Abstract Book of EAVLD2012 - eavld congress 2012

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S4 - O - 05 RAPID IDENTIFICATION OF BOVINE MASTITIS PATHOGENS USING MALDI TOF Gudrun Overesch 1 , Andreas Thomann 1 , Vincent Perreten 1 1 Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland Maldi Tof, mastitis, identification, veterinary pathogens Introduction Correct and rapid identification of pathogens is the main objective of bacteriological diagnostic. Phenotypical identification procedures using biochemical parameters have been widely used in the last decades. Although automatic systems were developed, these methods are still time consuming. Therefore molecular identification methods, i. e. real time PCR, were established to replace biochemical approach when applicable. The exclusive use of molecular based diagnostic does not allow performing antibiotic susceptibility tests. The recently established matrixassisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI TOF MS) approach overcomes the disadvantages of the methods mentioned above. It is faster than PCR and, while working with cultured strains, it is possible to perform antimicrobial susceptibility testing. The databases of MALDI TOF MS are usually based on spectra generated from human isolates [1]. Its use in veterinary bacteriology needs validation with livestock derived strains and update of the databases with spectra from relevant pathogens of veterinary interest. This study presents the establishment of MALDI TOF MS technology for the direct identification to the species level of relevant bovine mastitis pathogens. Materials & methods 156 bovine mastitis milk samples from individual cows were included. Ten microliter of whole milk was cultured on trypticase soy agar plates with 5% sheep blood (Becton Dickinson) at 37 °C for 24 hours under aerobic conditions. Colonies were identified phenotypically by routine diagnostic procedures [2] [3]. In parallel, the same colonies were identified by MALDI TOF MS (Biotyper 3.0, Bruker) using the direct transfer protocol recommended by the manufacturer. Brieflly, material from a single colony was taken with a toothpick, smeared on a steel plate in dublicates and overlaid with 1 μl of HCCA matrix solution. After air drying, the samples were measured using standard settings in the “Flex control” software. For calibration and as an internal reference the “bacterial test standard” (BTS, Bruker) was applied in triplicate on every plate measured. Analysis of spectra with the Biotyper 3.0 included a comparison against the internal commercial database in combination with the institute database. The latter was generated using veterinary field strains, which include coagulasenegative staphylococci (Table 1), S. pseudintermedius, Aerococcus viridans and Streptococcus uberis. All strains were identified with VITEK 2 (Biomérieux) and 16SrDNA sequence. New profiles were introduced into MALDI TOF MS database. Identification was accepted when a score value > 2.000 was achieved. All identified strains isolated from mastitis milk included in this study are listed in Table 1. Results After updating the commercial database with veterinary field strains (i. e. coagulase-negative staphylococci and. S. uberis) 155 out of 156 biochemical defined strains were identified from a 24 hour direct plating culture by MALDI TOF MS analyses (Table 1). These strains include all highly relevant mastitis pathogens, i. e. staphylococci and streptococci species. MALDI TOF MS identified all alpha- double- and non-hemolytic Staphylococcus aureus. Trueperella (Arcanobacterium) pyogenes, Histophilus somni, E. coli and Klebsiella oxytoca were also clearly identified. Members of the Streptococcus-, Lactococcus-, Enterococcusgroup, which cannot be easily characterized using classical methods could also be identified to the species level using MALDI TOF MS. Only Corynebacterium bovis could not be directly identified by MALDI TOF MS, since this species has not yet been introduced in the new database. Discussion & conclusion MALDI-TOF MS was shown to represent an excellent method for rapid species identification of relevant bovine mastitis pathogens by direct analysis of overnight cultures. However, the commercial database needed to be expanded with profiles of important bovine mastitis pathogens. Further improvement such has extraction prior identification should also be engaged to increase the identification spectrum. A more detailed identification of bacteria present in mastitis milk will also allow to increase the knowledge regarding species diversity involved in mastitis. Table 1: Biochemical identification and Maldi Tof identification of bacterial strains isolated from mastitis milk Isolates (N) Biochemical identification Maldi Tof identification (number) 39 Streptococcus uberis Streptococcus uberis 39 16 15 14 9 Non haemolytic staphylococci Double haemolytic S. aureus Streptococcus-, Lactococcus-, Enterococcus-group Alpha haemolytic staphylococci Streptococcus dysgalactiae subsp. dysgalactiae 8 Enterococcus spp. 5 Trueperella (Arcanobacterium) pyogenes S*. xylosus (19) S. chromogenes (7) S. sciuri (4) S. epidermidis (2) S. vitulinus (2) S. equorum (2) S. warneri (1) S. devriesei (1) S. aureus (1) S. aureus (16) Lactococcus garvieae (9) Aerococcus viridans (3) Lactococcus lactis (1) Enterococcus devriesei (1) Streptococcus uberis (1) S. aureus (6) S. hemolyticus (5) S. xylosus (3) Streptococcus dysgalactiae subsp. dysgalactiae Enterococcus faecalis (4) Aerococcus viridans (2) Streptococcus equinus (1) Enterococcus faecium (1) Trueperella (Arcanobacterium) pyogenes 4 Escherichia coli Escherichia coli 2 Streptococcus agalactiae Streptococcus agalactiae 1 Histophilus somni Histophilus somni 1 Klebsiella oxytoca Klebsiella oxytoca *S. = Staphylococcus Acknowledgments This study was partially financed by grant 1.11.21 of the Federal Veterinary Office (FVO), Switzerland. References 1. Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, et al. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis 2009;49(4):543-551. 2. Guélat-Brechbuehl M, Thomann A, Albini S, Moret-Stalder S, Reist M, Bodmer M, et al. Cross-sectional study of Streptococcus species in quarter milk samples of dairy cows in the canton of Bern, Switzerland. Vet Rec 2010;167(6):211-215. 3. Moret-Stalder S, Fournier C, Miserez R, Albini S, Doherr MG, Reist M, et al. Prevalence study of Staphylococcus aureus in quarter milk samples of dairy cows in the Canton of Bern, Switzerland. Prev Vet Med 2009;88(1):72-76.
S4 - O - 06 15 MINUTE ELISA USING A LOW COST COMMERCIAL BIOSENSOR Jason Sawyer 1 , Jennifer Cork 1 , Rebecca Jones 1 1 AHVLA (Weybridge), Technology Transfer Unit, New Haw, Addlestone, Surrey KT15 3NB, UK ELISA, IBR, Biosensor, Penside testing, Serology, Vantix Introduction The use of biosensors for diagnostic testing offers several potential advantages, but to date their use for routine diagnostic testing has been limited to specific markets (e.g. glucose testing). We evaluated the use of a commercially available potentiometric biosensor platform - Vantix - to carry out a serological assay for antibodies to infectious bovine rhinotracheitis (IBR), caused by Bovine Herpes Virus 1 (BoHV-1). The Vantix system 1 comprises a low cost reader unit and disposable potentiometric biosensors. The biosensors consist of a working and reference electrode coated in a conductive polymer (polypyrrole) and covered in a protective plastic film. Biochemical or enzymatic activity taking place on the surface of the working electrode as a result of immunocomplexes built up on the electrode, cause electrochemical changes in the conductive polymer layer on and around the working electrode thus generating a measurable change in electrical potential (measured in millivolts) relative to the reference electrode. The signal produced is proportional to the level of analyte under investigation. IBR is a highly contagious disease of cattle that is endemic in the UK and many other parts of the world 2 . ELISA is commonly used in the detection and control of this disease and AHVLA uses an in-house indirect ELISA. In this assay, BoHV-1 antigens, bound to the surface of a microtitre plate, capture BoHV-1 specific antibodies present in test samples. Bound antibodies are then detected using a horseradish peroxidase (HRP) labelled antibovine immunoglobulin and subsequent HRP catalysed oxidation of the chromogen substrate 3,3’,5,5’-tetramethylbenzidine (TMB) produces a colour change. The in-house ELISA for BoHV-1 used at AHVLA has a total assay time of over 3 hours which is typical of commercial ELISA tests. In this work, the well-characterised and established reagents used in the parent ELISA were used to construct an assay on the Vantix platform. A panel of serum samples, submitted for routine ELISA testing, were then tested with the Vantix assay and the results compared to the parent ELISA. Table 1: Comparison of methodology of the IBR antibody biosensor assay and parent ELISA. Biosensor assay Indirect ELISA Step Incubation Incubation Step Time Time Biosensors and ELISA plates pre coated with BoHV1 antigen and stored ready for use Incubation with diluted Incubation with diluted 5 min sample sample 2 h 8 Washes no soak 0.5 min 5 x washes 10 second soak 5 min Incubation with diluted Rabbit anti bovine HRP 5 min Incubation with diluted Rabbit anti bovine HRP 1 h Conjugate Conjugate 8 Washes no soak 0.5 min 5 x washes 10 second soak 5 min Probes added to TMB Plate incubated with 5 – 20 min substrate and read TMB substrate. 4 min immediately. Potential Addition of Stop 1 min difference reading. Colorimetric read 1 min Total assay time 15 min Total assay time >3.25 h Materials & methods Table 1 summarises the steps and timings involved in the performing the Biosensor assay compared to the ELISA. Biosensor probes were prepared by coating with BoHV-1 antigen, and were then stored at +4 o C for up to 12 weeks. To perform the assay, the electrodes of the antigen coated biosensors were briefly placed in diluted serum, washed, and briefly incubated with conjugate. After a final wash step, the sensors were inserted into the Vantix reader unit and the working and reference electrode placed into TMB substrate. Changes in electrochemical potential difference were observed over a period of four minutes and recorded using Vantix software. The biosensor assay was used to test 194 serum samples, comprising 90 positive and 104 negative as designated by the in house ELISA. % signal relative to weak positive control 250 200 150 100 50 0 IBR Biosensor 2 x 2 box analysis assay Serum Samples Positive Negative Totals IBR ELISA Negative Positive 86 4 90 Negative 2 102 104 Totals 88 104 194 Positive True positive rate (Sensitivity) 0 0 25 50 75 100 False positive rate (100 - Specificity) Fig.1 Comparison of results obtained when testing serum samples using the parent IBR antibody ELISA and the biosensor assay. 2x2 table (Top); Box and whisker plot showing biosensor results for ELISA negative and positive samples (bottom left) and ROC analysis (bottom right). Results Fig 1 shows the resulting 2X2 Table, Box and Whisker plot and ROC curve when the biosensor assay was compared to the ELISA. Using the optimal cut-off value (determined by ROC analysis and based on a percentage signal relative to the weak positive control serum run alongside the test samples) the biosensor assay had a sensitivity of 98% and a specificity of 96% when compared to the indirect ELISA. Only 6 of 194 samples resulting in a different categorisation (positive or negative) compared to the ELISA results. Overall, the results demonstrate that the biosensor assay produces test results that closely match those obtained with the parent ELISA. Discussion & conclusion This work has shown that results equivalent to the ELISA can be obtained using the Vantix biosensor platform in 15 min. The relatively low cost of the biosensors (~ few Euro each) and reader (~ few thousand euro) make this an interesting and promising technology. The rapid and simple nature of the assays, and the fact that established ELISA reagents can readily be converted to the platform, suggest Vantix could be useful in a variety of diagnostic testing scenarios. These may include rapid testing of samples which require fast turnaround or testing in low tech laboratories. The generation of an electronic signal offers the potential for transmission across mobile networks. Vantix are currently developing a hand held device which uses the same biosensors but offers the potential for more rapid (
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2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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Welcome Dear colleagues, Welcome to
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