Kun-Wei Chan

Development of a loop-mediated isothermal amplification for rapid detection of orf virus.

A loop-mediated isothermal amplification (LAMP) assay using six primers targeting a highly conserved region of the B2L gene has been developed to diagnose orf virus. The assay produces a ladder-like pattern of products on an agarose gel that can be specifically digested with BsrGI enzyme. The sensitivity of the LAMP assay, which was determined to be a single copy of the standard plasmid, was 100 fold and 10 fold higher than PCR and nested PCR, respectively; furthermore, no cross-reactivity was founded with the other tested viruses. By staining the products directly in the tube with PicoGreen or ethidium bromide, the products can be visualized with a similar sensitivity as by gel electrophoresis. Clinical samples were tested using PCR, nested PCR and LAMP assay and the positive rates were 60%, 70% and 70%, respectively. The LAMP assay allows easy, rapid, accurate and sensitive detection of infection with orf virus and is especially applicable in a resource-limited situation.

Identification and phylogenetic analysis of orf virus from goats in Taiwan

An outbreak of contagious ecthyma in goats in central Taiwan was investigated. The disease was diagnosed by physical and histopathologic examinations, and the etiology of the disease was identified as orf virus by electron microscopy and polymerase chain reaction (PCR) and sequence of major envelope protein (B2L) gene. The entire protein-coding region of B2L gene were cloned and sequenced. Phylogenetic analysis of B2L amino acid sequences showed that the orf virus identified in this outbreak was closer to the Indian ORFV-Mukteswar 59/05 isolate. This is the first report on the molecular characterization of orf virus in Taiwan.

Phylogenetic analysis of parapoxviruses and the C-terminal heterogeneity of viral ATPase proteins.

Two outbreaks of orf virus (a parapoxvirus) infection in goats found in Nantou and Taiping of central Taiwan were investigated. The nucleotide and the amino acid sequences of viral B2L, E3L and A32L genes in these two outbreaks were analyzed, and each of their phylogenetic trees were also constructed. In the A32L gene, an unexpected deletion of 24 nucleotides was found in the Taiping strain. The A32L gene can encode an ATPase and is supposed to be involved in virion DNA packaging. The 24 nucleotides correspond to 8 amino acids residues of the viral ATPase, which are located near the C-terminal region of the enzyme. Moreover, two copies of the RGD sequence at C-terminal region of ATPase were found in the Nantou strain. The 24-nucleotide difference in the A32L gene indicated that the Nantou strain and the Taiping strain were two separate strains, and it can be used in differential molecular diagnosis. Moreover, the C-terminal heterogeneity was found to be a general feature of the viral ATPase. Lastly, similar functional motifs of the ATPase and the Ras proto-oncoprotein (a GTPase) are discussed.

Antimicrobial resistance to cefotaxime and ertapenem in Enterobacteriaceae: the effects of altering clinical breakpoints

INTRODUCTION The Clinical and Laboratory Standards Institute (CLSI) updated its antimicrobial susceptibility testing interpretation criteria for Enterobacteriaceae. This study assessed the effects of clinical breakpoint changes in the CLSI 2009 to 2012 guidelines on antibiotic susceptibility testing reports. METHODOLOGY In total, 2,076 non-duplicate clinical isolates of Enterobacteriaceae were analyzed. The disk diffusion method was used for susceptibility testing. The CLSI 2009-12 clinical breakpoints were applied to determine susceptibility of cefotaxime and ertapenem. Combined-disk testing was used for phenotypic confirmation of extended-spectrum beta-lactamase (ESBL) production. RESULTS In total, Enterobacteriaceae resistance rates to cefotaxime increased from 13.1% using the CLSI 2009 guidelines to 23.6% with the CLSI 2010-12 guidelines, and the resistance rates to ertapenem were 0.4%, 1.0% and 0.8% with CLSI 2009, 2011 and 2012, respectively. Based on the 2010-12 CLSI criteria, all ESBL-producing Escherichia coli and Klebsiella pneumoniae were resistant to cefotaxime. Marked differences in susceptibility to ertapenem between the 2009 CLSI criteria and 2012-12 CLSI criteria were noted in ESBL-producing K. pneumoniae. CONCLUSIONS Breakpoints changes in the updated CLSI guidelines resulted in higher resistance rates to cefotaxime and ertapenem. In addition, the effects were different in individual Enterobacteriaceae species. As a result, clinicians may opt to use alternative antimicrobial agents. Upon implementation of the newer CLSI guidelines, laboratories should be aware of the possible consequences and closely monitor the effects.

Shewanella infection of snake bites: a twelve-year retrospective study

OBJECTIVE: Infections of snake bite wounds by Shewanella are rarely discussed in the medical literature. This study aims to characterize the presentation and management of Shewanella infections in snake bite wounds. METHOD: We retrospectively investigated the microbiology, clinical features, and outcomes of patients with Shewanella infected snake bite wounds admitted to a tertiary medical center from January 1998 to December 2009. RESULTS: Ten patients with Shewanella-infected snake bite wounds were identified. All of the snake bites were caused by cobras. The majority of patients had moderate to severe local envenomation and polymicrobial infections. Shewanella isolates are susceptible to ampicillin-sulbactam, piperacillin-tazobactam, third- and fourth-generation cephalosporins, carbapenems, aminoglycosides, and quinolones but are resistant to penicillin and cefazolin. All of the patients examined had favorable outcomes. CONCLUSION: It is recommended that Shewanella infection be considered in snake bite patients, especially when patients present with moderate to severe local envenomation.

Purification and functional motifs of the recombinant ATPase of orf virus

Our previous study showed that the recombinant ATPase encoded by the A32L gene of orf virus displayed ATP hydrolysis activity as predicted from its amino acids sequence. This viral ATPase contains four known functional motifs (motifs I-IV) and a novel AYDG motif; they are essential for ATP hydrolysis reaction by binding ATP and magnesium ions. The motifs I and II correspond with the Walker A and B motifs of the typical ATPase, respectively. To examine the biochemical roles of these five conserved motifs, recombinant ATPases of five deletion mutants derived from the Taiping strain were expressed and purified. Their ATPase functions were assayed and compared with those of two wild type strains, Taiping and Nantou isolated in Taiwan. Our results showed that deletions at motifs I-III or IV exhibited lower activity than that of the wild type. Interestingly, deletion of AYDG motif decreased the ATPase activity more significantly than those of motifs I-IV deletions. Divalent ions such as magnesium and calcium were essential for ATPase activity. Moreover, our recombinant proteins of orf virus also demonstrated GTPase activity, though weaker than the original ATPase activity.

A novel loop-mediated isothermal amplification approach for sex identification of Columbidae birds

Because it is difficult to differentiate male and female Columbidae birds (e.g., Columba livia) on the basis of morphology, detection of DNA fragments associated with Chromobox-Helicase-DNA binding genes or female-specific genes have been widely used. The objective was to establish a loop-mediated isothermal amplification system involving the 18S ribosomal RNA gene and a female-specific gene for sex identification of Columba livia birds. Unlike polymerase chain reaction (PCR), random amplification polymorphic DNA-PCR and amplified fragment length polymorphism-PCR, target DNA was amplified under isothermal conditions (the entire process was completed in <60 min). By modulating various parameters involved in amplification, e.g., concentrations of MgSO(4), betaine, Bst polymerase, and deoxynucleotide triphosphates, as well as the relative ratio of outer/inner primers and temperatures, optimal conditions for both targets were established that had equal detection limits (62.5 ng). To simplify sex determination, direct observations of the presence of white precipitate (derived from magnesium pyrophosphates) were used for positive samples, which was compared with the whitish ring which formed in a negative sample after addition of CuSO(4). This approach was a rapid alternative to electrophoresis or turbidimetry. DNA extracted from the blood and feathers of various birds were tested using loop-mediated isothermal amplification; results were consistent with a standard PCR. Thus, the assay was a simple, accurate, fast, and economical alternative suitable for veterinary practice.

Functional expression of the recombinant ATPase of orf virus

Nucleotide sequence analysis has indicated that the A32L gene of orf virus can encode an ATPase (Chan et al. in Gene 432:44–53, 2009). In this work, we cloned the A32L gene into a prokaryotic expression vector, and the recombinant protein was expressed and purified. The antigenicity of recombinant ATPase was examined by immunoblotting, and its identity was confirmed by mass spectrometry. The ATP hydrolysis function of the purified recombinant protein was examined, and our results showed that it exhibited the ATPase activity. Similar to other viral ATPases, the ATPase of orf virus remained active in the presence of different divalent ions; nevertheless, unlike other viral ATPases, our recombinant ATPase exhibited similar enzymatic activity in reaction buffers of different pH.

Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus.

Canine distemper (CD) is a widely distributed disease of dogs, caused by the canine distemper virus (CDV). In the present study, the gene encoding the hemagglutinin (H) protein of a CDV isolate from central Taiwan was sequenced and compared with other strains. Sequence variations were noticed in the H gene from the field CDV strain that had previously been implicated in the increasing incidence of CD. To establish a serology-based diagnostic test, the full-length H protein, as well as five deletion mutants of a recombinant H protein of the local isolate, were produced using an E. coli expression system. Three truncated recombinant proteins with relatively high expression levels, designated HM3, HM4 and HM5, were used as antigens to examine their reactivity with canine sera. By using three negative sera and 17 CD-positive sera, the high specificity of recombinant H proteins was observed by ELISA. In addition, immunoblotting demonstrated that all three purified recombinant proteins exhibit an antigenic property recognized by the serum of a CD-suspected dog.

The genotyping of Infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction

Infectious bronchitis virus (IBV; Avian coronavirus) causes acute respiratory and reproductive and urogenital diseases in chickens. Following sequence alignment of IBV strains, a combination of selective primer sets was designed to individually amplify the IBV wild-type and vaccine strains using a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) approach. This system was shown to discriminate the IBV wild-type and vaccine strains. Moreover, an ARMS real-time RT-PCR (ARMS qRT-PCR) was combined with a high-resolution analysis (HRMA) to establish a melt curve analysis program. The specificity of the ARMS RT-PCR and the ARMS qRT-PCR was verified using unrelated avian viruses. Different melting temperatures and distinct normalized and shifted melting curve patterns for the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were detected. The new assays were used on samples of lung and trachea as well as virus from allantoic fluid and cell culture. In addition to being able to detect the presence of IBV vaccine and wild-type strains by ARMS RT-PCR, the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were distinguished using ARMS qRT-PCR by their melting temperatures and by HRMA. These approaches have acceptable sensitivities and specificities and therefore should be able to serve as options when carrying out differential diagnosis of IBV in Taiwan and China.