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.     

Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs.

The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, cough, and dyspnea. Mycoplasma hyopneumoniae is among the most prevalent and important infectious agents associated with PRDC. Understanding of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available tests are relatively insensitive or nonspecific when used in a diagnostic laboratory setting or are too costly or difficult for routine diagnostic use. Several polymerase chain reaction (PCR) assays have been described, but they are not sensitive enough to detect the microorganisms in live pigs, from either nasal or tracheal swabs. A nested PCR using 2 species-specific sets of primers from the 16S ribosomal DNA gave positive results with as little as 80 microorganisms and did not cross-react with other mycoplasma species or with other microorganisms commonly found in the respiratory tract of pigs. This assay was better suited for detection of M. hyopneumoniae from nasal swabs than was conventional PCR. Nasal swab samples were taken at different time periods following experimental challenge of 10 susceptible pigs. Only 2 of the 55 swabs examined gave a positive result with conventional PCR, whereas 30 of the 55 swabs gave a positive result using the nested PCR. Twenty of 40 (50%) nasal swabs from pigs experiencing a respiratory disease outbreak where M. hyopneumoniae had been diagnosed also gave a positive result with the nested PCR. To confirm that the amplified product was specific, 4 nested PCR products were purified, sequences were determined and aligned, and they were confirmed to be from M. hyopneumoniae.     

Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 by using polymerase chain reaction DNA amplification.

Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 (MDV1) was attempted by polymerase chain reaction (PCR) using the primers chosen from the sequence within the long inverted repeats of MDV1 DNA. PCR of the DNAs extracted from oncogenic-strain-infected cells and Marek's disease tumor cell lines produced a major product containing two or three copies of 132-base-pair (bp) repeat units, whereas PCRs of the DNAs extracted from nononcogenic-strain-infected cells yielded amplified products with various sizes corresponding to the number of 132-bp repeat units. The primers chosen from the glycoprotein A genes of MDV1 and herpesvirus of turkeys also were used for determination of their serotype specificity. The PCR procedure was found to be a simple and sensitive procedure for identification of MDV1 and herpesvirus of turkeys and for estimation of oncogenicity of MDV1.     

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.     

Use of polymerase chain reaction to detect latent channel catfish virus.

Polymerase chain reaction was used to detect an economically important herpesvirus, channel catfish virus (CCV). A segment of the viral DNA was sequenced and oligonucleotide primers were produced from that sequence. After the primers were tested for the possibility of hybridization to catfish DNA, they were used to prime the polymerase chain reaction, using pure CCV DNA, CCV DNA added to catfish DNA, and DNA from catfish infected and not infected with CCV. In all cases, the method proved to be simple and sensitive in its detection by CCV DNA. When catfish DNA was present, less than 0.1 pg of CCV DNA was detectable. Channel catfish virus DNA in a latent carrier of CCV was readily detectable.     

A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus.

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.     

鸡马立克氏病病毒在鸡淋巴细胞和羽髓中的复制动力学分析

马立克氏病（MD）是由鸡马立克氏病病毒血清1型（MDV1）引起的鸡高度接触性淋巴细胞增生性疾病,MDV1在体内的复制状况与其致病性强弱及传播能力直接相关。本实验选择近几年从国内不同地区MD暴发鸡场分离的6株MDV1强毒株、弱毒疫苗“814”株和国内标准MDV1强毒J-1株,分别人工感染SPF鸡,采用双重实时荧光定量PCR（FQ—PCR）方法,检测感染后1d～28d病毒在淋巴细胞和羽髓中的复制状况。结果显示,接种1d后即可在淋巴细胞中检测到MDV1（10^2.6 copies～10^5.2 copies／10^6 cells）;在检测期间,淋巴细胞中病毒载量略有上升的趋势,总体呈现不规律变化,而且变化并不明显。接种7d后羽髓中病毒载量开始显著增加,14d～21d达到峰值,超强毒株峰值处病毒载量可达到10^7 copies／10^6 cells,峰值期病毒载量是感染前期（1d～7d）的100～10000倍。强毒株在体内的病毒载量高于弱毒株,即复制能力高于弱毒株。研究表明,MDV1国内流行的毒株有增殖速度快,病毒载量高的新特点;MDV1致病性的高低与在其在体内的复制能力的高低呈正相关。     

Use of a polymerase chain reaction assay to detect bovine leukosis virus in dairy cattle

OBJECTIVE: To evaluate the use of a polymerase chain reaction (PCR) assay in detecting bovine leukosis virus (BLV) in adult dairy cows. DESIGN: Prospective study. ANIMALS: 223 adult dairy cows. PROCEDURE: Cows were tested for BLV status by use of an ELISA and a PCR assay. Sensitivity, specificity, predictive values of positive and negative tests, and the percentage of cows correctly classified by PCR assay were calculated. Ninety-five percent confidence intervals were calculated for sensitivity and specificity. RESULTS: Sensitivity and specificity were 0.672 and 1.00, respectively. Prevalence of BLV in this herd was 0.807. Predictive value of a positive test was 1.00, and predictive value of a negative test was 0.421. The percentage of cows correctly classified by PCR assay was 73.5%. CONCLUSIONS AND CLINICAL RELEVANCE: A positive PCR assay result provided definitive evidence that a cow was infected with BLV. Sensitivity and negative predictive value for PCR assay were low. Consequently, PCR assay alone is unreliable for routine detection of BLV in herds with high prevalence of the disease.     

Application of the polymerase chain reaction to detect fowl adenoviruses.

The possibility of using the polymerase chain reaction (PCR) for the detection of fowl adenoviruses (FAdV) was tested. The optimal reaction parameters were evaluated and defined for purified genomic DNA of type 8 fowl adenovirus (FAdV-8), and then the same conditions were applied for nucleic acid extracted from infected cells. One hundred picograms of purified viral DNA, or 250 FAdV-8-infected cells, were detected by ethidium bromide staining of the PCR products in agarose gels. The sensitivity was increased to 10 pg purified viral DNA, or 25 infected cells, when the PCR products were hybridized with a specific labeled probe. Several field isolates of FAdV and the CELO virus (FAdV serotype 1) could be amplified by the same primers and conditions, but the size of the amplicons was smaller than that for the FAdV-8 PCR product. Other avian viruses and uninfected cell cultures tested negative.     

Development of a SYBR Green-based real-time quantitative polymerase chain reaction assay to detect enzootic nasal tumor virus in goats

Enzootic nasal adenocarcinoma is a contagious respiratory disease in goats that is caused by the enzootic nasal tumor virus 2 (ENTV-2). In order to increase the number of available detection methods for ENTV-2, we developed a SYBR Green real-time polymerase chain reaction (SGrPCR) assay that targets the gag gene of ENTV-2. The low limit of detection of the assay was 3.68 × 10¹ copies/μL, a hundredfold more sensitive than conventional PCR. The melt curve showed a single sharp melt peak at 83°C, which indicated that there was no non-specific amplification or primer dimer formation. The intra-assay and inter-assay coefficients of variation were 1.58% and 1.82%, respectively. There was no cross-reactivity with closely related goat viruses (i.e., orf virus, peste des petits ruminants virus, goatpox virus, foot-and-mouth disease virus) and endogenous retroviruses. In conclusion, the SGrPCR assay is specific for the gag gene of ENTV-2 and provides a rapid and sensitive approach for detecting ENTV-2 in clinical samples.     

Development of a polymerase chain reaction to differentiate avian leukosis virus (ALV) subgroups: detection of an ALV contaminant in commercial Marek's disease vaccines

Avian leukosis viruses (ALVs) are common in many poultry flocks and can be detected using an enzyme-linked immunosorbent assay or any other test designed to identify p27, the group-specific antigen located in gag. However, endogenous retroviruses expressing p27 are often present and can be confused with exogenous ALVs. A more specific and informative assay involves targeting the variable envelope glycoprotein gene (gp85) that is the basis for dividing ALVs into their different subgroups. We designed polymerase chain reaction (PCR) primers that would specifically detect and amplify viruses from each of the six ALV subgroups: A, B, C, D, E, and J. Subgroup B and D envelopes are related, and our B-specific primers also amplified subgroup D viruses. We also designed a set of common primers to amplify any ALV subgroup virus. To demonstrate the usefulness of these primers, we obtained from the Center for Veterinary Biologics in Iowa culture supernatant from chicken embryo fibroblasts infected with an ALV that was found to be a contaminant in two commercial Marek's disease vaccines. Using our PCR primers, we demonstrate that the contaminant was a subgroup A ALV. We cloned and sequenced a portion of the envelope gene and confirmed that the ALV was a subgroup A virus. Unlike typical subgroup A viruses, the contaminant ALV grew very slowly in cell culture. We also cloned and sequenced a portion of the long terminal repeat (LTR) from the contaminant virus. The LTR was found to be similar to those LTRs found in endogenous ALVs (subgroup E) and very dissimilar to LTRs normally found in subgroup A viruses. The E-like LTR probably explains why the contaminant grew so poorly in cell culture.     

Detection and identification of Mycoplasma from bovine mastitis infections using a nested polymerase chain reaction.

A polymerase chain reaction (PCR) test was compared with culture for the detection and diagnosis of bovine Mycoplasma intramammary infection. The PCR test was applied to 24-hour Mycoplasma enrichment cultures of milk from cows with suspected mastitis and from bulk tank milk. In comparison to culture, the sensitivity and specificity of the PCR method were 96.2% and 99.1% for individual cow milk and 100% and 99.8% for the bulk tank milk, respectively. However, in discrepant cases where PCR was positive and culture was negative, the PCR test was correct; subsequent PCR tests and culturing of the individual cow's milk yielded positive results. The PCR test simultaneously detected and differentiated among 11 bovine Mycoplasma species.     

Pooled polymerase chain reaction to detect Tritrichomonas foetus in beef bulls.

Preputial scraping samples from 305 mixed breed beef bulls were examined for the detection of Tritrichomonas foetus infection. All samples were collected by veterinarians and transported in commercial media to an accredited lab. Upon arrival samples underwent microscopic examination for the presence of Tritrichomonas foetus and were then incubated until 5 days postcollection before final microscopic examination. Culture detected 14 samples with Trichomonad spp.; all were confirmed to be Tritrichomonas foetus by polymerase chain reaction (PCR). After final examination samples were randomly placed in groups of 5 samples; technicians were blinded as to culture results of the individual samples constituting each pool. From each sample within a group, a portion of the fluid sediment was removed and pooled with the other samples of the group to form 61 pools. From each of the formed pools an aliquot was removed for PCR. PCR detected 16 positive pools; an additional 2 positive samples were then identified on individual PCR on samples previously diagnosed as culture negative. Relative to culture, the 95% confidence intervals for sensitivity and specificity of PCR pools to detect Tritrichomonas foetus were 76.8% to 100% (mean value: 100%) and 85.5 to 99.5% (mean value: 93.4%), respectively.     

Use of a polymerase chain reaction assay to detect infection with Eperythrozoon wenyoni in cattle.

OBJECTIVE: To determine whether a polymerase chain reaction (PCR) assay could be used to detect Eperythrozoon wenyoni in the blood of cattle. DESIGN: Prospective study. ANIMALS: 95 cattle from various herds in Alabama and Georgia and 96 bulls enrolled in Auburn University's Alabama Beef Cattle Improvement Association Bull Test program. PROCEDURE: Blood samples were collected by means of venipuncture of the median caudal vein and submitted for a CBC and PCR assay. Blood smears were made immediately after blood collection and examined by means of light microscopy. RESULTS: Three of 95 cattle from herds in Alabama and Georgia and 5 of 96 bulls enrolled in the Bull Test program had positive PCR assay results. Organisms were seen in blood smears from only 5 of these 8 animals. Organisms were not seen in blood smears from any animals for which results of the PCR assay were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a PCR assay may be an effective method for detecting E wenyoni infection in cattle and that the PCR assay may be a more sensitive test than evaluation of blood smears.     

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.