Posted by Dr. Steve Dudley on June 19 2009 08:11

Summary of Intervention Strategies for the Prevention of Mechanical Transmission of PRRSV during Cold Weather.

Background: The "snowball" study demonstrated the ease at which PRRSV could be mechanically transmitted by a number of different fomites during periods of cold weather. High-risk fomites and sites were determined to be truckwash floors, boots, vehicle interior (floormats), anteroom floors within swine facilities, and shipping containers.

Objective: The purpose of this study was to test a series of intervention strategies designed to interrupt mechanical spread of PRRSV throughout a coordinated sequence of events during cold weather. The selected strategies were:

• The use of disposable plastic boots at a truckwash.
• The value of Lysol spray disinfectant for decontaminating vehicle floormats.
• Chemical disinfection of boots by footbaths.
• The use of polygrate flooring for preventing contamination of anteroom floors.
• The use of "bag-in-a-box" technology for preventing contamination of animal health products following entry to a farm.

The methods and results of each strategy will be explained individually:

The use of disposable plastic boots at a truckwash

• PRRSV-infected snowballs were created as described in the previous study 1. (1ml PRRSV strain MN 30-100, 1x10 4 TCID/50 per snowball)
• Snowballs were placed on concrete truckwash floor.
• Study personnel donned disposable plastic boots over the existing footwear (duck boots). Personnel then crushed snowball, contaminating ventral surface of boots.
• Personnel sat in vehicle with legs still outside of the cab and manually removed plastic boots, placing them in a garbage bag that was sealed
• Personnel then completely entered the cab and drove out of the truckwash.
• The garbage bag containing the boots was deposited into a waste receptacle outside of the truckwash.
• Swabs were collected from the truckwash floor where the crushed snowball was placed, the ventral surface of plastic boots, the ventral surface of existing footwear, the floormats, hands, vehicle steering wheel, and the outside of the garbage bag.
• Samples were tested by PCR/VI.

A total of 5 replicates were conducted. The truckwash floor was disinfected between virus-positive replicates using 4.5 % bleach (manufacturer's recommended dilution), while the existing footwear, floormats, and steering wheel were disinfected with Lysol spray, and allowed to dry. The drying process was accelerated through the use of a portable hair dryer.

Positive controls: The process was repeated without disposable boots (5 replicates). Materials were sanitized as described between each positive control replicate.

Negative controls: The process was repeated 5 times using sham inoculated (no virus) snowballs. A sham inoculated replicate was conducted between each virus positive replicate.

Results (PCR only)

PRRSV RNA was detected in 5/5 swabs collected from crushed snowballs on the truckwash floor.

PRRSV RNA was detected in 5/5 swabs collected from ventral surface of plastic boots after stepping on snowballs.

PRRSV RNA was detected in 0/5 swabs collected from ventral surface of existing footwear and vehicle floormats after removal of plastic boots.

PRRSV RNA was detected in 2/5 swabs collected from hands (fingertips/palms) following removal of plastic boots.

PRRSV was detected in 2/5 swabs collected from steering wheel following contact with hands.

PRRSV was detected in 0/5 swabs collected from exterior of garbage bag containing contaminated plastic boots.

Positive controls

PRRSV RNA was detected in 5/5 swabs from truckwash floor, ventral surface of exiting footwear and floormats when plastic boots were not used.

Negative controls (sham-inoculated replicates)

All samples were negative.

Preliminary conclusions/observations

• The trial was conducted at daytime temperatures of -11 to -10 degrees C (10-13 degrees F)
• The use of plastic boots is an effective means to prevent contamination of existing footwear and vehicle floormats.
• However, the manual removal of plastic boots can results in accidental contamination of hands and the resulting contamination of other components of the vehicle cab, such as the steering wheel.
• As a follow-up to the steering wheel observation, following the last replicate, PRRSV was inoculated directly onto hands, which then contacted the steering wheel of the vehicle. The steering wheel was swabbed at 0, 2 and 4 hours post-contact. PRRSV RNA was detected at all 3 sampling times.

The Value of Lysol Spray Disinfectant for Decontaminating Vehicle Floormats

• PRRSV-positve snowballs were created as described above, crushed wearing existing footwear (duck boots), and existing footwear placed on floormats for 15 minutes at 10 degrees C (50 degrees F).
• After 15 minutes, floormats were shaken to remove any visible snow, water, and ice.
• Floormats were then sprayed with Lysol disinfectant and stored at 10 degrees C for a 4-hour drying period.
• Floormats were swabbed at 10 minutes, 2 hours, and 4 hours following the application of Lysol.

Five replicates were conducted and samples were tested by PCR and VI.

All materials were sanitized between replicates as described above.

Positive controls

As above, except no Lysol was applied.

Negative controls

Sham-inoculated mats with Lysol application.

Results (PCR only)

PRRSV RNA was detected in 5/5 swabs collected at 10 minutes, 2/5 swabs collected at 2 hours, and 0/5 swabs collected at 4 hours following the application of Lysol.

Positive controls

PRRSV RNA was detected in 5/5 swabs collected at 10 minutes, 5/5 swabs collected at 2 hours, and 2/5 swabs collected at 4 hours following the application of Lysol.

Negative controls

All replicates were PCR-negative.

Preliminary conclusions/observations

• Mats were not visibly dry after 2 hours, while were visibly dry after 4 hours, post-inoculation.
• Replicates involving the application of Lysol spray in conjunction with a 4-hour drying period resulted in fewer positive samples than did replicates that utilized a 4-hour drying period without the application of Lysol.
• However, the simple application of Lysol was not enough to inactivate PRRSV within 10 minutes and 2 hours post-application. Therefore, it appears that a combination of disinfecting and drying is important for proper sanitation.

Chemical Disinfecting of Boots by Footbaths

• Footbaths were set up using a 4.5% solution of sodium hypochlorite (bleach) and water according to manufacturer's recommendations.
• A PRRSV-positive snowball was created as before and allowed to melt on a linoleum anteroom floor at 10 degrees C (50 degrees F).
• Plastic boots were donned over existing footwear.
• Personnel walked through wet anteroom floor and the ventral surface of the plastic boots was swabbed.
• Personnel stepped in footbath for 5 seconds, then walked to neutral point.
• Plastic boots were removed and the ventral surface swabbed.
• 10 1-ml aliquots of footbath liquid were collected and pooled 10:1 after the final replicate.
• Samples were tested by PCR/VI.
• replicates were conducted and a new set of plastic boots was used for each replicate.

All materials were sanitized as above between replicates.

Positive controls

• The process was repeated without a footbath.
• The process was repeated using a footbath void of bleach.
• Footbath liquid was collected, pooled and tested as above.

Negative controls

The process was repeated using a sham inoculated snowball.

Results (PCR only)

PRRSV RNA was detected in 10/10 swabs collected from the ventral surface of boots after walking on the wet anteroom floor and prior to entering the footbath.

PRRSV RNA was detected in 1/10 swabs from the ventral surface of plastic boots after using the footbath.

PRRSV RNA was not detected in water samples collected from the footbath containing bleach.

Positive controls

PRRSV RNA was detected in 10/10 swabs collected from the ventral surface of plastic boots after walking on the wet anteroom floor and not using a footbath.

PRRSV RNA was detected in 8/10 swabs collected from the ventral surface of boots after walking on the wet anteroom floor and after using a footbath that was void of bleach.

PRRSV RNA was recovered from water samples collected from the footbath void of bleach.

Negative controls

All samples were negative.

Preliminary conclusions/observations

• Footbaths that contain 4.5% bleach are more effective at reducing PRRSV-contamination of plastic boots in comparison to not using a footbath at all, or using a footbath that is void of disinfectant. However, the footbath was not 100% effective.
• Only a 5-second disinfecting time was used in this trial. A longer period (10-seconds) may have eliminated all contamination.
• The effect of other disinfectants was not tested and the impact of fecal material in the footbath was not assessed.

The Use of Polygrate Flooring for Preventing Contamination of Anteroom Floors

• PRRSV snowballs were created as described, and crushed by personnel wearing existing footwear (duck boots).
• One boot was placed on the linoleum anteroom floor for 15 minutes at 10 degrees C (50 degrees F).
• The second boot was placed on a sheet of polygrate nursery flooring, approximately 5 cm (2 inches) above the linoleum floor for 15 minutes.
• Swabs were collected from the linoleum and polygrate surfaces that were exposed to boots.
• Swabs were also collected from linoleum underneath the polygrate floor section.
• Swabs were tested by PCR/VI.
• replicates were conducted.

All materials were sanitized as above between replicates.

Positive controls

1 ml of straight virus was placed on separate sections of both floor types.

Negative controls

Boots contacted sham inoculated snow and floor sections were tested as above.

Results (PCR only)

PRRSV RNA detected in 5/5 swabs collected from linoleum floor following 15 minutes of boot contact.

PRRSV RNA detected in 5/5 swabs collected from polygrate floor following 15 minutes of boot contact.

PRRSV RNA detected in 5/5 swabs collected from linoleum floor located underneath polygrate floor.

Positive controls

All samples positive.

Negative controls

All samples negative.

Preliminary conclusions/observations

The use of polygrate flooring is of no value for reducing the risk of contamination of the anteroom area if footwear has been previously exposed to PRRSV-infected snow. The melting of snow/ice on the ventral surface of the boots resulted in accumulation of PRRSV RNA on, and under, the polygrate.

The Use of "Bag-in-a-Box" Technology (Newport laboratories Snowball Pack) for Preventing Contamination of Animal Health Products Following Entry to the Farm.

• PRRSV-snowballs (created as above) were allowed to melt over time (10 degrees C) on the linoleum anteroom floor.
• Ten plastic bottles, filled with cell culture fluid to simulate an order of animal health products (vaccines) were placed in a plastic bag, deposited into a cardboard box, and the box was sealed. 3.The cardboard box was placed onto the wet anteroom floor and opened (Phase 1).
• The bag containing the animal health products was manually removed from the inside of the box and transferred to a separate area representing the farm office (Phase 2).
• Swabs were collected from the ventral surface of the cardboard box, the interior of the box, the exterior of the plastic bag, the hands of personnel, and the plastic bottles housed in the separate room.
• Samples were tested by PCR/VI.

10 replicates were conducted and a new Snowball Pack was used for each replicate.

Positive controls

• Following exposure to the wet floor, the cardboard box was directly transferred unopened to the farm office where the plastic bag was opened and the bottles removed.
• Swabs were collected from the exterior and interior of the box, the bag, the bottles and the hands of personnel.

Negative controls

Sham inoculated snow employed.

Results (PCR only)

PRRSV RNA was detected in 10/10 swabs collected from ventral surface of the box in the anteroom (Phase 1).

No PRRSV RNA detected from any other samples in the farm office or on the hands of personnel(Phase 2).

Positive control

PRRSV RNA detected from 10/10 swabs collected from the ventral surface of the box and 10/10 swabs collected from hands, the plastic bag, and bottles that were handled in the farm office (Phase 2).

Negative controls

All samples were negative.

Preliminary results/observations

The use of "Bag-in-a-Box" technology, in conjunction with the physical separation of the exterior of the box and its internal components resulted in the introduction of PRRSV-free animal health products into the farm office despite contamination of the cardboard box during Phase 1.

However, the use of this technology without the physical separation of Phases 1 and 2 can result in transfer of the contaminated container into the clean area of the farm and contamination of the animal health products.

Overall Summary (VI data pending)

• The use of plastic boots clearly prevented the introduction of virus to the floor of the vehicle cab; however, contamination was observed via accidental introduction of PRRSV by contaminated hands.
• A combination of the application of a disinfectant and sufficient drying time is necessary for sanitizing vehicle floormats.
• The use of bleach-based footbaths can reduce the level of PRRSV contamination when wearing plastic boots, but at a 5-second immersion time are not 100% effective.
• The use of raised porous flooring materials is of no benefit for the reduction of contamination of the anteroom area.
• Bag-in-a box shipping containers are very effective for preventing the entry of contaminated animal health products into the clean area of a farm, but must be used in conjunction with physical separation of the interior and exterior components of the container.