Dr. Paul Thomas - Porcine Epidemic Diarrhea Virus (PEDv)

Information about Dr. Paul Thomas - Porcine Epidemic Diarrhea Virus (PEDv)

Published on July 8, 2015

Author: trufflemedia

Source: slideshare.net

Content

1. Like AMVC on Facebook Follow us on Twitter @AMVCLLC Porcine Epidemic Diarrhea Virus (PEDV) – history, impacts, control PaulThomas, DVM, MS AMVC Management Services ISU Swine Medicine Education Center

2. Presentation Overview • PEDV characteristics • Introduction and spread in the US • Impacts of PEDV • Controlling PEDV

3. PEDV characteristics • PEDV is a coronavirus – Related to TGE virus in pigs – Enveloped virus – Prominent spike proteins • Survives best in cool, moist environments • Affects pigs of all ages – Disease is more severe in young pigs • Infection occurs via fecal-oral route

4. PEDV replication and lesions • PEDV infects enterocytes of small intestine and cecum – Intracellular viral replication destroys enterocytes – Results in severe “villous blunting” – Intestines appear thin-walled, grossly • Loss of enterocytes destroys the absorptive capabilities of the small and large intestine.

5. PEDV – gross lesions 7/8/2015 5 Small intestine is thin-walled

6. PEDV’s effect on pigs • Loss of absorptive capabilities of the intestine leads to: 1. Pig is unable to extract energy and nutrients from its feed or milk 2. Pig develops a severe, watery diarrhea • Malabsorptive, and osmotic • This occurs in all ages of pigs

7. PEDV is not a new or emerging disease • Europe • Asia / China • USA / North and South America

8. Likely came from China • Most closely related to virus isolate in Anhui Province in China • Has some genetic features that are same as a coronavirus found in bats – Suggests virus strain that is now in U.S. may have originated from bats and crossed species Source: Huang YW, Dickerman AW, Piñeyro P, Li L, Fang L, Kiehne R, Opriessnig T, Meng XJ. 2013. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. mBio 4(5):00737-13. doi:10.1128/mBio.00737-13.

9. And again! • Second introduction from China • Prior to January, 2014 all PEDV sequences (S1 region of the virus) were within 99-100% similar In late January, 2014 5 viruses were sequenced that were very different – Only 93.9-94.6% similar to the previous viruses – Closely matched a virus in the international database that was found in China PEDV dendogram based on entire PEDV genome

10. PEDV – USA How did it get in? • It is unknown how it entered the United States – Feed? – Supplies? – People? • Short timeframe of diagnosis in multiple unrelated sow farms across the US suggests it may have been here awhile before it was diagnosed – Perhaps was circulating in some finishing barns (Ohio case)

11. PEDV – USA How did it spread quickly? • Rapid spread of PEDV throughout US is likely due (in part) to contaminated livestock trailers – Terminal markets: for every contaminated trailer arriving at a plant, ~2 will leave the plant contaminated (Lowe et al., 2014) • Shortage of livestock trailers, drivers, and sanitation facilities results in little to no decontamination procedures between loads of market hogs Finishing barn  contaminated markets  Finishing barn

12. PEDv Diagnostic Tools available through Iowa State University VDL 1. Real-Time PCR – to detect presence of virus in feces or oral fluids (specifically viral RNA) 2. Immunohistochemistry (IHC) – to detect virus associated with lesions in small intestines 3. Indirect Fluorescent Antibody and ELISA (Serology) – to detect antibodies in blood 4. Electron microscopy (EM) – to detect virus (was used to determine PEDV was in U.S., not routinely done 5. Virus Isolation (VI) – to detect virus (for research) 6. Sequencing – to determine genetic “signature” of the virus (primarily for research at present) Source: Dr. Rodger Main, Iowa State University Veterinary Diagnostic Lab

13. PEDV in Sow Farms • Sows get infected and expose piglets to large amounts of virus • Sows are naïve to PEDV, so there is no protection through milk • Effect of the virus is the same, but piglets get dehydrated and weak extremely fast and die – Body size is too small to keep up with diarrhea – Milk diet may exacerbate the osmotic diarrhea

14. PEDV in Sow Farms PEDV is devastating to sow farms • Piglet mortality is 100% for 3-5 weeks minimum • Expect increased mortality and decreased quality of pigs for at least 2 more weeks Farm A Farm A

15. PEDV in finishing pigs • In finishing barns, PEDV will cause severe diarrhea within the whole group • Diarrhea is self-limiting – Pigs will recover if supported during this time – Easy, regular access to water – Gruel / mat feed, get pigs up several times per day – Provide plenty supplemental heat – +/- Antibiotic therapy to control bacterial enteritis

16. PEDV in Wean-Finish • Mortality is usually very low (1-3% in youngest pigs) • During the time of infection, pigs will not grow and will likely lose weight – Pigs will not make this growth up later • Group will be delayed 2 – 3 weeks • Pigs are shedding a HUGE amount of virus during this time – risk to other farms!

17. Controlling PEDV These numbers assume rapid and successful control of an outbreak • Proper steps must be taken in order to achieve this – Clean up environment / decrease amount of PEDV in farm – Get protection to piglets through the sows’ milk

18. Breakdown in biosecurity • Barn layout may make perfect biosecurity measures difficult to implement • Breakdown in communication to all barn employees can result in uneven implementation of biosecurity measures Farm B Farm B

19. Feedback • Once PEDV is diagnosed, immediate feedback of the whole herd should occur – Feedback material should be obtained from acutely infected animals – Exposing the whole herd will get all animals sick and over the disease at the same time – Expect lactogenic (milk) protection ~3 weeks after feedback begins • Piglets will continue become infected and die until then

20. Sanitation • Entire sow farm should be cleaned and disinfected – Farrowing rooms should especially be sanitized – Wash sows before entering farrowing – Wash and disinfect hallways every time an animal is moved – Stop movements between litters – Process piglets at 24 hours of age

21. Cleanliness • Some animals will continue to shed virus for an extended period even after they appear to be completely recovered – This means that after everything seems to be back to normal, piglets look good, scouring has stopped, and weaning numbers are back to normal --- PEDV is still on the farm • The minute you forget this and get lax with cleaning and sanitation, PED will flare back up. – To give sow and piglet immunity the best chance to be successful, we must minimize their challenge from the environment

22. Disease is a balance between challenge and protection • Dirty farrowing crates, hallways, poor job powerwashing • Animals shedding virus • Good exposure of all sows • +/- PEDV vaccine • Sows milking well

23. Cleanliness • The best immunity from the sow won’t protect piglets in a dirty environment

24. Prevention of PEDV Spread • Excellent biosecurity is critical to preventing PEDV from entering your sow farm – It is easy to get complacent with biosecurity when health on the farm is good. • Excellent internal biosecurity is critical to shutdown movement of the virus around the farm after recovery

25. Prevention of PEDV introduction • Minimize unnecessary outside visitors • Strict shower-in-shower-out at all times • Strict adherence to required down times • Cleaning and disinfecting of vehicles • Require feed drivers to wear disposable boots before stepping out onto your farm • Wash and disinfectant boots when entering “risky areas” – isolation barn, dead removal area, boots used for outdoor chores

26. Prevention of PEDV movement on an infected farm • Internal biosecurity – Wash sows before loading farrowing – Stop all piglet movements – Change gloves between litters – Disinfect equipment and eliminate unnecessary equipment – Separate farrowing and gestation employees – Wash and disinfectant boots between gestation and farrowing hallways

27. Prevention of PED outbreak • All livestock trailers must be washed, disinfected and baked between loads of animals. – In the presence of feces, PEDV must be heated to 160F to inactivate the virus – You should assume there is still feces in a cleaned trailer • When a trailer arrives at your farm, inspect it to ensure it is being properly cleaned – Empower employees to turn away improperly cleaned trailers

28. What’s known about sanitation properties • When it comes to PEDV….. Not much yet. • We can look at other viruses that are similar to PEDV to draw some conclusions • Proper disinfection is critical to successful inactivation in trailers and barns – Disinfectant choice – Concentration – Contact time – Environment • Proper heating parameters is important as well

29. Gluteraldehydes • 5 minutes at 2% decreased TGEV infectivity significantly (Hulkower et al., 2011). – This is higher concentration than commonly used – Increased contact time and concentration was required in the presence of serum proteins (feces) (Rabenau et al., 2005b). • 0.002% and 0.001% was ineffective against CCV even after 3 days of contact time at 4°C (Pratelli, 2008). – However, when temperature was increased to 25°C and 37°C both concentrations were very effective – Additionally, Darnell et al. (2004) showed this could be overcome by increasing gluteraldehyde concentration

30. Quaternary Ammoniums • 5 minutes of contact of two different products, one at 0.24% and the other at 0.12% concentration were both effective at reducing infectivity titers of TGEV by 4.5 log10 (Brown, 1981). • Again, the presence of serum proteins appeared to decrease disinfectant efficacy, with 0.1125% - 0.2% concentrations becoming ineffective at contact times of 10 minutes against HCoV-OC43 (Wood and Payne, 1997). – These are the concentrations we are using today! – At higher concentrations (0.5% or ~1:50 dilution) and longer times (30-60 minutes), inactivation of SARS-CoV in the presence of serum proteins was effective

31. Effect of Temperature • TGEV required 50-60 minutes at 54°C to be inactivated, multiple hours at temperatures below 50°C, and weeks at 5°C (Laude et al., 1981; Haas et al., 1995) • Similar results were observed for CCV, but inactivation required only 15 minutes when temperature was increased to 60°C, 5 minutes at 65°C, and was even more effective at 75°C (Pratelli, 2008; Saknimit et al., 1988) – Similar results were observed for HCoV-229E, HCoV- OC43, MERS-CoV, and SARS-CoV (Leclerq et al., 2014; Sizun and Talbot, 2000; Rabenau et al., 2005a)

32. Effect of Temperature • At cool temperatures coronaviruses are stable for days to weeks (Haas et al., 1995; Sizun and Talbot, 2000) • The effect of heat on virus inactivation was enhanced at alkaline pH • The effect of heat on virus inactivation was reduced in the presence of serum proteins (Rabenau et al., 2005a). – This was also true of disinfectants

33. Guidelines for Facility Sanitation What’s already known regarding other Coronaviruses: • Disinfection, heat, and pH control may all be effective means for inactivating PEDV in livestock trailers – RH is not a viable method as too much time is required – Disinfectant use should focus on correct concentrations applied at correct temperature – Heat inactivation should focus on attaining as high of temperatures as possible >65°C (150°F) • The removal of environmental protein (feces) is critical to increasing the effectiveness of either heat or disinfectants

34. Properties of PEDV inactivation • Summary of four studies evaluating trailer decontamination measures with and without washing for ability to inactivate or eliminate PEDV. – Not everyone can wash all livestock trailers – Washing does not remove 100% of organic material • Started with PEDV-positive feces • Evaluated the treatments in conditions similar to a trailer environment • Recollected the feces • Evaluated infectivity via pig bioassay – PCR+ does not indicate infectious virus is present

35. Study 1: Evaluation of Time & Temperature • Object was to evaluate a range of time and temperature combinations that might represent Thermo-Assisted Drying and Decontamination (TADD) systems or no use of TADD – 71°C (160°F), 10 minutes – 63°C (145°F), 10 minutes – 54°C (130°F), 10 minutes – 38°C (100°F), 12 hours – 20°C (68°F), 24 hours – 20°C (68°F), 7 days

36. Study 2: Evaluation of Stalosan® F disinfectant powder • Object was to evaluate a commercially-available, environmentally-applied disinfectant that is efficacious in the presence of feces and at freezing temperatures – Product is a fine, red powder – Applied to animal environments – Causes lysis of cell membranes • Evaluated efficacy of Stalosan® F Disinfectant after 1 hour of contact time • Three groups – Negative Control (n=8) – Positive control (n=8) – Stalosan® F treatment group (n=8)

37. Study 3: Evaluation of Accel® liquid foaming disinfectant • Object was to evaluate the efficacy of a commercially- available disinfectant in the presence of fecal material. • Accel® (accelerated hydrogen peroxide) is supposed to have longer residual activity due to enhanced contact. • Four treatment groups: – Light feces (5mL) @ 1:32 concentration for 30m – Heavy feces (10mL) @ 1:32 concentration for 30m – Light feces (5mL) @ 1:16 concentration for 30m – Heavy feces (10mL) @ 1:16 concentration for 30m – Transmission Control group

38. Study 4: Evaluation of various combinations of TADD following a wash and Synergize® • Object was to evaluate the efficacy of various TADD configurations following a wash and disinfection with Synergize® disinfectant @ 1:256. – Wash, 10m Synergize®, 68°C (154°F) for 10m – Wash, 10m Synergize®, 66°C (151°F) for 10m – Wash, 10m Synergize®, 60°C (140°F) for 20m – Wash, 10m Synergize®, 49°C (120°F) for 20m – Wash, 10m Synergize®, 20°C (68°F) for 12h – Wash, 60m Synergize® – Wash, 10m Synergize®

39. Source of virus and feces • PEDV-positive feces was obtained from 3-week old pigs that were experimentally infected with PEDV – Feces from multiple pigs was collected and pooled – Pooled feces was analyzed via PCR to ensure presence of virus – Frozen at -70C until time of challenge – Feces was retested following thawing on day of challenge • PEDV-negative feces was obtained from the negative control pigs from this same study – Negative feces was handled, stored, and evaluated for virus similarly

40. Tray Preparation • Four (4) trays per treatment group – Each tray was the experimental unit – Eight (8) trays per group in Study 2 • 5 or 10 mL of feces was placed on each tray depending upon the study / treatment group

41. Tray Preparation • Feces was spread in a thin layer to represent a trailer floor that had been scraped or swept, but not washed • A sample of the feces on the tray was taken for PEDV PCR prior to experimental treatment

42. Timed Thermal Application • Timed thermal application for the high temperature groups was performed using an incubator capable of reaching and holding temperatures of 75°C

43. Room temperature treatment groups • Replicates from the low temperature groups were placed in an insulated cooler indoors.

44. Collection of Treated Feces • Feces was re- suspended with 10 mL of sterile saline and drawn up for inoculation of the swine bioassays • A second sample of the feces was collected following re-suspension and tested via PEDV PCR

45. Swine Bioassay • A 14 Fr rubber catheter was used to pass the challenge material directly into each pig’s stomach • Each pig corresponded to an individual tray

46. Individual Housing 1 tub per room, 1 treatment group per tub.

47. Transmission Control Group Three negative pigs, one positive pig

48. Clean Sample Collection • Pigs were monitored daily for signs of PEDV infection • Rectal swabs were collected on day 3 and day 7 post- challenge to determine bioassay status

49. Results of tray swabs – Time & Temperature Table 1: PCR results for swabs of trays before and after treatment was applied in Study 1. Treatment Group Pre-Treatment RT-PCR: mean CT (± SD) Post-Treatment RT-PCR: mean CT (± SD) Negative Control >40 N/A Positive Control 15.22 (0.73) N/A 71C-10M 13.40 (0.30) 24.10 (0.76) 63C-10M 13.16 (0.48) 21.56 (0.71) 54C-10M 13.41 (0.32) 20.83 (0.53) 38C-12H 13.28 (1.21) 20.19 (0.09) 20C-24H 14.45 (0.57) 15.07 (0.18) 20C-7D 12.94 (0.55) 17.71 (0.41)

50. Bioassay Results – Time & Temperature Table 2. Description of treatment groups and bioassay outcomes for Study 1. Treatment Group Temperature & Time applied Percentage of PEDV positives (out of 4) Negative Control No heat, no time 0% (0/4) a Positive Control No heat, no time 100% (4/4) b 71C-10M 71°C (160°F), 10 minutes 0% (0/4) a 63C-10M 63°C (145°F), 10 minutes 25% (1/4) a, b 54C-10M 54°C (130°F), 10 minutes 25% (1/4) a, b 38C-12H 38°C (100°F), 12 hours 50% (2/4) a, b 20C-24H 20°C (68°F), 24 hours 25% (1/4) a, b 20C-7D 20°C (68°F), 7 days 0% (0/4) a Groups with different superscripts indicate statistically significant differences (P ≤0.05)

51. Results - Stalosan® F treatment Table 3. PCR results for swabs of trays before and after treatment was applied in Study 2. Treatment Group Pre-Treatment RT-PCR: mean CT (± SD) Post-Treatment RT-PCR: mean CT (± SD) Negative Control >40 N/A Positive Control 14.4 (0.67) 15.35 (0.73) Stalosan® F Treatment 15.2 (0.42) 18.4 (1.51) Table 4. Description of treatment groups and bioassay outcomes for Study 2. Treatment Group Description of Treatment Percentage of PEDV positives (out of 8) Negative Control No Stalosan® F contact 0% (0/8) a Positive Control No Stalosan® F contact 100% (8/8) b Stalosan® F Treatment One hour of Stalosan® F contact time 100% (8/8) b Groups with different superscripts indicate statistically significant differences (P ≤0.05)

52. Results of tray swabs – Accel® disinfectant Table 5: PCR results for swabs of trays before and after treatment was applied in Study 3. Treatment Group Pre-Treatment RT-PCR: mean CT (± SD) Post-Treatment RT-PCR: mean CT (± SD) Negative Control >35 N/A Positive Control 14.15 (0.41) N/A 5mL-1:16 13.43 (0.57) >35 10mL-1:16 13.83 (0.46) >35 5mL-1:32 14.30 (0.22) >35, (one tray @ 32.2) 10mL-1:32 13.83 (0.27) >35, (one tray @ 34.3) Transmission Control >35 (3 negative animals), 14.4 NA

53. Table 6. Description of treatment groups and bioassay outcomes for Study 3. Treatment group Description of treatment Percentage of PEDV positives (out of 4) Negative Control No treatment, pigs received a gavage of PEDV-negative feces 0% (0/3) a Positive Control No treatment, pigs received a gavage of PEDV-positive feces 100% (4/4) b 5mL-1:16 A 1:16 concentration of Accel® disinfectant was applied to 5 ml of PEDV- positive feces for 30 minutes 0% (0/4) a 10mL-1:16 A 1:16 concentration of Accel® disinfectant was applied to 10 ml of PEDV- positive feces for 30 minutes 0% (0/4) a 5mL-1:32 A 1:32 concentration of Accel® disinfectant was applied to 5 ml of PEDV- positive feces for 30 minutes 0% (0/4) a 10mL-1:32 A 1:32 concentration of Accel® disinfectant was applied to 10 ml of PEDV- positive feces for 30 minutes 0% (0/4) a Transmission Control 1/4 pigs in the group was gavaged with PEDV-positive feces, 3/4 were gavaged with PEDV-negative feces 25% (1/4) Groups with different superscripts indicate statistically significant differences (P ≤0.05). Results from the transmission control group were not included in statistical analysis. Bioassay Results – Accel® disinfectant

54. Results of tray swabs – Wash, Synergize®, & TADD PCR results for swabs of trays before and after treatment was applied in Study 4. Treatment Group Pre-Treatment RT-PCR: mean CT (± SD) Post-Treatment RT-PCR: mean CT (± SD) Negative Control >35 N/A Positive Control 20.93 (0.35) N/A WD-68C-10 21.85 (0.19) 34.43 (0.32), >35* WD-66C-10 21.08 (0.69) 33.6 (1.41), >35* WD-60C-20 21.33 (0.50) 32.83 (0.45), >35* WD-49C-20 21.55 (0.40) 33.93 (0.81), >35* WD-20C-12 28.48 (0.74) >35 WD60 20.68 (0.39) 31.65 (2.83) WD10 20.43 (0.25 31.6 (0.67) * One or more trays in this group had a CT >35, these trays were not included in calculation of the mean CT value.

55. Table 8. Description of treatment groups and bioassay outcomes for study 4. Treatment group Description of treatment Percentage of PEDV positives (out of 4) Negative Control No treatment, pigs received a gavage of PEDV-negative feces 0% (0/4) a Positive Control No treatment, pigs received a gavage of PEDV-positive feces 100% (4/4) b WD-68C-10 PEDV-positive feces power washed with detergent, application of 1:256 concentration of Synergize® disinfectant for 10 minutes, heated to 68° C (155° F) in an incubator and held at this temperature for 10 minutes 0% (0/4) a WD-66C-10 PEDV-positive feces power washed with detergent, application of 1:256 concentration of Synergize® disinfectant for 10 minutes, heated to 66° C (150° F) in an incubator and held at this temperature for 10 minutes 0% (0/4) a WD-60C-20 PEDV-positive feces power washed with detergent, application of 1:256 concentration of Synergize® disinfectant for 10 minutes, heated to 60° C (140° F) in an incubator and held at this temperature for 20 minutes 0% (0/4) a WD-49C-20 PEDV-positive feces power washed with detergent, application of 1:256 concentration of Synergize® disinfectant for 10 minutes, heated to 49° C (120° F) in an incubator and held at this temperature for 20 minutes 0% (0/4) a WD-20C-12 PEDV-positive feces power washed with detergent, application of 1:256 concentration of Synergize® disinfectant for 10 minutes, left at 20° C (room temperature) for 12 hours 0% (0/4) a WD60 PEDV-positive feces power washed with detergent, application of 1:256 concentration of Synergize® disinfectant for 60 minutes 0% (0/4) a WD10 PEDV-positive feces power washed with detergent, application of 1:256 concentration of Synergize® disinfectant for 10 minutes 0% (0/4) a Groups with different superscripts indicate statistically significant differences (P ≤0.05) Bioassay Results – Wash, Synergize®, & TADD

56. Conclusions • Only two combinations from Study 1 were 100% successful: – 71° C (160° F) for 10 minutes • Increase TADD temps to 160° F for 10 minutes to inactivate PEDV – 20°C (68° F) (room temperature) for 1 week • This is not practical for many farms or transporters • These samples dried out completely • Humidity / moisture would likely play a role in survival

57. Conclusions • Stalosan® F is ineffective at inactivating PEDV from contaminated hog trailers • Accel® (accelerated hydrogen peroxide) appears to effective at inactivating PEDV in presence of feces – This may have applications in situations where washing isn’t possible or practical – Further study demonstrated that Accel was effective at cold temperatures if you add 10% propylene glycol to prevent freezing

58. Conclusions • A wider range of TADD configurations were found to be effective following wash and disinfection with Synergize® – 20°C for 12h to 68°C for 10m – Synergize® and wash alone were also effective – Underscores the importance and utility of wash and disinfection steps in trailer sanitation – DOES NOT MEAN HEAT STEP SHOULD BE SKIPPED • Environmental swabs could not differentiate infectious from non-infectious PEDV – Implications for environmental sampling

59. Final Conclusions • A number of temperature and time combinations are effective at inactivating coronaviruses • Disinfectants commonly used in swine production and the concentrations they are used at are also effective • However, the effectiveness of both of these can be reduced by factors common to livestock trailers – The presence of in feces – Reduced temperatures • These points are summarized in review and supported by original research findings

60. Final Conclusions • An effective livestock trailer decontamination process should capitalize on these positive properties and seek to minimize the effects of the negative properties • A sound decontamination process will: 1. Remove all organic material first via high pressure washing 2. Utilize an appropriate concentration (label) of disinfectant for at least 10 minutes in a warm environment (disinfectants don’t work well when cold) 3. Following disinfection, trailers should be heated to 71°C for 10 minutes – this will inactivate any virus missed through washing and disinfection

61. Final Conclusions • These recommendations are consistent with today’s best-practices trailer decontamination practices for PRRSV and other pathogens – This means they can be immediately implemented by transporters and pork producers – Temperatures should be increased • Consider the increased risk due to PEDV-laden feces, and decreased infectious dose of PEDV.

62. Acknowledgments National Pork Board Preserve International PIC Iowa Select Farms AMVC Management Services SMEC staff and students

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