A polymerase chain reaction assay for the detection of Leptospira spp. in bovine semen.

A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

Development of a polymerase chain reaction and restriction typing assay for the diagnosis of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections.

A multiplex polymerase chain reaction (PCR) method coupled with a restriction analysis of PCR products (PCR with restriction fragment length polymorphism) was developed for the simultaneous detection of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections. The specificity, sensitivity, and practical diagnostic applicability of this method were evaluated. This assay may be also adapted to the diagnosis of suid herpesvirus 1 and equine herpesviruses 1 and 3 and could become a powerful diagnostic tool.     

Detection of leptospires in bovine semen by polymerase chain reaction

OBJECTIVE: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. DESIGN: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. RESULT: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. CONCLUSION: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.     

Development of a shuttle polymerase chain reaction for the detection of bovine herpesvirus 2

Three different polymerase chain reaction (PCR) protocols were evaluated for their ability to detect bovine herpesvirus 2 (BoHV-2): single-step PCR with 3 reaction stages (denaturation, annealing and extension), 2 reaction stages (denaturation and annealing/extension; shuttle PCR), and semi-nested PCR with 3 reaction stages. All the PCR protocols showed the same sensitivity (detection limit of 0.4 TCID(50)). A non-specific band sometimes appeared in mock cell DNA at annealing temperatures below 64 degrees C. The shuttle PCR was found to be superior to the other protocols under consideration because of the speed of its application. Furthermore, no non-specific band was detected in DNAs of eight other DNA viruses. Thus, the shuttle PCR seems to be an excellent diagnostic tool for BoHV-2 infections.     

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.     

Early detection of bovine leukosis virus DNA in infected sheep using the polymerase chain reaction.

The early diagnosis of bovine leukosis virus (BLV) infection, the aetiological agent in enzootic bovine leukosis, is important for the implementation of control measures. BLV infection is currently assessed by the detection of circulating antibodies against the viral envelope protein, gp51. However, this approach has shortcomings in the time taken to detect anti-BLV antibodies (three to four weeks after infection), and in the failure to detect antibodies in some animals. Clearly a technique such as the polymerase chain reaction (PCR), which directly detects the presence of viral DNA, has advantages over methods designed to measure host antibodies. The use of PCR for the detection of proviral DNA in an affected DNA sample with as little as 10(-5) micrograms of host DNA using agarose gel electrophoresis followed by ethidium bromide staining is described here. It was possible to improve the sensitivity of this assay by using hybridisation analysis with a BLV gene probe. PCR used in combination with hybridisation analysis will provide a sensitive diagnostic assay to detect BLV when antibody tests give weakly positive or equivocal results.     

Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen.

Bovine semen samples spiked with bovine herpesvirus 1 (BHV-1) were used to compare dot blot hybridization, polymerase chain reaction (PCR), and virus isolation for detection of BHV-1 in bovine semen. The PCR amplification used primers targeting the BHV-1 thymidine kinase gene and a nucleic acid releasing cocktail (GeneReleaser); the PCR product was used as the DNA probe in dot blot hybridization; virus isolation was done in primary bovine fetal testis (BFT) cell cultures. Semen diluted 1:20 in tissue culture medium had the least cytotoxicity and inhibition of viral cytopathic effects in BFT cells, allowing detection of 1 TCID50/100 microL of BHV-1 suspension by virus isolation. The presence of foreign DNA such as bovine sperm DNA or salmon sperm DNA increased the sensitivity of dot blot hybridization in detecting BHV-1, allowing detection of 20,000 TCID50/100 microL of neat semen. The inhibition of PCR amplification of BHV-1 DNA in bovine semen was eliminated by diluting the samples 1:20 in tissue culture medium. The best PCR amplification was obtained when semen was diluted 1:20 and when a reaction buffer of pH 9.0, with 1.0 mM MgCl2 was used. Under these conditions, the PCR followed by ethidium bromide staining of agarose gels could detect 1 TCID20/100 microL of sample, whereas PCR followed by Southern blot hybridization could detect 0.01 TCID50/100 microL of sample.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Brucella melitensis in semen using the polymerase chain reaction assay

An evaluation of the polymerase chain reaction (PCR) for detection of Brucella melitensis DNA in bovine and ovine semen was performed. Since semen contains different components that inhibit PCR amplification, a protocol was used to purify Brucella-DNA from bovine and ovine semen samples prior to conducting amplification of the targeted DNA. When separated fractions of naturally Brucella contaminated semen were analyzed by the PCR, most of B. melitensis DNA were present in the seminal fluid and non-sperm fractions.The PCR examination results for detection of B. melitensis DNA in different semen fractions were compared with the results for traditional cultural methods of Brucella from semen. The PCR was more sensitive than the traditional cultural methods since it detected Brucella-DNA in 12 (10%) out of 120 semen samples while direct culture detected only 7 (5.8%) in the same semen samples. The limit of detection by PCR was 100 CFU/ml of semen. In addition, the results of PCR were available in one day, whereas isolation and identification of Brucella organisms required days or even weeks. The PCR may be used as a supplementary test for detection of B. melitensis in semen.     

用改良的RT-PCR技术快速检测传染性法氏囊病病毒

本研究将最新的病毒核酸纯化技术和单管ＲＴ－ＰＣＲ技术相结合,应用于ＩＢＤＶ的诊断研究,对４０余个病毒样品进行扩增反应,均取得令人满意的效果。该技术灵敏快速,从核酸纯化到电脉检测ＰＣＲ产物只需５～６ｈ,反应灵敏度比传统方法至少提高１００倍。所设计的引物对毒株的适用范围较广,对１２个ＩＢＤＶ毒株和１２个病变法氏囊样品均得到分子量一致并与设计相符的扩增产物     

牛新孢子虫病PCR检测方法的建立和应用

犬新孢子虫感染是导致妊娠母牛流产的主要原因之一，准确快速地诊断是有目的治疗该病的前提。本研究根据已知的犬新孢子虫种属特异性基因片段Nc-5基因序列，设计了一对特异性引物，建立了检测犬新孢子虫的PCR技术。利用本方法可以从感染牛胎儿的脑脊液及实质脏器中扩增出1条大小为350bp的特异性核酸片段。在吉林省延边龙井地区的应用检测结果表明，流产牛胎儿中新孢子虫感染率为17％（15／90）。本方法在实际应用中快速、灵敏、特异性高，可以用于牛和其它动物新孢子虫病的快速诊断和流行病学调查。     

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.     

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.     

An international inter-laboratory ring trial to evaluate a real-time PCR assay for the detection of bovine herpesvirus 1 in extended bovine semen

Six laboratories participated in a ring trial to evaluate the reliability of a real-time PCR assay for the detection of bovine herpesvirus 1 (BoHV-1) from extended bovine semen. Sets of coded samples were prepared and distributed to each of the laboratories. The sample panel contained semen from naturally and artificially infected bulls, serial dilutions of positive semen with negative semen, semen from uninfected seronegative bulls, negative semen spiked with virus, as well as serial dilutions of reference virus. The samples were tested using a previously validated real-time PCR assay for the detection of BoHV-1 in each participating laboratory. The PCR tests were conducted with four different real-time PCR amplification platforms, including RotorGene 3000, Stratagene MX 3000/4000, ABI 7900, and Roche LightCycler 2.0. Virus isolation using one set of samples was performed in one laboratory. The results of the laboratories were compared with one another, and with those of virus isolation. It was found that the sensitivity and specificity of the real-time PCR test was greater than those of virus isolation (82.7% versus 53.6% and 93.6% versus 84.6%, respectively). A high level of agreement on PCR testing results between the laboratories was achieved (kappa value 0.59-0.95). The results of this study indicate that the real-time PCR assay is suitable for the detection of BoHV-1 in extended semen, and would be a good substitute for the slow and laborious virus isolation, for the screening testing at artificial insemination centres and for international trade.     

Noninfectivity of semen from bulls infected with bovine leukosis virus

An opportunity for study of the potential role of semen in the transmission of bovine leukosis virus (BLV) was provided when a Jersey herd was found to be BLV-seronegative. This was a closed herd; new genetic material had been introduced by artificial insemination (AI), using semen collected and processed at 7 AI centers in the United States. Of 66 donor bulls from which semen had been collected for AI use in the herd during the 5 years the herd remained seronegative, 24 were consistently BLV-seropositive and 2 became seropositive for BLV during the study. Semen collected from the BLV-seropositive bulls accounted for 1,019 semen units, representing 48.3% of the semen purchased. The maintenance of BLV seronegativity in this herd for 5 years, when semen from BLV-seropositive bulls was used for AI, provided evidence for the lack of infectivity of BLV in bovine semen.     

Use of a polymerase chain reaction assay for detection of Haemobartonella canis in a dog.

A polymerase chain reaction (PCR) originally developed for detection of Haemobartonella felis in cats was successfully used for detection of H canis in an 8-year-old spayed Great Dane. The dog had been splenectomized and was undergoing immunosuppressive chemotherapy at the time of diagnosis. Sequence analysis of the 16S ribosomal RNA gene revealed that the Haemobartonella spp infecting this dog was 97% homologous to the sequence previously reported for the Ohio strain of H felis. Clinical and hematologic abnormalities as well as identification of the organisms by use of light and electron microscopy supported the diagnosis of H canis. The PCR assay used for detection of H felis may be useful for the detection of H canis in dogs prior to blood donation, splenectomy, or treatment with immunosuppressive drugs.