Optimized clade 2.3.2.1c H5N1 recombinant-vaccine strains against highly pathogenic avian influenza

A/Puerto Rico/8/34 (PR8)-derived recombinant viruses have been used for seasonal flu vaccines; however, they are insufficient for vaccines against some human-fatal H5N1 highly pathogenic avian influenza (HPAI) viruses (HPAIV) due to low productivity. Additionally, the polymerase basic 2 (PB2) protein, an important mammalian-pathogenicity determinant, of PR8 possesses several mammalian-pathogenic mutations. We previously reported two avian PB2 genes (01310 and 0028) related to efficient replication in embryonated chicken eggs (ECEs) and nonpathogenicity in BALB/c mice. In this study, we generated PR8-derived H5N1 recombinant viruses harboring hemagglutinin (attenuated) and neuraminidase genes of a clade 2.3.2.1c H5N1 HPAIV (K10-483), as well as the 01310 or 0028 PB2 genes, and investigated their replication and immunogenicity. Compared with a control virus harboring six internal PR8 genes (rK10-483), the recombinant viruses possessing the 01310 and 0028 PB2 genes showed significantly higher replication efficiency in ECEs and higher antibody titers in chickens. In contrast to rK10-483, none of the viruses replicated in BALB/c mice, and all showed low titers in Madin-Darby canine kidney cells. Additionally, the recombinant viruses did not induce a neutralization antibody but elicited decreased protective immune responses against K10-483 in mice. Thus, the highly replicative and mammalian nonpathogenic recombinant H5N1 strains might be promising vaccine candidates against HPAI in poultry.

Surveillance and molecular epidemiology of avian influenza viruses from birds in zoos, backyard flocks and live bird markets in Korea / 대한수의학회지

The circulation and infection of avian influenza virus (AIV) in zoos and backyard flocks has not been systematically investigated. In the present study, we surveyed the birds including those in live bird markets (LBMs) and evaluated co-circulation of AIVs among them. Overall, 26 H9N2 AIVs and one H6N2 AIV were isolated from backyard flocks and LBMs, but no AIVs were isolated from zoo birds. Genetic analysis of the HA and NA genes indicated that most of the H9N2 AIVs showed higher similarities to AIVs circulating in domestic poultry than to those in wild birds, while the H6N2 AIV isolate from an LBM did to AIVs circulating in migratory wild birds. In serological tests, 15% (391/2619) of the collected sera tested positive for AIVs by competitive-ELISA. Among them, 34% (131/391) of the sera tested positive for AIV H9 antigen by HI test, but only one zoo sample was H9 positive. Although AIVs were not isolated from zoo birds, the serological results indicated that infection of AIVs might occur in zoos. It was also confirmed that H9N2 AIVs continue to circulate and evolve between backyard flocks and LBMs. Therefore, continuous surveillance and monitoring of these flocks should be conducted to control further epidemics.

Clinico-pathological Features of Chlamydophila psittaci Infection in Parrots and Genetic Characterization of the Isolates / 대한수의학회지

Avian chlamydiosis is caused by Chlamydophila psittaci and considered as one of an important zoonotic disease throughout the world. Among more than 400 avian species including poultry and pet birds susceptible to the disease, psittacine birds were known to be mostly susceptible hosts. In Korea, no outbreak of the disease and genetic analysis of the agent in poultry and pet birds have been reported. With histopathological findings and genetic identification of a causative agent, avian chlamydiosis was identified in parrots submitted from the same pet bird farm in 2006 and 2009 for the diagnosis. Based on genetic sequences and phylogenetic analysis of ompA gene, the two isolates of Chlamydophila psittaci showed 100% of genetic similarity and belonged to genotype A, suggesting that the same agent might be continuously circulated in the farm. This result indicates that serological survey of the disease in pet bird farms and impact of the disease on significance in public health may be further studied.

Development of immunoassays for the detection of kanamycin in veterinary fields

Monoclonal antibody against kanamycin was prepared, and competitive direct ELISA and immunochromatographic assay were developed using the antibody to detect kanamycin in animal plasma and milk. The monoclonal antibody produced was identified to be IgG1, which has a kappa light chain. No cross-reactivity of the antibody was detected with other aminoglycosides, indicating that the monoclonal antibody was highly specific for kanamycin. Based on competitive direct ELISA, the detection limits of kanamycin were determined to be 1.1 ng/ml in PBS, 1.4 ng/ml in plasma, and 1.0 ng/ml in milk. The concentration of intramuscularly injected kanamycin was successfully monitored in rabbit plasma with competitive direct ELISA. Based on the colloidal gold-based immunochromatographic assay, the detection limits of kanamycin were estimated to be about 6-8 ng/ml in PBS, plasma, and milk. The immunochromatographic assay would be suitable for rapid and simple screening of kanamycin residues in veterinary medicine. Screened positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could be used to complement each other as well as other laboratory findings. Moreover, instead of slaughtering the animals to obtain test samples, these methods could be applied to determine kanamycin concentration in the plasma of live animals.

Evaluation of the Manual Method of Liquid-Based Uterine Cervicovaginal Cytology - By The Manual Method Based on SurePathTM Methodology / 대한세포병리학회지

Liquid-Based Uterine Cervicovaginal Cytology is known to be a sensitive and effective screening method for cervical neoplasm. MonoPrepTM, ThinPrepTM, and SurePathTM methods have been recently used as Liquid- Based Uterine Cervicovaginal Cytology techniques, and the SurePathTM method has been used in Sung-Yoon Reference Laboratory since 2003. The goal of Liquid-Based Uterine Cervicovaginal Cytology is to separate cervical epithelial cells from non-target cells, red blood cells and neutrophils. This report describes a study which evaluated cellularity, stainability, and cellular changes of epithelial cells in samples processed using a manual technique as compared to samples processed using SurePathTM automated method. The samples processed by means of a manual technique contained a cellularity of epithelial cells similar to that of the samples processed using the SurePathTM automated method. In addition, we compared variable density gradient reagents, including dextran, dextrose, and sucrose, to SurePathTM gradient media in order to evaluate cell fractionation and cellularity of epithelial cells. 10% dextran of gradient media shows good fractionation. The samples processed with 10% dextran demonstrated sufficient cellularity of epithelial cells and shows the fewest cellular changes. In conclusion, using a manual technique on these samples is easier to read than those results obtained using the SurePathTM automated method.