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Elizabethkingia miricola is a Gram-negative rod which has been incriminated in severe infections in humans. Recently, a serious infectious disease was identified in Chinese spiny frogs (Quasipaa spinosa), in the Sichuan Province of China; the disease was characterized by corneal opacity, the presence of ascites and neurological symptoms. A Gram-negative bacillus was isolated from the liver, spleen and kidney of the diseased frogs. Experimental infection test revealed that the bacillus could infect the frogs Q. spinosa and the LD50 value was 1.19 × 106 cfu per frog. The isolated Gram-negative bacillus was identified as E. miricola according to phenotypic characteristics, 16S rRNA and gyrB gene sequence analysis. The isolated strain was only susceptible to florfenicol among all investigated chemotherapeutic agents. Histological examination revealed that E. miricola infection caused pathological lesions to multiple organs and tissues, especially in the liver, brain, kidney. These results confirmed that E. miricola is an emerging pathogen of Chinese spiny frogs.

In this study, lung tissue was collected from nursery piglets suspected of being infected with porcine reproductive and respiratory syndrome virus (PRRSV) in a large‑scale pig farm in Sichuan, China. Polymerase chain reaction (PCR) and reverse transcription quantitative‑polymerase chain reaction (RT‑qPCR) methods were used to ensure that no other pathogens were present. Virus isolation was also carried out where the presence of PRRSV was determined by indirect immunoinfluscent assay (IFA). Compared with the common PRRSV strain, the isolate did not produce evident Cytopathic effect (CPE) in the early stage of isolation. CPE was found in the late stage, and the titer was 104.17 TCID50/0.1 mL. The strain was named CJS01. Bioinformatics analysis showed that it was a NADC30‑Like strain. The virus load was determined by measuring the nucleic acid load during the proliferation of the strain on Marc‑145 cells. The strain showed good adaptability on cells, and the virus proliferated on cells for 84 hr when the highest nucleic acid load was achieved. By recombinant analysis of ORF3~7 genes and prediction of its epitope, it was found that CJS01 strain might interfere with the immunesystem of the infected animals.

Forest musk deer (FMD

To detect Ibaraki virus (IBAV) sensitively and quickly, a rapid detection technique of reverse transcriptase isothermal nucleic acid amplification (RT-LAMP) for the virus was established, through the main steps as follows: the design of 4 specific primers basing on the 6 conserved regions of IBAV VP3 protein gene, the optimization of reaction time and temperature, sensitivity test and specific test. The results showed that when the annealing temperature was 62 ℃ and the reaction time was 60 min, the electrophoresis band was the most obvious. The method has good specificity for IBAV without cross reaction to bovine epidemic heat virus (BEFV) and bovine viral diarrhea virus (BVDV). At the same time, the sensitivity is 100 times higher than that of RT-PCR, and the concentration of IBAV gene can be detected to 10 copies/μL. The method can complete the amplification reactiononly with a water bath within 1 hour, simply and efficiently. It provides a new detection method for the preliminary screening and detection of IBAV at the base level.

In order to establish the RT-LAMP detectionmethod of bluetongue virus (BTV), 4 specific LAMP primers were designed and synthesized by using the conservative sequence of the BTV VP7 genome published by GenBank. Through the synthesis of BTV artificial genes, the optimization of the reaction conditions of LAMP, the LAMP sensitivity experiment of BTV and the specific experiments, a preliminary method of BTV LAMP detection was established. The results showed that the BTV gene was successfully synthesized and the LAMP reaction conditions were optimized preliminarily, and the detection limit of the developed BTV LAMP method system could reach 43.7 copies/ ?L of target gene fragment, which could be detected with good sensitivity and specificity. In the experiment, the BTV virus could be amplified with a good specific amplification at 62? and 60min, and the results were negative after the amplification of DNA of Escherichia coli and Escherichia coli under the system constructed by the experiment.

Klebsiella pneumoniae is an important pathogen commonly associated with opportunistic infections. In this study, lung pathogenic K. pneumoniae (LPKP) was isolated and identified from suppurative pneumoniae in forest musk deer by conventional methods and by 16S ribosomal RNA sequence analysis. Median lethal dose and histopathologic analysis were used to demonstrate pathogenicity of the organism in mice. Furthermore, a draft genome of LPKP was sequenced, and its virulence genes were detected. One hundred and twenty-two virulence genes encoded determinant of capsule polysaccharide (CPS), lipopolysaccharide, fimbriae, outer membrane proteins, iron acquisition, and urease. In particular, 20 CPS-related genes were highly conserved in LPKP, K. pneumoniae U, K. pneumoniae NTUH-KP35, and K. pneumoniae KP-1. All of the strains were identified as capsular type K54. This is the first report of capsular type K54 K. pneumoniae causing suppurative pneumonia in an animal. The results of this study provided the basis for understanding the pathogenicity of LPKP and laid a foundation for the development of vaccines for the capsular type K54 K. pneumoniae disease.

A new loop-mediated isothermal amplification (LAMP) method for the detection of Cyprinid herpesvirus 2 (CyHV-2) was developed. Following the synthesis in vitro of a 413 bp DNA fragment of CyHV2 DNA-dependent DNA polymerase gene by overlap extension PCR using 11 overlapping oligo primers designed according to the gensome sequences information of CyHV2 published in GenBank, a set of 6 LAMP primers were designed based on the synthesized DNA fragment, and the LAMP assay was primarily established after preparation of target gene fragments, optimization of reaction conditions, products identification, sensitivity and specificity tests. Results showed the developed LAMP assay was a rapid, specific and sensitive method for CyHV2 detection, with a ladder-like pattern of amplification bands from about 177 bp incubation at 64°C for 30 min by using agarose gel electrophoresis. The detection limit could reach about 15 copies of cloned viral genomic fragments, and no cross reaction with Aeromonas veronii, Aeromonas hydrophila, Pseudomonas fluorescens and Streptococcus common pathogens isolated from fish detection by this approach.

To develop the reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of European brown hare syndrome virus (EBHSV), 3 specific primers and 9 overlapping oligo primers were designed to amplify a fragment of 335 nucleotides from the genome of EBHSV according to the genome sequences published in GenBank, and a 359 bp DNA fragment of the capsid protein(VP60)gene of EBHSV was synthesized in vitro by using overlap extension PCR to construct the recombinant plasmids pGM-T-EBHSV, then the RT-PCR method was described through the optimization of reaction conditions, sensitivity and specificity tests. Results showed the RT-PCR method was supplied for the detection of EBHSV with good specificity and sensitivity. The sensitivity of the RT-PCR could reach about 50 copies of cloned viral genomic fragments (pGM-T-EBHSV), and no amplifications for RHDV, Pasteurella multocida, E.coliandSalmonellafrom rabbits detection by this approach.