Non-target Pathogens Research Articles

Quantitative detection of Exserohilum turcicum in northern leaf blight diseased sorghum and maize leaves

Exserohilum turcicum is the causal agent of northern leaf blight (NLB) disease in sorghum and maize. Early detection of this economically important pathogen is essential for effective disease management to limit yield losses. Here we present a real-time quantitative PCR (qPCR) assay specific for E. turcicum detection and biomass quantification in sorghum and maize. In planta fungal quantification was achieved through amplification of a cytochrome P450 oxidoreductase (cpr1) gene fragment and subsequent normalisation to the host glutathione S-transferase III gene (gst3). The assay could specifically detect E. turcicum in sorghum and maize, but the cpr1 gene fragment was not amplified in non-target fungal pathogens. Application of the assay with NLB diseased sorghum and maize leaf material revealed a significant increase in E. turcicum DNA in leaves with lesion symptoms when compared to leaves with early stage chlorotic fleck symptoms in both hosts. Furthermore, E. turcicum was detected at levels as low as 1 pg in infected sorghum and maize leaves. The assay enables rapid detection and quantification of E. turcicum in sorghum and maize and has useful applications in crop breeding programmes and disease management where cultivar selection and early detection of the pathogen are essential to limit disease spread.

Development of a SYBR green I-based duplex real-time PCR assay for detection of pseudorabies virus and porcine circovirus 3

In the present study, a specific and reliable duplex SYBR green I-based quantitative real-time polymerase chain reaction assay was established to detect pseudorabies virus (PRV) and porcine circovirus 3 (PCV3) simultaneously. Viral genomes of PRV and PCV3 in one specimen were identified by their different melting temperatures with melting peaks at 87 °C and 81 °C for PRV and PCV3 respectively, whilst other non-targeted swine pathogens exhibited no fluorescent signals. The assay displayed a high degree of linearity (R2 > 0.997), and the limits of detection were 37.8 copies/μL, 30.6 copies/μL and 60 copies/μL for PRV, PCV3 and the mixture of two recombinant plasmids, respectively. It had good repeatability and reproducibility, and the coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. In this research, the duplex assay was further evaluated using 117 clinical tissue specimens from diseased pigs in the field. The results revealed the infection rates of PRV and PCV3 were 23.08% (27/117) and 55.56% (65/117) respectively, and PRV and PCV3 co-infection rate was 14.53% (17/117). The assay could be utilized as a diagnostic tool with specificity, sensitivity, and reliability for molecular epidemiological surveillance of PRV and PCV3.

Impedimetric Detection of Haemophilus Influenzae on Antibodies-Modified Boron-Doped Carbon Nanowall Electrodes

Haemophilus influenzae (Hi) is a gram-negative pathogenic bacterium. Type b bacteria (Hib) and non-enveloped strains are of greatest clinical importance. Hib causes systemic infections by hematopoietic dissemination from the respiratory system to distant sites such as meninges.Nowadays the most reliable method for Hib infections diagnosis is isolation in microbiological culture. A cerebrospinal fluid sample from a patient suspected of having meningitis is stained and microbiologically cultured. Isolation efficiency and quality depend on many factors also in addition, carrying out the complete test procedure is time consuming and expensive. Thus it is crucial to develop techniques that allow detecting pathogenic bacteria in fast and qualitative way before the first symptoms appear.In the following work the electrochemical impedance methods were used to detect Hi bacteria and specific protein (protein D) appearing on its surface. Hi immunosensor was developed on boron-doped carbon nanowall (B:CNW) electrodes modified with anti-protein D antibodies.The B:CNW modification steps included electrochemical reduction of the aminobenzoic acid diazonium salt and antibody binding via EDC/NHS chemistry. Many scientists deal with the topic of use bovine serum albumin (BSA) for the blockage of unmodified electrode. Here this step was abandoned due to high resistance occurring on the sample after the electrografting stage, which indicates a very effective modification of the electrode surface. An additional argument confirming the correctness of skipping the BSA stage are the results of the sensor's cross-reactivity with other pathogens. For the determination of the sensor selectivity, three non-target pathogens were selected: S. pyogenes, S. pneumoniae, B. parapertussis. For each pathogen used for the selectivity study, the impedimetric responses were significantly lower than for Hi at similar concentrations.Electrochemical impedance spectroscopy measurements were performed on anti-protein D antibodies (ap. D) for protein D (Fig. 1a) and Hi. Then calibration curve was constructed as a relative change in the electron transfer resistance (Rct) (Fig. 1b). Since the calibration curve shows the logarithmic relationship of the antigen concentration to the impedimetric sensor response, linear regression cannot be used to calculate the LOD. It is therefore necessary to modify the calculation procedure so that it can be applied in the present case. This requires changing the equation's layout and then deriving the formula for the deviation of the logarithm of the concentration. The LOD value calculated in this way for Hi bacteria was 19.45 CFU/ml which is a reliable value.For the sensor’s selectivity studies, the electrodes were prepared identically to the electrodes intended for Hi detection. The response of sensor for S. pyogenes (64750 CFU/ml) and B. parapertussis (17200 CFU/ml) were less than 5%, which are almost unnoticeable in comparison to the response for Hi at level of 50% (47910 CFU/ml).This work was supported by the National Centre for Research and Development TECHMATSTRATEG 1/347324/12/NCBR/ 2017 Figure 1

Efficient Electrochemical Sensing Using Hybrid Boron-Doped Diamond/Graphene Nanowall Electrodes

Electrochemical sensors have attracted increasing interest due to their low cost, high sensitivity of detected analytes, and the possibility of sensor miniaturization, which allows for portable and field applications. These devices provide information about the system composition in real-time by combining a chemically selective layer with an electrochemical transducer. Thereby, the chemical energy of the specific interaction between the chemical substances and the sensor is converted into an analytically useful signal.Herein we demonstrate the electrochemical sensor based on boron-doped diamond/graphene nanowall electrodes (B:DGNW). A B:DGNW electrode is a hybrid electrode, which combines the extraordinary features of boron-doped diamond and a graphene nanowall on the same surface. B:DGNW is a sp2 -rich phase material with multilayered graphene walls oriented vertically to the substrate. Moreover, the presence of the boron-doped diamond phase enhances the electrochemical performance and kinetics of the electrode surface when compared with typical carbon nanowalls. The electrodes were fabricated in a one-step growth process using chemical vapor deposition without any additional modifications (thin films were grown on (100)-oriented silicon substrates).Two different sensing platforms will be presented: i) impedimetric biosensor for detection of pathogenic bacteria (e.g., Haemophilus influenzae), and ii) voltammetric sensor for determination of important nitroaromatic explosive compounds (2,4,6-trinitrotoluene (TNT), 2,4,6-trinitroanisole (TNA), 4,4',5,5'-tetranitro-1H,1'H-2,2'-biimidazole (TNBI)). The proposed B:DGNW electrodes were revealed to have a high electroactive area towards the voltammetric detection of various nitroaromatic compounds.The biosensor platform was obtained by B:DGNW surface functionalization firstly by electrografting of diazonium salt and secondly by the attachment of antibodies. The electrochemical biosensor shows high sensitivity and selectivity towards Haemophilus influenzae. The measurements were conducted in the presence of three non-target pathogens S. pyogenes, S. pneumoniae, and B. parapertussis. The proposed B:DGNW nonmodified carbon electrodes were revealed to have a high electroactive area towards the voltammetric determination of various nitroaromatic compounds, thus appearing to be an attractive nanocarbon surface for further applications. The B:DGNW electrode exhibits high sensing performance for TNT detection with linearity between 0.05 to 2 ppm and 4 to 15 ppm, and a limit of detection (LOD) of 73 ppb. The LOD for 2,4,6-trinitroanisole, in turn, is equal to 0.27 ppm, whereas the limit of detection of TNBI is equal to 0.52 ppm. To the authors' knowledge, this is the first time B:DGNW has been used to detect the nitroaromatic compounds electrochemically [1].AcknowledgmentThis work was supported by the Science for Peace Programme of NATO [Grant no. G5147], Polish National Science Centre [2016/22/E/ST7/00102], and the National Centre for Science and Development [347324/12/NCBR/2017]. The DS funds of the Faculty of Electronics, Telecommunications, and Informatics of the Gdansk University of Technology are also acknowledged.Reference:[1] A. Dettlaff et al., J. Hazard. Mater [In press]. Doi: 10.1016/j.jhazmat.2019.121672.

Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR

In the present study, the SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of classical swine fever virus (CSFV) and porcine circovirus 3 (PCV3). The assay was used to detect both CSFV and PCV3 in one sample by their distinct melting temperatures (melting peaks at 87°C for CSFV and 81.5 °C for PCV3), and no specific fluorescence signals were detected for other non-targeted porcine pathogens. The assay had a high degree of linearity (R2 > 0.998) with the detection limits of 23 copies/μL for CSFV and 36 copies/μL for PCV3, and exhibited high repeatability and reproducibility with a low coefficient of variation below 2.0% in both intra- and inter-assay. In this study, 130 clinical samples collected from sick pigs in the field were tested by this assay with the positive rates of 9.23% (12/130) for CSFV and 21.54% (28/130) for PCV3 respectively, and the positive rate of CSFV and PCV3 co-infection was 6.92% (9/130). Our results showed that the developed method was a reliable diagnostic tool to monitor and survey CSFV, PCV3 and CSFV/PCV3 co-infection in the field.

Ciprofloxacin removal: BDD anode coupled with solid polymer electrolyte and ultrasound irradiation

Ciprofloxacin (CIP) is a broad-spectrum antibiotic of the fluoroquinolone group. The low biodegradability of this compound enhances its accumulation in the environment, with associated interference on non-target pathogens, photosynthesis of plants, alterations in the structure of algae and increasing risk of resistant bacteria development. Production plants and hospitals are the main sources of water contamination. Hence, the development of efficient technologies for the treatment of wastewaters is recommended. Here, ultrasound irradiation coupled with the electrochemical generation of hydroxyl radicals by Nb/BDD anode were investigated to remove CIP from aqueous solutions. A solid polymer electrolyte (SPE) was used to overcome the low conductivity of the solution treated. Response Surface Methodology (RSM) was applied with the aim to evaluate the effects of electric current, ultrasound irradiation and stirring rate on the removal and the energy requirements associated to the treatment. Up to 91.36 % of CIP disappearance was attained within 20 min. at 1.16 A and 520 rpm. Ultrasound irradiation at 40 kHz enhanced the removal of the compound only at low current intensity, while strongly affected the energy demand. A treatment time of 30 min. at 0.642 A and 660 rpm has been suggested to minimize the specific energy consumption, estimated to be <1.91 10−3 kWh mg−1. Further research is encouraged, given the promising potential of a zero-discharge removal of several emerging pollutants as phytosanitary, pharmaceuticals and personal care products.

Rapid point-of-care diagnostics for the detection of Chlamydia pecorum in koalas (Phascolarctos cinereus) using loop-mediated isothermal amplification without nucleic acid purification.

Infectious disease, predominately chlamydiosis, contributes significantly to the decline in health of wild koala (Phascolarctos cinereus) populations in some regions of Australia. In this study, we describe the development and evaluation of a simple, sensitive, and specific loop‐mediated isothermal amplification (LAMP) assay for the detection of Chlamydia pecorum in koalas as a point‐of‐care diagnostic tool that can be used in any wildlife hospital and in the field on specialized instrumentation. A set of primers targeting a 188‐bp region of the C. pecorum genome was designed. 100% specificity of the LAMP assay was revealed by demonstrating no cross‐reactivity with 33 nontarget pathogens, and complete correlation with qPCR results for 43 clinical swabs collected opportunistically from wildlife hospitals. In sensitivity evaluations, the technique successfully detected serial dilutions of extracted C. pecorum DNA with a detection limit of 44 IFU/ml.

Performance of the T2Bacteria Panel for Diagnosing Bloodstream Infections: A Diagnostic Accuracy Study.

Blood cultures, the gold standard for diagnosing bloodstream infections (BSIs), are insensitive and limited by prolonged time to results. The T2Bacteria Panel (T2 Biosystems) is a direct-from-blood, nonculture test that identifies the most common ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli). To assess performance of the T2Bacteria Panel in diagnosing suspected BSIs in adults. Prospective patient enrollment (8 December 2015 through 4 August 2017). Eleven U.S. hospitals. 1427 patients for whom blood cultures were ordered as standard of care. Paired blood culture and T2Bacteria testing. Performance of T2Bacteria compared with a single set of blood cultures in diagnosing proven, probable, and possible BSIs caused by T2Bacteria-targeted organisms. Blood culture and T2Bacteria results were positive for targeted bacteria in 3% (39 of 1427) and 13% (181 of 1427) of patients, respectively. Mean times from start of blood culture incubation to positivity and species identification were 38.5 (SD, 32.8) and 71.7 (SD, 39.3) hours, respectively. Mean times to species identification with T2Bacteria were 3.61 (SD, 0.2) to 7.70 (SD, 1.38) hours, depending on the number of samples tested. Per-patient sensitivity and specificity of T2Bacteria for proven BSIs were 90% (95% CI, 76% to 96%) and 90% (CI, 88% to 91%), respectively; the negative predictive value was 99.7% (1242 of 1246). The rate of negative blood cultures with a positive T2Bacteria result was 10% (146 of 1427); 60% (88 of 146) of such results were associated with probable (n= 62) or possible (n= 26) BSIs. If probable BSIs and both probable and possible BSIs were assumed to be true positives missed by blood culture, per-patient specificity of T2Bacteria was 94% and 96%, respectively. Low prevalence of positive blood cultures, collection of a single set of culture specimens, and inability of T2Bacteria to detect nontargeted pathogens. The T2Bacteria Panel rapidly and accurately diagnoses BSIs caused by 5 common bacteria. T2 Biosystems.

Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System.

Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

Development of a TB green II-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine circovirus 2 and 3

Porcine circovirus 3 (PCV3), as a newly emerged circovirus, is widely distributed in pig populations worldwide. Co-infection of PCV2 and PCV3 has been reported frequently in clinical samples. In the present study, a TB Green II-based duplex real-time polymerase chain reaction (qPCR) was developed to rapidly and differentially detect PCV2 and PCV3. The assay specifically detected PCV2 and PCV3, with no fluorescence signals being detected for other non-targeted pig pathogens. The duplex qPCR showed a high degree of linearity (R2 > 0.998), and its limits of detection were 10 and 78 copies/μL for PCV2 and PCV3, respectively. The duplex qPCR could detect and differentiate PCV2 (melting peaks at 85.5 °C) and PCV3 (melting peaks at 82.5 °C), and showed high repeatability and reproducibility, with intra- and inter-assay coefficients of variation of less than 2.0%. Fifty-six tissue samples from 18 pig farms were used to evaluate the duplex qPCR method. The results revealed infection rates of 66.07% (37/56) and 39.28% (22/56) for PCV2 and PCV3, respectively. The PCV2 + PCV3 co-infection rate was 39.28% (22/56). The developed method could be used as an efficient molecular biology tool for epidemiological investigations of PCV2 and PCV3.

Development of a SYBR green I-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine epidemic diarrhea virus and porcine circovirus 3

The development of a rapid, specific, and sensitive SYBR Green I-based duplex real-time quantitative PCR assay is described for the simultaneous detection of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 3 (PCV3). The assay specifically detected PEDV and PCV3, with no fluorescence detected for other non-targeted pig pathogens. The assay showed a good linear relationship, and the limits of detection for this assay were 34.6 copies/μL and 61.2 copies/μL for PEDV and PCV3, respectively. The assay exhibited high repeatability and reproducibility, with intra-assay and inter-assay variation coefficients less than 2.0%. A clinical evaluation using intestinal tissue and fecal samples from piglets suffering from diarrhea at different pig farms in China revealed that the singular infection rates of PEDV and PCV3 were 43.94% (29/66) and 16.67% (11/66), respectively, while the co-infection rate of PCV3 with PEDV was 27.27% (18/66). The results indicate this assay is a rapid and reliable diagnostic tool for PEDV and PCV3 monitoring and surveillance in the field, and provides technical support for the quantitative detection of clinical samples infected or co-infected with PEDV and PCV3.

Specific detection of Muscovy duck parvovirus infection by TaqMan-based real-time PCR assay

BackgroundMuscovy duck parvovirus (MDPV) causes high mortality and morbidity in Muscovy ducks, with the pathogenesis of the virus still unknown in many respects. Specific MDPV detection is often rife with false positive results because of high identity at the genomic nucleotide level and antigenic similarity with goose parvovirus (GPV). The objective of this study was to develop a sensitive, highly specific, and repeatable TaqMan-based real-time PCR (qPCR) assay for facilitating the molecular detection of MDPV.ResultsThe specific primers and probe were designed based on the conserved regions within MDPVs, but there was a variation in GPVs of the nonstructural (NS) genes after genetic comparison. After the optimization of qPCR conditions, the detection limit of this qPCR assay was 29.7 copies/μl. The assay was highly specific for the detection of MDPV, and no cross-reactivity was observed with other non-targeted duck-derived pathogens. Intra- and inter-assay variability was less than 2.21%, means a high degree of repeatability. The diagnostic applicability of the qPCR assay was proven that MDPV-positive can be found in cloacal swabs samples, Muscovy duck embryos and newly hatched Muscovy ducklings.ConclusionsOur data provided incidents that MDPV could be possible vertically transmitted from breeder Muscovy ducks to Muscovy ducklings. The developed qPCR assay in the study could be a reliable and specific tool for epidemiological surveillance and pathogenesis studies of MDPV.

Development of duplex fluorescence-based loop-mediated isothermal amplification assay for detection of Mycoplasma bovis and bovine herpes virus 1

Mycoplasma bovis (MB) and bovine herpes virus 1 (BHV-1) are two important pathogens that cause bovine respiratory disease in the beef feedlot and dairy industries. The aim of this study was to develop and validate a duplex fluorescence-based loop-mediated isothermal amplification (DLAMP) assay for simultaneous detection of MB and BHV-1. Two sets of specific primers for each pathogen were designed to target the unique sequences of the MB uvrC gene and the BHV-1 gB gene. The inner primer for BHV-1 was synthesized with the fluorophore FAM at the 5′ end to detect the BHV-1 gB gene, and the inner primer for MB was synthesized with the fluorophore CY5 at the 5′ end to detect the MB uvrC gene. The DLAMP reaction conditions were optimized for rapid and specific detection of MB and BHV-1. The DLAMP assay developed here could specifically detect MB and BHV-1 without cross-reaction with other known non-target bovine pathogens. The sensitivity of this DLAMP assay was as low as 2 × 102 copies for recombinant plasmids containing the MB and BHV-1 target genes. In a detection test of 125 clinical samples, the positive rates for MB, BHV-1 and co-infection were 44.8%, 13.6% and 1.6%, respectively. Furthermore, the sensitivity and specificity of DLAMP were determined as 95%–96.6% and 100%, respectively, of those of field sample detection by the real-time polymerase chain reaction (PCR) assay recommended by the World Organisation for Animal Health. Overall, DLAMP provides a rapid, sensitive and specific assay for the identification of MB and BHV-1 in clinical specimens and for epidemiological surveillance.

On-site detection of equid alphaherpesvirus 3 in perineal and genital swabs of mares and stallions

Equine coital exanthema (ECE) is an infectious, venereally transmitted muco-cutaneous disease affecting mares and stallions, caused by equid alphaherpesvirus 3 (EHV3). Diagnostic tools for rapid identification of EHV3 are of primary importance to diminish the risk of EHV3 dissemination at the time of breeding. In the last years, it has been shown that the performance of the insulated-isothermal polymerase chain reaction (iiPCR) is comparable to virus isolation, nested PCR and real-time PCR (qPCR) in detecting pathogens of various animal species. Analytical sensitivity and specificity of the iiPCR were compared with a qPCR, using a plasmid containing the target region of the EHV3 glycoprotein G gene and an Argentinian EHV3 isolate (E/9283/07 C3A). In order to evaluate the diagnostic performance of the iiPCR, nucleic acids of 85 perineal and genital swabs (PGS) of mares and stallions were extracted by tacoTM mini and tested by both techniques. EHV3 was detected in 46 and 45 of the 85 PGS by the iiPCR and qPCR, respectively. There was almost perfect agreement between the two diagnostic methods (98.82%; 95% CI: 95.03–100%; κ = 0.98). The iiPCR had a limit of detection of 95.00% at 6 genome equivalents per reaction and a detection endpoint for viral DNA comparable to that of the qPCR, and did not react with six non-targeted equine pathogens. The iiPCR represents a sensitive and specific method for the rapid on-site diagnosis of EHV3 infection. Its routinely implementation in breeding facilities, and artificial insemination and embryo transfer centers, will contribute to prevent the dissemination of this venereal, highly contagious disease in horses.

Simultaneous Detection of Key Bacterial Pathogens Related to Pneumonia and Meningitis Using Multiplex PCR Coupled With Mass Spectrometry.

Pneumonia and meningitis continue to present an enormous public health burden and pose a major threat to young children. Among the causative organisms of pneumonia and meningitis, bacteria are the most common causes of serious disease and deaths. It is challenging to accurately and rapidly identify these agents. To solve this problem, we developed and validated a 12-plex PCR coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method (bacterial pathogen-mass spectrometry, BP-MS) that can be used to simultaneously screen for 11 key bacterial pathogens related to pneumonia and meningitis. Forty-six nasopharyngeal swabs and 12 isolates were used to determine the specificity of the method. The results showed that, using the BP-MS method, we could accurately identify the expected bacteria without cross-reactivity with other pathogens. For the 11 target bacterial pathogens, the analytical sensitivity of the BP-MS method was as low as 10 copies/reaction. To further evaluate the clinical effectiveness of this method, 204 nasopharyngeal swabs from hospitalized children with suspected pneumonia were tested using this method. In total, 81.9% (167/204) of the samples were positive for at least one of the 11 target pathogens. Among the 167 bacteria-positive samples, the rate of multiple infections was 55.7% (93/167), and the most frequent combination was Streptococcus pneumoniae with Haemophilus influenzae, representing 46.2% (43/93) two-pathogen mixed infections. We used real-time PCR and nested PCR to confirm positive results, with identical results obtained for 81.4% (136/167) of the samples. The BP-MS method is a sensitive and specific molecular detection technique in a multiplex format and with high sample throughput. Therefore, it will be a powerful tool for pathogen screening and antibiotic selection at an early stage of disease.

Evaluation of microplate immunocapture method for detection of Vibrio cholerae, Salmonella Typhi and Shigella flexneri from food

BackgroundImproved methods with better separation and concentration ability for detection of foodborne pathogens are in constant need. The aim of this study was to evaluate microplate immunocapture (IC) method for detection of Salmonella Typhi, Shigella flexneri and Vibrio cholerae from food samples to provide a better alternative to conventional culture based methods.ResultsThe IC method was optimized for incubation time, bacterial concentration, and capture efficiency. 6 h incubation and log 6 CFU/ml cell concentration provided optimal results. The method was shown to be highly specific for the pathogens concerned. Capture efficiency (CE) was around 100% of the target pathogens, whereas CE was either zero or very low for non-target pathogens. The IC method also showed better pathogen detection ability at different concentrations of cells from artificially contaminated food samples in comparison with culture based methods. Performance parameter of the method was also comparable (Detection limit- 25 CFU/25 g; sensitivity 100%; specificity-96.8%; Accuracy-96.7%), even better than culture based methods (Detection limit- 125 CFU/25 g; sensitivity 95.9%; specificity-97%; Accuracy-96.2%).ConclusionThe IC method poses to be the potential to be used as a method of choice for detection of foodborne pathogens in routine laboratory practice after proper validation.

Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis

BackgroundDogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs and difficulties in implementing them for field diagnostics. Therefore, we developed a simplified technique, loop-mediated isothermal amplification (LAMP), for the rapid visual detection of N. caninum.MethodsLAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand.ResultsSpecificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697).ConclusionsThis is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis.

Rapid and sensitive insulated isothermal PCR for point-of-need feline leukaemia virus detection.

ObjectivesFeline leukaemia virus (FeLV), a gamma retrovirus, causes diseases of the feline haematopoietic system that are invariably fatal. Rapid and accurate testing at the point-of-need (PON) supports prevention of virus spread and management of clinical disease. This study evaluated the performance of an insulated isothermal PCR (iiPCR) that detects proviral DNA, and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA, for FeLV detection at the PON.MethodsMycoplasma haemofelis, feline coronavirus, feline herpesvirus, feline calicivirus and feline immunodeficiency virus were used to test analytical specificity. In vitro transcribed RNA, artificial plasmid, FeLV strain American Type Culture Collection VR-719 and a clinical FeLV isolate were used in the analytical sensitivity assays. A retrospective study including 116 clinical plasma and serum samples that had been tested with virus isolation, real-time PCR and ELISA, and a prospective study including 150 clinical plasma and serum samples were implemented to evaluate the clinical performances of the iiPCR-based methods for FeLV detection.ResultsNinety-five percent assay limit of detection was calculated to be 16 RNA and five DNA copies for the RT-iiPCR, and six DNA copies for the iiPCR. Both reactions had analytical sensitivity comparable to a reference real-time PCR (qPCR) and did not detect five non-target feline pathogens. The clinical performance of the RT-iiPCR and iiPCR had 98.82% agreement (kappa[κ] = 0.97) and 100% agreement (κ = 1.0), respectively, with the qPCR (n = 85). The agreement between an automatic nucleic extraction/RT-iiPCR system and virus isolation to detect FeLV in plasma or serum was 95.69% (κ = 0.95) and 98.67% (κ = 0.85) in a retrospective (n = 116) and a prospective (n = 150) study, respectively.Conclusions and relevanceThese results suggested that both RT-iiPCR and iiPCR assays can serve as reliable tools for PON FeLV detection.

Harnessing the beneficial heterologous effects of vaccination.

Clinical evidence strongly suggests that certain live vaccines, in particular bacille Calmette-Guérin (BCG) and measles vaccines, can reduce all-cause mortality, most probably through protection against non-targeted pathogens in addition to the targeted pathogen. The underlying mechanisms are currently unknown. We discuss how heterologous lymphocyte activation and innate immune memory could promote protection beyond the intended target pathogen and consider how vaccinologists could leverage heterologous immunity to improve outcomes in vulnerable populations, in particular the very young and the elderly.

Comparative susceptibility of peanut genetically engineered for sclerotinia blight resistance to non-target peanut pathogens

Field trials were conducted from 2006 to 2008 at the Tidewater Agricultural Research and Extension Center (TAREC) in Suffolk, Virginia to determine whether Blight Blocker transgenic peanut lines showed possible increased or decreased susceptibility to common peanut pathogens. Disease susceptibility was evaluated for seven transgenic lines containing a barley oxalate oxidase gene and their corresponding parental cultivars (Perry, Wilson, NC 7). In addition to Sclerotinia blight, the peanut diseases evaluated included: i) early leaf spot caused by Cercospora arachidicola, ii) Cylindrocladium black rot caused by Cylindrocladium parasiticum, iii) southern stem rot caused by Sclerotium rolfsii, iv) tomato spotted wilt virus, and v) aflatoxin levels in seeds caused by Aspergillus flavus or A. parasiticus. Results demonstrated that the susceptibility of Blight Blocker transgenic lines to common peanut pathogens was similar to that of non-transgenic cultivars, while transgenic lines provided resistance to Sclerotinia blight caused by S. minor. Transgenic lines consistently provided high levels of resistance to S. minor in all three years, however, the barley oxalate oxidase had little or no effect on the disease susceptibility to other organisms on peanut. The results of this research should provide additional evidence needed to petition for deregulation of Blight Blocker peanut lines.