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Livestock Nutrition 2015 11-12 August, 2015 Franfurt, Germany Non-specific antiviral components in plasma can contribute to the safety of SDPP towards PEDV © Veos NV Quist-Rybachuk GV1, Nauwynck HJ1, Kalmar ID2 1Laboratory of Virology, Ghent University, BE 2Veterinary Natural ingredients R&D, Veos group, BE Background - Porcine Epidemic Diarrhea Virus (PEDV) 1st case: 1971 ● swine alfa-corona virus since April 2013 7 million piglets ● major infection route: Faecal-Oral 3 80’s - 90 ’s after 2000: rare 1 ● highly enteropathogenic 2 1st case: 1973 prevalent in Asia since ‘10: severity small intestinal villus atrophy acute, watery diarrhea (malabsorptive & maldigestive) After Opriessnig, 2014 ● Severity strongly depends on age at infection (also on virus strain, lactogenic immunity, co-infections, …) Neonatal & Suckling Weanling to Adult Gestation up to 80-100 M% self-limiting (mild) Reproduction o fetid, watery diarrhea o anorexia o vomiting o o o o o diarrhea depression loss of apetite weight loss (vomiting) o FR - RR - AR o TB - BA - Mu o especially gilts, d1-d30 (Orlanratmanee et al., 2010) Background - Transmission of PEDV Direct Infected pigs ● Faecal-Oral Indirect Faecally contaminated fomites equipement, clothing, lorries,… Live pig collection points (USA July ‘14) 6,6% of trailers: RNA-pos at arrival 5,2% of RNA neg trailers: left RNA-pos (Lowe et al., 2014) ? Milk-borne RNA detected in sow milk Infectivity not tested (Sun et al, 2011; Li et al, 2011; Chen et al, 2013) ? Aerosolised RNA in air samples 16 km downwind infected farms Field samples : Not infectious Exp. samples : Infectious (Alonso et al., 2014) lactating sows - faeces - vomit - saliva Background - Transmission of PEDV Direct Infected pigs ● Faecal-Oral Indirect Faecally contaminated fomites equipement, clothing, lorries,… FEED - INGREDIENTS Proven field cases (USA Jan ‘14) - 3 breeding herds: “emergency feed” - Origin diets: different mills - clinical sings 2 d post-delivery - Ingredients: corn - soybean meal - Vit&Min Ct feed = 19,5-22,2 Bioassay: infectious PEDV (Dee et al., 2014) Adequate biosafety measures should be in place for feed and its ingredients Background - What about SDPP ? Ontario-cases, 2014 US-SDPP Feed : common factor (Ct 37-40) SDPP : Ct 36-37 (Ct 37-40) bioassay CFIA bioassay US FDA : POS (sampled at feedmill) (Pasick et al., 2014) : NEG (sampled at production plant) (US FDA, 2014) Ontario-cases occured > 10 weeks post-production of SDPP Epidemiology Brazil & West-Canada remained neg (Crenshaw et al., 2014) Despite large imports of PCR-pos US-SDPP (2.5-3.5 M Brazilian pigs/3.5-4.0 M West-Canadian pigs) Timing PEDV is sensitive to dessication (Pujols & Segalés, 2014) Inactivation of 2.8 log10 TCID50/g (Ct 22) 3 wk at 4°C (Ct 31.5) ; 2 wk at 12°C (Ct 24); 1 wk at 21°C (Ct 21) Processing PEDV is sensitive to spray-drying (Gerber et al, 2014; Pujols & S, 2014) Inactivation of 4.2 log10 TCID50/ml (Ct 13.9) Spraydrying at 80 °C or 70 °C outlet temp (Ct 23.3-23.8) Present Trial Why ? Prevention of further geographical spread of PEDV is critical Adequate biosafety measures should be in place for feed and feed ingredients, including SDPP Extremely High Fecal Shedding Clothing Lorries RNA detected in sow milk Air ? PIGS Feed (vegetal) Replication in lung macrophages RNA detected in nasal/oral secretions (? faecal contamination) RNA detected in acute phase serum Equipm. SDPP (US) Present Trial Research Questions 1. Does porcine plasma contain non-specific anti-PEDV activity ? 3-5 dpi : 2.3-3.2 log PEDV ge/ml serum (Opriessnig et al. 2014) Bio-assay: 3-5 dpi, seronegative serum (body temp) : non-infectious (Gerber et al., 2014) 2. Are anti-PEDV effects in plasma temperature dependent ? [refrigerated plasma (4°C) vs living pig (37.8-40°C°)] Bio-assay: PEDV spiked, seronegative serum (Gerber et al., 2014) (4°C) : infectious (but high spike) 3. Is PEDV inactivation facilitated in porcine plasma ? ingredient-specific PEDV survival at outdoor storage (-25 to 20 °C) SDPP, VTM, soy oil, corn :≤7d DDGS, M&B, Lys, stock-virus : > 30 d Soybean meal : >180 d (≤210 d) non-infectious (in vitro and in bio-assay) (Dee et al., 2015) Materials & Methods - Spike Inactivation Assays 90% 10% SPIKE INACTIVATION Test Matrix PEDV Spike Test Sample 90:10 matrix (MEM or porcine plasma):virus sterile filtered heat inactivated seronegative for PEDV ASSAY In vitro infectivity Materials & Methods - Spike Inactivation Assays 90% 10% SPIKE INACTIVATION Test Matrix PEDV Spike ASSAY Test Sample Inactivation (treatment) Condition pH: neutral (pH 7.2) temperature: 4, 37 or 40 °C (body temp: 37.8 - 40 °C) Time up to 180 min Matrix MEM or porcine plasma In vitro infectivity Materials & Methods - Spike Inactivation Assays 90% 10% SPIKE Test Matrix PEDV Spike ASSAY INACTIVATION In vitro infectivity Test Sample virus titration of whole test sample 1 ml test samples PFU count 1:10 in 175 cm² tissue culture flasks (log10) Titer 100 ul test samples TCID50 in 96-well plates (survival curves, n=3) Survival Curve spike 1 D-value Time Results & Discussion - Survival Curves 4°C 4 °C (log10 TCID50/ml) Surviving PEDv titer Spike-Inactivation Assay Ph: 7.248°C 44°C LDL below LDL 6 4 2 0 0 15 30 4°C 4 °C (log10 TCID50/ml) 45 60 75 90 105 120 time Ph: (min) 7.2 Incubation time (min) pHIncubation 7.2 MEM Surviving PEDv titer 40°C Sensitivity of PEDV 40°C 44°C Ph: 7.248°C LDL below LDL 6 4 2 0 0 Plasma 15 30 45 60 75 90 105 120 (min) 7.2 Incubation time (min) pHIncubation 7.2 Ph: time Stable in MEM at 4°C Stable in plasma at 4°C Results & Discussion - Survival Curves 4°C 4 °C (log10 TCID50/ml) Surviving PEDv titer Spike-Inactivation Assay 44°C 40°C Ph: 7.248°C LDL below LDL 6 4 2 0 0 15 30 45 60 75 90 105 120 time Ph: (min) 7.2 Incubation time (min) pHIncubation 7.2 4°C 4 °C (log10 TCID50/ml) Stable in MEM at 4°C Stable in plasma at 4°C Stable in MEM at 40°C Sensitive to 40°C in plasma (tailing effect in plasma) MEM Surviving PEDv titer 40°C Sensitivity of PEDV 40°C 44°C 40°C Ph: 7.248°C LDL below LDL 6 D40°C,MEM = 120 ± 17 min D40°C,plasma = 16 ± 5 min 7.5 x faster “in vivo” conditions 4 2 0 0 Plasma 15 30 45 60 75 90 105 120 (min) 7.2 Incubation time (min) pHIncubation 7.2 Ph: time Results & Discussion - Survival Curves 4°C 4 °C (log10 TCID50/ml) Surviving PEDv titer Spike-Inactivation Assay 44°C 40°C Ph: 7.248°C LDL below LDL 6 4 2 0 0 15 30 45 60 75 90 105 120 time Ph: (min) 7.2 Incubation time (min) pHIncubation 7.2 4°C 4 °C (log10 TCID50/ml) Stable in MEM at 4°C Stable in plasma at 4°C Stable in MEM at 40°C Sensitive to 40°C in plasma (tailing effect in plasma) MEM Surviving PEDv titer 40°C Sensitivity of PEDV 44°C LDL Ph: 40°C n48°C 7.2 44°C 40°C below 48LDL °C D40°C,MEM = 120 ± 17 min D40°C,plasma = 16 ± 5 min 6 7.5 x faster “in vivo” conditions 4 HAT-pasteurisation plasma [48°C - pH 10.2 - 10 min] 2 0 0 Plasma 15 30 45 60 75 90 105 120 (min) 7.2 Incubation time (min) pHIncubation 10.2Ph: time D-plasmaUCL95 : 35 sec 17.4 log PEDV TCID50/ml (in MEM: DUCL95=114 sec 5.3 log) Quist-Rybachuck, Nauwynck, Kalmar, subm Results & Discussion - Confirmation Assay Acute phase serum has been reported to contain PEDV Does PEDV remain infectious in plasma at in vivo conditions? Spike-inactivation assay in tissue culture flasks HAT determinants: H = 37°C; A = pH 7.2; T = 120 or T = 180 min D value at in vivo conditions Matrix Spike Vol pH Temp Time (log10 TCID50) Plasma 5.80 1 ml 7.2 Surviving PEDV Measured D value (sec or min) (°C) (min) # 37 120 3 23.2 min Expected D value mean [UCL95] 30.5 [55.8] min Flask assay at in vivo conditions: D37°C, pH 7.2 = 23.2 min Even in absence of anti-PEDV IgG, infective PEDV is inactivated in porcine plasma at in vivo conditions (normal pig body temperature = 37.8-40°C) Results & Discussion - Confirmation Assay Acute phase serum has been reported to contain PEDV Does PEDV remain infectious in plasma at in vivo conditions? Spike-inactivation assay in tissue culture flasks HAT determinants: H = 37°C; A = pH 7.2; T = 120 or T = 180 min obtainment of PEDV sterility at in vivo conditions Matrix Spike Vol pH Temp Time (log10 TCID50) Plasma 5.80 1 ml 7.2 (°C) (min) 37 180 Surviving Sterility PEDV obtained ? # PFU 0 Expected time to sterility (yes/no) mean YES 177 [UCL95] [324] min Flask assay at in vivo conditions: D37°C, pH 7.2 = 23.2 min Even in absence of anti-PEDV IgG, infective PEDV is inactivated in porcine plasma at in vivo conditions (normal pig body temperature = 37.8-40°C) In vivo conditions result in PEDV sterility of a 5.8 log10 TCID50 per ml spike within 180 min Conclusions 1. Porcine plasma at body temperature shows anti-PEDV activity but refrigerated plasma does not tailing effect when inactivation at 37 - 40 °C no tailing effect at 48 °C / important dilution effect (further trials) PEDV inactivation in seronegative porcine plasma at 37°C ± 0,44 million infectious particles /ml plasma (5.8 log TCID50/ml) reduced to 3 (0) infectious particles in 2 h (3 h) viraemic PEDV would not remain infectious for prolonged times 2. Inactivation of PEDV is facilitated in plasma non-specific anti-viral acitivity of plasma increases safety of SDPP 3. Viral inactivation assays should take matrix-effects into account 4. Biosafety measures: needed at all points of the distribution chain Acknowledgements - Questions PEDV syncytium cell nuclei PEDV virions © Veos NV Vero-Ba cell line Additional Slides - In vitro PEDV culture Infective PEDV forms plaques in cell culture Vero-Ba cells © Veos NV PEDV Replication © Veos NV Actin filaments (cytoplasm proteins) Cell nucleus Attached PEDV particle PEDV Syncytium © Veos NV Additional Slides - In vitro PEDV culture Infective PEDV forms plaques in cell culture © Veos NV Macroscopic plaques Microscopic CPE Confocal image of syncytium Additional Slides - PEDV detection methods 1. RT-qPCR (Ct value; ge/ml or g) amplification of a section of the viral genome fast & relatively cheap Intact (infectious) virus Inactivated virus Incomplete virus POS ≠ “contagious” NEG = “below detection limit” 2. Cell culture (PFU/ml or g; TCID50/ml or g) Intact (infectious) virus cytopathogenic effects: syncytia (plaques) only detects infectious virus: POS = “contagious” know-how is limited to selected labs ideal for in vitro assays using lab-strains less suitable for clinical samples © Veos NV 3. Bioassay (animal trial) Intact (infectious) virus only detects infectious virus: POS = “contagious time-consuming & high costs PEDV is sensitive to dessication (infectivity drops in time retrospective analyses) VS Additional Slides - HAT pasteurisation of plasma Spike-Inactivation Assay (log10 TCID50/ml) Surviving PEDv titer 4°C 44°C 48°C LDL below LDL pH 7.2 6 4 40 5 4 4 40 48 0 0 15 (log10 TCID50/ml) 30 45 60 75 90 105 2. Medium Plasmaas is Not sensitive (R) 120 4. Temp Sensitivity Incubation time (min) 4°C 7 1. Temp x pH 4 °C x 7.2 Not sensitive (R) 3. Treatment x Medium No interaction at 4°C / pH 7.2 2 MEM Surviving PEDv titer 40°C Sensitivity of PEDV 40°C 44°C 48°C LDL below LDL pH 7.2 6 5. Temp Sensitivityplasma InteractionT x medium 6. Plasma40 °C 4 7. Plasma44-48 Tailing effect 5 2 0 0 Plasma 15 Tailing effect 30 45 60 75 Incubation time (min) 90 105 120 Additional Slides - HAT pasteurisation of plasma Spike-Inactivation Assay (log10 TCID50/ml) Surviving PEDv titer 4°C 44°C 48°C LDL below LDL pH 10.2 6 Not sensitive (R) 3. Treatment x Medium No interaction at 4°C / pH 7.2 2 0 0 15 30 45 60 75 90 105 120 4. Temp Sensitivity Incubation time (min) 4°C (log10 TCID50/ml) 1. Temp x pH 4 °C x 7.2 Not sensitive (R) 2. Medium Plasmaas is 4 MEM Surviving PEDv titer 40°C Sensitivity of PEDV 40°C 44°C 48°C LDL below LDL pH 10.2 6 5. Temp Sensitivityplasma InteractionT x medium 6. Plasma40 °C 4 Tailing effect 7. Plasma44-48 Tailing effect 2 8. pH 10.2 Not sensitive 0 0 Plasma 15 30 45 60 75 Incubation time (min) 90 105 120 9. pH SensitivityTemp 10. pH Sensitivityplasma Additional Slides - HAT pasteurisation of plasma Spike-Inactivation Assay (log10 TCID50/ml) Surviving PEDv titer 4°C 44°C 48°C LDL below LDL pH 7.2 6 4 40 4 40 48 0 0 15 30 45 60 75 90 105 2. Medium Plasmaas is Not sensitive (R) 120 4. Temp Sensitivity Incubation time (min) 4°C (log10 TCID50/ml) 1. Temp x pH 4 °C x 7.2 Not sensitive (R) 3. Treatment x Medium No interaction at 4°C / pH 7.2 2 MEM Surviving PEDv titer 40°C Sensitivity of PEDV 40°C 44°C 48°C LDL below LDL pH 10.2 6 5. Temp Sensitivityplasma InteractionT x medium 6. Plasma40 °C 4 Tailing effect 7. Plasma44-48 Tailing effect 2 8. pH 10.2 Not sensitive 0 0 Plasma 15 30 45 60 75 Incubation time (min) 90 105 120 9. pH SensitivityTemp 10. pH Sensitivityplasma