Establishment of a p30-based lateral flow assay for African swine fever virus detection.

African swine fever virus (ASFV) has continuously devastated the global pig industry. Viral persistence causes problems in large pig farms and kills small farms. Timely diagnostic tools play an important role in controlling outbreaks and minimizing losses. In this study, we developed a lateral flow assay to detect ASFV on-site. The VDRG® ASFV Ag Rapid Kit was established using two monoclonal antibodies (mAbs) against the p30 protein. The conjunction pad of the kit was coated with a mixture of the mAb and colloidal gold. This rapid kit was capable of detecting 11.5 ng of antigen and 0.16 HAD50 of virus from samples, in 20 min for the entire procedure. It passed cross-specific tests using common viruses that cause infectious diseases in pigs. ASFV was detected after 4 days in experimental infection in pigs by the kit. The specificity and sensitivity of the kit for clinical samples were 99.88% and 84.52% (93.8% for samples with a Ct value below 30), respectively. Finally, the kit can detect 100% positive herd outbreaks. The VDRG® ASFV Ag Rapid Kit presents a useful point-of-care tool for ASFV detection.

Development of a novel real-time PCR assay targeting p54 gene for rapid detection of African swine fever virus (ASFV) strains circulating in Vietnam.

African swine fever (ASF) continues to cause outbreaks throughout regions of Africa, Europe and Asia. The disease can cause severe morbidity and mortality resulting in serious economic losses. Since there is no vaccine available to control ASF, early detection is critical to contain and control the disease. The aim of this study was to develop a novel real-time PCR assay based on highly conserved ASFV gene E183L (p54). The limit of detection of the assay, VNUA-p54 real-time PCR, was 2.63 copies/reaction and 2 Log10 HAD50 /ml. The VNUA-p54 real-time PCR was able to detect fifteen different ASFV reference strains representing p72 genotypes I, II and V. The assay was specific and did not amplify other swine viruses including CSFV, FMDV, PRRSV and PEDV. The diagnostic sensitivity of the real-time PCR assay was evaluated using 200 field clinical specimens collected from swine farms located in different provinces in Vietnam. The VNUA-p54 real-time PCR assay is an additional tool for ASF diagnostics and can be used in combination with other p72 based ASFV real-time PCR assays as a rapid confirmatory assay.

Comparing recombinant MPB70/SahH and native 20-kDa protein for detecting bovine tuberculosis using ELISA.

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. Test-and-cull protocols and gross pathological examinations of abattoir animals as well as milk pasteurization have been implemented to prevent the spread of tuberculosis from animals to humans worldwide. Despite the importance of precise and rapid diagnostic tests, conventional methods including intradermal skin tests and γ-interferon assays are limited by the high rate of false-negative results for cattle in the late infectious stage and due to laborious and time-consuming procedures. Therefore, antibody detection methods such as enzyme-linked immunosorbent assay (ELISA) are urgently needed to supplement the established approaches and expand the diagnostic window. This study was conducted to develop a bTB ELISA by evaluating recombinant and native proteins and various assay parameters. We produced recombinant MPB70 and SahH (M70S) and a native 20-kDa protein (20K) and optimized the ELISA protocol. The 20K ELISA showed 94.4% sensitivity and 98.2% specificity with an optimal sample-to-positive ratio cut-off of 0.531. The sensitivity and specificity of M70S ELISA were 94.4% and 97.3%, respectively, with an optimal sample-to-negative ratio cut-off of 1.696. Both assays showed acceptable diagnostic efficiency and could be used for bTB diagnosis in combination with established methods for herd screening and to expand the diagnostic window.

O/SEA/Mya-98 lineage foot-and-mouth disease virus was responsible for an extensive epidemic that occurred in late 2018 in Vietnam.

Since late 2018, foot-and-mouth disease (FMD) has reemerged and rapidly swept through pig farms in North and Central Vietnam, despite widespread use of commercial FMD vaccines. To investigate the FMD virus (FMDV) strains responsible for the current epidemics, 40 FMDV samples were collected from 17 provinces during November-December 2018, and the VP1 coding genes were sequenced and analyzed. Phylogenetic analysis and sequence comparisons revealed that all of the reemerging Vietnamese FMDVs belonged to the Mya-98 lineage of the O/Southeast Asia topotype (O/SEA/Mya-98) and shared high nucleotide (99.06-100% identity) and amino acid (97.65-100% identity) sequence similarity with each other. The study results suggested that the reemerging FMDVs originated from local Vietnamese strains. Field viruses had different amino acids in the antigenic sites of VP1 when compared to the strains used in the vaccines. The present study provides an important basis for vaccine selection in the battle against FMD in Vietnam.

Outbreak of African Swine Fever, Vietnam, 2019.

African swine fever is one of the most dangerous diseases of swine. We confirmed the 2019 outbreak in Vietnam by real-time reverse transcription PCR. The causative strain belonged to p72 genotype II and was 100% identical with viruses isolated in China (2018) and Georgia (2007). International prevention and control collaboration is needed.

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.

Prolonged shedding of the canine influenza H3N2 virus in nasal swabs of experimentally immunocompromised dogs.

PURPOSE: The avian origin canine influenza virus H3N2 has been recently isolated and found to be currently in dog population in South Korea and China. The purpose of this study was to clarify the relationship between immunosuppressive glucocorticoids used in veterinary clinical practice and viral shedding pattern of influenza in dogs. MATERIALS AND METHODS: Eight conventional beagle dogs were divided into control infection group and immunocompromised group. Dogs of both groups were infected with H3N2 canine influenza virus (2×10(6.0) EID50/0.1 mL). Dogs in immunocompromised group were given orally 3.0 mg/kg prednisolone for 7 days. Virus shedding was monitored using real-time polymerase chain reaction. After necropsy, histopathologic lesions were compared. RESULTS: We found that immunocompromised dogs exhibited more prolonged (8 days vs. 13 days) and higher magnitude viral shedding than control group (peak titer of viral shedding 4.6 vs. 5.5 EID50). CONCLUSION: Restricted use of immunosuppressive drugs in the clinical setting might help control the rapid spread of H3N2 through local dog populations.

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.

Association between nasal shedding and fever that influenza A (H3N2) induces in dogs.

BACKGROUND: Avian origin canine influenza virus was reported in Korea. The dog to dog contact transmission of the avian origin canine influenza virus (CIV) H3N2 and CIV H3N8 was shown by experimental contact transmission. This study was focused on viral excretion and fever in order to elucidate the epidemiological associations which might be helpful to control the disease transmissions in CIV outbreak in dogs. METHODS: An influenza seronegative 10-week-old Beagle dog was experimentally inoculated with the canine influenza virus A/canine/01/2007, subtype H3N2. Eight hours after inoculation, the infected dog was cohoused with seven uninfected Beagle dogs. Clinical signs including fever were recorded for 14 days post inoculation. RESULTS: The infected dog and four of seven contact dogs in the study showed clinical signs (sneezing, nasal discharge and coughing) during the study. Viral shedding occurred in all of the animals tested and began on 1 to 6 DPI in dogs with clinical signs. Elevated body temperatures above 39.5 °C (geometric mean temperature of 39.86 °C ± 0.49) were observed in all symptomatic dogs. The mean viral titer during fever was 2.99 log EID50/ml, which was significantly higher than the viral titer detected in the non fever. CONCLUSIONS: The data show that contact dogs with a canine influenza infected dog shed different levels of virus in their nasal excretions and demonstrate that clinical signs, including fever, significantly correlate with the viral shedding.

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.

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.

Protective efficacy and immunogenicity of an inactivated avian-origin H3N2 canine influenza vaccine in dogs challenged with the virulent virus.

Transmission of avian-origin influenza A virus (H3N2) to dogs had been reported and since then the H3N2 virus infection across South Korea has been occurred repeatedly in the country's animal clinics and kennels. Dog-to-dog transmission of the virus had also been experimentally demonstrated by direct contact. In this study, immunogenicity and protective efficacy against challenge exposure of the formalin-inactivated H3N2 influenza virus vaccine with a synthetic polymer adjuvant was investigated in dogs. The beagle puppies received two inactivated vaccine injections intramuscularly 2 weeks apart. Serological investigation by a hemagglutination inhibition (HI) test and an ELISA assay indicated that a significant increase in antibody titer was displayed 2 weeks after the second vaccination. Clinical signs, virus shedding and histopathological lesions in the lungs were exhibited in unvaccinated beagle puppies directly challenged through an intranasal route with the virus 2 weeks after the second vaccination. However, the vaccinated animals did not show any clinical signs and showed milder pathological lung lesions and shorter shedding duration with lower loads than controls'. These results indicated that the synthetic polymer-adjuvant avian-origin canine influenza virus (CIV) vaccine had produced antibody response and protection from avian-origin CIV challenge in dogs.

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.

A serological survey of avian origin canine H3N2 influenza virus in dogs in Korea.

Canine H3N2 influenza viruses of avian origin were recently isolated and found to induce disease in dogs. Results of serologic analysis indicate that avian origin canine influenza virus can spread rapidly through local dog populations, which indicates its potential for becoming established in dogs throughout Korea.

Experimental infection of dogs with avian-origin canine influenza A virus (H3N2).

Susceptible dogs were brought into contact with dogs experimentally infected with an avian-origin influenza A virus (H3N2) that had been isolated from a pet dog with severe respiratory syndrome. All the experimentally infected and contact-exposed dogs showed elevated rectal temperatures, virus shedding, seroconversion, and severe necrotizing tracheobronchitis and bronchioalveolitis.

A simple and rapid immunochromatographic strip test for detecting antibody to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome is rapidly gaining worldwide importance as one of the most economically significant diseases of swine. The antibody of Porcine reproductive and respiratory syndrome virus (PRRSV) is detected currently by the combined use of an enzyme-linked immunosorbent assay, serum neutralization test, immunoperoxidase monolayer assay, indirect immunofluorescent antibody test. These methods are time-consuming and require specialized equipment operated by trained technicians. The purpose of this study was to evaluate a simple strip assay (based on a chromatographic and immunogold system) for specific detection of PRRSV antibody in swine sera. This "immunochromatographic strip" test uses Escherichia coli-expressed viral recombinant membrane protein antigen in combination with recombinant nucleocapsid protein as capture protein for detecting antibodies against PRRSV. In this study, the performance of this assay was evaluated with sera from both clinical samples and experimentally infected piglets. Detection by immunochromatographic strip test was compared with detection by a standard, available commercially, indirect enzyme-linked immunosorbent assay and an immunoperoxidase monolayer assay. The immunochromatographic test strip detected antibodies in sera known to contain antibodies to PRRSV in 95.7% sensitivity of samples from pigs infected experimentally and 98.6% sensitivity of clinical serum samples. For sera that did not contain antibodies to PRRSV, the specificity was 97.8% and 98.2% for clinical and experimental serum samples, respectively.

Transmission of avian influenza virus (H3N2) to dogs.

In South Korea, where avian influenza virus subtypes H3N2, H5N1, H6N1, and H9N2 circulate or have been detected, 3 genetically similar canine influenza virus (H3N2) strains of avian origin (A/canine/Korea/01/2007, A/canine/Korea/02/2007, and A/canine/Korea/03/2007) were isolated from dogs exhibiting severe respiratory disease. To determine whether the novel canine influenza virus of avian origin was transmitted among dogs, we experimentally infected beagles with this influenza virus (H3N2) isolate. The beagles shed virus through nasal excretion, seroconverted, and became ill with severe necrotizing tracheobronchitis and bronchioalveolitis with accompanying clinical signs (e.g., high fever). Consistent with histologic observation of lung lesions, large amounts of avian influenza virus binding receptor (SAalpha 2,3-gal) were identified in canine tracheal, bronchial, and bronchiolar epithelial cells, which suggests potential for direct transmission of avian influenza virus (H3N2) from poultry to dogs. Our data provide evidence that dogs may play a role in interspecies transmission and spread of influenza virus.

Evaluation of a rapid immunodiagnostic test kit for rabies virus.

A rapid immunodiagnostic test kit for rabies virus detection was evaluated using 51 clinical samples and 4 isolates of rabies virus. The quick detection of rabies virus under field conditions may be helpful in determining if post-exposure prophylaxis is needed, thereby avoiding unnecessary treatments, as well as undue economic burden. There are several widely used diagnostic methods for rabies, including fluorescent antibody tests, reverse transcription polymerase chain reaction, and electron microscopy; however, these methods include time-consuming, intricate, and costly procedures. The rapid immunodiagnostic test was able to detect rabies virus in clinical samples, including brain tissue and saliva, in addition to 10(3.2) 50% lethal dose (LD(50))/mL cell-adapted rabies virus. The assay was not cross-reactive with non-rabies virus microbes. When the performance of the rapid immunodiagnostic test was compared to a fluorescent antibody test, the rapid immunodiagnostic test had a sensitivity of 91.7% and specificity of 100% (95.8% CI).