H3N2 canine influenza virus with the matrix gene from the pandemic A/H1N1 virus: infection dynamics in dogs and ferrets.

After an outbreak of pandemic influenza A/H1N1 (pH1N1) virus, we had previously reported the emergence of a recombinant canine influenza virus (CIV) between the pH1N1 virus and the classic H3N2 CIV. Our ongoing routine surveillance isolated another reassortant H3N2 CIV carrying the matrix gene of the pH1N1 virus from 2012. The infection dynamics of this H3N2 CIV variant (CIV/H3N2mv) were investigated in dogs and ferrets via experimental infection and transmission. The CIV/H3N2mv-infected dogs and ferrets produced typical symptoms of respiratory disease, virus shedding, seroconversion, and direct-contact transmissions. Although indirect exposure was not presented for ferrets, CIV/H3N2mv presented higher viral replication in MDCK cells and more efficient transmission was observed in ferrets compared to classic CIV H3N2. This study demonstrates the effect of reassortment of the M gene of pH1N1 in CIV H3N2.

Development and characterization of mouse monoclonal antibodies reactive with chicken IL-1ß.

Interleukin-1ß proteins from chicken, duck, goose, turkey, and pigeon share 77 to 99% amino acid sequence similarity among themselves, and only 31 to 35% sequence similarity is shared between avian and mammalian IL-1ß. There have been no antibodies that specifically detect avian IL-1ß, and the current study was conducted to develop mouse monoclonal antibodies (mAb) against chicken IL-1ß (chIL-1ß) to further define its biochemical and immunological properties. In this study, 2 mouse mAb that are specific for chIL-1ß were produced and characterized. Both mAb identified a 66.0 kDa recombinant chIL-1ß protein expressed in Escherichia coli by Western blot analysis that corresponded to the expected molecular weight of a recombinant fusion protein containing the full-length 23.0 kDa chIL-1ß protein and a 43.0 kDa maltose binding protein tag. Immunohistochemical analysis identified cells producing endogenous chIL-1ß in the cecal tonsils, bursa of Fabricius, and spleen. Purified recombinant chIL-1ß dose-dependently stimulated the proliferation and nitric oxide production by thymocytes, and both activities were inhibited by co-incubation with the 2 chIL-1ß mAb described in this paper. These mAb will be important immune reagents for basic and applied poultry research of IL-1ß in poultry.

Isolation and genetic characterization of naturally NS-truncated H3N8 equine influenza virus in South Korea.

Equine influenza virus (EIV) causes a highly contagious respiratory disease in equids, with confirmed outbreaks in Europe, America, North Africa, and Asia. Although China, Mongolia, and Japan have reported equine influenza outbreaks, Korea has not. Since 2011, we have conducted a routine surveillance programme to detect EIV at domestic stud farms, and isolated H3N8 EIV from horses showing respiratory disease symptoms. Here, we characterized the genetic and biological properties of this novel Korean H3N8 EIV isolate. This H3N8 EIV isolate belongs to the Florida sublineage clade 1 of the American H3N8 EIV lineage, and surprisingly, possessed a non-structural protein (NS) gene segment, where 23 bases of the NS1-encoding region were naturally truncated. Our preliminary biological data indicated that this truncation did not affect virus replication; its effect on biological and immunological properties of the virus will require further study.

Prevalence of classical swine fever virus in domestic pigs in South Korea: 1999-2011.

The major policy for eradication of classical swine fever (CSF) in South Korea has focused on the implementation of compulsory vaccination of the susceptible pig population. A vaccine strain of CSF virus, the LOM strain, is used to maintain high herd seroconversion, a practice complementary to the 'stamping-out policy' and restriction of animal movement during disease outbreaks. To survey for the prevalence of CSF in domestic pigs in South Korea over the past 13 years (1999-2011), we tested 4 193 782 and 1 162 645 samples for antibodies and antigens, respectively. Whereas seropositivity for CSF antibodies has been maintained at over 95% in the mainland, in Jeju Island, where no-vaccination has been administered since 1999, seroprevalence has been below 1% during the last 3 years of study (2009-2011). The highest number of outbreaks in South Korea occurred in 2002 and 2003; since then, outbreaks have decreased each year, with the last CSF outbreak recorded in 2009. No outbreaks have occurred during the past 3 years, and a high level of herd immunity has been maintained in the mainland pig population for 8 years; therefore, South Korea could now switch to a no-vaccination policy throughout the country. However, the constant threat of the re-emergence of the disease in the susceptible pig population should be the main consideration in planning and carrying out the last phase of the CSF eradication process.

Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene.

Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) µg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

Clostridium perfringens alpha-toxin and NetB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens.

Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs, pathologic symptoms, and age of onset. The primary virulence factors of C perfringens are its four major toxins (alpha, beta, epsilon, iota) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C perfingens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C perfringens alpha-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C perfringens alpha-toxin and NetB toxin that were increased in birds experimentally coinfected with Eimeria maxima and C perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.

Avian necrotic enteritis: experimental models, host immunity, pathogenesis, risk factors, and vaccine development.

The increasing trends of legislative restrictions and voluntary removal of antibiotic growth promoters worldwide has already affected, and will continue to affect, poultry production and animal health. Necrotic enteritis (NE) is being considered among the most important infectious diseases in the current poultry production system globally, with an estimated annual economic loss of more than $2 billion, largely attributable to medical treatments and impaired growth performance. Thus, there is an urgent need to develop rational, alternative, and integrated management strategies not only to control NE, but also to prevent it. In both humans and many warm-blooded animals and birds, NE is caused by Clostridium perfringens, a gram-positive, anaerobic, spore-forming bacterium. To accomplish these goals, better understanding of host- and environmentally related factors on the development of NE and potential vaccination strategies against C. perfringens infection will be necessary. Furthermore, a reliable and reproducible NE disease model is needed for characterization of C. perfringens pathogenesis and host protective immunity. This review summarizes recent developments in NE disease models, pathogenesis, host immunity, risk factors, and vaccine development for C. perfringens-associated NE in poultry.