen disease in chickens. At least three genotypes of avian HEV have been identified from chickens worldwide. A total of 297 serum samples collected from chickens in 35 flocks in Korea were tested for avian HEV antibody with an enzyme-linked immunosorbent assay. The results showed that approximately 57 % of chicken flocks and 28 % of chickens from Korea were positive for antibodies to avian HEV. Thirteen pooled fecal samples from chickens were tested for avian HEV RNA by RT-PCR, and three fecal samples were positive. The partial helicase and capsid genes of the Korean avian HEV isolates were determined, and sequence analyses revealed that the Korean avian HEV isolates were clustered together and closely related to the genotype 1 avian HEV from Australia. The complete genomic sequence of a Korean avian HEV strain HH-F9 from a broiler breeder was determined, and shown to be 6,653 nt in length, excluding the poly (A) tail, which is 1 nt shorter than the prototype avian HEV from chicken with HS syndrome in the United States. Compared to the full-length sequences of other 5 known avian HEV strains worldwide, the Korean avian HEV shared approximately 83-97 % nucleotide sequence identity. The finding that Korean avian HEV belongs to genotype 1 avian HEV which was previously identified only from chickens in Australia has significant implication in understanding the global epidemiology of avian HEV.

Construction of an infectious cDNA clone of avian hepatitis E virus (avian HEV) recovered from a clinically healthy chicken in the United States and characterization of its pathogenicity in specific-pathogen-free chickens.

A genetically distinct strain of avian hepatitis E virus (avian HEV-VA strain) was isolated from a healthy chicken in Virginia, and thus it is important to characterize and compare its pathogenicity with the prototype strain (avian HEV-prototype) isolated from a diseased chicken. Here we first constructed an infectious clone of the avian HEV-VA strain. Capped RNA transcripts from the avian HEV-VA clone were replication-competent after transfection of LMH chicken liver cells. Chickens inoculated intrahepatically with RNA transcripts of avian HEV-VA clone developed active infection as evidenced by fecal virus shedding, viremia, and seroconversion. To characterize the pathogenicity, RNA transcripts of both avian HEV-VA and avian HEV-prototype clones were intrahepatically inoculated into the livers of chickens. Avian HEV RNA was detected in feces, serum and bile samples from 10/10 avian HEV-VA-inoculated and 9/9 avian HEV-prototype-inoculated chickens although seroconversion occurred only in some chickens during the experimental period. The histopathological lesion scores were lower for avian HEV-VA group than avian HEV-prototype group in the liver at 3 and 5 weeks post-inoculation (wpi) and in the spleen at 3 wpi, although the differences were not statistically significant. The liver/body weight ratio, indicative of liver enlargement, of both avian HEV-VA and avian HEV-prototype groups were significantly higher than that of the control group at 5 wpi. Overall, the avian HEV-VA strain still induces histological liver lesions even though it was isolated from a healthy chicken. The results also showed that intrahepatic inoculation of chickens with RNA transcripts of avian HEV infectious clone may serve as an alternative for live virus in animal pathogenicity studies.

Mice as potential carriers of infectious bursal disease virus in chickens.

To investigate the role of mice as potential carriers of infectious bursal disease virus (IBDV), three mice were inoculated with a very virulent strain of IBDV and allowed to have contact with three uninoculated mice. Faeces, intestine and pooled liver and spleen collected from inoculated mice 12 and 24 h post-inoculation were positive for IBDV by reverse transcriptase-polymerase chain reaction (PCR)-nested PCR (RT-PCR-nPCR). IBDV was detected by RT-PCR-nPCR in 3/3 samples of intestine and 2/3 samples of pooled liver and spleen from uninoculated in-contact mice at 24 h after exposure. Chickens developed clinical signs of IBD and died when inoculated with faecal extracts prepared from mice 24 h after inoculation with IBDV. Bursae were atrophied and positive for IBDV by RT-PCR-nPCR.

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-gamma.

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-gamma were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-gamma (ChIFN-gamma) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killedvaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-gamma did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.

Sequence analysis of the ORF 7 region of transmissible gastroenteritis viruses isolated in Korea.

Three (KT2, 133, and DAE) transmissible gastroenteritis viruses (TGEVs) were isolated from pigs suspected of having TGE in Korea. One, KT2 (KT2-L), was passaged 128 times (KT2-H) in swine testicular cells. The open reading frame 7 (ORF 7) gene from each of the four TGEVs (KT2-L, KT2-H, 133, and DAE), which is located at the 3' end of the TGEV genome, was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). Amplified PCR products were cloned, sequenced, and compared with published sequences of non-Korean TGEV strains. Differences in replication and cytopathic effect (CPE) between the KT2-L and KT2-H strains in swine testicular cells were investigated. Korean TGEV field strains had 94.8-99.6% nucleotide and 92.1-98.7% amino acid sequence similarity with each other, and 87.8-100.0% nucleotide and 84.2-100.0% amino acid sequence similarity with non-Korean TGEV strains. Compared to the original KT2-L strain, the KT2-H strain differed by 2.2 and 3.9% in nucleotide and amino acid sequences, respectively. Specifically, the KT2-H had six nucleotide and two amino acid deletions compared to the original KT2-L strain. In phylogenetic analysis of the ORF 7 gene, Korean TGEV strains were clustered into two groups. One group (KT2-L, KT2-H, 133) was related to TGEV strains isolated in Japan. Another Korean TGEV isolate (DAE) was related to a strain from China and one from the USA. The Korean TGEV isolates appear to have evolved from a separate lineage of TGEV strain. Differences in growth rate and CPE between the KT2-L and KT2-H strains were discovered in swine testicular cells (STCs). The KT2-H strain exhibited a higher replication rate than KT2-L and produced a CPE distinctly different from that of the KT2-L strain.

Sequence analysis of the S1 glycoprotein gene of infectious bronchitis viruses: identification of a novel phylogenetic group in Korea.

Twelve Korean infectious bronchitis viruses (IBVs) were isolated in the field from chickens suspected of being carriers of infectious bronchitis between 2001 and 2003. The S1 glycoprotein genes of these IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RTPCR) and analyzed by restriction fragment length polymorphism (RFLP) analysis. These Korean IBV isolates were classified into three groups according to their RFLP patterns obtained using the restriction enzyme HaeIII. Half of the twelve isolates were similar to the KM91 RFLP pattern, which is a common pattern in Korea. Three more isolates were related to the Arkansas strain pattern, but with some unique variations. The other three viruses showed variant RFLP patterns. For a comparison with the published sequences for non-Korean IBV strains, amplified PCR products from the twelve isolates were cloned and sequenced. The Korean IBV field isolates had 71.2-99.7% nucleotide sequence homology with each other and 45.9-80.7% nucleotide sequence homology with non-Korean IBV strains. With respect to the deduced amino acid sequence, the Korean IBV isolates had 71.5-99.3% similarity with each other and 44.9-80.3% similarity with non-Korean IBV strains. Phylogenetic tree analysis revealed that some of the IBV isolates appear to belong to a new group, different from the non-Korean IBV strains or from previously isolated Korean IBV strains. Specifically, the new Korean IBV isolates K10217-03, K3-3 and K1255-03 represented a separate group. These findings suggest that the Korean IBVs appear to be continuously evolving.

Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-gamma on protective immunity by a DNA vaccine against IBDV in chickens.

This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-gamma (ChIFN-gamma) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-ã was constructed. Twice at 2-week intervals, two-week-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-gamma were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-gamma was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-gamma groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-gamma had no significant effect.

Comparison of tissue and fluid samples for the early detection of canine distemper virus in experimentally infected dogs.

The clinical utility of various specimens was examined for the early diagnosis of canine distemper (CD). Seven healthy dogs at 17 weeks of age were experimentally infected with a field isolate of canine distemper virus. The RT-PCR was carried out to detect CDV NP gene. Dogs showed mild fever and leukopenia, however, typical clinical signs of CD were not seen through the experimental period. CDV amplicons were detected more, earlier and for longer period in the conjunctival swabs than in the other samples employed. These results suggested that conjunctival swab samples, which are easy to obtain and non-invasive, would be the most suitable and practical specimen for the early antemortem diagnosis of CDV infection.