Multiplex polymerase chain reaction for distinguishing Taylorella equigenitalis from Taylorella equigenitalis-like organisms.

It is difficult to distinguish isolates of Taylorella equigenitalis, the cause of contagious equine metritis, from a T. equigenitalis-like organism isolated from asymptomatic donkeys and horses. Although T. equigenitalis is responsible for a severe, contagious disease of the reproductive tract of equids, the T. equigenitalis-like organism, although contagious, does not appear to produce disease. Because of the economic consequences of correctly distinguishing isolates of these 2 microorganisms, a polymerase chain reaction (PCR)-based assay was developed that will distinguish isolates of T. equigenitalis from the T. equigenitalis-like microorganism. The primers used in the PCR assay were designed to amplify unique regions of the gene encoding the 16S ribosomal RNA.     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds

In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.     

Identification of Taylorella equigenitalis responsible for contagious equine metritis in equine genital swabs by direct polymerase chain reaction

A direct-PCR assay was developed for the rapid detection of Taylorella equigenitalis, a Gram-negative bacterium responsible for contagious equine metritis (CEM) in Equidae. The bacteria may be detected in equine genital swabs without need for a preliminary step of DNA extraction or bacterial isolation. Specificity was determined with 125 isolates of T. equigenitalis, 24 isolates of Taylorella asinigenitalis, five commensal bacteria of the genital tract and a facultative intracellular pathogen of foals found in large concentration in soil. Our PCR is specific and amplified a 413-bp 16S ribosomal DNA product only in all T. equigenitalis.     

A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses

: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.     

Evaluation of a newly developed real-time PCR for the detection of Taylorella equigenitalis and discrimination from T. asinigenitalis

A 'culture-LightCycler PCR' assay has been developed for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM) in horses. The primers and hybridisation probes were derived from the 16S rDNA sequence. Their specificity was determined in two closely related organisms and six commensal bacteria of the genital tract. The assay was specific for T. equigenitalis and discriminates T. asinigenitalis isolates. It also avoids misidentifications of morphologically and phenotypically similar organisms. The sensitivity was evaluated in comparison to a standard bacteriological culture method. It detected T. equigenitalis in 10 of 52 samples that had not been identified bacteriologically. The results indicated that the assay had a greater sensitivity. This is the first real-time PCR for the detection of T. equigenitalis and avoids PCR carry-over contamination. The 'culture-LightCycler PCR' assay is specific, sensitive and reproducible, and can be used effectively for the detection of T. equigenitalis infections.     

Comparison of nested PCR and real-time PCR for the detection of Toxoplasma gondii in biological samples from naturally infected cats

There are different protocols of molecular diagnosis methods available including DNA extraction methods to diagnose of Toxoplasma gondii, being necessary to perform comparative studies in biological samples. The aim of this study is to compare real-time PCR (rtPCR) and nested PCR (nPCR) to evaluate the detection of T. gondii in naturally infected cats. Biological samples of Toxoplasma-seropositive cats were assayed for detection of T. gondii DNA - extracted by both the lysis buffer and proteinase K (LB proteinase K) method and the acid guanidinium thiocyanate (GuSCN) method - using rtPCR and nPCR. T. gondii DNA was detected by nPCR in 43.6% and 40.8% of the samples from which it was extracted by the LB proteinase K and the GuSCN method, respectively. With rtPCR these figures fell significantly to 33.8% and 14.1%. Despite of nPCR showed higher sensitivity, the agreement observed between two PCRs was good; this agreement, however, was affected by the DNA extraction method used, LB proteinase K method showed better results.     

Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi

OBJECTIVE: To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. SAMPLE POPULATION: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. PROCEDURE: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture and standard polymerase chain reaction methods. RESULTS: The QPCR assay detected the vapA gene in pure culture of R. equi and in tracheobronchial fluid samples that contained as few as 20 CFUs of virulent R. equi/mL and accurately quantitated virulent R. equi to 10(3) CFUs/mL of fluid. The assay was highly specific for detection of the vapA gene of virulent R. equi and was more sensitive than standard polymerase chain reaction for detection of R. equi in tracheobronchial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R. equi. The accuracy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitivity of the QPCR method in detection of virulent R. equi should facilitate rapid and accurate diagnosis of R. equi pneumonia in foals.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene.

METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay.

RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 10⁴ cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%.

CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene. METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay. RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 104 cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%. CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

Development of quantitative real-time polymerase chain reaction for duck enteritis virus DNA

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, we introduce a quantitative real-time polymerase chain reaction (PCR) assay for DEV DNA using TaqMan technology and a two-step protocol. It was confirmed to be rapid, sensitive, and specific for DEV detection. The primers and probe were designed and directed to the DNA polymerase gene of DEV. The method will provide a valuable tool for rapid laboratory diagnosis of DEV infection. By virtue of its high-throughput format and its ability to accurately quantify the viral DNA, the method may be useful for large epidemiological surveys and clarification of pathogenesis, such as latency and reactivation of the virus.     

Development of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from Taylorella asinigenitalis

A discriminatory real time PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and the related species T. asinigenitalis was developed for the direct examination of genital swabs. The 112bp amplicons produced from the two species were discriminated from each other using TaqMan probes labelled with different fluorophores. The TaqMan PCR was shown to be specific for the 16S ribosomal DNA of the two species of taylorella and did not cross-hybridise with the 16S ribosomal DNA of other bacteria tested. Direct amplification from genital swabs was shown to be equally sensitive to that of culture methods. Prevalence in a sample set from The Netherlands was shown to be equivalent to that demonstrated by culture. A companion real time PCR that amplified a fragment of the 16S rDNA gene of equine commensal bacteria was developed to ensure bacterial DNA was extracted from swab material supplied for testing. The use of a rapid and reliable real time PCR for the organism causing CEM should aid the control of this disease.     

Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples

PCR assay has been shown to be a promising option for the diagnosis of brucellosis. However, few studies have been performed with field samples in order to evaluate the assay as a diagnostic tool. In this study, routine use of a species-specific PCR assay previously developed for the identification of Brucella cultures was assessed for the detection of Brucella DNA directly from the stomach contents of aborted sheep fetuses. The assay is based on the insertion sequence IS711 in the Brucella chromosome. In the study, during 3 successive lambing seasons (1998-1999, 1999-2000 and 2000-2001) 126 aborted fetus samples each from different flocks and locations were examined. Brucella strains were isolated from 39 (31%) of the samples and all of the strains were identified as Brucella melitensis by biochemical characteristics, agglutination with monospecific A and M sera and PCR. Thirty-seven of 39 B. melitensis isolates were biotyped as biotype 3, and 2 isolates as biotype 1. From 38 of 39 culture positive fetal stomach contents B. melitensis-specific DNA was detected by PCR. PCR was found negative in all of the culture negative samples. Compared with culture, sensitivity and specificity of PCR were determined as 97.4 and 100%, respectively. The results indicate that this PCR procedure has a potential for use in routine diagnosis of sheep brucellosis.     

Rapid detection of pseudorabies virus genomic sequences in biological samples from infected pigs using polymerase chain reaction DNA amplification

The presence of the pseudorabies virus (PRV) genome in infected hosts has previously been studied by standard hybridization techniques, which showed the viral genome to be present at very low levels in infected tissues. The recently introduced polymerase chain reaction (PCR) procedure provides an alternative and rapid means of amplifying small quantities of specific DNA sequences. We applied this technique to a study of pigs infected by PRV. The sequence selected for amplification consisted of 222 base pairs lying in the gene coding for the glycoprotein gp50. We used a pair of 20-mer oligonucleotides flanking this sequence as primer and a cloned Stu-Nde fragment containing the sequence as target DNA. To avoid the tedious DNA extraction procedure we performed PCR directly on disrupted cells and detected specific amplification after 25 cycles of PCR with the thermostable Taq DNA polymerase. Amplified products were detected by gel electrophoresis directly. Nasal samples from experimentally and naturally infected pigs were tested by this PCR technique. When compared with tissue culture and serological tests, detection by gel electrophoresis of PCR amplified fragments provided excellent specificity and sensitivity. We concluded that PCR amplification will be a valuable tool for rapid diagnosis of PRV infection in pigs, taking less than 1 h to complete.     

Evaluation of a Campylobacter fetus subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples

The detection and subspeciation of Campylobacter fetus subsp. venerealis (CFV) from veterinary samples is important for both clinical and economic reasons. Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a venereal disease that can lead to serious reproductive problems in cattle, and strict international regulations require animals and animal products to be CFV-free for trade. This study evaluated methods reported in the literature for CFV detection and reports the translation of an extensively tested CFV-specific polymerase chain reaction (PCR) primer set; including the VenSF/VenSR primers and a real-time, quantitative PCR (qPCR) platform using SYBR Green chemistry. Three methods of preputial sample preparation for direct qPCR were evaluated and a heat lysis DNA extraction method was shown to allow for CFV detection at the level of approximately one cell equivalent per reaction (or 1.0 × 10³ CFU/mL) from prepuce. The optimized sample preparation and qPCR protocols were then used to evaluate 3 western Canadian bull cohorts, which included 377 bulls, for CFV. The qPCR assay detected 11 positive bulls for the CFV-specific parA gene target. DNA sequence data confirmed the identity of the amplified product and revealed that positive samples were comprised of 2 sequence types; one identical to previously reported CFV parA gene sequences and one with a 9% sequence divergence. These results add valuable information towards our understanding of an important CFV subspeciation target and offer a significantly improved format for an internationally recognized PCR test.     

Comparison of various primer sets for detection of Toxoplasma gondii by polymerase chain reaction in fetal tissues from naturally aborted foxes

Tissues from 4 aborted polar foxes (3 samples of brain and 4 samples of liver) were selected for Toxoplasma gondii PCR assay. Positive results of serological tests of mothers and immunofluorescence test (IFT) of fetal organ smears were the criteria of sample selection. Five sets of primers designed from B1 gene and ITS1 sequences of T. gondii were used for detection of the parasite in fetal fox tissues. All used primer sets successfully amplified T. gondii DNA in PCR from organs which were positive by IFT. Single tube nested PCR also showed positive result from a sample negative by IFT, but this product was not confirmed. The studies showed usefullness of PCR for routine diagnosis of toxoplasmosis in carnivores.     

Comparison of serological tests for equine trypanosomosis in naturally infected horses from Kazakhstan

In this study, we compared the complement fixation test (CFT), the horse complement fixation test (HCFT) and a card agglutination test for trypanosomosis (CATT/T. evansi) for the diagnosis of equine trypanosomosis in the Republic of Kazakhstan. Cohen's kappa test was used to evaluate the concordance between the three tests. Kappa scores for CFT versus HCFT and CATT are both 0.6165 (95% Confidence Interval CI 0.414--0.819) indicating a "substantial" agreement between CFT and HCFT or CATT, respectively. Kappa for HCFT versus CATT is 0.395 (CI 0.142--0.648) indicating a "fair" agreement between the two tests. In the absence of a golden standard, seroprevalence and sensitivity and specificity of the three tests were estimated using maximum likelihood estimation. CFT has a sensitivity of 57.2% (CI 31.5--79.5%) and a specificity of 95.8% (CI 89.2--98.5%), HCFT has a sensitivity of 80.6% (CI 44.1--95.6%) and a specificity of 99.5% (CI 90.7--100%), CATT has a sensitivity of 80.2% (CI 44.5--95.2%) and a specificity of 98.5% (CI 79.5--99.9%). The seroprevalence of equine trypanosomosis in Kazakhstan was estimated at 16.4% (CI 9.4--27.0%). The data suggest that for epidemiological studies and the control of equine trypanosomosis serological tests prove useful since they have a high specificity and a satisfactory sensitivity. Field applicable tests, such as CATT/T. evansi may be used to replace laboratory-based tests, such as CFT and HCFT.     

Nested polymerase chain reaction detection and duration of porcine circovirus type 2 in semen with sperm morphological analysis from naturally infected boars.

A nested polymerase chain reaction (nPCR) protocol was applied to porcine semen to demonstrate the porcine circovirus type 2 (PCV2) shedding patterns and duration in naturally infected boars. Sperm morphology analysis was performed on a subset of samples to determine if the presence of PCV2 DNA in semen was associated with reduced semen quality. Semen was collected serially from 43 boars representing 6 breeds, aged 33.9 to 149.3 weeks. Of the 903 semen samples collected, 30 samples (3.3%) were positive for PCV2 DNA by nPCR from 13 boars. Boars shedding PCV2 DNA in semen ranged between 35.9 and 71.0 weeks of age, and shedding occurred during a period of up to 27.3 weeks. A semen nPCR test was 2.6 times more likely to be positive when collected from pigs that were < or =52 weeks of age, and 3.0 times more likely to be positive when collected from pigs that were < or =26 weeks from time of entry into the stud main unit (generalized estimating equations: P = 0.02; 95% confidence interval [CI] of the odds ratio 1.2 to 5.5, and P = 0.01; 95% CI of the odds ratio 1.3 to 6.9, respectively). These results demonstrate a sporadic and long-term shedding pattern of PCV2 DNA in semen from naturally infected boars. PCV2 DNA in semen does not appear to have detrimental effects on sperm morphology; however, boar age and, possibly, breed may contribute to the persistence of PCV2-shedding in semen.