GUDP-projekt: ” Diagnostic tests for veterinary practice (VetDiagnostics)” 01-10-2016 to 31-03-2020. 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”.

Our vision:

That the veterinarian can collect, transport and analyse disease material using a standardized, “one protocol for all purposes”, diagnostic-package, and that such information, when interpreted in connection with clinical signs, will improve treatment outcomes.

qPCR for detection of pathogens associate with pneumonia in calf

Pneumo 4

Filter Target bacteria

CY5 Mannheimia haemolytica

ROX Pasteurella multocida

HEX Histophilus somni

FAM Mycoplasma bovis

ATTO IAC

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

Test on 44 target strains and 135 non-target strains

Positive strains detection

M. haemolytica 15 100%

P. multocida 16 100%

H. somni 10 100%

M. bovis 3 100%

Actinobacillus arthritidis Actinobacillus equuli Actinobacillus lignieresii

Actinobacillus rossi Actinobacillus seminis Aerococcus viridans

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

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

Amplification plots

M. haemolytica (CY5) IAC (ATTO) P. multocida (ROX) IAC (ATTO)

H. somni (HEX) IAC (ATTO) M. bovis (FAM) IAC (ATTO)

Pneumo 4 Instrument: Agilent Mx3005 qPCR: 40 cycles

N positive

Pneumo 4

positive

culture

positive

PCR (DTU)

M. haemolytica 65 20 4

P. multocida 65 34 19

H. somni 65 4 0

M. bovis 64 30 - 16

65 clinical samples from 10 farms

M. haemolytica Culture

Pneumo 4 Pos Neg

Pos 4 16

Neg 0 45

Comparison results of multiplex PCR, bacterial cultivation and Mycoplasma bovis PCR in 65 clinical samples

H. somni Culture

Pneumo 4 Pos Neg

Pos 0 4

Neg 0 61

P. multocida Culture

Pneumo 4 Pos Neg

Pos 18 16

Neg 1 30

M. bovis PCR - DTU

Pneumo 4 Pos Neg

Pos 15 15

Neg 1 33

Limit of detection of qPCR for copy number of targets

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

Filter Target virus

CY5 Bovine parainfluenzavirus 3

ROX Bovine coronavirus

HEX Bovine respiratory syncytial virus

FAM IBR/BVD

ATTO Internal amplication control

RT-qPCR for dection of virus associate with pneumonia in calf

Testing of virus RNA in 12 of 64 DNA lysate samples

Virus N Positive

Bovine coronavirus 64 19

Bovine respiratory syncytial virus 64 10

Bovine parainfluenza virus 3 64 0

Sample BRSV PI-3 BCoV

DTU DNA-D DTU DNA-D DTU DNA-D

1 - - - - Pos Pos

2 - - - - Pos Pos

3 - - - - Pos Pos

4 - - - - Pos Pos

5 Pos - - - - -

6 Pos Pos - Pos - -

7 Pos Pos - - - -

8 Pos Pos - - - -

9 Pos Pos - - - -

10 Pos Pos - - - -

11 Pos Pos - - Pos -

12 - - - - - -

Ongoing work

RNA templates using synthesized RNA by cloning and in vitro transcription

Primers and probes have been designed

Further work

Test of specificity of primers and probes

RT-qPCR multiplex PCR optimization

Validation 65 tracheal aspirate samples

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