猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌复合PCR检测方法的建立

目的建立可以同时检测猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速而可靠的PCR检测方法。方法和结果根据胸膜肺炎放线杆菌的Apx-VIA基因序列、多杀性巴氏杆菌和副猪嗜血杆菌的16SrRNA基因序列设计5条引物。猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌模板的PCR扩增产物大小分别为342bp,485bp和1258bp。复合PCR对1～12型猪胸膜肺炎放线杆菌标准株,6株多杀性巴氏杆菌标准株,1～15型副猪嗜血杆菌以及25株经生化鉴定确认为上述三种细菌的分离株的基因组DNA作为模板进行检测,均获得预期大小的扩增产物。以猪放线杆菌、吲哚放线杆菌等14种常见细菌作为阴性对照进行PCR检测,结果仅有支气管败血波氏杆菌产生了可以和上述三个特异性条带明显区分的PCR产物。复合PCR针对胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的敏感性分别为14pg、34pg和37pg。结论本研究建立的复合PCR特异性好,敏感性高,可以用于猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速检测。     

Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates

In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.     

Development and application of a multiplex polymerase chain reaction for avian respiratory agents

A multiplex polymerase chain reaction (PCR) was developed and optimized to simultaneously detect 6 avian respiratory pathogens. Six sets of specific oligonucleotide primers for infectious bronchitis virus (IBV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were used respectively in the test. With the use of agarose gel electrophoresis for detection of the PCR-amplified DNA products, the sensitivity of detection was between 10 pg for IBV, AIV, MG, and ILTV and 100 pg for NDV and MS after 35 cycles of PCR. Similar sensitivity of these primers was achieved with chickens experimentally infected with respiratory pathogens. In experimental infections, the multiplex PCR was able to detect all the infected chickens in each group at I and 2 wk postinfection as compared with serologic tests at 2 wk postinfection that confirmed the presence of specific antibodies. The multiplex PCR was also able to detect and differentiate coinfections with two or more pathogens. No specific DNA amplification for respiratory avian pathogens was observed among noninoculated birds kept separately as a negative control group.     

Development of a polymerase chain reaction assay for the detection of antibiotic resistance genes in community DNA.

Many methods are used to detect antibiotic resistance genes in samples. The objective of the study reported here was to compare polymerase chain reaction (PCR) analysis of community DNA with fecal culturing for detecting antibiotic resistance genes in cattle samples. In the laboratory-based portion of this study, known concentrations of an Escherichia coli strain with 3 antibiotic resistance genes (cmy-2, flo, and cat) were added to feces from dairy cattle. These genes were used to assess the effect of various primer pairs, chromosomally versus plasmid-encoded genes, and gene copy number on the sensitivity of PCR amplification. Gene-specific PCR amplification was performed on the community DNA extracted from the feces. Feces were cultured for the inoculated strain. In the field-based portion of the study, 80 cattle fecal samples of unknown gene status were compared by use of similar methods. Culture and PCR amplification from community DNA extractions produced variable results, and this variability was most noticeable at dilutions that approached the detection limit of the assay. Typically, PCR amplification had a higher sensitivity than did culture for detecting the gene of interest. However, the sensitivity of culture was improved by plating on selective media containing antibiotics. The community DNA approach enables assessment of bacterial communities in complex samples such as feces, a task that can be prohibitive by budget or time constraints associated with culture methods. Through a strategic combination of culture and community DNA approaches, the relationship between specific selection pressures and the persistence and dissemination of specific resistance genes can be elucidated.     

猪胸膜肺炎放线杆菌PCR检测方法的建立及临床应用

为建立直接检测可疑病料中胸膜肺炎放线杆菌（APP）的PCR诊断方法，对用以扩增apxⅣA毒力基因3’端长约442bp的核苷酸片段的引物、模板、Taq DNA聚合酶、dNTPs的最适剂量及退火温度进行了优化筛选，并进行了特异性及重复性试验；以筛选出的优化条件，对临床可疑病料进行了PCR检测。结果，在25μL体系中，以5pmol／μL引物、0．25μL Taq DNA聚合酶、2mmol／L dNTPs、56℃退火温度对APP标准菌株apxⅣA毒力基因的扩增效果最好，重复性和稳定性高；而对类症菌株的扩增结果则为阴性，表明其特异性强。对分离到APP的病料PCR阳性率为100％（5／5）；未分离到APP的纤维渗出性病料中，新鲜与陈旧病料PCR阳性率分别为70．59％（12／17）、50．00％（4／8），而非纤维渗出性病料的PCR结果均为阴性（0／45、0／9）。结果表明，建立的PCR方法特异、快速、稳定、敏感，优于细菌学方法，它可作为APP可疑病料的理想诊断方法。     

猪肺炎支原体PCR诊断方法的建立

建立了一个PCR检测猪肺炎支原体(Mhp)的方法。根据国外发表Mhp 16sr RNA基因设计了一对特异性引物，扩增出一个大小为653bp的特异性片段。将PCR产物克隆并测序表明，与GenBank的Mhp的序列的同源性为89．2％。而对于常见的猪呼吸道疾病有关的病原胸膜肺炎放线杆菌、支气管败血波氏杆菌、多杀性巴氏杆菌以及牛支原体、羊支原体不能扩增出特异性片段；PCR的敏感性实验显示这对引物能够检测到1ng的DNA，结果表明此方法特异、敏感。     

CSFV、PRV和PRRSV多重PCR检测方法的建立及其应用

由猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)和猪繁殖与呼吸综合征病毒(PRRSV)引起的繁殖障碍性疫病在中国的不断暴发,增加了猪的发病率和死亡率。为了建立同时检测这3种病毒的多重PCR方法,本研究根据GenBank中这3种病毒的参考基因序列设计引物,并对反应中的影响因素进行优化,建立了同时检测CSFV、PRV和PRRSV的多重PCR方法。该方法扩增的基因片段大小分别为570(CSFV),232 (PRRSV)和173bp(PRV)。敏感性和特异性试验结果显示,该方法对3种病毒的核酸最低检出量分别为23.88(CSFV),13.10(PRRSV)和14.60pg(PRV),对大肠杆菌(Ec.oli)、沙门菌(Salmonella)、猪乙型脑炎病毒(JEV)及猪圆环病毒2型(PCV2)的检测结果均为阴性。对2015年5月至2016年1月收集的168份临床样本检测结果显示,CSFV阳性率为24.4%,PRRSV阳性率为21.4%,提示河北省该段时间内引起猪繁殖障碍性疫病的主要病原为CSFV和PRRSV。经多重PCR检测为PRRSV、PRV或CSFV阳性的临床样本,再次使用商品化的试剂盒进行检测,符合率为100.0%。这些结果表明,本试验建立的多重PCR方法省时、高效,可用于PRRSV、PRV和CSFV感染的临床诊断。     

胸膜肺炎放线杆菌ApxⅡ毒素结构基因的原核表达以及重组蛋白的免疫原性分析

猪传染性胸膜肺炎（Porcine infectious pleuropneumonia）是由胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）引起的猪的一种呼吸道疾病。本实验以APP血清7型的一株现地分离株L25—4株为研究对象，对其分泌的ApxⅡ毒素的结构基因apxⅡA进行了全基因克隆和测序，并利用原核表达载体pGEX-6P-1在E．coli中进行了原核表达。表达产物以包涵体形式存在，包涵体蛋白经过洗涤后作为抗原用于Western—blotting免疫印迹实验，结果表明重组的ApxⅡA蛋白具有良好的免疫原性。     

Evaluation of a multiplex polymerase chain reaction assay for simultaneous detection of Rhodococcus equi and the vapA gene

OBJECTIVE: To evaluate sensitivity and specificity of a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Rhodococcus equi and differentiation of strains that contain the virulence-associated gene (vapA) from strains that do not. SAMPLE POPULATION: 187 isolates of R equi from equine and nonequine tissue and environmental specimens and 27 isolates of bacterial species genetically or morphologically similar to R equi. PROCEDURE: The multiplex PCR assay included 3 gene targets: a universal 311-bp bacterial 16S ribosomal RNA amplicon (positive internal control), a 959-bp R equi-specific target in the cholesterol oxidase gene (choE), and a 564-bp amplicon of the vapA gene. Duplicate multiplex PCR assays for these targets and confirmatory singleplex PCR assays for vapA and choE were performed for each R equi isolate. An additional PCR assay was used to examine isolates for the vapB gene. RESULTS: Results of duplicate multiplex and singleplex PCR assays were correlated in all instances, revealing high specificity and reliability (reproducibility) of the vapA multiplex assay. Of the pulmonary isolates from horses with suspected R equi pneumonia, 97.4% (76/78) yielded positive results for vapA. Seven of 50 (14%) human isolates of R equi yielded positive results for vapA. Six human R equi isolates and 1 porcine isolate yielded positive results for vapB. No isolates with vapA and vapB genes were detected. CONCLUSIONS AND CLINICAL RELEVANCE: The multiplex PCR assay is a sensitive and specific method for simultaneous confirmation of species identity and detection of the vapA gene. The assay appeared to be a useful tool for microbiologic and epidemiologic diagnosis and research.     

Development of multiplex polymerase chain reaction assays for detecting enterotoxigenic Escherichia coli and their application to field isolates from piglets with diarrhea.

Fimbriae and enterotoxins are major virulence factors associated with enterotoxigenic Escherichia coli (ETEC). In this study, 3 sets of multiplex polymerase chain reaction (mPCR) assays targeting fimbriae, enterotoxins, and other adherence factors were developed for detecting ETEC. A total number of 188 E. coli field isolates were examined, and percentages of E. coli strains carrying each virulence factors were as follows: F4 (7.45%), F5 (29.79%), F6 (6.38%), F18 (15.43%), F41 (3.72%), STa (10.11%), STb (20.74%), LT (9.57%), Stx2e (2.13%), EAST1 (42.02%), F1 (67.55%), AIDA-I (2.66%), and pAA (7.45%). Of the 188 E. coli field isolates examined, 25.53% were found to be pathogenic ETEC, having both fimbriae and enterotoxins. However, the ratio increased to 44.68% when the presence of other adhesins was considered as criteria for virulence. Among the adherence factors, F1 was found to be the most prevalent. AIDA-I and pAA were also found with similar ratio as compared with other virulence factors. In addition, virulence patterns carrying these alternate adhesive genes with enterotoxins were detected with significant ratio. Therefore, it is desirable that alternate adhesins be considered as markers for diagnosis of ETEC.     

Development of a polymerase chain reaction to detect Vietnamese isolates of duck virus enteritis.

A polymerase chain reaction (PCR) method for the detection of duck virus enteritis (DVE) virus in tissues of infected and affected ducks, and in cell culture was developed. This required us to obtain specific nucleotide sequence information as we could not find any specific data about the genome of the virus. We found the assay to be highly effective in detecting the virus under experimental conditions and to be easily transferred to laboratories in Vietnam where it is being used in studies on the epidemiology of the disease. We have applied this simple and rapid diagnostic method to the detection of DVE isolates grown in cell culture and tissues from infected birds. The assay was also able to differentiate DVE from other avian herpesviruses, such as Marek's disease, infectious laryngotracheitis virus and goose herpesvirus.     

Development and field validation of a Mycoplasma iowae real-time polymerase chain reaction assay

A Mycoplasma iowae real-time polymerase chain reaction (PCR) assay using primers and probes targeting the 16S rRNA gene was developed and field-validated in this study. The assay specifically identified M. iowae with a detection limit of 80 colony-forming units (cfu) per turkey cloacal swab sample (3.2 cfu per PCR reaction). It was validated by testing 154 field turkey cloacal swab samples in parallel with culture isolation. The diagnostic sensitivity of the PCR was 97.6%, and the specificity was 95.5%. The real-time PCR developed in this study is a rapid, sensitive, and cost-effective alternative to culture isolation for detecting M. iowae from cloacal swab samples.     

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.     

Development of a quantitative-competitive polymerase chain reaction assay for serotype 1 Marek's disease virus

We have developed a quantitative-competitive (QC) polymerase chain reaction (PCR) for the detection of Marek's disease virus (MDV) DNA. The assay utilizes a competitor DNA that differs from the viral DNA of interest by having a small insertion. The competitor DNA acts as an internal standard for the estimation of viral DNA in an unknown sample. The amount of viral DNA in a sample is quantitated by coamplification in the presence of a known amount of competitor DNA. The same PCR primers that amplify the viral DNA also amplify the competitor DNA. When the amount of competitor is equal to the amount of viral DNA in a sample, there is equal amplification of the competitor and the virus. Thus, we are able to quantitate the viral DNA in an unknown sample. To establish the utility of this assay, in vivo correlations between virulence and virus replication were studied. Our data demonstrated that a more virulent strain of MDV (648A) replicated better in thymus during cytolytic infection than did a less virulent strain (GA). However, no differences in virus titer were observed when these two viruses were propagated in tissue culture. Our data are consistent with the generally held idea that "hot" strains of MDV replicate earlier and better in birds. Thus, QC-PCR is extremely specific and sensitive to measure MDV DNA over a wide range and can be applied to in vivo studies of viral pathogenesis.     

Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey

By the end of 2004, the Canadian swine population had experienced a severe increase in the incidence of Porcine circovirus-associated disease (PCVAD), a problem that was associated with the emergence of a new Porcine circovirus-2 genotype (PCV-2b), previously unrecovered in North America. Thus, it became important to develop a diagnostic tool that could differentiate between the old and new circulating genotypes (PCV-2a and PCV-2b, respectively). Consequently, a multiplex real-time quantitative polymerase chain reaction (mrtqPCR) assay that could sensitively and specifically identify and differentiate PCV-2 genotypes was developed. A retrospective epidemiologic survey that used the mrtqPCR assay was performed to determine if cofactors could affect the risk of PCVAD. From 121 PCV-2-positive cases gathered for this study, 4.13%, 92.56%, and 3.31% were positive for PCV-2a, PCV-2b, and both genotypes, respectively. In a data analysis using univariate logistic regressions, the PCVAD-compatible (PCVAD/c) score was significantly associated with the presence of Porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV viral load, PCV-2 viral load, and PCV-2 immunohistochemistry (IHC) results. Polytomous logistic regression analysis revealed that PCVAD/c score was affected by PCV-2 viral load (P = 0.0161) and IHC (P = 0.0128), but not by the PRRSV variables (P > 0.9), which suggests that mrtqPCR in tissue is a reliable alternative to IHC. Logistic regression analyses revealed that PCV-2 increased the odds ratio of isolating 2 major swine pathogens of the respiratory tract, Actinobacillus pleuropneumoniae and Streptococcus suis serotypes 1/2, 1, 2, 3, 4, and 7, which are serotypes commonly associated with clinical diseases.     

Development of a multiplex PCR test for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and 12

A PCR assay for simultaneous species identification and separation of Actinobacillus pleuropneumoniae serovars 1, 7 and 12 was developed. Primers specific for genes involved in biosynthesis of the capsular polysaccharides (cps genes) of serovars 1, 7, and 12 were combined with a species-specific PCR test based on the omlA gene. The PCR test was evaluated with the serovar reference strains of A. pleuropneumoniae as well as 183 Danish field isolates. For all typable strains, a complete correspondence was found between results obtained with the multiplex PCR test and results from the traditional serotyping methods. Among eight serologically cross-reacting strains designated K1:O7, seven isolates produced amplicons of similar sizes as serovar 1 and one isolate produced amplicons of similar sizes as serovar 7. The species specificity of the assay was evaluated using a collection of 126 strains representing 25 different species within the family Pasteurellaceae including 45 field strains of the phylogenetically affiliated species Actinobacillus lignieresii. All these isolates tested negative for the cps genes by the multiplex PCR test except for 6 isolates of A. lignieresii. Five of these isolates produced an amplicon identical to the cps gene of serovar 7, whereas one isolate produced an amplicon identical to the cps gene of serovar 1. In addition, four isolates of Actinobacillus genomospecies 1 tested positive for the omlA gene but negative for the cps genes. The test represents a convenient and specific method for serotyping A. pleuropneumoniae in diagnostic laboratories.     

Development of a polymerase chain reaction test for Entamoeba invadens.

Entamoeba invadens is a protozoal parasite of reptiles that causes colitis, abscesses of liver and other organs, and sometimes acute death. It is generally considered a commensal of chelonians but has also been implicated as a cause of colitis, diarrhea, and death in gopher (Gopherus polyphemus) and leopard (Geochelone pardalis) tortoises. Diagnosis of E. invadens is currently by detection of trophozoites and/or cysts upon direct fecal examination. However, definitive diagnosis of E. invadens has been difficult due to the very similar morphology of nonpathogenic Entamoeba spp., including E. ranarum, E. insolita, E. barreti, and E. terrapinae. Definitive speciation of Entamoeba spp. is important to avoid misdiagnosis or overtreatment for nonpathogenic protozoa. It is also important for consideration of mixed species reptile collections to avoid exposing snakes and lizards to E. invadens. In this study, we developed polymerase chain reaction (PCR) primers for E. invadens, E. ranarum, E. terrapinae, and E. insolita and conducted PCR amplification of purified DNA from cell cultures, as well as purified DNA from reptile stool samples with E. invadens trophozoites added. As a result of this study, a naturally occurring infection of E. invadens was confirmed in a giant South American river turtle (Podocnemis expansa). This study has developed successful PCR primers for four species of Entamoeba and demonstrates that PCR is a promising diagnostic tool for the definitive identification of E. invadens.     

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.     

Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum

Pythium insidiosum is an important cause of cutaneous and gastrointestinal disease in horses and dogs in the southeastern United States. Culture-based diagnosis of pythiosis is rarely definitive because production and identification of reproductive structures is difficult. The purpose of this study was to develop a polymerase chain reaction (PCR)-based assay for the identification of P insidiosum. Genomic DNA was extracted from 3 clinical isolates of P insidiosum and I isolate each of Pythium graminicola and Pythium arrhenomanes. The ITS I region of the ribosomal RNA gene of each isolate was amplified and sequenced, and the resultant sequences were aligned with published sequences for Pythium aphanidermatum, P acanthicum, and P myriotylum. A pair of P insidiosum-specific primers (PI-1 and PI-2) were designed from variable regions within the ITSI region. A nested PCR assay was developed in which the 1st round amplified the ITSI region by use of universal fungal primers. Second-round amplification utilized the internal P insidiosum-specific primers PI-1 and PI-2. Specificity of the assay was tested with DNA extracted from cultures of the following: 10 clinical isolates of P insidiosum and 1 isolate each of P graminicola, P irregulare, P arrhenomanes, P myriotylum, P deliense, Basidiobolus ranarum, Conidiobolus coronatus, Aspergillus terreus, Lagenidium giganteum, and a canine-pathogenic Lagenidium species. Nested PCR produced a single 105-base pair amplicon for each of the P insidiosum isolates, but did not produce amplicons for any of the other isolates. Results of this study suggest that PCR is a useful tool for the identification of P insidiosum.     

胸膜肺炎放线杆菌在不同培养基中生长、生物膜形成及Apx毒素分泌能力的差异研究

为比较胸膜肺炎放线杆菌(APP)血清1型～12型菌株在不同培养基中的生长、生物膜形成以及Apx毒素分泌能力的差异,选用5种细菌培养基BHI、LB、MHB、PPLO和TSB,首先测定APP不同血清型菌株在5种培养基中的生长曲线,进而测定不同血清型菌株在5种培养基中的生物膜形成能力,同时测定这些菌株对20种抗菌药物的耐药性...