Download Animal Nutrition

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 