A PCR-based assay for discriminating Cervus and Rangifer (Cervidae) antlers with mitochondrial DNA polymorphisms

This study describes a method for discriminating Rangifer antlers from true Cervus antlers using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination. Specific primers labeled with fluorescent tags were designed to amplify fragments from the mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus differentially. A 466-bp fragment that was observed for both Cervus and Rangifer antlers served as a positive control, while a 270-bp fragment was specifically amplified only from Rangifer antlers. Allelic discrimination was used to differentiate between Cervus and Rangifer antlers, based on the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for forensic and quantitative analyses of Cervus and Rangifer antlers in a single step, without having to obtain any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and are especially useful in this study for the identification of original Cervus deer products from fraudulent Rangifer antlers.     

Potential of auxotrophic Edwardsiella tarda double-knockout mutant as a delivery vector for DNA vaccine in olive flounder (Paralichthys olivaceus)

To evaluate potential of an auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) as a delivery vehicle for DNA vaccine in fish, olive flounder (Paralichthys olivaceus) were immunized with the E. tarda mutant harboring plasmids (pG02-ASD-CMV-eGFP) for eukaryotic expression of the enhanced green fluorescent protein (eGFP) gene through either intraperitoneal (i.p.) or oral route, and the expression of eGFP in the internal organs and generation of antibody against eGFP in fish were analyzed. In fish i.p. injected with 2×10(7)CFU/fish of Δalr Δasd E. tarda harboring pG02-ASD-CMV-eGFP, expression of eGFP was detected in liver, kidney, and spleen from 1 day to 28 days post-injection. In fish orally administered with 1×10(9)CFU/fish of the bacteria, the eGFP band was detected in liver, kidney, and spleen from 1 day to 14 days post-administration, whereas, in intestine, the band was detected only at 1 day post-administration. Either oral or i.p. immunization of olive flounder with recombinant E. tarda that carried eGFP-expressing eukaryotic plasmids was successful to induce humoral adaptive immunity against not only E. tarda that was used as a delivery vehicle but also eGFP that was used as the reporter protein of DNA vaccine, suggesting attenuated E. tarda-vectored DNA vaccine has a potential to be used as a combined vaccine against infectious diseases in fish.     

Evaluation of the efficacy of immunocastration vaccine composed of gonadotrophin-releasing hormone conjugated with Salmonella typhimurium flagellin in rats

Immunocastration is an alternative method to replace surgical castration that is commonly performed in domestic and pet animals. In this study, a new immunocastration vaccine was developed, and its efficacy was evaluated in male rats. Six tandem copies of gonadotrophin-releasing hormone (GnRH) peptide were genetically fused to Salmonella typhimurium flagellin fljB (STF2) that is a ligand of toll-like receptor 5 (TLR5). The recombinant STF2-GnRH protein expressed in Escherichia coli was used as the immunocastration vaccine. Sixteen male rats were equally assigned to four groups. Excluding the control rats, three groups were immunized with 100, 200 and 400 μg of the STF2-GnRH vaccine, respectively. All of the immunized rats developed significantly higher titres of antibodies to GnRH than the control rats. The size and weight of both testes and epididymides from the immunized rats were significantly smaller than those of the control rats. Testicular tissues in the immunized rats demonstrated atrophy of seminiferous tubules and decreased numbers of both spermatogonia and spermatocytes. These data indicate that the newly developed STF2-GnRH vaccine has a potent immunogenicity to GnRH and efficiently suppresses the development of testes in rats.     

Phenotypic characterization and random amplified polymorphic DNA (RAPD) analysis of Pasteurella multocida isolated from Korean pigs

Pasteurella multocida causes various respiratory disease symptoms in pigs, including atrophic rhinitis and pneumonia. In the present study, 69 strains of P. multocida were isolated from 443 pigs with respiratory clinical symptoms at 182 farms located throughout South Korea from 2009 to 2010. A multiplex capsular PCR typing assay revealed that 69 strains of P. multocida isolated in this study had the biosynthetic locus of the capsules of either serogroup A (47 strains, 68.1%) or serogroup D (22 strains, 31.9%). The 22 strains positive for serogroup D-specific primers were divided into four clusters and the 47 strains positive for serogroup A-specific primers were divided into 12 clusters according to the results of Random Amplified Polymorphic DNA (RAPD) analysis. P. multocida strains in the present study were susceptible to most of the antimicrobial agents used. An analysis of antimicrobial resistance and virulence gene pattern combined with RAPD indicated that a certain P. multocida strain appeared to be genetically identical, implying the persistence of the strain within a single farm.     

Seroprevalence of subtype H3 influenza A virus in South Korean cats

To investigate the potential transmission of subtype H3 influenza virus to cats, a serological survey was carried out in South Korea. Serum samples (n = 1027) were obtained from 809 pet cats and 218 domesticated cats living in urban colonies (D-cats) from 2008 to 2010, and tested using an influenza anti-nucleoprotein (NP)-specific enzyme-linked immunosorbent assay (ELISA) and the haemagglutination inhibition (HI) test, which was recommended by the World Organization for Animal Health. Anti-influenza virus antibodies were detected in 3.12% and 2.43% of cat sera tested using the NP-specific ELISA and HI test, respectively. Anti-H3 antibodies were also identified when the HI assay was used for influenza virus serotyping. These data may indicate the sporadic transmission of subtype H3 influenza virus from other infected species to cats in South Korea.     

Sequence variations of the bovine prion protein gene (PRNP) in native Korean Hanwoo cattle

Bovine spongiform encephalopathy (BSE) is one of the fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) caused by infectious prion proteins. Genetic variations correlated with susceptibility or resistance to TSE in humans and sheep have not been reported for bovine strains including those from Holstein, Jersey, and Japanese Black cattle. Here, we investigated bovine prion protein gene (PRNP) variations in Hanwoo cattle [Bos (B.) taurus coreanae], a native breed in Korea. We identified mutations and polymorphisms in the coding region of PRNP, determined their frequency, and evaluated their significance. We identified four synonymous polymorphisms and two non-synonymous mutations in PRNP, but found no novel polymorphisms. The sequence and number of octapeptide repeats were completely conserved, and the haplotype frequency of the coding region was similar to that of other B. taurus strains. When we examined the 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP, Hanwoo cattle had a lower deletion allele and 23-bp del/12-bp del haplotype frequency than healthy and BSE-affected animals of other strains. Thus, Hanwoo are seemingly less susceptible to BSE than other strains due to the 23-bp and 12-bp indel polymorphisms.     

Dietary sophorolipid accelerates growth by modulation of gut microbiota population and intestinal environments in broiler chickens

Background:Gut is a crucial organ for the host’s defense system due to its filtering action of the intestinal membrane from hazardous foreign substances.One strategy to strengthen the gut epithelial barrier function is to upregulate beneficial microflora populations and their metabolites.Sophorolipid(SPL),which is a glycolipid biosurfactant,could increase beneficial microflora and decrease pathogenic bacteria in the gastrointestinal tract.Therefore,herein,we conducted an experiment with broiler chickens to investigate the fortifying effects of SPL on the host’s gut defense system by modulating the microbiota population.Methods:A total of 5401-day-old chicks(Ross 308)were used,and they were immediately allotted into three treatment groups(6 replications with 30 chicks/pen)according to their initial body weight.The dietary treatments consisted of CON(basal diet),BAM(10 mg/kg bambermycin),and SPL(10 mg/kg SPL).During the experiment,birds freely accessed feed and water,and body weight and feed intake were measured at the end of each phase.On d 35,birds(one bird/pen)were sacrificed to collect jejunum and cecum samples.Results:Dietary SPL and BAM supplementation significantly accelerated birds’growth and also significantly improved feed efficiency compared to CON.Intestinal microbial community was significantly separated by dietary SPL supplementation from that of CON,and dietary SPL supplementation significantly increased Lactobacillus spp.and Akkermansia muciniphila.Moreover,birds fed with dietary SPL also showed the highest concentration of cecal butyrate among all treatment groups.Gut morphological analysis showed that dietary SPL significantly increased villus height,ratio of villus height to crypt depth,goblet cell numbers,and the gene expression levels of claudin-1 and mucin 2.Additionally,dietary SPL significantly decreased the mRNA expression level of pro-inflammatory cytokine,interleukin-6,and increased that of anti-inflammatory cytokine,interleukin-10,compared to other treatments.Conclusions:Dietary SPL increases the beneficial bacterial population and butyrate concentration,which leads to a strengthened gut barrier function.In addition,the intestinal inflammation was also downregulated by dietary SPL supplementation.     

Insulin sensitivity decreases with obesity, and lean cats with low insulin sensitivity are at greatest risk of glucose intolerance with weight gain

This study quantifies the effects of marked weight gain on glucose and insulin metabolism in 16 cats which increased their weight by an average of 44.2% over 10 months. Significantly, the development of feline obesity was accompanied by a 52% decrease in tissue sensitivity to insulin and diminished glucose effectiveness. In addition, glucose intolerance and abnormal insulin response occurred in some cats. An important finding was that normal weight cats with low insulin sensitivity and glucose effectiveness were at increased risk of developing impaired glucose tolerance with obesity. High basal insulin concentrations or low acute insulin response to glucose also independently increased the risk for developing impaired glucose tolerance. Male cats gained more weight relative to females and this, combined with their tendency to lower insulin sensitivity and higher insulin concentrations, may explain why male cats are at greater risk for diabetes. Results suggest an underlying predisposition for glucose intolerance in some cats, which is exacerbated by obesity. These cats may be more at risk of progressing to overt type 2 diabetes mellitus.