The effect of TLR9 agonist CpG oligodeoxynucleotides on the intestinal immune response of cobia (Rachycentron canadum).

Cytosine-guanine oligodeoxynucleotide (CpG ODN) motifs of bacterial DNA are recognized through toll-like receptor 9 (TLR9) and are potent activators of innate immunity. However, the interaction between TLR9 and CpG ODN in aquatic species has not been well characterized. Hence, cobia TLR9 isoform B (RCTLR9B) was cloned and its expression and induction in intestine were investigated. RCTLR9B cDNA consists of 3113bp encoding 1009 amino acids containing three regions, leucine rich repeats, transmembrane domain, and toll/interleukin-1 receptor (TIR) domain. Intraperitoneal injection of CpG ODN 2395 upregulated RCTLR9 A and B and MyD88 and also induced the expressions of Mx, chemokine CC, and interleukin IL-1 ß . Cobia intraperitoneally injected with CpG ODN 1668 and 2395 had increased survival rates after challenge with Photobacterium damselae subsp. piscicida. In addition, formulation of CpG ODN with formalin-killed bacteria (FKB) and aluminum hydroxide gel significantly increased expressions of RCTLR9 A (50 folds) and B (30 folds) isoforms at 10 dpi (CpG ODN 1668) and MyD88 (21 folds) at 6 dpv (CpG ODN 2395). Subsequently, IL-1 ß increased at 6 dpv in 1668 group. No histopathological damage and inflammatory responses were observed in the injected cobia. Altogether, these results facilitate CpG ODNs as an adjuvant to increase bacterial disease resistance and efficacy of vaccines in cobia.

Alkylphenol polyethoxylate derivatives in groundwater and blood samples collected from pig herds in Taiwan.

Alkylphenol polyethoxylate (APEO) derivatives, such as nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), nonylphenol (NP) and octylphenol (OP), have been detected in the surface water, sediment, food and groundwater of numerous countries. Because groundwater is the main source of water for pig herds, the aim of this study was to measure the concentrations of APEO derivatives in groundwater and blood samples that were collected from pig herds raised near the Wuluo River in Southern Taiwan. The mean concentrations of NP, OP, NP1EO and NP2EO in the groundwater supply for 10 pig herds were 0.04 µg/l, 0.26 ± 0.23 µg/l, 0.74 ± 0.69 µg/l and 0.17 ± 0.22 µg/l, respectively. NP was detected in all blood samples collected from 5 of the 10 pig herds. The highest concentrations detected in the blood samples collected from six-week-old piglets and sows were 12.00 µg/l and 56.94 µg/l, respectively. Blood samples from 4 of the 5 herds showed OP contamination. The highest OP concentrations detected in 6-week-old piglets and sows were 275.58 µg/l and 566.32 µg/l, respectively. These results indicate that APEO derivatives accumulated in the groundwater supply and the bloodstreams of the pigs.

Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus.

There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity.

Imprinted genes and satellite loci are differentially methylated in bovine somatic cell nuclear transfer clones.

In mammals, genome-wide epigenetic reprogramming systems exist in primordial germ cells and zygotes. These reprogramming systems play crucial roles in regulating genome functions during critical stages of embryonic development, and they confer the stability of gene expression during mammalian development. The frequent unexpected loss of progeny from somatic cell nuclear transfer (SCNT) is an ongoing problem. In this study, we used six cloned bovines (named NT-1 to NT-6), which were created by ear fibroblast nuclear transfer and displayed short life spans with multiple organ defects, as an experimental model. We focus here on three imprinted genes (IGF2, H19, and XIST) and four satellite loci (Satellite I, Satellite II, Art2, and VNTR) to investigate their methylation changes. The results revealed that aberrant methylation frequently occurred in the analyzed imprinted genes, but not in the satellite loci, of the cloned bovines. After the bovine fibroblast cells were treated with the 5-aza-2(')-deoxycytidine (5-Aza-dc) demethylation agent, the methylation percentages of the XIST and H19 putative differentially methylated region (DMR) were significantly decreased (XIST, p<0.01; H19, p<0.05) followed by an increase in their mRNA expression levels (p<0.01). Furthermore, we found that five short-lived cloned bovines (NT-1 to NT-5) exhibited more severe aberrant methylation changes in the three imprinted genes examined than the little longer-lived clone (NT-6) compared with wild-type (WT) cows. Our data suggest that the reprogramming of the methylation-controlled regions between the imprinted genes and satellite loci are differences and may be involved with additional mechanisms that need further elucidation.

Genotypic change and phylogenetic analysis of porcine circovirus type 2 in Taiwanese pig herds.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome. Two major PCV2 genotypes, PCV2a and PCV2b, have been identified. To explore the prevalence of different subgroups of PCV2 in Taiwan, 37 PCV2 isolates collected during 2002-2011 were analyzed. The genotypes of the PCV2 isolates collected before 2007 belonged to either PCV2a or PCV2b. However, all of the isolates collected after 2008 were PCV2b. Most of the isolates obtained since 2008 have been classified into a novel genotype within a subgroup of PCV2b based on complete ORF2 sequence analysis. Moreover, analysis of the PCV2 isolates from the same pig farm but from different years revealed that the viruses shifted from a PCV2b genotype to a novel subgroup of the PCV2b genotype. Collectively, PCV2b was the dominant PCV2 genotype in Taiwan currently, and the viruses have shifted into a new emerging subgroup of the PCV2b genotype.

Serotypes, antimicrobial susceptibility, and minimal inhibitory concentrations of Actionbacillus pleuropneumoniae isolated from slaughter pigs in Taiwan (2002-2007).

In total, 211 isolates of A. pleuropneumoniae were collected from pigs with hemorrhagic pneumonia at slaughterhouses during 2002-2007. Serotypes, antimicrobial susceptibility and minimum inhibitory concentration (MIC) values were determined for each isolate of A. pleuropneumoniae to 10 antimicrobial agents. Serovar 1 of A. pleuropneumoniae was predominant in Taiwan in 138 of the 211 isolates, followed by serovars 2 and 5. More than 90% of collected isolates were sensitive to ceftiofur, cephalothin, and chloramphenical. However, lincospectin and gentamicin were relatively less susceptible with sensitivities of only 2.4 and 5.7%, respectively. Additionally, ceftiofur had the highest in vitro activity with an MIC(50) of 2.2 µg/ml, followed by cephalothin (2.7 µg/ml) and chloramphenicol (7.9 µg/ml). Lincospectin had the least activity with MIC(50) and MIC(90) values of 73.9 and 114.5 µg/ml, respectively. The data indicate that ceftiofur and cephalothin were extremely active against A. pleuropneumoniae and with minimum MIC values. These drugs are suitable for controlling and treating hemorrhagic pleuropneumonia outbreaks in swine.

Shedding pattern and serological profile of porcine circovirus type 2 infection in cesarean-derived, colostrum-deprived and farm-raised pigs.

Six 5-week-old porcine circovirus type 2 (PCV2)-free, cesarean-derived, colostrums-deprived (CDCD) pigs were inoculated intranasally with 10(6) TCID(50) of PCV2. Four CDCD pigs were untreated cohabitants. Forty farm-raised pigs from two PCV2-contaminated herds were randomly selected for PCV2 trace investigations. Blood, nasal, oropharyngeal and fecal samples were collected from all tested pigs weekly. The PCV2 DNA shed at 6-11 and 7-12 weeks of age for PCV2-inoculated pigs and cohabitants, respectively. All the CDCD pigs exhibited seroconversion after PCV2 exposure. In the farm-raised animals, PCV2 shed at 9-15 weeks of age and seroconversion started at 11 weeks of age. Collectively, the pigs had a prolonged PCV2 shedding period following viral exposure, and growing pigs were the source of horizontal PCV2 transmission in PCV2-infected herds.

Pathological and molecular studies on mycobacteriosis of milkfish Chanos chanos in Taiwan.

investigated in milkfish Chanos chanos, which had a cumulative mortality of up to 66.7% over the course of 1 yr. Gross reddish- or greyish-white nodules appeared on the peritoneal surface, spleen, kidney, liver and gastrointestinal (GI) tract. Epithelioid granulomas with the formation of Langhan's type giant cells were the prominent histopathological changes. Despite large numbers of acid-fast bacilli in the granulomas, neither caseous necrosis nor dystrophic calcification were observed. Using degenerate primers that targeted the heat shock protein 65 kDa gene of Mycobacterium spp., a 441 bp product was amplified. When compared with published sequences, our products were identical to those of Mycobacterium abscessus Type II (GenBank accession number AY603554). This is the first report of M. abscessus infection in milkfish.