Upload
john-blue
View
110
Download
1
Tags:
Embed Size (px)
Citation preview
Like AMVC on FacebookFollow us on Twitter @AMVCLLC
Porcine Epidemic Diarrhea Virus(PEDV) – history, impacts, control
Paul Thomas, DVM, MS
AMVC Management ServicesISU Swine Medicine Education Center
Presentation Overview
• PEDV characteristics
• Introduction and spread in the US
• Impacts of PEDV
• Controlling PEDV
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
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.
04/15/2023
PEDV – gross lesions
Small intestine is thin-walled
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 milk2. Pig develops a severe, watery diarrhea• Malabsorptive, and osmotic
• This occurs in all ages of pigs
PEDV is not a new or emerging disease
• Europe
• Asia / China
• USA / North and South America
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 speciesSource: 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.
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
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)
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
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
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
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 weeksFarm A
Farm A
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
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!
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
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
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
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
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
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
Cleanliness
• The best immunity from the sow won’t protect piglets in a dirty environment
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
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
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
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
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
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
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
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)
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
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
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
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
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)
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
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®
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
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
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
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
Room temperature treatment groups
• Replicates from the low temperature groups were placed in an insulated cooler indoors.
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
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
Individual Housing
1 tub per room, 1 treatment group per tub.
Transmission Control Group
Three negative pigs, one positive pig
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
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)
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)
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)
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
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
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.
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-10PEDV-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-10PEDV-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-20PEDV-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-20PEDV-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
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
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
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
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
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 washing2. 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
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.
Acknowledgments
National Pork BoardPreserve International
PICIowa Select Farms
AMVC Management ServicesSMEC staff and students