A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the ‘Bruce ladder’ multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.     

Recombinant OMP28 antigen-based indirect ELISA for serodiagnosis of bovine brucellosis

Brucellosis is a zoonosis of both public health and economic importance in many developing countries including India. Early detection and segregation of the infected animals are important in order to control the disease. Serodiagnostic tests for brucellosis is mainly based on detection of antibodies developed against lipopolysaccharide (LPS) component of cell. In this study we evaluated a protein antigen, 28 kDa outer membrane protein (OMP28), of Brucella melitensis as an alternative to LPS. Recombinant OMP28 was produced in Escherichia coli system. The efficacy of purified OMP28 was studied in an indirect enzyme-linked immunosorbent assay (ELISA) for diagnosis of brucellosis in field sera collected from different regions of country. Using known negative and known positive serum samples it was found that OMP28 is immunoreactive to Brucella infected cattle, sheep, goat and dog sera. Three hundred and eighty two cattle sera were screened by OMP28 antigen-based ELISA and the results were compared to rose Bengal plate agglutination Test (RBPT). Recombinant OMP28 antigen-based ELISA has shown sensitivity of 88.7%, specificity of 93.8% and accuracy of 92.9%. It was concluded that recombinant B. melitensis OMP28 could be used as a protein antigen for diagnosis of brucellosis in domestic animals.     

A real-time PCR assay for detection of emerging infectious Elizabethkingia miricola

Elizabethkingia miricola, a Gram-negative bacillus, is emerging as a life-threatening pathogen in both humans and animals. However, no specific and rapid diagnostic method exists to detect E. miricola. Here, we established a real-time PCR assay for the rapid, sensitive, and specific detection of E. miricola with a wide dynamic range of 10⁸ copies/μL to 10² copies/μL. The detection limit of the real-time assay was 145 copies/μL, which was 100 times more sensitive than conventional PCR. All clinical isolates E. miricola from different host species yield very close Tm (80.25 ± 0.25 °C). Additionally, no cross-reaction or false positives were observed in the assay for non-target bacterial species. The performance of this assay was primarily assessed by testing frog tissue samples. Overall, our study provided a real-time PCR assay, which is a rapid, sensitive, and specific diagnostic method that could be used for early diagnosis and epidemiological investigation of E. miricola.     

The development of a selective medium for the Brucella abortus strains and its comparison with the currently recommended and used medium

The Brucella spp. are fastidious and relatively slow-growing organisms. The isolation of such strains in a variety of specimens often requires the use of a selective medium to reduce or eliminate the growth of unexpected microorganisms. The modified Brucella selective (MBS) medium, which contains improved antibiotic mixtures, erythritol as the only carbon source, and neutral red as a pH indicator, showed good selectivity for the Brucella abortus strains, including the RB51 vaccine strain. Erythritol in the MBS medium was able to promote and/or recover the delayed growth of the B. abortus strains through the antibiotic mixtures. The Brucella colonies, which assumed a pinkish color at their central part, were easily differentiated from other organisms. The MBS medium also allows the isolation of the Brucella strains even in contaminated specimens and/or in specimens containing small numbers of viable organisms. Moreover, this medium can be applied to environmental samples for the isolation of the Brucella strains, and it can thus offer epidemiologic traceback sources for the dissemination or transfer of diseases. Therefore, the MBS medium can be applied as a useful tool of important control measures in the eradication programs.     

Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus.     

The evaluation of a user-friendly lateral flow assay for the serodiagnosis of human brucellosis in Kazakhstan

Serum samples from all patients with culture-confirmed brucellosis including those with chronic disease from Kazakhstan tested positive in the serum agglutination test for titers ≥1:25 and reacted in the Brucella immunoglobulin M/immunoglobulin G lateral flow assay (LFA) confirming the high sensitivity of these assays. The strong reactivity in the LFA observed for the majority (92.1%) of the samples from the patients with culture-confirmed brucellosis together with the user-friendliness of the assay procedure makes the LFA ideal for the confirmation of brucellosis in endemic areas in Kazakhstan. The Rose Bengal test lacked sensitivity in particular for patients with chronic brucellosis therefore limiting its value as a quick screening assay. The study emphasizes the importance of the LFA as a useful, rapid, and easy-to-perform tool in the diagnostic testing of brucellosis.     

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples

Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen.A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T_m = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C_q) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea.The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples.     

Indirect fluorescent antibody test versus enzyme-linked immunosorbent assay and agglutination tests in the serodiagnosis of patients with brucellosis

Anti-Brucella IgG, IgM and IgA in sera from patients with blood culture positive for B. melitensis and controls were measured by indirect fluorescent antibody (IFA) test and the findings compared with those of enzyme-linked immunosorbent assay (ELISA) and microagglutination test (MAT). Brucella melitensis and B. abortus antigens from three vendors (BioMerieux, Wellcome and Oxoid) and from reference strains (Ames, Iowa) were used in IFA and MAT while a whole cell heat-killed B. melitensis antigen was used in ELISA. Statistical analysis showed comparable results when using B. melitensis or B. abortus antigen, in IFA, from the same manufacturer but there were subtle differences among antigens from different manufacturers. Correlation between IFA and ELISA titers was poor, due to differences in the levels of these titers. However, the percentage of sensitivity, specificity, predictive positive, and predictive negative at different titers indicated the most reliable discriminative titers to be as follows: ELISA IgG 1 : 800 (100% for all), IgM 1 : 400 (100%, 93%, 100%, 100%, respectively) and IgA 1 : 200 (95%, 100%, 100%, 94%, respectively); IFA IgG 1 : 320 (95%, 93%, 95%, 93%, respectively) and IgM 1 : 80 (95%, 100%, 100%, 94%, respectively). IFA IgA showed either poor sensitivity or specificity at all titers. These findings and the subjective reading of IFA limit its value in Brucella diagnosis while the MAT showed high false negatives (5%–40%). Thus, ELISA proves to be the most reliable test for the diagnosis of patients with brucellosis.     

Development and Evaluation of PCR Assay Based on Outer Membrane Protein 22 Gene for Genus Specific Diagnosis of Brucella spp

Brucellosis is a worldwide zoonosis and a significant cause of loss of health in human and animals. Conventionally, definitive diagnosis is done by isolation of brucellae which is time consuming, technically demanding and may be hazardous. The present study was aimed to develop PCR assay based on evolutionarily conserved outer membrane protein 22 gene for rapid diagnosis of Brucella spp. A set of primer was designed after multiple alignment of outer membrane protein 22 gene sequences by clustalW. PCR amplification resulted in single specific amplicon of 639 bp in all the Brucella strains tested. The primers were found to be highly sensitive and specific. PCR assay developed may be used for clinical diagnosis of Brucella infection.     

Multiplex real-time polymerase chain reaction for rapid detection of β-lactamase–negative,ampicillin-resistant Haemophilus influenzae

We evaluated a multiplex real-time quantitative polymerase chain reaction (PCR) method for quantification of Haemophilus influenzae and rapid detection of β-lactam–resistant strains. We designed 5 PCR primer sets to simultaneously detect the β-lactam–resistant genes and quantify the pathogen. To demonstrate the validity of this assay, we used 191 clinical isolates, including 141 H. influenzae strains, and 100 purulent sputum samples, including 30 samples from which H. influenzae had been isolated. This assay showed 92.9% sensitivity and 91.8% specificity for detecting β-lactam–resistant genes, relative to the conventional phenotypic method, and this assay correlated well with conventional quantitative culture counts. By using this assay, we could quantify H. influenzae and identify β-lactam susceptibility in only 3 h and with only one tube. This method will be helpful for the rapid detection of H. influenzae infections and the selection of appropriate antibiotics.     

Loop-mediated isothermal amplification method targets to the phoP gene for detection of Yersinia enterocolitica

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of Yersinia enterocolitica. The assay used specifically designed primers to target within the phoP gene and correctly identified all 37 strains of Y. enterocolitica and 50 non-Y. enterocolitica strains. The probability of detection was 100%, when the DNA of extracted from 10¹ CFU Y. enterocolitica was used as template in LAMP assay. Prior to the LAMP assay, a sample preparation protocol was applied that included a pre-enrichment step in Luria–Bertani broth, followed by extraction and purification of DNA. In this way, 102 various food samples were investigated for Y. enterocolitica including 79 minced pork samples and 23 powdered milk samples. The accuracy of LAMP was shown to be 100% when compared to the standard method, ISO 10273. This combination of sample enrichment, and LAMP assay can detect 2.2 CFU per 100 g food samples. The overall analysis time for the LAMP assay was approximately 24 h. This is in contrast to 5 days of analysis time required for the traditional culture method. Consequently, the LAMP described here, has the potential to become a standardized method for the rapid detection of Y. enterocolitica in diagnostic laboratories once further validated by inter-laboratory studies.     

Loop-mediated isothermal amplification assay targeting the ompA gene for rapid detection of Riemerella anatipestifer

A novel loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of Riemerella anatipestifer (RA) infection. The LAMP assay exhibited a higher sensitivity than conventional polymerase chain reaction (PCR) and microbial isolation. The specificity of the assay was determined by restriction enzyme digestion of the LAMP products and detection of Escherichia coli, Salmonella enterica and Pasteurella multocida. The LAMP assay was able to detect RA effectively in samples of the reference strains, isolated strains and infected duck brains. This assay is a useful tool for the diagnosis of RA infection in the clinical setting.     

Rapid diagnosis of largemouth bass ranavirus in fish samples using the loop-mediated isothermal amplification method

Largemouth bass ranavirus (LMBV) has been recognized as the causative pathogen responsible for infectious skin ulcerative syndrome in cultured largemouth bass in China. A fast and simple LMBV detection method is urgently needed. Here, a loop-mediated isothermal amplification (LAMP) assay was established for the detection of this virus using primers targeting the major capsid protein gene of LMBV. The amplification conditions were optimized; the assay was specific for the diagnosis of LMBV, as there was no cross-reactivity with other four Iridoviridae viruses (large yellow croaker iridovirus, Singapore grouper iridovirus, tiger frog virus, and soft-shelled turtle iridovirus), grass carp reovirus, white spot syndrome virus, or healthy largemouth bass. The sensitivity of the LAMP assay was found to be 8.55 × 10¹ copies/μL of LMBV DNA, which was 10-fold higher than that of the conventional PCR. Application of the LAMP assay was evaluated using 10 clinical samples, and the results indicated the reliability of the test as a rapid, field diagnostic tool for LMBV detection. Thus, the simplicity and nearly instrument-free LAMP method provides an alternative for rapid and sensitive detection of LMBV and has great potential for early diagnosis of LMBV infection in the farm.     

Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens

Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni are considered important pathogens causing the most food-related human illnesses worldwide. Current methods for pathogen detection have limitations in the effectiveness of identifying multiple foodborne pathogens. In this study, a pathogen detection microarray was developed using various 70-mer oligonucleotides specifically targeting the above pathogens. To reduce the cost of detection, each microarray chip was designed and fabricated to accommodate 12 identical arrays which could be used for screening up to 12 different samples. To achieve high detection sensitivity and specificity, target-specific DNA amplification instead of whole genome random amplification was used prior to microarray analysis. Combined with 14-plex PCR amplification of target sequences, the microarray unambiguously distinguished all 4 pathogens with a detection sensitivity of 1 × 10^?4 ng (approximately 20 copies) of each genomic DNA. Applied the assay to 39 fresh meat samples, 16 samples were found to be contaminated by either 1 or 2 of these pathogens. The co-occurrences of Salmonella and E. coli O157:H7, Salmonella and L. monocytogenes in the same meat samples were also observed. Overall, the microarray combined with multiplex PCR method was able to effectively screen single or multiple pathogens in food samples and to provide important genotypic information related to pathogen virulence.     

Development of real-time PCR assays for specific detection of hmsH,hmsF, hmsR,and irp2 located within the 102-kb pgm locus of Yersinia pestis

Virulent isolates of three pathogenic Yersinia species (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) harbor a 102-kb chromosomal region which encodes elements critical for virulence. A 35-kb high pathogenicity island is contained in this region, is a known virulence determinant, contains irp1 and irp2 iron-regulating genes. An additional segment, the 68-kb high pathogenicity island, contains genetic elements responsible for conferring the Y. pestis pigmentation phenotype on Congo red agar at 28 °C. Collectively, these contiguous segments are referred to as the pigmentation (pgm) locus, the absence of which results in strain attenuation and exemption from CDC Select Agent status. In this study, we developed a set of four real-time PCR assays to detect the presence or absence of multiple virulence genes located within this region. Specifically, we designed TaqMan^® PCR assays to individually detect three hemin storage genes (hmsH, hmsF, and hmsR) which are genetic elements that confer the pigmentation phenotype, as well as the iron-regulating status of 25 Y. pestis isolates (representing 23 different strains), thus establishing a molecular based assay capable of determining the pgm status of candidate Y. pestis isolates. Included in the validation process, was a comparison of these real-time PCR assays and newly developed conventional PCR assays targeting much larger areas of the 102-kb region (including one assay spanning hmsR and hmsF, one spanning hmsH and hsmF, one targeting hmsF, and one targeting irp2). There was high concordance between the conventional and real-time PCR assays for all Y. pestis strains tested. The results from the comparative analysis document the specificity and sensitivity of the real-time PCR assays and further solidify the ostensible benefits of real-time PCR over conventional PCR.     

Administration of a Triple versus a Standard Double Antimicrobial Regimen for Human Brucellosis More Efficiently Eliminates Bacterial DNA Load

The effects of doxycycline-streptomycin-rifampin versus a standard doxycycline-streptomycin regimen on residual Brucella DNA were compared in 36 acute brucellosis patients. At admission, all patients given triple (n = 22) and double (n = 14) regimens had detectable Brucella DNA with similar mean loads (P = 0.982). At follow-up, 14 to 20 months postpresentation, significantly more patients receiving triple than double regimens had undetectable Brucella DNA (P = 0.026). The doxycycline-streptomycin-rifampin regimen eliminates Brucella DNA more efficiently than doxycycline-streptomycin, which may result in superior long-term clearance of Brucella.     

Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus

The similar clinical characteristics of canine circovirus (CaCV) and canine astrovirus (CaAstV) infections and high frequency of co-infection make diagnosis difficult. In this study, a duplex SYBR Green I-based real-time polymerase chain reaction (PCR) assay was established for the rapid, simultaneous detection of CaCV and CaAstV. Two pairs of specific primers were designed based on the Rep gene of CaCV and the Cap gene of CaAstV. By using the real-time PCR assay method, the two viruses can be distinguished by the difference in melting temperatures, 79 °C and 86 °C for CaCV and CaAstV, respectively. This assay had high specificity, showing no cross-reaction with other common canine viruses, as well as high sensitivity, with minimum detection limits of 9.25 × 10¹ copies/μL and 6.15 × 10¹ copies/μL for CaCV and CaAstV, respectively. Based on the mean coefficient of variation, the method had good reproducibility and reliability. In a clinical test of 57 fecal samples, the rates of positive detection by real-time PCR were 14.04% (8/57) and 12.28% (7/57) for CaCV and CaAstV, respectively, and the rate of co-infection was 8.77% (5/57). In conclusion, the newly established duplex SYBR Green I-based real-time PCR assay is sensitive, specific, reliable, and rapid and is an effective tool for the detection of co-infections with CaCV and CaAstV.     

A comparison of Brucella IgG and IgM ELISA assays with agglutination methodology

Despite brucellosis having a low incidence rate in developed nations, it still remains the leading zoonotic disease in the world. Culturing of Brucella spp. provides good specificity but in cases where the fever is intermittent, sensitivity is problematic. This has led to the development of serological methods of detection. Brucella agglutination methods have been considered the serological gold‐standard since their inception, although commercial Brucella IgG and IgM enzyme‐linked immunosorbentassays are available to potentially aid in the diagnosis of the disease. In our study, anti‐Brucella IgG and IgM assays were compared with agglutination. Individually the IgG assay tested had an accuracy of 56% and the IgM assay had an accuracy of 77%. These poor accuracies reinforce Centers for Disease Control's conclusion that nonagglutination tests should not be used to confirm brucellosis. J. Clin. Lab. Anal. 24:160–162, 2010. © 2010 Wiley‐Liss, Inc.     

Duplex TaqMan real-time PCR assay for simultaneous detection and quantification of Anaplasma capra and Anaplasma phagocytophilum infection

Anaplasma capra and A. phagocytophilum, two species of the family Anaplasmataceae, are zoonotic tick-borne obligate intracellular bacteria affecting wild and domestic ruminants, dogs, cats, horses and humans. A. capra and A. phagocytophilum infections have been steadily increasing in both number and geographic distribution, and the accurate diagnosis of these infections is challenging. This study aimed to develop a rapid, sensitive and reliable duplex real-time PCR assay for the specific detection and differentiation of these Anaplasma species.We designed primers and probes against the conserved regions of A. capra groEL and A. phagocytophilum 16S rRNA genes. A range of PCR-related parameters were evaluated such as the dosage of primers and probes, and annealing temperature. The specificity, sensitivity and repeatability of this assay were evaluated. Assay performance was further evaluated using samples collected from 124 goats in four regions of Henan, China. This set of samples was also tested using conventional PCR under conditions previously described.The developed duplex real-time PCR assay allowed the simultaneous detection of A. capra and A. phagocytophilum in a reasonably short time at levels as small as 10² copies/μL, respectively, with optimal specificity and reproducibility. In addition, this duplex real-time PCR assay is the first DNA-based method designed to detect A. capra and A. phagocytophilum, and will be valuable for timely diagnosis and treatment of these infections.