Broad-spectrum neutralization of avian influenza viruses by sialylated human milk oligosaccharides: in vivo assessment of 3'-sialyllactose against H9N2 in chickens.

Two sialylated human milk oligosaccharides (SHMOs) 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) were accessed for their possible antiviral activity against six different subtypes of thirteen avian influenza (AI) viruses in vitro. 3'-SL exhibited promising antiviral activity against almost all subtypes of tested AI viruses in hemagglutination inhibition assay, whereas 6'-SL showed activity against few selected H1N1, H1N2, and H3N2 subtype strains. 3'-SL has minimum inhibitory concentration values of 15.62 mM or less in more than half of the viruses examined. 3'-SL also showed effective inactivation of H9N2 Korea isolate (A/Chicken/Korea/MS96/1996) at 12.5 mM concentration in Madin Darby Canine Kidney (MDCK) cell line. Thus, 3'-SL was further studied for in vivo study against H9N2 virus in pathogen free chicken experiment models. In vivo study exhibited improved clinical symptoms on H9N2 infected chickens when treated with 3'-SL. Moreover, treating chickens with 3'-SL resulted in complete elimination of H9N2 viruses within 24 h of virus infection (0.8 HAU of H9N2). Indirect ELISA assay confirmed complete wash-out of H9N2 viruses from the colon after neutralization by 3'-SL without entering the blood stream. These in vivo results open up possible applications of 3'-SL for the prevention of AI virus infections in birds by a simple cleansing mechanism.

Genetic Characteristics and Immunogenicity of Pandemic H1N1 Influenza Virus Isolate from Pig in Korea.

A pandemic influenza A (H1N1) virus strain was isolated from a pig farm in Korea in December 2009. The strain was propagated in and isolated from both the Madin-Darby canine kidney cell line and embryonated eggs. The partial and complete sequences of the strain were identical to those of A/California/04/2009, with >99% sequence similarity in the HA, NA, M, NS, NP, PA, PB1, and PB2 genes. The isolated strain was inactivated and used to prepare a swine influenza vaccine. This trial vaccine, containing the new isolate that has high sequence similarity with the pandemic influenza A (H1N1) virus, resulted in seroconversion in Guinea pigs and piglets. This strain could therefore be a potential vaccine candidate for swine influenza control in commercial farms.

Usefulness of a Helicobacter pylori stool antigen test for diagnosing H. pylori infected C57BL/6 mice.

Among several diagnostic tests, a Helicobacter pylori stool antigen (HpSA) test may offer a useful noninvasive method for diagnosing infection without sacrificing animals. In this study, male C57BL/6 mice (n=6) were infected with H. pylori ATCC 49503 (1×10(8) CFU/mouse) by intragastric inoculation three times at 2-day intervals, and H. pylori infected stool specimens were collected 1, 3, 5, 7, 14, 21 days after infection to assess reliability of the HpSA test. Five of six specimens were positive at 5-21 days after infection, and the sensitivity of the HpSA test was 83.33%. The presence of H. pylori infection was confirmed by the rapid urease test and genomic DNA polymerase chain reaction (PCR), and showed the same results as the HpSA. However, the rapid urease test and genomic DNA PCR are invasive tests and require animal sacrifice to detect H. pylori in gastric biopsy samples. We suggest that an HpSA test kit would be useful and effective for monitoring H. pylori in various laboratory animals, as H. pylori can be easily monitored without sacrificing animals.

A novel reassortant canine H3N1 influenza virus between pandemic H1N1 and canine H3N2 influenza viruses in Korea.

During recent canine influenza surveillance in South Korea, a novel H3N1 canine influenza virus (CIV) that is a putative reassortant between pandemic H1N1 2009 and H3N2 CIVs was isolated. Genetic analysis of eight genes of the influenza virus revealed that the novel H3N1 isolate presented high similarities (99.1-99.9 %) to pandemic influenza H1N1, except for in the haemagglutinin (HA) gene. The HA gene nucleotide sequence of the novel CIV H3N1 was similar (99.6 %) to that of CIV H3N2 isolated in Korea and China. Dogs infected with the novel H3N1 CIV did not show any notable symptoms, in contrast to dogs infected with H3N2 CIV. Despite no visible clinical signs of disease, nasal shedding of virus was detected and the infected dogs presented mild histopathological changes.

A serological survey of canine respiratory coronavirus and canine influenza virus in Korean dogs.

The relationship between canine respiratory coronavirus (CRCoV) and canine influenza virus (CIV) seropositivity in dogs in Korea was examined. Sixty-two of the 483 samples (12.8%) were seropositive for CRCoV by indirect fluorescent antibody (IFA) analysis. Nineteen animals were seropositive for CIV by ELISA out of the 385 samples tested. Serum antibodies for both viruses were detected in 6 of the 483 dogs sampled, suggesting that these viruses are present in dogs in Korea. Although the role of CRCoV in canine infectious tracheobronchitis has not been fully elucidated, co-infection with CIV may synergistically worsen respiratory clinical signs and result in more severe canine tracheobronchitis.

Evaluation of new rapid antigen test for detection of pandemic influenza A/H1N1 2009 virus.

We evaluated the SD Bioline Influenza Ag A/B/A(H1N1) Pandemic test kit and compared it with real-time reverse transcriptase PCR (RT-PCR) for its ability to detect H1N1 2009. The sensitivity and specificity of the test kit for H1N1 2009 were 77% and 100%, respectively.

Validation of egg yolk antibody based C-ELISA for avian influenza surveillance in breeder duck.

Active surveillance for avian influenza virus (AIV) has expanded from chicken to various poultry species including duck. To further effective antibody screening in laying breeder ducks, we validated the egg yolk antibody as alternative source to serum for AIV antibody. Sera and eggs were collected at weekly intervals after two types of AIV vaccination, H5N3 and H9N2. The antibody levels were determined by an agar gel immunodiffusion (AGID) test, haemagglutination inhibition (HI) test and the competitive enzyme-linked immunosorbent assay (C-ELISA). AGID test did not detect antibodies in egg yolk, and the agreement between AGID test and either HI test or C-ELISA in serum was slight and fair based on kappa statistics (kappa value (kappa)< or =0.19 in H5N3 group and kappa< or =0.37 in H9N2 groups). However, there was almost perfect agreement between HI test and C-ELISA (kappa>0.9 in all group). The C-ELISA was as sensitive and specific as the HI test, and could be used as a pre-screening test for the detection of type A avian influenza virus antibody. Comparison was made between egg yolk and serum antibody titers by a regression analysis. A high correlation was observed between serum and yolk antibody titers (r=0.8762 for H5N3 and 0.8914 for H9N2 in HI test; r=1 for H5N3 and 0.9686 for H9N2 in ELISA test), although egg yolk antibodies were detected later and remained lower levels than serum antibodies. In field trials involving 54 duck flocks, the positive rate of egg yolk and serum samples showed agreement for the detection of AIV antibody. We concluded that as an alternative to serum, antibody monitoring of laying breeder duck using egg yolk with C-ELISA is feasible and is recommended.

Comparative measurement of cell-mediated immune responses of swine to the M and N proteins of porcine reproductive and respiratory syndrome virus.

The principal objectives of this study were to develop autologous antigen-presenting cells (APCs) and to characterize the antigen-specific T-cell responses to the M and N proteins of porcine reproductive and respiratory syndrome virus (PRRSV) by using those APCs in outbred pigs. The orf6 and orf7 genes fused with porcine granulocyte-macrophage colony-stimulating factor (GM-CSF) were cloned into the mammalian expression vector to generate two plasmid DNAs, namely, pcDNA3.1-GM-CSF-PRRSV-M and pcDNA3.1-GM-CSF-PRRSV-N. Three of six pigs in two groups were repeatedly immunized with either plasmid DNA construct, and four pigs were used as controls. The recombinant M and N proteins fused with the protein transduction domain (PTD) of the human immunodeficiency virus type 1 transactivator of transcription protein were employed to generate major histocompatibility complex-matched autologous APCs from each pig. The levels of T-cell proliferation and gamma interferon (IFN-gamma) synthesis were compared between pigs immunized with the two plasmid DNAs after stimulation of the peripheral blood mononuclear cells (PBMCs) of each pig with the autologous antigen-presenting dendritic cells and PBMCs. Higher levels of T-cell proliferation and IFN-gamma synthesis were identified in PBMCs isolated from the pigs immunized with pcDNA3.1-GM-CSF-PRRSV-M than in those isolated from the pigs immunized with pcDNA3.1-GM-CSF-PRRSV-N. By way of contrast, serum antibodies were detected only in pigs immunized with pcDNA3.1-GM-CSF-PRRSV-N. However, no T-cell response or antibody production was detected in the control pigs. These results suggest that the M protein of PRRSV is a more potent T cell-stimulating antigen than the N protein. Nevertheless, it should be emphasized that the N protein substantially induces both cellular and humoral immune responses. The newly developed protocol for generating self APCs may prove effective in further efforts to characterize additional PRRSV proteins involved in the induction of cell-mediated immunity.

Elevated serum levels of S100 calcium binding protein A8 (S100A8) reflect disease severity in canine atopic dermatitis.

A monoclonal antibody to canine S100 calcium binding protein A8 (S100A8) was developed to determine the association between S100A8 and the disease severity of canine atopic dermatitis. Serum S100A8 concentrations were studied in dogs with canine atopic dermatitis (n=213) and healthy dogs (n=213). Statistical correlations between these indices and atopic dermatitis activity were established, and dermatitis severity was assessed according to the CADESI score. Serum S100A8 concentrations were measured with an enzyme-linked immunosorbent assay (ELISA). S100A8 serum levels were significantly higher in canine atopic dermatitis patients than in healthy dogs. A strong positive correlation was identified between S100A8 levels and canine atopic dermatitis patients. Our findings suggested that S100A8 is actively involved in the pathogenesis and clinical picture of canine atopic dermatitis.

Evaluation of a competitive ELISA for antibody detection against avian influenza virus.

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.

Development and validation of an immunogold chromatographic test for on-farm detection of PRRSV.

An immunochromatographic test strip was developed to detect porcine reproductive and respiratory syndrome virus (PRRSV). The test uses two gold-labeled monoclonal antibodies: D5 against recombinant nucleocapsid protein (rN) and E9 against recombinant M protein (rM). In the test, PRRSV binds to a mixture of D5 and E9 labeled with colloidal gold; the complexes move through a membrane and are captured by rabbit anti-rM and anti-rN antibodies at a test line, producing a reddish-purple band because of the increased concentration of gold. Unbound monoclonal antibodies move past the test line to be captured by goat anti-mouse antibodies, producing a band at a control line. In samples without PRRSV or with low virus concentration, a band appears only at the control line. A crossover-trial demonstrated that the test strip was highly specific for PRRSV. The test strip detection limit was between 7.8x10(3) and 1.6x10(4) TCID(50)/ml. Analysis of 100 clinical samples indicated that the sensitivity, specificity, and accuracy of the immunochromatographic test strip relative to reverse transcription polymerase chain reaction (RT-PCR) were 97.0, 93.9, and 96.0%, respectively. Because the test is simple and rapid, it can be used by an unskilled person to detect PRRSV in the field.

Evaluation of immunochromatographic assay for serodiagnosis of Brucella canis.

Canine brucellosis is a contagious disease with venereal and oral modes of transmission that produces late abortion in females, epididymides and prostates in males. Diagnosis is difficult because of unstable serum antibody titers that vary from individual to individual as well as between different methods used for their detection. The objective of this work was to evaluate the clinical utility of the immunochromatographic assay (ICA) for serodiagnosis of dogs suspected of having brucellosis, and results were compared with those obtained for hemoculture (HC) and the rapid screening agglutination with 2-mercaptoethanol (2-ME RSAT). The all experimentally infected dogs were positive in ICA, HC and 2-ME RSAT from 5 weeks, 7 weeks, and 3 weeks after infection, respectively. Also, among dogs selected from 10 different breed kennels occurred brucellosis, 24.8%, 39.5% and 39.1% of dogs tested were detected as positive with HC, 2-ME RSAT and ICA, respectively. The kappa value between 2-ME RSAT and ICA was 0.89. The results of this study showed that sensitivity and specificity of the ICA are comparable with those obtained using conventional serological and bacteriological test for brucellosis. In conclusion, the ICA kit provides a handy and accurate tool for the rapid serodiagnosis of canine brucellosis.

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.

Serologic surveillance of swine H1 and H3 and avian H5 and H9 influenza A virus infections in swine population in Korea.

Influenza A is a respiratory disease common in the swine industry. Three subtypes, H1N1, H1N2 and H3N2 influenza A viruses, are currently co-circulating in swine populations in Korea. An outbreak of the highly pathogenic avian influenza H5N1 virus occurred in domestic bird farms in Korea during the winter season of 2003. Pigs can serve as hosts for avian influenza viruses, enabling passage of the virus to other mammals and recombination of mammalian and avian influenza viruses, which are more readily transmissible to humans. This study reports the current seroprevalence of swine H1 and H3 influenza in swine populations in Korea by hemagglutination inhibition (HI) assay. We also investigated whether avian H5 and H9 influenza transmission occurred in pigs from Korea using both the HI and neutralization (NT) tests. 51.2% (380/742) of serum samples tested were positive against the swine H1 virus and 43.7% (324/742) were positive against the swine H3 virus by HI assay. The incidence of seropositivity against both the swine H1 virus and the swine H3 virus was 25.3% (188/742). On the other hand, none of the samples tested showed seropositivity against either the avian H5 virus or the avian H9 virus by the HI and NT tests. Therefore, we report the high current seroprevalence and co-infectivity of swine H1 and H3 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 and H9 influenza transmission to Korean pigs.

One-step immunochromatography assay kit for detecting antibodies to canine parvovirus.

This study was performed to determine the feasibility of using whole serum to detect antibodies to canine parvovirus (CPV) under nonlaboratory conditions and to evaluate the performance characteristics of an immunochromatography assay kit. Precise detection of levels of antibody against CPV in puppies can be used to determine a vaccination schedule, because maternal antibodies frequently result in the failure of protective vaccination, and can also be used to determine the antibody levels of infected puppies. Several methods for the titration of CPV antibodies have been reported, including the hemagglutination inhibition (HI) assay, which is considered the "gold standard." These methods, however, require intricate and time-consuming procedures. In this study, a total of 386 serum specimens were tested. Compared to the HI assay, the rapid assay had a 97.1% sensitivity and a 76.6% specificity (with a cutoff HI titer of 1:80). This single-step assay could be performed rapidly and easily without special equipment. The kit provides a reliable method for detection of anti-CPV antibody where laboratory support and personnel are limited.

Use of an internal control in a quantitative RT-PCR assay for quantitation of porcine epidemic diarrhea virus shedding in pigs.

Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, has caused a devastating enteric disease in the Korean swine industry. Previously, the differences between virulent field PEDV strains and a Vero cell culture adapted PEDV DR13 strain were determined using restriction fragment length polymorphism analysis (RFLP), and PEDV shedding patterns in pigs were reported. In an extension to these studies, an internal control was constructed and quantitative analysis of virus shedding after oral inoculation was established. A parent field PEDV and a cell culture adapted PEDV DR13 were inoculated orally to colostrum-deprived 1-day-old piglets, commercial 2-week-old pigs, and sows (1-5 ml dose, 10(5.8)-10(6.0) TCID(50)/0.1 ml). PEDV shedding was monitored every day and virus levels were measured using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method. In fecal samples from experimentally-inoculated pigs, the level of virus excreted peaked at 2 days after oral inoculation and gradually decreased thereafter. In addition, PEDV from field specimens was quantified using the same RT-PCR assay to determine shedding viral load. This suggests that measurement of PEDV shedding viral load in pigs, by quantitative RT-PCR, may be a useful tool for estimating the transmission potential of PEDV in the swine population.

Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection.

An indirect porcine epidemic diarrhea (PED) virus (PEDV) enzyme-linked immunosorbent assay (ELISA) was compared with the serum neutralization (SN) test by testing 46 samples from experimentally infected sows, 73 samples from naive sows, and 1,024 field sow samples from 48 commercial swine farms of undefined PED status. The SN test and the ELISA were performed using PEDV, KPEDV-9 strain. Viral proteins as a coating antigen of PEDV ELISA were extracted from the cytoplasm of PEDV-infected Vero cells using a non-ionic detergent, Triton X-100, and a simple protocol of PEDV ELISA was followed. The presence of antibodies in these experimental samples was confirmed by SN and ELISA in which the sensitivity of the ELISA was 89.1%, and the corresponding specificity was 94.5%. On testing 1,024 field samples, an overall agreement of 84.2% was generated between the SN and ELISA. This study demonstrates that the PEDV ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against PEDV.

Effect of soluble porcine aminopeptidase N on antibody production against porcine epidemic diarrhea virus.

A few members of coronavirus group I which includes porcine epidemic diarrhea virus (PEDV) use porcine aminopeptidase N (pAPN) as a cellular receptor. Cellular receptors play an important role in virus attachment and entry. However, the low permissiveness of PEDV to APN-expressing porcine cell lines has made it difficult to elucidate the role of pAPN in vitro. The purpose of this study was to prove whether the treatment of soluble pAPN could enhance the antibody production against PEDV in guinea pigs, rabbits and sows. The animals (20 guinea pigs, 8 rabbits and 20 sows) were divided into 4 groups. Group A was injected intramuscularly (IM) with soluble pAPN at one hour before intramuscular infection of PEDV on the same site, group B for IM simultaneous injection of pAPN and PEDV, and group C for IM injection of PEDV only. Group D served as a control of pAPN treatment or PEDV infection. Antibody production against PEDV was compared among groups at regular intervals. The results suggested that pAPN could enhance the antibody production against PEDV in guinea pigs and rabbits which are free of pAPN, however, the effect of pAPN treatment in sows was not clearly elucidated.

Identification of a putative cellular receptor 150 kDa polypeptide for porcine epidemic diarrhea virus in porcine enterocytes.

Porcine epidemic diarrhea virus (PEDV) causes an acute enteritis in pigs of all ages, often fatality for neonates. PEDV occupies an intermediate position between two well characterized members of the coronavirus group I, human coronavirus (HCoV-229E)and transmissible gastroenteritis virus (TGEV) which uses aminopeptidase N (APN), a 150 kDa protein, as their receptors. However, the receptor of the PEDV has not been identified yet. A virus overlay protein binding assay (VOPBA) was used to identify PEDV binding protein in permissive cells. The binding ability of PEDV to porcine APN (pAPN) and the effects of pAPN on infectivity of PEDV in Vero cells were also investigated. VOPBA identified a 150 kDa protein, as a putative PEDV receptor in enterocytes and swine testicle (ST) cells. Further the PEDV binding to pAPN was blocked by anti-pAPN and pAPN enhanced PEDV infectivity in Vero cells. In conclusion, these results suggested that pAPN may act as a receptor of PEDV.