Isolation of Campylobacter jejuni and Campylobacter coli from the gall bladder samples of sheep and identification by polymerase chain reaction

In this study, 100 gall bladder samples of sheep slaughtered at an abattoir in Elazi? province were examined for Campylobacter jejuni and Campylobacter coli by culture and polymerase chain reaction (PCR). Preston Campylobacter Agar supplemented with 7% horse blood and Preston Selective Supplement (Oxoid, Hampshire, UK) were used for isolation of the agents. Campylobacter spp. were isolated in 66 samples, and they were identified as 34% C. jejuni and 32% C. coli. A multiplex PCR based upon the use of ceuE gene-specific primers was applied on DNA samples extracted from C. jejuni and C. coli isolates. All C. jejuni and C. coli strains that were positive by culture were also detected to be positive by PCR. This study shows that PCR can be used an alternative, rapid and sensitive test for the identification of C. jejuni and C. coli which threaten human and animal health.     

应用RAPD分子标记探讨拟鹅观草属的种间关系

采用随机扩增多态性DNA(RAPD)技术,分析了拟鹅观草属(Pseudoroegneria)8种1亚种和鹅观草属(Roegneria)7种植物.35个引物产生的344条DNA扩增片段中,328条(95.35%)具有多态性,利用344个RAPD标记,在NTSYS软件中,计算Jaccard遗传相似系数,建立UPGMA聚类图.结果表明:1)物种间遗传差异明显,具有丰富的遗传多样性;2)R. elytrigioides、 R. alashanica和R. magnicaespes与拟鹅观草属的物种聚类在一起,表明它们与拟鹅观草属的亲缘关系较近,而与鹅观草属的亲缘关系较远;3)RAPD分子标记可以将拟鹅观草属的物种分开,而且形态相似,地理分布相同或相近的物种聚类在一起;4)RAPD结果与形态学和细胞学的分析结果一致,表明RAPD技术能为拟鹅观草属植物的系统学研究提供DNA水平上丰富的资料.     

Phenotypic characterization and random amplified polymorphic DNA (RAPD) analysis of Pasteurella multocida isolated from Korean pigs

Pasteurella multocida causes various respiratory disease symptoms in pigs, including atrophic rhinitis and pneumonia. In the present study, 69 strains of P. multocida were isolated from 443 pigs with respiratory clinical symptoms at 182 farms located throughout South Korea from 2009 to 2010. A multiplex capsular PCR typing assay revealed that 69 strains of P. multocida isolated in this study had the biosynthetic locus of the capsules of either serogroup A (47 strains, 68.1%) or serogroup D (22 strains, 31.9%). The 22 strains positive for serogroup D-specific primers were divided into four clusters and the 47 strains positive for serogroup A-specific primers were divided into 12 clusters according to the results of Random Amplified Polymorphic DNA (RAPD) analysis. P. multocida strains in the present study were susceptible to most of the antimicrobial agents used. An analysis of antimicrobial resistance and virulence gene pattern combined with RAPD indicated that a certain P. multocida strain appeared to be genetically identical, implying the persistence of the strain within a single farm.     

Differentiation of Moraxella bovoculi sp. nov. from other coccoid moraxellae by the use of polymerase chain reaction and restriction endonuclease analysis of amplified DNA.

Moraxella ovis was historically the only coccoid Moraxella identified in cultures of ocular fluid from cattle with infectious bovine keratoconjunctivitis (IBK) and could be morphologically and biochemically differentiated from Moraxella bovis. Moraxella bovoculi sp. nov. is a recently characterized Moraxella isolated from ulcerated eyes of calves with IBK in northern California in 2002. Like Moraxella ovis, M. bovoculi sp. nov. is a gram-negative coccus/diplococcus. All 18 original isolates of M. bovoculi sp. nov. possessed phenylalanine deaminase (PADase) activity and could therefore be differentiated from M. ovis and M. bovis. During the characterization of 44 additional isolates of hemolytic gram-negative cocci that were cultured from ulcerated eyes of IBK-affected calves, 2 PADase-negative isolates were identified that could not be differentiated biochemically from M. ovis; however, the DNA sequence of the 16S-23S intergenic spacer region (ISR) of the isolates matched the 16S-23S ISR DNA sequence of M. bovoculi sp. nov. To facilitate the identification of PADase-negative moraxellae, a polymerase chain reaction (PCR) coupled with restriction enzyme digestion analysis of amplified DNA was developed. Amplification of the 16S-23S ISR followed by AfaI digestion of amplified DNA could differentiate M. bovoculi sp. nov. from M. ovis and other moraxellae. The DNA sequence analysis of the amplified 16S-23S ISR from the 42 PADase-positive isolates of hemolytic gram-negative cocci indicated that all were M. bovoculi sp. nov. and all possessed an AfaI site. A PCR coupled with restriction analysis of amplified DNA can aid in identifying M. bovoculi sp. nov.     

Development of a polymerase chain reaction-based method to identify species-specific components in dog food

OBJECTIVES: To determine whether there is a relationship between species-specific mitochondrial DNA (mtDNA), especially canine and feline mtDNA, and detectable amounts of pentobarbital in previously analyzed dog food samples. SAMPLE POPULATION: 31 dog food samples previously analyzed for pentobarbital (limit of detection, 1 microg/kg). PROCEDURE: Polymerase chain reaction (PCR) analysis was performed on dog food samples by use of PCR primers specific for either canine, feline, equine, bovine, porcine, ovine, or poultry mtDNA. RESULTS: PCR amplicons specific for feline or canine mtDNA at a 0.007% (70 microg/g [wt/wt basis]) or 0.0007% (7 microg/g) level, respectively, were not found in the 31 dog food samples. Most of the 31 dog food samples had a PCR amplicon on PCR analysis when a PCR primer set capable of simultaneously detecting mtDNA of cows, pigs, sheep, goats, deer, elk, and horses was used. Results of PCR analysis by use of primers specific for bovine, swine, sheep and goat, or horse mtDNA revealed amplicons specific for bovine or swine mtDNA only in 27 of the 31 samples. Analysis of the remaining 4 samples failed to yield amplicons for any mammalian mtDNA. Pentobarbital was detected in 2 of these 4 samples. Results of PCR analysis correlated with the stated ingredient list for most, but not all samples. CONCLUSIONS AND CLINICAL RELEVANCE: Because canine and feline mtDNA were not found in a set of retail dog food samples, these results indicate that the source of pentobarbital in dog food is something other than proteins from rendered pet remains.     

Serogroups of Campylobacter jejuni from man and animals

A total of 186 campylobacter strains from aborted calf and sheep fetuses, from scouring dogs, rabbits and man, and from retailed poultry were isolated and examined biochemically and serologically for heat stable antigens. Immune sera were produced in rabbits against Penner reference strains from 1 to 60, and against two field isolates. Out of 186 biochemically tested strains 179 (96.2%) proved C. jejuni and only 6 (3.2%) C. coli. One strain has been identified as C. laridis. In cattle and sheep 3.2 and 21.7% respectively of all campylobacter abortions were due to C. jejuni infection. The same agent caused 12.7% of diarrhoea of dogs. The campylobacter infection rate of freshly slaughtered and dressed chicken varied between 25 and 64.3%. Out of the serologically examined 140 C. jejuni strains 118 (84.3%) could be assigned to 16 Penner serogroups and 13 (9.3%) to 2 further serogroups. Serogroups 8 (31.4%), 1 (19.3%) and 2 (12.1%) occurred most frequently. The human isolates represented the widest serotype distribution, as 32 tested strains belonged to 12 serogroups. All those serogroups which caused abortion or diarrhoea in animals or were isolated from poultry carcases were isolated also from man with diarrhoea, but some serogroups were found only in man.     

Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.     

应用随机扩增多态性进行山羊致病性大肠杆菌的分子多态性分析

为了解山羊致病性大肠杆菌广西分离株的分子多态性,应用随机扩增多态性方法(RAPD)对山羊致病性大肠杆菌进行分型研究.从8条随机引物中筛选出4条能在10株大肠杆菌中具有较好多态性扩增的随机引物,4条随机引物共扩增出18条DNA片段,10个菌株无共有带谱,显示出良好的扩增多态性.菌株Nx31与Nx32曾被认为是同一菌株的两次分离,但是在RAPD分析中,两株细菌的带谱存在明显的差别,表明RAPD比传统的血清学分型具有更高的分辨性.     

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH,SRY and AMEL genes

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.     

Detection of babesia species from infected dog blood by polymerase chain reaction

Polymerase chain reaction (PCR) was first applied to diagnosis of canine babesiosis in Japan. Blood samples from 13 dogs suffering from canine babesiosis were used for examination of specificity and sensitivity of the PCR diagnosis. Of the 13 dogs, three were experimentally infected, and ten were naturally infected with Babesia species in west part of Japan. We designed a nested PCR to amplify the babesial small subunit ribosomal RNA gene and found that only the nested PCR produced a visual band, which were not apparent by the first-round PCR to the positive samples. Specificity of the nested PCR was confirmed by amplification after the second-round PCR. Sensitivity of the nested PCR was examined by diluting the blood samples from infected and uninfected dogs. The nested PCR was found to show positive results on the most diluted blood at 0.0001% parasitemia. These results indicate that the nested PCR is highly sensitive and useful for diagnosis of canine babesiosis.     

猪链球菌河南分离株的生化分群与随机扩增DNA(RAPD)分型

从河南省11个猪场的送检病料中分离纯化到11株链球菌,将其进行了系统的生化分群,并应用16条随机引物做随机扩增多态性DNA(RAPD)分型。结果表明,11株猪链球菌分别属于C群(3/11)、D群(2/11)、E群(4/11)和L群(1/11),其中1株未定。在RAPD研究中,有3条引物呈现良好的多态性和稳定性,可产生稳定、复杂的指纹图谱;采用SPSSl0.0软件分析了不同菌株间的遗传距离,绘制出菌株间的亲缘关系树状图。依据遗传距离,分离的11株猪链球菌可被分为4个聚类群。     

用RAPD标记分析高羊茅的遗传多样性

采用RAPD分子标记技术对从国外引进的15个高羊茅品种的遗传多样性进行了研究。从60个随机引物中筛选出12个有效引物,它们共扩增出85条DNA带,其中59条为多态性带,占69.41%,平均每个引物扩增出多态性带4.92条;利用NTSYS-PC软件计算出的不同品种间Jaccard遗传相似系数(GS)变化范围较大,为0.373~0.932;根据得到的遗传相似性矩阵进行非加权组法(UPGMA)聚类分析,建立高羊茅品种的分子系统树状图;以相似系数0.68为标准,可将所有品种分为3类,品种翠丽和贝克各自聚为一类,其余13个品种聚成一类。     

Inter and intra-specific genetic variation of avian Eimeria isolated from Iran by random amplified polymorphic DNA--polymerase chain reaction

Eimeria species from poultry breeder farms without previous exposure to anticoccidial vaccines in five distinct geographical regions of Iran were examined for genetic relatedness by the random amplified polymorphic DNA (RAPD) assay. Eight different oligonucleotide decamers with arbitrary DNA sequences were tested as primers to amplify DNA from five isolates of each E. acervulina, E. tenella, and E. maxima. Depending on the species/isolate-primer combination, between 1 and 14 DNA fragments ranging in size from 240 to 3000 bp were amplified. The two isolates originated from Northeast and North parts of Iran showed minor differences and two isolates originated from Northeast and Southwest of Iran showed major differences in their amplified DNA patterns. The intra-specific similarity coefficient within five isolates of each species of, E. acervulina, E. tenella, E. maxima was 74, 82 and 72%, respectively. The distance indices observed between species were greater than those found between isolates (80-90%) with all examined primers. The inferred phylogenetic tree on the fingerprinting of all species revealed that the RAPD-PCR can easily differentiate within and between species and could be a useful and valuable tool in future epidemiological studies, designing and developing of vaccines against avian coccidosis, here in Iran and neighboring countries.     

Species-specific DNA probes for Campylobacter species isolated from pigs with proliferative enteritis

Cloned, chromosomal DNA probes from porcine isolates of Campylobacter hyointestinalis and C. mucosalis were developed for the detection and identification of these putative swine enteric pathogens. High molecular weight chromosomal DNA from each species was used to construct genomic libraries in plasmids. Recombinants were selected which hybridized strongly to the homologous organism, but not to any other species of Campylobacter. Species-specific recombinants were labeled with phosphorus-32 and tested for sensitivity by dot blot hybridization to various dilutions of DNA and bacteria from each swine species, including C. hyointestinalis, C. mucosalis, C. coli and C. jejuni. Specificity was tested by hybridizing these probes against various strains of C. hyointestinalis or C. mucosalis, and against reference strains of all other described Campylobacter species. A C. hyointestinalis-specific probe and a C. mucosalis-specific probe were identified which were capable of detecting 1 ng of DNA or 10(4) cfu by bacterial spot blotting on nylon membranes. These probes hybridized to intestinal mucosal scrapings containing C. hyointestinalis and C. mucosalis obtained from pigs with proliferative enteritis, but not to material from normal pigs. Thus, cloned, chromosomal DNA probes may be useful in the detection and identification of bacteria involved in swine proliferative enteritis.