GUDP-projekt: Diagnostic tests for veterinary practice (VetDiagnostics)

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1 GUDP-projekt: Diagnostic tests for veterinary practice (VetDiagnostics) to Colaboration: Københavns Universitet, Institut for Sygdomsbiologi, LVK-Dyrlæger and DNA Diagnostic Projektet har fået tilskud fra Grønt Udviklings- og Demonstrationsprogram (GUDP) under Miljø- og Fødevareministeriet.

2 Our vision: That the veterinarian can collect, transport and analyse disease material using a standardized, one protocol for all purposes, diagnosticpackage, and that such information, when interpreted in connection with clinical signs, will improve treatment outcomes.

3 qpcr for detection of pathogens associate with pneumonia in calf

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5 Pneumo 4 qpcr for detection of pathogenic bacteria in calf-pneumonia Same DNA extraction as for Mastit 4 Four bacteria test in one qpcr reaction of 40 cycles in 52 minutes DNA extraction qpcr Result in 3.5 hours Filter CY5 ROX HEX FAM ATTO Target bacteria Mannheimia haemolytica Pasteurella multocida Histophilus somni Mycoplasma bovis IAC

6 Test on 44 target strains and 135 non-target strains Positive strains detection M. haemolytica % P. multocida % H. somni % M. bovis 3 100% Actinobacillus arthritidis Actinobacillus equuli Actinobacillus lignieresii Arcanobacterium pyogenes Avibacterium gallinarum Bibersteinia trehalosi Citrobacter fruendii Cronobacter sakazakii Enterobacter asburiae Enterobacter cloacae Enterobacter intermedius Enterococcus faecalis Escherichia coli Frederiksenia canicola Fusobacterium nechrophorum Gallibacterium anatis Gallibacterium melopsittaci Gallibacterium salpingitidis Haemophilus haemoglobinophilus Klebsiella pneumoniae Lactococcus raffinolactis Mannheimia caviae Mannheimia glucosida Mannheimia granulomatis Mannheimia ruminalis Mannheimia varigena Mannheimmia granulomatis Mycoplasma alkalescens Mycoplasma arginini Mycoplasma bovigenitalium Mycoplasma bovirhinis Mycoplasma californicum Mycoplasma canadense Mycoplasma dispar Actinobacillus rossi Actinobacillus seminis Aerococcus viridans Neisseria zoodegmatis Pasteurella aerogenes Pasteurella caballi Pasteurella mairii Pasteurella oralis Proteus Prototheca Pseudomonas aeruginosa Salmonella enterica spp. Enterica Salmonella paratyphi Seratia marcescens Shigella sonnei Staph chromogenes Staphylococcus aureus Staphylococcus epidermidis Staphylococcus gallinarium Staphylococcus haemolyticus Staphylococcus lentus Staphylococcus sciuri Staphylococcus xylosus Streptoccoccus pluraninalium Streptococcus agalactiae Streptococcus alcaligenes faecalis Streptococcus dysgalactiae Streptococcus equi Streptococcus oralis Streptococcus suis Streptococcus uberis Streptococcus zooepidemicus Taxon 10 Bisgaard Truperella pyogenes

7 Amplification plots Pneumo 4 Instrument: Agilent Mx3005 qpcr: 40 cycles M. haemolytica (CY5) IAC (ATTO) P. multocida (ROX) IAC (ATTO) H. somni (HEX) IAC (ATTO) M. bovis (FAM) IAC (ATTO)

8 65 clinical samples from 10 farms N positive Pneumo 4 positive culture positive PCR (DTU) M. haemolytica P. multocida H. somni M. bovis

9 Comparison results of multiplex PCR, bacterial cultivation and Mycoplasma bovis PCR in 65 clinical samples M. haemolytica Culture Pneumo 4 Pos Neg Pos 4 16 Neg 0 45 H. somni Culture Pneumo 4 Pos Neg Pos 0 4 Neg 0 61 P. multocida Culture Pneumo 4 Pos Neg Pos Neg 1 30 M. bovis PCR - DTU Pneumo 4 Pos Neg Pos Neg 1 33

10 Limit of detection of qpcr for copy number of targets

11 CFU correlation to Ct value of each target bacteria 3 x 3

12 RT-qPCR for dection of virus associate with pneumonia in calf Filter Target virus CY5 Bovine parainfluenzavirus 3 ROX HEX FAM ATTO Bovine coronavirus Bovine respiratory syncytial virus IBR/BVD Internal amplication control

13 Testing of virus RNA in 12 of 64 DNA lysate samples Virus N Positive Bovine coronavirus Bovine respiratory syncytial virus Bovine parainfluenza virus Sample BRSV PI-3 BCoV DTU DNA-D DTU DNA-D DTU DNA-D Pos Pos Pos Pos Pos Pos Pos Pos 5 Pos Pos Pos - Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos - - Pos

14 Ongoing work RNA templates Primers and probes using synthesized RNA by cloning and in vitro transcription have been designed Further work Test of specificity of primers and probes RT-qPCR Validation multiplex PCR optimization 65 tracheal aspirate samples

15 Use of swabs Send samples Long distance days No cooling Sample dries out with silicat Use bronopol in the fluid or befor swab Same result after weeks

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