Search

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.

We present here a rapid immunochromatographic assay for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV) antigen in the nasal swabs of dromedary camels. The assay is based on the detection of MERS-CoV nucleocapsid protein in a short time frame using highly selective monoclonal antibodies at room temperature. The relative sensitivity and specificity of the assay were found to be 93.90% and 100%, respectively, compared to that of the UpE and open reading frame 1A (Orf1A) real-time reverse transcriptase PCR (RT-PCR). The results suggest that the assay developed here is a useful tool for the rapid diagnosis and epidemiological surveillance of MERS-CoV infection in dromedary camels.

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 (κ) ≤ 0.19 in H5N3 group and κ ≤ 0.37 in H9N2 groups). However, there was almost perfect agreement between HI test and C-ELISA (κ > 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.

An immunochromatograhy (IC) based infectious bursal disease virus (IBDV) detection kit, which employed two anti-IBDV VP2 monoclonal antibodies, was evaluated for rapid diagnosis of infectious bursal disease virus (IBD). The detection limit of the IC kit for IBDV was 10 3.1 to 10 3.9 EID??/mL, indicating that the IC kit detected IBDV sensitively as same as double antigen capture ELISA but less than a RT-PCR assay. The IC kit did not detect other viral pathogens such as Newcastle disease virus, infectious bronchitis, avian influenza virus, and infectious larynotracheitis virus. When applied to tissue samples of experimental chickens died 3 or 4 days post infection after very virulent IBDV (strain Kr/D62) infection, the IC kit detected IBDV in all samples of the bursa of Fabricius, spleen, kidney, cecal tonsil and in 87.5%, 37.5% and 0% of liver, thymus and proventriculus samples. In particular, BF tissue samples showed stronger signal bands than other tissues. Positive signal was observed. All except for one thymus sample of samples having negative results by the IC kit showed the same result with DAS-ELISA but RT-PCR assay detected IBDV in some of IC kit negative samples of thymus and proventriculus. When swab samples from the bursa of Fabricius of dead chickens (n=231) on field farms were tested, the sensitivity and specificity of the IC assay relative to RT-PCR was 100% (109/109) and 97.5% (119/122), respectively and kappa value between both assay was 0.97. The kit can provide a useful aid for rapid detection of IBDV in chickens under field circumstances.

Infection with and transmission of the virus was identified in pet dogs with severe respiratory disease., 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 (SAα 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.

The spike (S) gene of the attenuated porcine epidemic diarrhea virus (PEDV) DR13 was cloned and sequenced to further explore the functions of wild type PEDV and attenuated PEDV. Sequencing revealed a single large ORF of 4,149 nucleotides encoding a protein of 1,382 amino acids with predicted M r of 151 kDa. The coding region of the S gene of attenuated PEDV DR13 had 20 nucleotide changes that appeared to be significant determinants of function in that they produced changes in its predicted amino acid sequence. Notably, attenuated PEDV DR13 has previously been found to exhibit reduced pathogenicity in pigs. The regions containing these 20 nucleotide changes may therefore be crucial for PEDV pathogenicity. The attenuated PEDV DR13 S protein contains 28 Asn-Xaa-Ser/Thr sequons, 21 asparagines that are predicted to be N-glycosylated and a stretch of highly hydrophobic residues at positions 1,327–1,347, which is predicted to form an α-helix and to function as a membrane anchor. One (from N to K at 378) of the changes in the deduced amino acid sequence destroyed N-linked glycosylation sites, while another change (from N to S at 114) created a new one at a different location. These alterations in N-linked glycosylation sites reflected 3 nucleotide changes, which were related to the above-mentioned nucleotide changes and are suggested to influence the pathogenicity of attenuated PEDV DR13. Attenuated PEDV DR13 has 96.5, 96.4, 96.1, 93.9, 93.5 and 96.6% DNA sequence identities with CV777, Br1/87, JS-2004-2, Spk1, Chinju99 and parent DR13, respectively. Likewise, it shares 95.7, 95.4, 95.6, 92.0, 91.6 and 95.7% identity with those genes at the deduced amino acid sequence level. Phylogenetic analysis suggested that attenuated PEDV DR13 is closely related to CV777, Br1/87, JS-2004-2 and parent DR13, rather than to Spk1 and Chinju99 and is especially close to the Chinese PEDV strain JS-2004-2.

Porcine epidemic diarrhoea virus (PEDV) causes acute and severe watery diarrhoea and dehydration, as well as 50–100 per cent mortality in piglets. For the PEDV diagnosis, a rapid test kit that is specific and sensitive to PEDV is critical to monitor this disease at pig farms. The present study aimed to develop an immunochromatographic assay (ICA) strip test for detecting PEDV in faecal swabs. The newly developed diagnostic test showed a detection limit of 10 4.0 TCID 50 /ml of PEDV. Using faecal swab samples, the relative sensitivity and specificity of the ICA kit were 95.0 per cent and 98.6 per cent, respectively, compared with those of real-time RT-PCR. In samples from piglets experimentally infected with PEDV, the results showed 100 per cent agreement with those found by real-time RT-PCR. Our developed test strip will be useful for rapid diagnosis and can be used for epidemiological surveillance of PEDV infection.

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-γ) 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-γ 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.

Background International Standards or commercial panels used for performance validation of diagnostic kits might not reflect the viral characteristics common in Korea. Also, continuous use of these materials is difficult because of limited quantity and high cost. Objectives Establishment of HBsAg reference materials to be used as National Standards for validation of HBsAg diagnostic kits. Study design 568 plasma units with OD less than 2.0 on HBsAg EIA were collected. HBsAg testing with 3 EIAs and 1 CIA was performed on all units. HBsAg positive units were subjected to HBV DNA quantification, genotyping and subtyping. Candidates for the mixed titer performance panel and working standard were confirmed for HBsAg by neutralization. A collaborative study was conducted for the candidates of the mixed titer performance panel and the working standard. Results Based on the results of the collaborative study, a working standard (KFDA08/024) consisting of a series of four-fold dilutions of 2 materials, one with genotype/subtype C2/adr and the other with C1/adw, was established. A mixed titer performance panel composed of 2 negative and 16 positive samples was also established. A G1896A and a T/I126S mutant are included in the positive samples. Conclusions An HBsAg mixed titer performance panel and a working standard reflecting HBV genotypes/subtypes prevalent in Korea have been established as National Standards. This will enable consistent supply of validation materials, improve the validation system of HBsAg diagnostic kits in Korea and lead to quality improvement of diagnostic kits.

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.

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.

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.

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, 105.8–106.0 TCID50/0.1 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.

The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDS-polyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzyme-linked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by beta-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s).

Avian influenza (AI) is an infectious, low pathogenic virus that is endemic all over the world and poses a potential threat to the poultry industry. Vaccination is a widely used effective method to prevent avian influenza virus. Here we employed a comparative proteomics approach [two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF)] to characterize proteome in the sera from the specific pathogen free (SPF) chickens, the vaccinated chickens, and the naturally infected chickens. We identified total 58 proteins that were differentially expressed in the sera of three groups. Among them ovotransferrin and vitamin D-binding protein were more expressed in the sera of naturally infected chickens compare with other groups. Our results suggested that the level of these two proteins in the serum may help to discriminate the naturally infected chicken from the vaccinated chicken.

Avian influenza (AI) virus infects both animal and human. Low pathogenic AI virus infections (some H7 and H9 subtypes) have been reported all over the world and pose a potential threat to the poultry industry. Vaccination is the most effective way to prevent virus infection. However, vaccination makes it difficult to differentiate between vaccinated chickens and infected chickens. In order to differentiate vaccinated chickens from naturally infected chickens, we adopted synthetic peptide-based enzyme-linked immunosorbent assay (ELISA) using the peptide sequences from nonstructural protein 1 (NS1) of H9N2. Five synthetic peptides were designed using Protein Variability Sever (http://imed.med.ucm.es/PVS/) and synthesized. NS1-1 ~ NS1-4 peptides failed to detect serum antibodies from both vaccinated and naturally infected chickens. NS1-5 peptide from the C-terminal NS1 protein detected serum antibody from naturally infected chickens but not vaccinated chickens. These results imply that NS1-5 peptide may be a useful tool to differentiate naturally infected chicken from vaccinated chicken as being used in the synthetic peptide-based ELISA.