A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.
Animals ; Bacteria ; genetics ; isolation & purification ; Bacteriological Techniques ; methods ; Endometritis ; microbiology ; veterinary ; Female ; Multiplex Polymerase Chain Reaction ; standards ; Polymerase Chain Reaction ; veterinary ; Sensitivity and Specificity ; Sheep ; Sheep Diseases ; microbiology ; Tibet

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.
Animals ; Distemper Virus, Canine/genetics/*isolation & purification ; Dogs ; Polymerase Chain Reaction/*methods/*veterinary ; RNA, Viral/genetics/isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Attenuated ; Viral Vaccines

Bacillus (B.) anthracis, the etiological agent of anthrax, is one of the most genetically monomorphic bacteria species in the world. Due to the very limited genetic diversity of this species, classification of isolates of this bacterium requires methods with high discriminatory power. Single nucleotide repeat (SNR) analysis is a type of variable-number tandem repeat assay that evaluates regions with very high mutation rates. To subtype a collection of 21 isolates that were obtained during a B. anthracis outbreak in Korea, we analyzed four SNR marker loci using nucleotide sequencing analysis. These isolates were obtained from soil samples and the Korean Center for Disease Control and Prevention. The SNR analysis was able to detect 13 subgenotypes, which allowed a detailed evaluation of the Korean isolates. Our study demonstrated that the SNR analysis was able to discriminate between strains with the same multiple-locus variable-number tandem repeat analysis genotypes. In summary, we obtained SNR results for four SNR marker loci of newly acquired strains from Korea. Our findings will be helpful for creating marker systems and help identify markers that could be used for future forensic studies.
Bacillus anthracis/*classification/*genetics/isolation & purification ; *Genetic Variation ; *Minisatellite Repeats ; Polymerase Chain Reaction/veterinary ; Republic of Korea ; Sequence Analysis, DNA/*methods/veterinary ; *Soil Microbiology

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.
Aeromonas salmonicida/*genetics ; Animals ; DNA Primers/genetics ; Fish Diseases/*diagnosis/*microbiology ; Fishes ; Flavobacterium/*genetics ; Gram-Negative Bacterial Infections/diagnosis/*veterinary ; Polymerase Chain Reaction/methods/*veterinary ; Yersinia rucker/*genetics

The present study was to construct a parentage testing system for Thoroughbred (TB) horse. A total number of 1,285 TB horse samples including 962 foals for parentage testing, 9 sires and 314 dams for individual identification were genotyped. Genomic DNA was extracted from 5 hair roots and genotyped by using 14 microsatellite markers (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS3, HMS6, HMS7, HTG4, HTG10, LEX3 and VHL20). This method consisted of multiplexing PCR procedure and showed reasonable amplification of all PCR products. Genotypes were determined by genetic analyzer. The number of alleles per locus varied from 3 to 9 with a mean value of 6.36 in TB horse. The expected heterozygosity was ranged from 0.548 to 0.831 (mean 0.699), and the total exclusion probability of 14 microstellite loci was 0.9998. Of the 14 markers, ASB2, ASB17, ASB23, HMS7 and HTG10 loci have relatively high PIC value (> 0.7). Of the 962 foals, 960 foals were qualified by compatibility according to the Mendelism. These results suggest that the DNA typing method has high potential for parentage verification and individual identification of TB horses.
Alleles ; Animals ; DNA/chemistry/genetics ; DNA Fingerprinting/methods/*veterinary ; Female ; Genotype ; Horses/*genetics ; Korea ; Male ; Microsatellite Repeats/genetics ; Pedigree ; Polymerase Chain Reaction/veterinary ; Polymorphism, Genetic

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C.
Animals ; Foot-and-Mouth Disease/*diagnosis ; Foot-and-Mouth Disease Virus/genetics ; Nucleic Acid Amplification Techniques/*methods/veterinary ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Reverse Transcription/genetics ; Sensitivity and Specificity

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.
Animals ; Edwardsiella tarda/genetics/*isolation & purification ; Enterobacteriaceae Infections/diagnosis/microbiology/*veterinary ; Fish Diseases/*diagnosis/microbiology ; Fisheries/*methods ; *Flatfishes ; Multiplex Polymerase Chain Reaction/economics/*veterinary ; Sensitivity and Specificity ; Streptococcal Infections/diagnosis/microbiology/*veterinary ; Streptococcus/genetics/*isolation & purification

Using three reference strains of Brachyspira hyodysenteriae (B204, B234, B169), one B. pilosicoli (P43/6/78), one B. murdochii (56-150), one B. intermedia (PWS/A), one B. innocens (B256) and ten Korean isolates, PCR-RFLP analysis of DNA encoding 23S rRNA was performed to establish a rapid and accurate method for characterizing porcine intestinal spirochetes. Consequently, B. hyodysenteriae and B. pilosicoli revealed different restriction patterns; however, the other three species shared the same pattern. These findings are not consistent with a prior report. Differences in 23S rRNA gene sequences, between two B. murdochii strains, 56-150 and 155-20, were observed. These results indicate that 23S rRNA PCR-RFLP could be used as an identification method for pathogenic Brachyspira spp. (B. hyodysenteriae and B. pilosicoli) as well as an epidemiological tool for characterizing spirochetes isolated from swine.
Animals ; DNA, Bacterial/genetics ; Dysentery, Bacillary/diagnosis/microbiology/*veterinary ; Korea ; Phylogeny ; Polymerase Chain Reaction/methods/*veterinary ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 23S/chemistry/*genetics ; Spirochaetales/*genetics/*isolation&purification ; Spirochaetales Infections/diagnosis/microbiology/*veterinary ; Swine ; Swine Diseases/diagnosis/*microbiology

Streptococcus (S.) dysgalactiae subsp. equisimilis is responsible for severe diseases in humans, including primary bacteraemia, pneumonia, endocarditis, and toxic shock syndrome. Infection in some animal species can also occur, although a few studies have looked into cross-species infectivity. In horses, S. equisimilis is generally considered infrequent or opportunistic, but has recently been isolated from cases of strangles-like disease. Rapid and sensitive diagnostic techniques could enable epidemiological studies and effective investigation of outbreaks involving these bacteria. In this study, PCR protocols previously described in cattle and in humans to detect the species S. dysgalactiae and the subspecies equisimilis were evaluated to detect specific sequences in equine samples. For this purpose, 99 monolateral nasal swabs were collected from horses from stud farms with a history of S. equisimilis infection and were tested blindly by bacteriological isolation and by single and duplex PCR. DNA for PCR was extracted both from the colonies grown on agar media and from enrichment broth aliquots after incubation with nasal swab samples. S. equisimilis was identified by bacteriological isolation in 23 out of 99 swab samples, and PCR assays on these colonies were fully concordant with bacteriological identification (kappa statistic = 1.00). In addition, PCR of the enrichment broth aliquots confirmed the bacteriological results and detected S. equisimilis in 6 samples more than the bacteriological examination (kappa statistic = 0.84). The PCR protocols appeared to be reliable for the rapid identification of S. equisimilis in equine nasal swab samples, and could be useful for microbiological diagnosis.
Animals ; NA, Bacterial/chemistry/genetics ; Horse Diseases/diagnosis/*microbiology ; Horses ; Limit of Detection ; Male ; Nasopharynx/microbiology ; Polymerase Chain Reaction/methods/*veterinary ; Respiratory Tract Infections/diagnosis/microbiology/*veterinary ; Sensitivity and Specificity ; Streptococcal Infections/diagnosis/microbiology/*veterinary ; Streptococcus/genetics/*isolation & purification

A multiplex PCR assay was developed for the simultaneous detection of the etiologic agents associated with porcine proliferative enteropathies (PPE), swine dysentery (SD)and porcine salmonellosis (PS)in a single reaction using DNA from swine intestinal samples. Single and multiplex PCR amplification of DNA from Lawsonia intracellularis, Salmonella typhimurium and Brachyspira hyodysenteriae with each primer set produced fragments of the predicted size without any nonspecific amplification, 210-bp, 298-bp and 403-bp bands, respectively. The single PCR assay could detect as little as 100 pg of purified DNA of S. typhimurium and L. intracellularis, and 50 pg of B.hyodysenteriae, respectively. However, multiplex PCR turned out to be 10 times lower sensitivity with S. typhimurium compared with single PCR. With 23 swine intestinal specimens suspected of having PPE, SD and/or PS, the multiplex PCR assay showed identical results with conventional methods except one. In conclusion, this multiplex PCR is a feasible alternative to standard diagnostic methods for detection of L. intracellularis, B. hyodysenteriae and Salmonella spp. from swine intestinal specimens.
Animals ; Desulfovibrionaceae Infections/microbiology/veterinary ; Intestines/microbiology ; Lawsonia Bacteria/*isolation&purification ; Polymerase Chain Reaction/*methods/veterinary ; Salmonella/*isolation&purification ; Salmonella Infections, Animal/diagnosis ; Sensitivity and Specificity ; Spirochaetales/*isolation&purification ; Spirochaetales Infections/microbiology/veterinary ; Swine ; Swine Diseases/*diagnosis/*microbiology