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Wei B.,Chonbuk National University | Cha S.-Y.,Chonbuk National University | Kang M.,Chonbuk National University | Park I.-J.,Chonbuk National University | And 3 more authors.
Poultry Science | Year: 2013

Infections with Pasteurella multocida, Salmonella enterica, Riemerella anatipestifer, and Escherichia coli result in high morbidity and mortality, which cause significant economic loss in the poultry industry. It can be difficult to distinguish these pathogens based on clinical signs because these pathogens can cause similar clinical signs and coinfections can occur. Thus, rapid and sensitive detection of these 4 major bacterial pathogens are important in ducks. The aim of this study was to develop a multiplex PCR (mPCR) assay for simultaneously detecting and identifying these 4 pathogenic bacteria in a single tube reaction. The target genes used were KMT1 of P. multocida, the invasion protein gene of S. enterica, 16S rDNA of R. anatipestifer, and the alkaline phosphatase gene of E. coli. The detection limit of the assay for all bacterial DNA was 10 pg. The mPCR did not produce any nonspecific amplification products when tested against other related pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, Mycoplasma gallinarum, Mycoplasma synoviae, and Mycoplasma gallisepticum, which can also infect ducks. We applied mPCR to field samples, and the results were the same as the single PCR results. These results suggest that mPCR for the 4 bacteria is a useful and rapid technique to apply to field samples. © 2013 Poultry Science Association Inc.


Cha S.-Y.,Chonbuk National University | Kang M.,Chonbuk National University | Yoon R.-H.,Chonbuk National University | Park C.-K.,Kyungpook National University | And 2 more authors.
Comparative Immunology, Microbiology and Infectious Diseases | Year: 2013

An investigation was carried out to determine the prevalence and antibiotic resistance of Salmonella serotypes at South Korean duck farms. A total of 7119 samples collected from 72 duck farms in five provinces were examined from 2011 to 2012. The overall prevalence of Salmonella serotypes was 43.4% (69/159) in duck flocks from 65.2% (47/72) of the duck farms. Eighty-five strains were isolated from 69 duck flocks. Three serotypes of Salmonella enterica were identified such as S. Typhimurium (39/85), S. Enteritidis (44/85), and S. London (2/85). The prevalence of Salmonella infection decreased significantly in 3-week-old ducks compared to that in 1-week-old ducks (P< 0.05). All isolates except one were resistant to at least one antimicrobial and 27% of the isolates were resistant to 5-16 antimicrobials. Our findings provide baseline information on the degree of Salmonella infection and distribution of Salmonella serotypes in ducks and indicate that ducks should be considered an important source of foodborne pathogens. © 2013 Elsevier Ltd.


Lee K.-J.,Plant and Fisheries Quarantine and Inspection Agency QIA | Choi J.-G.,Plant and Fisheries Quarantine and Inspection Agency QIA | Kang H.-M.,Plant and Fisheries Quarantine and Inspection Agency QIA | Kim K.-I.,Plant and Fisheries Quarantine and Inspection Agency QIA | And 2 more authors.
Clinical and Vaccine Immunology | Year: 2013

Outbreaks of avian influenza A virus infection, particularly the H5N1 strains that have affected birds and some humans for the past 15 years, have highlighted the need for increased surveillance and disease control. Such measures require diagnostic tests to detect and characterize the different subtypes of influenza virus. In the current study, a simple method for producing reference avian influenza virus antisera to be used in diagnostic tests was developed. Antisera of nine avian influenza A virus neuraminidases (NA) used for NA subtyping were produced using a recombinant baculovirus. The recombinant NA (rNA) proteins were expressed in Sf9 insect cells and inoculated intramuscularly into specific-pathogen-free chickens with the ISA70 adjuvant. The NA inhibition antibody titers of the rNA antiserum were in the ranges of 5 to 8 and 6 to 9 log2 units after the primary and boost immunizations, respectively. The antisera were subtype specific, showing low cross-reactivity against every other NA subtype using the conventional thiobarbituric acid NA inhibition assay. These results suggest that this simple method for producing reference NA antisera without purification may be useful for the diagnosis and surveillance of influenza virus. Copyright © 2013, American Society for Microbiology. All Rights Reserved.


Park J.-W.,Korea University | Jin Lee S.,Korea University | Choi E.-J.,Plant and Fisheries Quarantine and Inspection Agency QIA | Kim J.,Plant and Fisheries Quarantine and Inspection Agency QIA | And 2 more authors.
Biosensors and Bioelectronics | Year: 2014

In this study, we successfully developed a ssDNA aptamer pairs by using an advanced immobilization-free SELEX method with affinity-based selection and counter-screening process at every round. By implementing this method, two different aptamers specifically binding to bovine viral diarrhea virus type 1(BVDV type 1) with high affinity were successfully screened. This aptamer pair was applied to ultrasensitive detection platform for BVDV type 1 in a sandwich manner. The ultrasensitive detection of BVDV type 1 using one of aptamers conjugated with gold nanoparticles was obtained in aptamer-aptamer sandwich type sensing format, with the limit of detection of 800. copies/ml, which is comparable to a real-time PCR method. © 2013 Elsevier B.V.


Chae H.S.,Seoul Metropolitan Government Research Institute of Public Health and Environment | Park G.N.,Catholic University of Pusan | Kim S.H.,Catholic University of Pusan | Jo H.J.,Catholic University of Pusan | And 7 more authors.
Poultry Science | Year: 2012

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. © 2012 Poultry Science Association Inc.


So J.H.,Plant and Fisheries Quarantine and Inspection Agency QIA | Kim J.,Yonsei University | Bae I.K.,Yonsei University | Jeong S.H.,Yonsei University | And 4 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2012

This study was performed to investigate the prevalence of rectal colonization with multidrug-resistant Escherichia coli in dogs hospitalized at veterinary hospitals in Korea and to assess the molecular epidemiologic traits of this organism. A total of 63 unique E. coli isolates obtained from the rectal swabs of hospitalized dogs were analyzed. Genes encoding CTX-M extended-spectrum β-lactamases (ESBLs) and AmpC enzymes were detected in 21 (33.3%) and 15 (23.8%) canine E. coli isolates, respectively. Twelve canine E. coli isolates harbored both the genes encoding the CTX-M and AmpC enzymes. Six ESBL-producing E. coli isolates also carried the rmtB gene. All 24 E. coli isolates producing CTX-M ESBL and/or CMY-2 were resistant to ciprofloxacin. Furthermore, mutations were found in the gyrA and the parC genes. In most cases, the bla genes of the CTX-M ESBL and AmpC enzymes and the rmtB gene were localized to incompatibility group F (IncF) plasmids. Possible small clonal outbreaks are suggested because some E. coli isolates recovered in the same veterinary hospital were identified as identical sequence types and showed identical banding patterns in repetitive sequence-based polymerase chain reaction. The horizontal transfer of IncF plasmids and the clonal transfer of E. coli strains are suggested to play a role in the dissemination of antimicrobial resistance genes, and this transfer may occur across host species (i.e., between humans and dogs). © 2012 Elsevier Inc.


Tamang M.D.,Plant and Fisheries Quarantine and Inspection Agency QIA | Nam H.-M.,Plant and Fisheries Quarantine and Inspection Agency QIA | Kim S.-R.,Plant and Fisheries Quarantine and Inspection Agency QIA | Chae M.H.,Plant and Fisheries Quarantine and Inspection Agency QIA | And 3 more authors.
Foodborne Pathogens and Disease | Year: 2013

The purpose of this study was to determine the prevalence and characteristics of CTX-M β-lactamases in Escherichia coli among healthy swine and cattle in Korea. A total of 1212 fecal samples obtained from healthy pigs (n=558) and cattle (n=654) were screened for CTX-M-type extended spectrum β-lactamase (ESBL)-producing E. coli isolates. One hundred and twenty-one E. coli that produced ESBL were subjected to phenotypic and genotypic characterization. A high number (120/558, 21.5%) of swine fecal samples showed the presence of CTX-M β-lactamase-producing E. coli compared to cattle samples (1/654, 0.2%). The most predominant CTX-M-type identified was CTX-M-14 (n=82), followed by CTX-M-15 (n=16). Isolates producing CTX-M-3, CTX-M-27, CTX-M-55, and CTX-M-65 were also identified. Overall, the blaTEM-1 gene was associated with CTX-M β-lactamase in 55 E. coli isolates. Transfer of blaCTX-M gene was demonstrated from 76 out of 121 bla CTX-M-positive E. coli isolates to the recipient E. coli J53 by conjugation. Plasmid DNA isolation from the transconjugants revealed a large (90-120 Kb) conjugative plasmid. ISEcp1 and IS903 were detected upstream and downstream of blaCTX-M genes in 117 and 91 E. coli isolates, respectively. Our results demonstrated that a combination of clonal expansion and horizontal transmission is spreading blaCTX-M genes among swine E. coli. The horizontal dissemination of blaCTX-M genes among E. coli was mostly mediated by IncF or IncI1-Iγ plasmids. To the best of our knowledge, this study represents the first report of CTX-M-3, CTX-M-27, CTX-M-55, and CTX-M-65 β-lactamases in bacterial isolates from food animals in Korea. This study revealed that the CTX-M β-lactamase-producing E. coli are widely disseminated among healthy pigs but very rare in cattle in Korea. Increasing prevalence of blaCTX-M genes in intestinal E. coli of food animals is a matter of concern and should be carefully monitored. © Mary Ann Liebert, Inc.


Lee H.S.,Purdue University | Her M.,Plant and Fisheries Quarantine and Inspection Agency QIA | Levine M.,Purdue University | Moore G.E.,Purdue University
Preventive Veterinary Medicine | Year: 2013

Brucellosis is considered to be one of the most important zoonotic diseases in the world, affecting underdeveloped and developing countries. The primary purpose of brucellosis control is to prevent the spread of disease from animals (typically ruminants) to humans. The main objective of this study was to retrospectively develop an appropriate time series model for cattle-to-human transmission in South Korea using data from independent national surveillance systems. Monthly case counts for cattle and people as well as national population data were available for 2005-2010. The temporal relationship was evaluated using an autoregressive integrated moving average with exogenous input (ARIMAX) model [notated as ARIMA(p, d, q) - AR(p)] and a negative binomial regression (NBR) model.Human incidence rate was highly correlated to cattle incidence rate in the same month and the previous month (both r=0.82). In the final models, ARIMA (0, 1, 1) - AR (0, 1) was determined as the best fit with 191.5% error in the validation phase, whereas the best NBR model including lags (0, 1 months) for the cattle incidence rate yielded a 131.9% error in the validation phase. Error (MAPE) rates were high due to small absolute human case numbers (typically less than 10 per month in the validation phase). The NBR model however was able to demonstrate a marked reduction in human case immediately following a hypothetical marked reduction in cattle cases, and may be better for public health decision making. © 2013 Elsevier B.V.


PubMed | Plant and Fisheries Quarantine and Inspection Agency QIA
Type: Comparative Study | Journal: Journal of virological methods | Year: 2012

Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, the Korean sacbrood virus (KSBV) caused great losses in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting KSBV using total RNA extracted from honeybees (A. cerana) infected with SBV. The LAMP and the polymerase chain reaction (PCR) methods were then compared for their ability to detect KSBV in clinical samples. The virus was detected in RT-LAMP reactions containing 10(3) copies of pBX-KSBV within 30min, which was comparable to RT-PCR. In addition, the LAMP was able to distinguish between KSBV and other closely-related SBV strains, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay is a useful method for the rapid diagnosis of KSBV infection in honeybees.

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