When first detection of ASFV occurs in the farm, whole herd sampling and qPCR tests were carried out to evaluate the disease status in the herd. Then infected pigs were accurately removed and environment decontaminated by the precision removal process. After one or more rounds of this method were applied until the whole herd remained negative for 7-14days.
Four different farms were chosen in the time order. First ASFV detection was in February 7th 2019, June 2nd 2019, July 2nd 2o19, November 9th 2019 in Farm 1-4, respectively. For each farm, the study period last from the day of first ASFV infection detection to 7-14 days after the day of last positive qPCR result in the herd.
Farm 1 was a wean-finishing site holding 4231 growing pigs with average weight ranging from 7 kg to 130kg.
Farm 2, a typical commercial sow farm, which had newly developed a herd by introducing 1484 gilts in the breeding gestation room. The breeding gestation room is part of a typical uniformed production line, which was designed to hold 3000 sows and include two breeding gestation rooms each with 1296 stalls and 10 farrowing rooms each with 60 crates. Infection was firstly detected in one breeding gestation room.
Farm 3 was a commercial farrow-wean sow farm with 5167 in production sows kept in two independent uniformed production lines as described above. Infection was firstly detected in one breeding gestation room.
Farm4 was also a commercial farrow-wean sow farm with 3928 in production sows kept in two independent uniformed production lines as described above. Infection was firstly detected in one breeding gestation room.
Detailed maps showing the precise layout of each farm were produced to inform the sampling protocol. Pigs and the environment were sampled and if one pig was diagnosed as ASFV positive, a whole herd (WH) sampling of pigs and environment was performed, meaning sampling each pig and surfaces in the building within 24 hours. Subsequent whole herd pig samples were collected seven to ten days later.
Risk based early detection of infected pigs
Two kinds of samples from pigs were collected with a modified method based on those used for PRRSV monitoring 14. For early detection, all clinically abnormal pigs with signs including off-feed, fever, lethargy, hemorrhagic diarrhea, redness of skin, lameness, and abortion were sampled and tested. Swabs from nasal, oral, rectal (NOR), trough lips, and defecation area surface (pig contact area surface, PCAS) in the sow herd; or oral fluid (OF), trough lips, waterers, and defecation area surface and other floor surface (pig contact area surface, PCAS) swabs in the finishing pig herd were pooled together in a 2ml microtube as one sample. The pooled sample was only valid when at least three out of the five swab samples were successfully taken from the same pig. For confirmation of first ASFV DNA detection, especially when samples showed Ct values of higher than 35, lymph node (LN) samples were collected using an innovative lymph node sample collector (Figure s1). In brief, the pig was held down and the needle-like collector was pierced into an inguinal lymph node. The sample was taken out by the barb of the collector and was then injected into a 2 ml microtube as one lymph node sample.
Whole-herd sampling of pigs
The NOR samples from each sow in the farm were taken individually based on method described above after ASFV infection was confirmed.
Whole Surface (WS) sampling of supplies, personnel, and environment.
Whole surface sampling can be literally defined as sampling the surface of supplies, personnel, and environment (Figure s2). A 20 cm ×20 cm gauze soaked with 0.9% sodium chloride was used to wipe the surface of supplies, personnel and environment. For environmental sampling, a grid sampling frame was used. A grid represented 20~30 stalls in a gestation room, or a crate in a farrowing room, or a pen with solid walls in a wean-finishing site, or a functional room in the facility such as one dormitory or kitchen. Each pig and grid are clearly marked on the electronic map.
Ground surface, feeder, waterer, slats, and every object in the grid were wiped from top to the bottom. Each grid served as one sample. All samples from hair, face, nasal cavity, glasses, clothes, and boots of individual staff constituted a sample. For supplies, each category of incoming items was swabbed as one sample. The WS samples were put in a valve bag.
Sample processing and aggregation of samples
NOR or PCAS samples were diluted with 1ml of 0.9% sodium chloride and vortexed and then centrifuged at 4500 rpm for 30 seconds. Supernatant was collected and stored at -20℃. The WS samples were squeezed for 30 seconds until the dilution were homogenized. The dilutant was then poured into a 1.5ml microtube and centrifuged at 4500 rpm for 30 seconds. Supernatant was collected and stored at -20℃ for further use. lymph node samples were added with 500ul of 0.9% sodium chloride and homogenized with a homogenate machine. The homogenate was centrifuged at 4500 rpm for 30 seconds and supernatant was collected and stored at -20℃ until further use.
No more than 5 NOR and PCAS or WS samples were aggregated as one. Each lymph node sample was tested individually.
DNA extraction was performed using a DNA extraction machine Gene Pure Pro 96 from Bioer company (Hangzhou, China) according to the manufacturer’s instructions.
5 ul of extracted DNA was added to 20ul of qPCR mix from MRD company (Beijing, China) or Thermo Fisher Scientific (Waltham, USA) and qPCR was performed in a Gentier 48E machine from Tianlong company (Xian, China) or Step one Plus from Thermo Fisher Scientific (Waltham, USA) according to the manufacturers’ instructions. The procedure for qPCR test was as follows:
50℃ for 2 minutes, 1 cycle,
95℃ for 3minutes, 1 cycle,
95℃ for 10 seconds, 45 cycles
60℃ for 20 seconds,45 cycles
Materials and equipment used for precision removal
Facial masks, latex gloves, overalls, shoe covers, waterproof polyester clothing, vessels, and carts used for carrying dead pigs were purchased from local markets. Sodium hydroxide and sodium hypochlorite was purchased locally. Virkon was purchased from Lanxess (Cologne, Germany).
Number of removed pigs
The number of pigs removed was based on production type (gestation, farrowing, wean to finish or GDU), number of pigs infected in one grid, and Ct values of qPCR test.
In gestation, if the Ct value was lower than 30, and/ or more than two pigs were infected in one grid, the whole grid was depopulated. If the Ct value was ＞30, the infected pig and the two adjacent pigs were removed. In the farrowing room, regardless of Ct values, sows were removed by crate, and suckling piglets in the same crate were also removed. In the finishing site, if one pig was infected, the entire pen of pigs was removed. The two adjacent pens were only removed if the pens were not divided by solid walls.
Precision Removal of the pigs
Pigs were removed in a bio-secure manner. Sealed U-shape tunnel were made from waterproof polyester cloth (Figure s3) to move the pigs. The pigs were transferred using exclusive carts off the facility. After the pigs were removed, the supplies including gloves, overalls and cloth were incinerated. Afterwards, Virkon was applied in each grid.
Paired sampling & testing and subsequent daily monitoring and management
Sampling and testing continued afterwards using the same grid as the first round of WH sampling as recorded on the electronic map. At least one round of subsequent sampling & testing was required to eliminate the ASFV. More rounds were needed in the case of heavy contamination. Paired sampling and testing was ceased when previous round found no positive pigs.
After the herd (including pigs and the environment) remained negative for 21 consecutive days, clinically abnormal pigs with signs, such as being off-feed, fever, lethargy, hemorrhagic diarrhea, redness of skin, lameness, and abortion were tested daily. The NOR and PCAS samples were collected for ASFV qPCR test.
Validation of maximum number of sample aggregation for valid nucleoid acid detection
Verified LN, WS, NOR, and PCAS samples with initial Ct of ~20 and ~33 respectively were serially diluted 2 folds with PBS and quantified by qPCR for 2 replicates.
Data including qPCR result and TTNH (time to negative herd, in which both pigs and environment were negative ) were collected from each farm. TTNH was determined by calculating the days from the first ASFV positive qPCR result until the last positive result.