Development and application of Whole-herd-Sampling, qPCR-based- Testing, and Precision-Removal methods to Eliminate ASFV in Four Large Swine Herds in China

Development and application of Whole-herd-Sampling, qPCR-based- Testing, and Precision-Removal methods to Eliminate ASFV in Four Large Swine Herds in China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and application of Whole-herd-Sampling, qPCR-based- Testing, and Precision-Removal methods to Eliminate ASFV in Four Large Swine Herds in China Xiaowen Li, Peng Li, Bingzhou Zhang, Weisheng Wu, Junxian Li, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4515313/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Since the first ASFV case was reported in China in 2018, the conventional depopulation method to control ASF has proved unwieldy because of its high production intensity and complex trade network. To provide an alternative to conventional stamping out methods, we developed a” Whole-herd-Sampling, qPCR-based-Testing, and Precision-Removal” method by sampling every whole herd sampling and qPCR tests to determine the status of ASFV in herds and using a precision removal of identified sows. By developing and applying these methods, we successfully controlled ASF and eliminated the virus from 4 large swine herds from 2019 to 2020. The time to negative herd (TTNH) was 19, 28, 14, and 1 day from farm 1 to 4, respectively. Retention rates of pigs from farm 1 to farm 4 were 69.7%, 65%, 99.4%, and 99.72%, respectively. We anticipated that this innovative method would be a good alternative to the conventional stamping out method and greatly facilitate the control and eradication of ASFV in China and worldwide. African swine fever virus quantitative PCR test removal Figures Figure 1 Figure 2 1. Introduction The first outbreak of ASF in China was reported on 3rd August 2018 [ 1 ]. Without a commercial vaccine, the only available tool to prevent ASFV is the implementation of strict biosecurity measures at regional and farm levels. The response of the veterinary authorities was in accordance with the OIE guidelines. This means that the Ministry of Agriculture and Rural Affairs (MOARA) of China required depopulation of infected and proximal farms once ASFV was reported [ 2 ]. However, it soon became apparent, that these measures were not effective in China for several reasons. These include the high density of pig farms, of which the vast majority were small-scale producers that were connected with each other via highly complex pig and pork trade networks. There may also have been inefficiencies in the response by the industry and government in the early stage of the epidemic. Unlike other pig diseases such as pseudorabies, there is no scientific literature that describes the elimination of ASFV within a herd without stamping out. Field observations from affected farms indicate that the transmission pattern of ASFV in large herds differed from that of other major swine viral diseases. Published information also indicates that after infection, ASFV DNA detection in oral, nasal, and rectal swab samples occurred between 0 and 2 days before the onset of clinical signs[ 3 , 4 ], suggesting that early detection of viral DNA by qPCR before or around the onset of clinical signs may be possible as part of active within-herd ASF surveillance. It was observed that ASFV spreads relatively slowly within a herd following introduction, with an estimated within-pen basic reproduction ratio (R0) of approximately 2.8 and between-pen R0 of approximately 1.4 [ 5 ]. Moreover, environmental contamination of ASFV can be relatively light, as was shown that the introduction of negative pigs into contaminated pens 3, 5, and 7 days after infected pigs were removed did not cause infection in this study [ 4 ]. In addition, aerosol transmission of ASFV appears unlikely to occur as the half-life of ASFV in air was estimated to be 14 ~ 19 minutes[ 6 ]. We also observed that most infections within barns were associated with common routes of contact via direct contact, feces, or saliva. This published scientific information together with the field observations in China indicated that it might be possible to achieve ASFV eradication within a herd by using a test-and-removal method. Thus, in this study, we delineated the “Whole-herd-Sampling, qPCR-based-Testing, and Precision-Removal” method in eliminating ASFV Georgia 2007/1 Strain without stamping out the whole population in 1 finishing and 3 sow herds. 2. Materials and Methods After ASFV was detected in the farm, whole herd sampling and qPCR tests were carried out to evaluate the disease status in the herd. Then positive pigs were removed, and the environment was decontaminated by the precision removal process. One or more rounds of this method were applied until the whole herd remained negative for 7-14 days. The study was approved by the ethics committee of of Swine Research Institute of New Hope Liuhe Co., Ltd (attachment). 2.1. Farms Four different farms were included in the time order. The first ASFV detection was on February 7th, 2019, June 2nd, 2019, July 2nd, 2019, and November 9th, 2019 in Farm 1 to 4, respectively. For each farm, the study period started from the day of the first ASFV infection detection to 7-14 days after the day of the 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 breed-wean farm, which had established a new herd by introducing 1484 gilts in the breeding gestation room. The breeding gestation room is part of a uniformed production line, which was designed to hold 3000 sows and include two breeding gestation rooms each with 1296 stalls and 10 farrowing rooms, and each farrowing room with 60 crates. Infection was first detected in one breeding gestation room. Farm 3 was a commercial breed-wean sow farm with 5167 in production sows kept in two independent uniformed production lines as described above. Infection was first detected in one breeding gestation room. Farm 4 was also a commercial farrow-wean sow farm with 3928 sows kept in two independent uniformed production lines as described above. Infection was first detected in one breeding gestation room. 2.2. Risk-based early detection of infected pigs Two kinds of samples from pigs were collected with a modified method based on previously described methods for ASFV and PRRSV detection and monitoring[7, 8]. i) For early detection of ASFV, syndromic sampling, i.e., samples from clinically abnormal pigs with signs including off-feed, fever, lethargy, hemorrhagic diarrhea, redness of skin, lameness, and abortion were sampled and tested. Oropharyngeal swab (OPS) from each clinically abnormal sow was collected with an innovative oropharyngeal collector (Figure s1) with some modifications from the previously described method[7]. In short, the rod will be inserted into the sow’s mouth as deep as it can to the end of the oropharyngeal area and stirred for ten seconds. The qualified sample should be viscous and mucous-like. Oral fluid (OF) from each pen with clinically abnormal pigs was collected and pooled as one sample. In short, oral fluid was collected by hanging a cotton rope in the handrail of the pen, and oral fluid was squeezed and accumulated by stripping the wet rope into a sterile plastic bag[7, 8]. ii) For confirmation of the first ASFV DNA detection, especially when samples showed Ct values higher than 35, lymph node (LN) samples were collected using an innovative lymph node sample collector (Figure s2)[7, 8]. In brief, the pig was held down and the needle-like collector was pierced into the 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 (LN) sample. 2.4. Whole-herd sampling of pigs and environmental surfaces Individual OPS samples from each sow in the breed-to-wean herds and OF from each pen in the wean-to-finisher site were taken using the methods described above after ASFV infection was confirmed. Whole surface (WS) sampling can be defined as sampling the surface of supplies, personnel, and environment (Figure s4). 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 sharing the water trough in a gestation room, a crate in a farrowing room in a breed-to-wean site, 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. The ground surface, feeder, waterer, slats, and every object in the grid were wiped from top to 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. 2.5. Sample processing and pooling of samples OPS and OF vortexed and then centrifuged at 4500 rpm for 30 seconds. The supernatant was collected and stored at -20℃. The WS samples were squeezed for 30 seconds until the dilution was homogenized. The dilutant was then poured into a 1.5ml microtube and centrifuged at 4500 rpm for 30 seconds. The 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 the supernatant was collected and stored at -20℃ until further use. No more than 5 OPS, OF, or WS samples were pooled as one. Positive pools were opened and tested individually. Each lymph node sample was tested individually. 2.6. qPCR testing DNA extraction DNA extraction was performed using a DNA extraction kit Ex-DNA / RNA in viruses (4.0) (Xi'an Tianlong Science and Technology, co. ltd, Xi'an, China) on extraction machine GeneRotex 96 (Xi'an Tian-long Science and Technology, co. ltd, Xi'an, China) according to the manufacturer’s instructions as described by Zhang [9]. qPCR testing 5 ul of extracted DNA was added to 20ul of qPCR mix of MRD ASFV Real-Time PCR Test kit (MRD Technology Development co,.ltd, Beijing, China) and qPCR was performed in Step one Plus (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions. The procedure for the 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 According to the instructions of the manufacturer, Ct of under 40 was deemed as positive. 2.7. Precision removal i) 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 were purchased locally. Virkon was purchased from Lanxess (Cologne, Germany). ii) 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 the 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 and suckling piglets in the litter were removed by the crate. 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. iii) Precision Removal of the pigs Pigs were removed in a bio-secure manner. A sealed U-shape tunnel was made from waterproof polyester cloth (Figure s4) 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. 2.8. Paired whole herd sampling & testing and switch to risk-based sampling. Several subsequent paired whole herd sampling & testing were carried out at a week interval until all negative results. The same grid as recorded on the electronic map for the first whole-herd sampling was used to make sampling and test consistent. At least one round of negative results from subsequent sampling & testing was required to ensure the elimination of the ASFV. More rounds were needed in the case of heavy contamination. After the herd (including pigs and the environment) remained negative for the last one or two rounds of paired whole herd sampling, sampling was switched to risk-based mode again for daily ASFV surveillance. 2.9. Data collection and analysis Data including qPCR results 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. 3. Results 3.1. TTNH (Time to negative herd) of a finishing herd and three sow herds Time to negative herd (TTNH), which is defined as the time from the day of first ASFV detection (day 0) to the day of last ASFV detection in pigs and environment, in Farm 1 was 19 days (Fig. 1 A). On farm 1 (a finishing herd), Ct values of ASFV-qPCR in lymph nodes from 4 dead pigs were 20.97 (room 1), 20.39(room 3), 32.67(room 6), 29.85(room 3), respectively on day 0 (May 14th, 2019) (Figure S5). Considering the low Ct values, the whole pen was depopulated in a bio-secure manner on day 0. On day 2, samples from three dead pigs and two vehicles transporting pigs were found to be qPCR positive. The whole herd sampling yielded 241 pig samples and 135 environmental samples of which only one environmental sample from the pig transfer corridor was qPCR positive with a Ct value of 32.55. After the precision removal of pigs and thorough cleaning and disinfection of the pig contact area, the herd remained negative for 14 consecutive days until Pen 10 in Room 5 was found qPCR positive in oral fluid samples on day 19. A paired whole herd sampling was carried out again and the herd restored its negative status by removing a total of 1282 pigs and remaining ASFV negative since day 19. TTNH for Farm 2 was 28 days (Fig. 1 B). In Production Line 1 of Farm 2, from one out of twenty-one samples, ASFV was first detected by qPCR (Ct value of 22.7) in a lymph node sample from a dead pig in Stall G17 of Gestation Room 1 on July 11, 2019 (day 0). Later in the same day, whole herd sampling and testing of Line 1 were performed. Pooled samples of Row A in Gestation Room 1 and Row K of Gestation Room 2 were found ASFV qPCR positive with a Ct value of 34.71 and 34.87, respectively. We observed an intermittent mode of qPCR positive results. The whole surface environmental samples in Gestation Room 1 were found to be qPCR positive almost every day until day 28 when disinfectant sodium hypochlorite was applied. Pigs in Gestation Room 2 restored negative status from day 6, even though the whole surface of environmental samples were found ASFV qPCR positive on day 8 day 26, and day 27. Swab samples from 2 working employees were found to be qPCR positive on day 4 and day 23, respectively. The whole herd of Production Line 1 restored negative status from day 28 and remained negative ever since the last detection (data not shown). TTNH for Farm 3 was 14 days (Fig. 1 C). In Farm 3, an OPS sample from an off-feed sow was found ASFV qPCR positive with Ct values of 35.65 (Stall A-87) on day 0 (January 28th, 2020). The Ct value from the lymph node sample of the same sow was 32.39, which confirmed the case of infection. All samples from individual sows, the whole surface of the environment, personnel, and supplies were negative after precision removal and thorough cleaning and disinfection. One interesting finding was that the feeder outlet from A87 was shown qPCR positive with a Ct value of 39.53 on day 14. Farm 3 became negative since day 14 after the feeder was decontaminated by sodium hypochlorite. TTNH for Farm 4 was 1 day (Fig. 1 D). In farm 4, the OPS samples from an off-feed sow were ASFV qPCR positive with Ct values of 26.12 (Stall H26) on day 0 (June 4th, 2020). The Ct value of qPCR results from lymph node samples was 32.39 (data not shown). After precision removal and thorough cleaning and disinfection, the herd restored its negative status on day 1 and remained negative since then. 3.2. Retention rate The retention rate (= the number of retained sows/the number of sows before ASFV detection) of farm 1 to farm 4 was determined to be 69.7%, 65%, 99.4%, and 99.72% respectively. (Fig. 3 ) 3.2. Figures All figures are listed below. 4. Discussion Without vaccines, an innovative and practical way to deal with ASFV was needed due to the unsuccessful and expensive application of conventional standard culling measures. By combining the scientific knowledge of ASFV with field attempts, we successfully developed a systematic “Whole-herd Sampling, qPCR-based Testing, and Precision Removal” method that successfully eradicated the Georgia 2007/1 strain of ASFV in several swine herds. ASFV was found to be relatively slow in transmission after infection occurs. One study showed that the within pen R0 of ASFV was estimated to be 2.8 [ 5 ]. Our field practices and observations were consistent with these findings. As demonstrated in the field cases of Farm 1 to Farm 4, if strict biosecurity measures were taken, ASF could be contained in one area and systematically eliminated until the whole herd regained negative status. For example, in Farm 1, which was most heavily contaminated among four herds, 5 out of 10 barns (2, 4, 7, 8, 9) remained negative during the eradication process. This is also consistent with field observations that solid barriers like concrete walls can effectively block the transmission of ASFV between pens. This also supports the idea that ASF is not likely to be an airborne disease [ 10 ]. Environmental contamination of ASFV was relatively low, as was reported that the introduction of negative pigs into contaminated pens 3, 5, and 7 days after ASFV-infected pigs were removed did not result in subsequent infection[ 4 ]. This seemed contradictory to the results of Farm 2 but was consistent with the results of Farm 1, 3, and 4. In the case of Farm 2, WS environmental samples in Gestation Room 1 were found qPCR positive almost every day until day 28 when a new disinfection method using sodium hypochlorite was implemented. We hypothesize that the positive qPCR results were due to non-degraded ASFV DNA but not infectious ASFV viruses. This is also consistent with the findings of a recent report that sodium hypochlorite and chlorine work well in damaging ASFV DNA [ 1 ]. The fact that sodium hypochlorite was more effective in breaking down DNA of ASFV makes it more favorable to be used in ASFV elimination by avoiding confusing positive DNA with infectious ASFV especially when evaluating the cleaning and disinfection effect. These characteristics of ASFV constitute the scientific foundation for our method and vice versa, our results supported these findings. Our method has been constantly upgraded including the establishment of quick test labs and using sodium hypochlorite as disinfectants, etc., since the first case of ASFV detection. As shown by the TTNH and retention rate results, the latest removal in Farm 4 holds almost 99% of the herd. This can be attributed to several key points in the application and constant upgrading of the method. The key points are listed below. 1) Early detection is essential. Early detection means low levels of contamination and less likelihood of spread of the virus. Compared with the cases of Farm 1 and Farm 2 in 2019, the qPCR results of the first ASFV detection in Farm 3 and Farm 4 in 2020 were of higher CT values, in which farms had a smaller number of pigs infected (only 1 detected pig in Farm 3 and Farm 4 and retention rate was over 99%.). The early detection contributed to a great extent to the successful implementation of the method. Early detection can be attributed to several improvements in management. After 2019, each sow farm was equipped with a qPCR instrument and skilled personnel near the farm, which allows daily monitoring of clinically abnormal pigs, environment, personnel, and incoming supplies. In the cases of Farm 1 and Farm 2 in 2019, samples had to be sent to a qualified lab for analysis, which caused a day delay in results and higher chances of spread. OPS samples were chosen for early detection and lymph node for confirmation. Zhao et al reported that viral DNA appeared 1 ~ 3 days earlier in oral fluid than in blood in pigs infected by the Chinese strain via contact [ 11 ]. Moreover, OPS, OF, and WS samples were collected in a less invasive way and had less chance of contamination to the pigs and environment as compared to blood samples. 2) Precision evaluation of the herd. Upon detecting ASFV in the herd, a precision evaluation of the degree of contamination was carried out promptly. Four tools are useful in the evaluation: electronic maps, whole herd sampling, paired sampling & testing, and qPCR. Electronic maps and whole herd sampling together generate an accurate picture of ASFV distribution status in the herd (figure s5). This helps make informative decisions in precision removal, tracing sources of contamination, and restarting production. For example, in Farm 3, whole herd sampling suggested that only Stall A87 was positive for one day (day 0), but interestingly, the internal surface of the feeder outlet was found qPCR positive with a CT value of 39.53 on day 13. Epidemiological investigation suggested that the feed plant was contaminated on day 20, so the source of infection might be attributed to the contaminated feed. This example highlights the importance of whole herd sampling, which is not easy to perform, in epidemiological tracing and decisions of resuming normal operations. The concept of Paired sampling & testing means once again sampling & testing the same grid of the first round of whole herd sampling, which is marked on the electronic map. At least one or two rounds of paired sampling & testing were required to confirm ASFV negative status. We observed in the field that some farms experienced repeated ASFV infection. Infected pigs varied in time to manifest clinical signs [ 4 ], and some studies showed that virus shedding appeared earlier than clinical signs[ 1 ], so it is important to conduct paired sampling and testing to avoid misdiagnosing assumed infected animals. As was seen in the case of Farm 2, an intermittent mode of ASFV detection was found probably due to not fully implemented paired sampling. A similar method was used in the eradication of PRV. In the PRV eradication program, reestablishing negative breeding herd status after PRV infection requires repeated sampling and testing 30 days after the initial test[ 12 ]. The underlying mechanism was similar, but the time interval between paired sampling & testing in our practice stems precisely from the latent period of ASFV strain Georgia 2007, the prevalent strain in China[ 13 ]. The successful eradication of ASFV from herds proved the credibility of paired sampling & testing. Although qPCR was widely used in academic studies, it is uncommon to see the use of qPCR as a tool for swine disease diagnosis or monitoring in China. In less than two years of the ASFV outbreak in China, our large production systems were equipped with qPCR instruments and reagents. The qPCR test showed a superior advantage over traditional gel-based tests on aspects of sensitivity and promptness. Moreover, since Ct values are inversely proportional to the amount of nucleic acid in the sample, qPCR results are indicative of the level of viral shedding or contamination. 3) Precision removal was based on precision evaluation. After precision evaluation of the level of contamination, individual pigs can be removed in a bio-secure manner that has the least chance of spreading the virus (figure s5). For example, the number of pigs removed was based on production type (Gestation, Farrowing, and Wean to finish or GDU), the number of pigs infected in one grid, and Ct values. In the gestation, if the Ct value was lower than 30, and/ or more than two pigs were infected in the grid, the whole grid was depopulated. If the CT value was >30, the infected pig and two adjacent pigs were removed. This decision was made based on rough scientific estimation but gives staff clear operation instructions on site and makes the method easy to implement, which is essential in the eradication of ASFV from herds. Another example of precision removal based on precision evaluation is the design of pig removal routes. As is seen from Figure s5, the pig removal route was designed based on electronic maps and had the least chance of spreading the virus. 5. Conclusions In conclusion, we firstly developed an innovative, and systematic method, and successfully implemented it to eradicate ASFV in four farms with most herds retained (nearly 90% in recent two cases) for normal production. The successful eradication of ASFV in herds would greatly facilitate the control and eradication of ASFV in China and worldwide. Declarations Author Contributions: Conceptualization, Zhichun Yan, Xiaowen Li; Methodology, Zhichun Yan, Xiaowen Li, Xinglong Wang; Data curation, Xiaowen Li, Peng Li; Writing—original draft preparation, Xiaowen Li, Peng Li, Bingzhou Zhang; Writing, review and editing, Zhichun Yan; Project administration, Xiaowen Li, Weisheng Wu, Peng Li, Junxian Li, Wenchao Gao, Jincheng Yu, Mingyu Fan, Yunzhou Wang, Qiannan Yu, Jintao Li, Xiaoyang Zhang, Qingyuan Liu, Lili Wu; Funding acquisition, Xiaowen Li. All authors have read and agreed to the published version of the manuscript. Funding: The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by Taishan Industry Leadership Talent Project of Shandong Province in China(tscx202306093), and the earmarked fund for CARS (CARS-35). Institutional Review Board Statement: The animal study protocol was approved by the Swine Research Institute of New Hope Liuhe Co., Ltd. All methods were carried out in accordance with Nine Key Techniques for Prevention and Control of African Swine Fever and Resumption of Pig Production by National Pig Industry Technology System. All methods are reported in accordance with ARRIVE guidelines. Data Availability Statement: Not applicable. Acknowledgments: We thank Professor. John Deen from University of Minnesota and Professor Dirk U. Pfeiffer from City University of Hong Kong, Dr. Tang Hao from FAO, and Dr Cui Jixian for suggestions and proofreading. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References Gong, L., et al., African swine fever recovery in China. Vet Med Sci, 2020. 6 (4): p. 890-893. China, M.o.A.a.R.A.o., ASFV contigency plan of Repulic of China . 2017. Gallardo, C., et al., Experimental Infection of Domestic Pigs with African Swine Fever Virus Lithuania 2014 Genotype II Field Isolate. Transbound Emerg Dis, 2017. 64 (1): p. 300-304. Olesen, A.S., et al., Short time window for transmissibility of African swine fever virus from a contaminated environment. Transbound Emerg Dis, 2018. 65 (4): p. 1024-1032. Guinat, C., et al., Inferring within-herd transmission parameters for African swine fever virus using mortality data from outbreaks in the Russian Federation. Transbound Emerg Dis, 2018. 65 (2): p. e264-e271. de Carvalho Ferreira, H.C., et al., Quantification of airborne African swine fever virus after experimental infection. Vet Microbiol, 2013. 165 (3-4): p. 243-51. Guinat, C., et al., Dynamics of African swine fever virus shedding and excretion in domestic pigs infected by intramuscular inoculation and contact transmission. Vet Res, 2014. 45 (1): p. 93. Hernandez-Garcia, J., et al., The use of oral fluids to monitor key pathogens in porcine respiratory disease complex. Porcine Health Manag, 2017. 3 : p. 7. Yang, Q., et al., Westward Spread of Highly Pathogenic Avian Influenza A(H7N9) Virus among Humans, China. Emerg Infect Dis, 2018. 24 (6): p. 1095-1098. Gallardo, C., J. Fernández-Pinero, and M. Arias, African swine fever (ASF) diagnosis, an essential tool in the epidemiological investigation. Virus Res, 2019. 271 : p. 197676. Zhao, D., et al., Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerg Microbes Infect, 2019. 8 (1): p. 438-447. Lowell A. Anderson , N.B., Thomas J. Hagerty , John P. Kluge , Paul L. Sundberg , and United States. Animal and Plant Health Inspection Service, Pseudorabies (Aujeszky’s Disease) and Its Eradication: A Review of the U.S. Experience . 2008: U.S. Department of Agriculture, Animal and Plant Health Inspection Service. Bao, J., et al., Genome comparison of African swine fever virus China/2018/AnhuiXCGQ strain and related European p72 Genotype II strains. Transbound Emerg Dis, 2019. 66 (3): p. 1167-1176. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4515313","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":310315826,"identity":"62f13dd3-6422-43d7-8971-676f1f680f57","order_by":0,"name":"Xiaowen Li","email":"","orcid":"","institution":"College of Veterinary Medicine, Northwest A\u0026F University, Xianyang, Yangling, China","correspondingAuthor":false,"prefix":"","firstName":"Xiaowen","middleName":"","lastName":"Li","suffix":""},{"id":310315827,"identity":"54fd3bb8-511d-4f73-adc5-f4644d78f713","order_by":1,"name":"Peng Li","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Li","suffix":""},{"id":310315830,"identity":"8113cb17-58c0-4228-8918-07d5036b25e3","order_by":2,"name":"Bingzhou Zhang","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Bingzhou","middleName":"","lastName":"Zhang","suffix":""},{"id":310315831,"identity":"b26a315a-d2eb-4c03-a9cb-7c22f0707fda","order_by":3,"name":"Weisheng Wu","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Weisheng","middleName":"","lastName":"Wu","suffix":""},{"id":310315832,"identity":"d1b235fb-6c3e-43b0-88c0-2a21a775ea4e","order_by":4,"name":"Junxian Li","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Junxian","middleName":"","lastName":"Li","suffix":""},{"id":310315833,"identity":"711d1329-2db7-4d9b-9d33-588801a4f549","order_by":5,"name":"Wenchao Gao","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Wenchao","middleName":"","lastName":"Gao","suffix":""},{"id":310315834,"identity":"69e1ea64-817f-41a3-845d-6b113e333391","order_by":6,"name":"Jincheng Yu","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Jincheng","middleName":"","lastName":"Yu","suffix":""},{"id":310315835,"identity":"c11d7710-2794-438b-8af5-b0b816a02eaf","order_by":7,"name":"Mingyu Fan","email":"","orcid":"","institution":"College of Veterinary Medicine, Northwest A\u0026F University, Xianyang, Yangling, China","correspondingAuthor":false,"prefix":"","firstName":"Mingyu","middleName":"","lastName":"Fan","suffix":""},{"id":310315836,"identity":"5743eeb3-bb99-4072-8868-a50bd0f60adc","order_by":8,"name":"Yunzhou Wang","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Yunzhou","middleName":"","lastName":"Wang","suffix":""},{"id":310315837,"identity":"84efaa5d-0ac4-4ab0-a776-5582ecee3aa0","order_by":9,"name":"Qiannan Yu","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Qiannan","middleName":"","lastName":"Yu","suffix":""},{"id":310315841,"identity":"9e9c040e-0624-41cc-8860-d8392713761b","order_by":10,"name":"Jintao Li","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Jintao","middleName":"","lastName":"Li","suffix":""},{"id":310315842,"identity":"634c9cb9-51c2-4004-b01a-dd68f1a65cff","order_by":11,"name":"Xiaoyang Zhang","email":"","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyang","middleName":"","lastName":"Zhang","suffix":""},{"id":310315844,"identity":"c4c539d2-1479-4052-9fce-0edcfc2b8473","order_by":12,"name":"Qingyuan Liu","email":"","orcid":"","institution":"College of Veterinary Medicine, Northwest A\u0026F University, Xianyang, Yangling, China","correspondingAuthor":false,"prefix":"","firstName":"Qingyuan","middleName":"","lastName":"Liu","suffix":""},{"id":310315847,"identity":"002935c3-7258-45c7-aab0-01f621830468","order_by":13,"name":"Lili Wu","email":"","orcid":"","institution":"College of Veterinary Medicine, Northwest A\u0026F University, Xianyang, Yangling, China","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Wu","suffix":""},{"id":310315848,"identity":"8752745b-a8c1-4bae-a85b-59171b2f1c54","order_by":14,"name":"Xinglong Wang","email":"","orcid":"","institution":"College of Veterinary Medicine, Northwest A\u0026F University, Xianyang, Yangling, China","correspondingAuthor":false,"prefix":"","firstName":"Xinglong","middleName":"","lastName":"Wang","suffix":""},{"id":310315850,"identity":"99986151-c8de-460a-901b-aff37fef0229","order_by":15,"name":"Zhichun Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYBAC9gYIncDPzHzwAVFaGGFaJNvZkg1I02JwnsdMgDgt7ccfPi5su5NnfJjBjIGhxiaasJaeHGPjmW3Pis0OM6Q9YDiWlttA2GE5bNK8bYcTtx1mOG7A2HCYCC39z5+BtWxuZmyTIEqL4IwEM7CWDczMbMRpkZZ4Y2zMc+5w4ozDbMwGCcT4hY8//eFjnrLDif395z8++FBjQ1gLGDCyQRkJRCkHgz/EKx0Fo2AUjIIRCAB9YD+RvzKEHQAAAABJRU5ErkJggg==","orcid":"","institution":"Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China","correspondingAuthor":true,"prefix":"","firstName":"Zhichun","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2024-06-02 02:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4515313/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4515313/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58759681,"identity":"f8d8abfa-38c9-4dcf-8169-35f24c979b3e","added_by":"auto","created_at":"2024-06-20 18:45:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":81930,"visible":true,"origin":"","legend":"\u003cp\u003eTTNH (time to negative herds) in Farm 1(A), 2(B), 3(C), 4(D) respectively. The red box meant positive qPCR result and the green box represented negative result. White box meant not detected.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4515313/v1/6b11a0f3a7a5a3268259956f.png"},{"id":58759684,"identity":"dceaa43e-1e5b-42fc-a18e-4ad0f2152066","added_by":"auto","created_at":"2024-06-20 18:45:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24332,"visible":true,"origin":"","legend":"\u003cp\u003eRetention rate (=number of retained sows/ number of sows before detection) in 4 farms.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4515313/v1/e2dd52f3507087efc115ccb7.png"},{"id":59395546,"identity":"bfd79470-2a26-499b-9a38-76feca423255","added_by":"auto","created_at":"2024-07-01 09:03:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":484236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4515313/v1/1debea2f-f522-4c66-922c-ff1e71d0b30d.pdf"},{"id":58759647,"identity":"758acc42-6216-4327-8c55-5ca815ed27fd","added_by":"auto","created_at":"2024-06-20 18:45:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1999861,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4515313/v1/98e8e5806bfe880b1e700db2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and application of Whole-herd-Sampling, qPCR-based- Testing, and Precision-Removal methods to Eliminate ASFV in Four Large Swine Herds in China","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe first outbreak of ASF in China was reported on 3rd August 2018 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Without a commercial vaccine, the only available tool to prevent ASFV is the implementation of strict biosecurity measures at regional and farm levels. The response of the veterinary authorities was in accordance with the OIE guidelines. This means that the Ministry of Agriculture and Rural Affairs (MOARA) of China required depopulation of infected and proximal farms once ASFV was reported [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, it soon became apparent, that these measures were not effective in China for several reasons. These include the high density of pig farms, of which the vast majority were small-scale producers that were connected with each other via highly complex pig and pork trade networks. There may also have been inefficiencies in the response by the industry and government in the early stage of the epidemic. Unlike other pig diseases such as pseudorabies, there is no scientific literature that describes the elimination of ASFV within a herd without stamping out.\u003c/p\u003e\u003cp\u003eField observations from affected farms indicate that the transmission pattern of ASFV in large herds differed from that of other major swine viral diseases. Published information also indicates that after infection, ASFV DNA detection in oral, nasal, and rectal swab samples occurred between 0 and 2 days before the onset of clinical signs[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], suggesting that early detection of viral DNA by qPCR before or around the onset of clinical signs may be possible as part of active within-herd ASF surveillance.\u003c/p\u003e\u003cp\u003eIt was observed that ASFV spreads relatively slowly within a herd following introduction, with an estimated within-pen basic reproduction ratio (R0) of approximately 2.8 and between-pen R0 of approximately 1.4 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, environmental contamination of ASFV can be relatively light, as was shown that the introduction of negative pigs into contaminated pens 3, 5, and 7 days after infected pigs were removed did not cause infection in this study [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, aerosol transmission of ASFV appears unlikely to occur as the half-life of ASFV in air was estimated to be 14\u0026thinsp;~\u0026thinsp;19 minutes[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. We also observed that most infections within barns were associated with common routes of contact via direct contact, feces, or saliva. This published scientific information together with the field observations in China indicated that it might be possible to achieve ASFV eradication within a herd by using a test-and-removal method.\u003c/p\u003e\u003cp\u003eThus, in this study, we delineated the \u0026ldquo;Whole-herd-Sampling, qPCR-based-Testing, and Precision-Removal\u0026rdquo; method in eliminating ASFV Georgia 2007/1 Strain without stamping out the whole population in 1 finishing and 3 sow herds.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eAfter ASFV was detected in the farm, whole herd sampling and qPCR tests were carried out to evaluate the disease status in the herd. Then positive pigs were removed, and the environment was decontaminated by the precision removal process. One or more rounds of this method were applied until the whole herd remained negative for 7-14 days.\u0026nbsp;The study was approved by the ethics committee of of Swine Research Institute of New Hope Liuhe Co., Ltd (attachment).\u003c/p\u003e\n\u003cp\u003e2.1. Farms\u003c/p\u003e\n\u003cp\u003eFour different farms were included in the time order. The first ASFV detection was on February 7th, 2019, June 2nd, 2019, July 2nd, 2019, and November 9th, 2019 in Farm 1 to 4, respectively. For each farm, the study period started from the day of the first ASFV infection detection to 7-14 days after the day of the last positive qPCR result in the herd.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFarm 1 was a wean-finishing site holding 4231 growing pigs with average weight ranging from 7 kg to 130kg.\u003c/p\u003e\n\u003cp\u003eFarm 2, a typical commercial breed-wean farm, which had established a new herd by introducing 1484 gilts in the breeding gestation room. The breeding gestation room is part of a uniformed production line, which was designed to hold 3000 sows and include two breeding gestation rooms each with 1296 stalls and 10 farrowing rooms, and\u0026nbsp;each\u0026nbsp;farrowing room with 60 crates. Infection was first detected in one breeding gestation room.\u003c/p\u003e\n\u003cp\u003eFarm 3 was a commercial breed-wean sow farm with 5167 in production sows kept in two independent uniformed production lines as described above. Infection was first detected in one breeding gestation room.\u003c/p\u003e\n\u003cp\u003eFarm 4 was also a commercial farrow-wean sow farm with 3928 sows kept in two independent uniformed production lines as described above. Infection was first detected in one breeding gestation room.\u003c/p\u003e\n\u003cp\u003e2.2. Risk-based early detection of infected pigs\u003c/p\u003e\n\u003cp\u003eTwo kinds of samples from pigs were collected with a modified method based on previously described methods for ASFV and PRRSV detection and monitoring[7, 8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ei) For early detection of ASFV, syndromic sampling, i.e., samples from clinically abnormal pigs with signs including off-feed, fever, lethargy, hemorrhagic diarrhea, redness of skin, lameness, and abortion were sampled and tested. Oropharyngeal swab (OPS) from each clinically abnormal sow was collected with an innovative oropharyngeal collector (Figure s1) with some modifications from the previously described method[7]. In short, the rod will be inserted into the sow\u0026rsquo;s mouth as deep as it can to the end of the oropharyngeal area and stirred for ten seconds. The qualified sample should be viscous and mucous-like. Oral fluid (OF) from each pen with clinically abnormal pigs was collected and pooled as one sample. In short, oral fluid was collected by hanging a cotton rope in the handrail of the pen, and oral fluid was squeezed and accumulated by stripping the wet rope into a sterile plastic bag[7, 8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eii) For confirmation of the first ASFV DNA detection, especially when samples showed Ct values higher than 35, lymph node (LN) samples were collected using an innovative lymph node sample collector (Figure s2)[7, 8]. In brief, the pig was held down and the needle-like collector was pierced into the 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 (LN) sample.\u003c/p\u003e\n\u003cp\u003e2.4. Whole-herd sampling of pigs and environmental surfaces\u003c/p\u003e\n\u003cp\u003eIndividual OPS samples from each sow in the breed-to-wean herds and OF from each pen in the wean-to-finisher site were taken using the methods described above after ASFV infection was confirmed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhole surface (WS) sampling can be defined as sampling the surface of supplies, personnel, and environment (Figure s4). A 20 cm \u0026times;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 sharing the water trough in a gestation room, a crate in a farrowing room in a breed-to-wean site, 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.\u003c/p\u003e\n\u003cp\u003eThe ground surface, feeder, waterer, slats, and every object in the grid were wiped from top to 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.\u003c/p\u003e\n\u003cp\u003e2.5. Sample processing and pooling of samples\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOPS and OF vortexed and then centrifuged at 4500 rpm for 30 seconds. The supernatant was collected and stored at -20℃. The WS samples were squeezed for 30 seconds until the dilution was homogenized. The dilutant was then poured into a 1.5ml microtube and centrifuged at 4500 rpm for 30 seconds. The supernatant was collected and stored at -20℃\u0026nbsp;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 the supernatant was collected and stored at -20℃\u0026nbsp;until further use.\u003c/p\u003e\n\u003cp\u003eNo more than 5 OPS, OF, or WS samples were pooled as one. Positive pools were opened and tested individually. Each lymph node sample was tested individually.\u003c/p\u003e\n\u003cp\u003e2.6. qPCR testing\u003c/p\u003e\n\u003cp\u003eDNA extraction\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDNA extraction was performed using a DNA extraction kit Ex-DNA / RNA in viruses (4.0) (Xi\u0026apos;an Tianlong Science and Technology, co. ltd, Xi\u0026apos;an, China) on extraction machine GeneRotex 96 (Xi\u0026apos;an Tian-long Science and Technology, co. ltd, Xi\u0026apos;an, China) according to the manufacturer\u0026rsquo;s instructions as described by Zhang\u0026nbsp;[9].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eqPCR testing\u003c/p\u003e\n\u003cp\u003e5 ul of extracted DNA was added to 20ul of qPCR mix of MRD ASFV Real-Time PCR Test kit (MRD Technology Development co,.ltd, \u0026nbsp;Beijing, China) and qPCR was performed in Step one Plus \u0026nbsp;(Thermo Fisher Scientific, Waltham, USA) according to the manufacturer\u0026rsquo;s instructions. The procedure for the qPCR test was as follows:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e50℃\u0026nbsp;for 2 minutes, 1 cycle,\u003c/p\u003e\n\u003cp\u003e95℃\u0026nbsp;for 3minutes, 1 cycle,\u003c/p\u003e\n\u003cp\u003e95℃\u0026nbsp;for 10 seconds, 45 cycles\u003c/p\u003e\n\u003cp\u003e60℃\u0026nbsp;for 20 seconds,\u0026nbsp;45 cycles\u003c/p\u003e\n\u003cp\u003eAccording to the instructions of the manufacturer, Ct of under 40 was deemed as positive.\u003c/p\u003e\n\u003cp\u003e2.7. Precision removal\u003c/p\u003e\n\u003cp\u003ei) Materials and equipment used for precision removal\u003c/p\u003e\n\u003cp\u003eFacial 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 were purchased locally. Virkon was purchased from Lanxess (Cologne, Germany).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eii) Number of removed pigs\u003c/p\u003e\n\u003cp\u003eThe 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 the qPCR test.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn 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\u0026nbsp;＞30, the infected pig and the two adjacent pigs were removed. In the farrowing room, regardless of Ct values, sows and suckling piglets in the litter were removed by the crate. 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.\u003c/p\u003e\n\u003cp\u003eiii) Precision Removal of the pigs\u003c/p\u003e\n\u003cp\u003ePigs were removed in a bio-secure manner. A sealed U-shape tunnel was\u0026nbsp;made from waterproof polyester cloth (Figure s4) 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.8. Paired whole herd sampling \u0026amp; testing and switch to risk-based sampling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral subsequent paired whole herd sampling \u0026amp; testing were carried out at a week interval until all negative results. The same grid as recorded on the electronic map for the first whole-herd sampling was used to make sampling and test consistent. At least one round of negative results from subsequent sampling \u0026amp; testing was required to ensure the elimination of the ASFV. More rounds were needed in the case of heavy contamination.\u003c/p\u003e\n\u003cp\u003eAfter the herd (including pigs and the environment) remained negative for the last one or two rounds of paired whole herd sampling, sampling was switched to risk-based mode again for daily ASFV surveillance.\u003c/p\u003e\n\u003cp\u003e2.9. Data collection and analysis\u003c/p\u003e\n\u003cp\u003eData including qPCR results 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.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. TTNH (Time to negative herd) of a finishing herd and three sow herds\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTime to negative herd (TTNH), which is defined as the time from the day of first ASFV detection (day 0) to the day of last ASFV detection in pigs and environment, in Farm 1 was 19 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). On farm 1 (a finishing herd), Ct values of ASFV-qPCR in lymph nodes from 4 dead pigs were 20.97 (room 1), 20.39(room 3), 32.67(room 6), 29.85(room 3), respectively on day 0 (May 14th, 2019) (Figure S5). Considering the low Ct values, the whole pen was depopulated in a bio-secure manner on day 0. On day 2, samples from three dead pigs and two vehicles transporting pigs were found to be qPCR positive. The whole herd sampling yielded 241 pig samples and 135 environmental samples of which only one environmental sample from the pig transfer corridor was qPCR positive with a Ct value of 32.55. After the precision removal of pigs and thorough cleaning and disinfection of the pig contact area, the herd remained negative for 14 consecutive days until Pen 10 in Room 5 was found qPCR positive in oral fluid samples on day 19. A paired whole herd sampling was carried out again and the herd restored its negative status by removing a total of 1282 pigs and remaining ASFV negative since day 19.\u003c/p\u003e \u003cp\u003eTTNH for Farm 2 was 28 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In Production Line 1 of Farm 2, from one out of twenty-one samples, ASFV was first detected by qPCR (Ct value of 22.7) in a lymph node sample from a dead pig in Stall G17 of Gestation Room 1 on July 11, 2019 (day 0). Later in the same day, whole herd sampling and testing of Line 1 were performed. Pooled samples of Row A in Gestation Room 1 and Row K of Gestation Room 2 were found ASFV qPCR positive with a Ct value of 34.71 and 34.87, respectively. We observed an intermittent mode of qPCR positive results. The whole surface environmental samples in Gestation Room 1 were found to be qPCR positive almost every day until day 28 when disinfectant sodium hypochlorite was applied. Pigs in Gestation Room 2 restored negative status from day 6, even though the whole surface of environmental samples were found ASFV qPCR positive on day 8 day 26, and day 27. Swab samples from 2 working employees were found to be qPCR positive on day 4 and day 23, respectively. The whole herd of Production Line 1 restored negative status from day 28 and remained negative ever since the last detection (data not shown).\u003c/p\u003e \u003cp\u003eTTNH for Farm 3 was 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In Farm 3, an OPS sample from an off-feed sow was found ASFV qPCR positive with Ct values of 35.65 (Stall A-87) on day 0 (January 28th, 2020). The Ct value from the lymph node sample of the same sow was 32.39, which confirmed the case of infection. All samples from individual sows, the whole surface of the environment, personnel, and supplies were negative after precision removal and thorough cleaning and disinfection. One interesting finding was that the feeder outlet from A87 was shown qPCR positive with a Ct value of 39.53 on day 14. Farm 3 became negative since day 14 after the feeder was decontaminated by sodium hypochlorite.\u003c/p\u003e \u003cp\u003eTTNH for Farm 4 was 1 day (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In farm 4, the OPS samples from an off-feed sow were ASFV qPCR positive with Ct values of 26.12 (Stall H26) on day 0 (June 4th, 2020). The Ct value of qPCR results from lymph node samples was 32.39 (data not shown). After precision removal and thorough cleaning and disinfection, the herd restored its negative status on day 1 and remained negative since then.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Retention rate\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe retention rate (=\u0026thinsp;the number of retained sows/the number of sows before ASFV detection) of farm 1 to farm 4 was determined to be 69.7%, 65%, 99.4%, and 99.72% respectively. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Figures\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll figures are listed below.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWithout vaccines, an innovative and practical way to deal with ASFV was needed due to the unsuccessful and expensive application of conventional standard culling measures. By combining the scientific knowledge of ASFV with field attempts, we successfully developed a systematic \u0026ldquo;Whole-herd Sampling, qPCR-based Testing, and Precision Removal\u0026rdquo; method that successfully eradicated the Georgia 2007/1 strain of ASFV in several swine herds.\u003c/p\u003e \u003cp\u003eASFV was found to be relatively slow in transmission after infection occurs. One study showed that the within pen R0 of ASFV was estimated to be 2.8 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Our field practices and observations were consistent with these findings. As demonstrated in the field cases of Farm 1 to Farm 4, if strict biosecurity measures were taken, ASF could be contained in one area and systematically eliminated until the whole herd regained negative status. For example, in Farm 1, which was most heavily contaminated among four herds, 5 out of 10 barns (2, 4, 7, 8, 9) remained negative during the eradication process. This is also consistent with field observations that solid barriers like concrete walls can effectively block the transmission of ASFV between pens. This also supports the idea that ASF is not likely to be an airborne disease [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Environmental contamination of ASFV was relatively low, as was reported that the introduction of negative pigs into contaminated pens 3, 5, and 7 days after ASFV-infected pigs were removed did not result in subsequent infection[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This seemed contradictory to the results of Farm 2 but was consistent with the results of Farm 1, 3, and 4. In the case of Farm 2, WS environmental samples in Gestation Room 1 were found qPCR positive almost every day until day 28 when a new disinfection method using sodium hypochlorite was implemented. We hypothesize that the positive qPCR results were due to non-degraded ASFV DNA but not infectious ASFV viruses. This is also consistent with the findings of a recent report that sodium hypochlorite and chlorine work well in damaging ASFV DNA [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The fact that sodium hypochlorite was more effective in breaking down DNA of ASFV makes it more favorable to be used in ASFV elimination by avoiding confusing positive DNA with infectious ASFV especially when evaluating the cleaning and disinfection effect.\u003c/p\u003e \u003cp\u003eThese characteristics of ASFV constitute the scientific foundation for our method and vice versa, our results supported these findings. Our method has been constantly upgraded including the establishment of quick test labs and using sodium hypochlorite as disinfectants, etc., since the first case of ASFV detection. As shown by the TTNH and retention rate results, the latest removal in Farm 4 holds almost 99% of the herd. This can be attributed to several key points in the application and constant upgrading of the method. The key points are listed below.\u003c/p\u003e \u003cp\u003e1) Early detection is essential. Early detection means low levels of contamination and less likelihood of spread of the virus. Compared with the cases of Farm 1 and Farm 2 in 2019, the qPCR results of the first ASFV detection in Farm 3 and Farm 4 in 2020 were of higher CT values, in which farms had a smaller number of pigs infected (only 1 detected pig in Farm 3 and Farm 4 and retention rate was over 99%.). The early detection contributed to a great extent to the successful implementation of the method.\u003c/p\u003e \u003cp\u003eEarly detection can be attributed to several improvements in management. After 2019, each sow farm was equipped with a qPCR instrument and skilled personnel near the farm, which allows daily monitoring of clinically abnormal pigs, environment, personnel, and incoming supplies. In the cases of Farm 1 and Farm 2 in 2019, samples had to be sent to a qualified lab for analysis, which caused a day delay in results and higher chances of spread. OPS samples were chosen for early detection and lymph node for confirmation. Zhao et al reported that viral DNA appeared 1\u0026thinsp;~\u0026thinsp;3 days earlier in oral fluid than in blood in pigs infected by the Chinese strain via contact [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, OPS, OF, and WS samples were collected in a less invasive way and had less chance of contamination to the pigs and environment as compared to blood samples.\u003c/p\u003e\u003cp\u003e2) Precision evaluation of the herd. Upon detecting ASFV in the herd, a precision evaluation of the degree of contamination was carried out promptly. Four tools are useful in the evaluation: electronic maps, whole herd sampling, paired sampling \u0026amp; testing, and qPCR.\u003c/p\u003e\u003cp\u003eElectronic maps and whole herd sampling together generate an accurate picture of ASFV distribution status in the herd (figure s5). This helps make informative decisions in precision removal, tracing sources of contamination, and restarting production. For example, in Farm 3, whole herd sampling suggested that only Stall A87 was positive for one day (day 0), but interestingly, the internal surface of the feeder outlet was found qPCR positive with a CT value of 39.53 on day 13. Epidemiological investigation suggested that the feed plant was contaminated on day 20, so the source of infection might be attributed to the contaminated feed. This example highlights the importance of whole herd sampling, which is not easy to perform, in epidemiological tracing and decisions of resuming normal operations.\u003c/p\u003e \u003cp\u003eThe concept of Paired sampling \u0026amp; testing means once again sampling \u0026amp; testing the same grid of the first round of whole herd sampling, which is marked on the electronic map. At least one or two rounds of paired sampling \u0026amp; testing were required to confirm ASFV negative status. We observed in the field that some farms experienced repeated ASFV infection. Infected pigs varied in time to manifest clinical signs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and some studies showed that virus shedding appeared earlier than clinical signs[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], so it is important to conduct paired sampling and testing to avoid misdiagnosing assumed infected animals. As was seen in the case of Farm 2, an intermittent mode of ASFV detection was found probably due to not fully implemented paired sampling.\u003c/p\u003e \u003cp\u003eA similar method was used in the eradication of PRV. In the PRV eradication program, reestablishing negative breeding herd status after PRV infection requires repeated sampling and testing 30 days after the initial test[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The underlying mechanism was similar, but the time interval between paired sampling \u0026amp; testing in our practice stems precisely from the latent period of ASFV strain Georgia 2007, the prevalent strain in China[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The successful eradication of ASFV from herds proved the credibility of paired sampling \u0026amp; testing.\u003c/p\u003e \u003cp\u003eAlthough qPCR was widely used in academic studies, it is uncommon to see the use of qPCR as a tool for swine disease diagnosis or monitoring in China. In less than two years of the ASFV outbreak in China, our large production systems were equipped with qPCR instruments and reagents. The qPCR test showed a superior advantage over traditional gel-based tests on aspects of sensitivity and promptness. Moreover, since Ct values are inversely proportional to the amount of nucleic acid in the sample, qPCR results are indicative of the level of viral shedding or contamination.\u003c/p\u003e \u003cp\u003e3) Precision removal was based on precision evaluation. After precision evaluation of the level of contamination, individual pigs can be removed in a bio-secure manner that has the least chance of spreading the virus (figure s5). For example, the number of pigs removed was based on production type (Gestation, Farrowing, and Wean to finish or GDU), the number of pigs infected in one grid, and Ct values. In the gestation, if the Ct value was lower than 30, and/ or more than two pigs were infected in the grid, the whole grid was depopulated. If the CT value was \u0026gt;30, the infected pig and two adjacent pigs were removed. This decision was made based on rough scientific estimation but gives staff clear operation instructions on site and makes the method easy to implement, which is essential in the eradication of ASFV from herds. Another example of precision removal based on precision evaluation is the design of pig removal routes. As is seen from Figure s5, the pig removal route was designed based on electronic maps and had the least chance of spreading the virus.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn conclusion, we firstly developed an innovative, and systematic method, and successfully implemented it to eradicate ASFV in four farms with most herds retained (nearly 90% in recent two cases) for normal production. The successful eradication of ASFV in herds would greatly facilitate the control and eradication of ASFV in China and worldwide.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, Zhichun Yan, Xiaowen Li; Methodology, Zhichun Yan, Xiaowen Li, Xinglong Wang; Data curation, Xiaowen Li,\u0026nbsp;Peng Li; Writing\u0026mdash;original draft preparation, Xiaowen Li,\u0026nbsp;Peng Li, Bingzhou Zhang; Writing, review and editing, Zhichun\u0026nbsp;Yan; Project administration, Xiaowen Li,\u0026nbsp;Weisheng Wu, Peng Li, Junxian Li, Wenchao Gao, Jincheng Yu, Mingyu Fan,\u0026nbsp;Yunzhou Wang, Qiannan Yu, Jintao Li, Xiaoyang Zhang,\u0026nbsp;Qingyuan Liu, Lili Wu; Funding acquisition, Xiaowen Li. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by Taishan Industry Leadership Talent Project of Shandong Province in China(tscx202306093), and the earmarked fund for CARS (CARS-35).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe animal study protocol was approved by the Swine Research Institute of New Hope Liuhe Co., Ltd. All methods were carried out in accordance with Nine Key Techniques for Prevention and Control of African Swine Fever and Resumption of Pig Production by National Pig Industry Technology System. All methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Professor. John Deen from University of Minnesota and Professor Dirk U. Pfeiffer from City University of Hong Kong, Dr. Tang Hao from FAO, and Dr Cui Jixian for suggestions and proofreading.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGong, L., et al., \u003cem\u003eAfrican swine fever recovery in China.\u003c/em\u003e Vet Med Sci, 2020. \u003cstrong\u003e6\u003c/strong\u003e(4): p. 890-893.\u003c/li\u003e\n\u003cli\u003eChina, M.o.A.a.R.A.o., \u003cem\u003eASFV contigency plan of Repulic of China\u003c/em\u003e. 2017.\u003c/li\u003e\n\u003cli\u003eGallardo, C., et al., \u003cem\u003eExperimental Infection of Domestic Pigs with African Swine Fever Virus Lithuania 2014 Genotype II Field Isolate.\u003c/em\u003e Transbound Emerg Dis, 2017. \u003cstrong\u003e64\u003c/strong\u003e(1): p. 300-304.\u003c/li\u003e\n\u003cli\u003eOlesen, A.S., et al., \u003cem\u003eShort time window for transmissibility of African swine fever virus from a contaminated environment.\u003c/em\u003e Transbound Emerg Dis, 2018. \u003cstrong\u003e65\u003c/strong\u003e(4): p. 1024-1032.\u003c/li\u003e\n\u003cli\u003eGuinat, C., et al., \u003cem\u003eInferring within-herd transmission parameters for African swine fever virus using mortality data from outbreaks in the Russian Federation.\u003c/em\u003e Transbound Emerg Dis, 2018. \u003cstrong\u003e65\u003c/strong\u003e(2): p. e264-e271.\u003c/li\u003e\n\u003cli\u003ede Carvalho Ferreira, H.C., et al., \u003cem\u003eQuantification of airborne African swine fever virus after experimental infection.\u003c/em\u003e Vet Microbiol, 2013. \u003cstrong\u003e165\u003c/strong\u003e(3-4): p. 243-51.\u003c/li\u003e\n\u003cli\u003eGuinat, C., et al., \u003cem\u003eDynamics of African swine fever virus shedding and excretion in domestic pigs infected by intramuscular inoculation and contact transmission.\u003c/em\u003e Vet Res, 2014. \u003cstrong\u003e45\u003c/strong\u003e(1): p. 93.\u003c/li\u003e\n\u003cli\u003eHernandez-Garcia, J., et al., \u003cem\u003eThe use of oral fluids to monitor key pathogens in porcine respiratory disease complex.\u003c/em\u003e Porcine Health Manag, 2017. \u003cstrong\u003e3\u003c/strong\u003e: p. 7.\u003c/li\u003e\n\u003cli\u003eYang, Q., et al., \u003cem\u003eWestward Spread of Highly Pathogenic Avian Influenza A(H7N9) Virus among Humans, China.\u003c/em\u003e Emerg Infect Dis, 2018. \u003cstrong\u003e24\u003c/strong\u003e(6): p. 1095-1098.\u003c/li\u003e\n\u003cli\u003eGallardo, C., J. Fern\u0026aacute;ndez-Pinero, and M. Arias, \u003cem\u003eAfrican swine fever (ASF) diagnosis, an essential tool in the epidemiological investigation.\u003c/em\u003e Virus Res, 2019. \u003cstrong\u003e271\u003c/strong\u003e: p. 197676.\u003c/li\u003e\n\u003cli\u003eZhao, D., et al., \u003cem\u003eReplication and virulence in pigs of the first African swine fever virus isolated in China.\u003c/em\u003e Emerg Microbes Infect, 2019. \u003cstrong\u003e8\u003c/strong\u003e(1): p. 438-447.\u003c/li\u003e\n\u003cli\u003eLowell A. Anderson , N.B., Thomas J. Hagerty , John P. Kluge , Paul L. Sundberg , and United States. Animal and Plant Health Inspection Service, \u003cem\u003ePseudorabies (Aujeszky\u0026rsquo;s Disease) and Its Eradication: A Review of the U.S. Experience\u003c/em\u003e. 2008: U.S. Department of Agriculture, Animal and Plant Health Inspection Service.\u003c/li\u003e\n\u003cli\u003eBao, J., et al., \u003cem\u003eGenome comparison of African swine fever virus China/2018/AnhuiXCGQ strain and related European p72 Genotype II strains.\u003c/em\u003e Transbound Emerg Dis, 2019. \u003cstrong\u003e66\u003c/strong\u003e(3): p. 1167-1176.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"African swine fever virus, quantitative PCR, test removal","lastPublishedDoi":"10.21203/rs.3.rs-4515313/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4515313/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSince the first ASFV case was reported in China in 2018, the conventional depopulation method to control ASF has proved unwieldy because of its high production intensity and complex trade network. To provide an alternative to conventional stamping out methods, we developed a\u0026rdquo; Whole-herd-Sampling, qPCR-based-Testing, and Precision-Removal\u0026rdquo; method by sampling every whole herd sampling and qPCR tests to determine the status of ASFV in herds and using a precision removal of identified sows. By developing and applying these methods, we successfully controlled ASF and eliminated the virus from 4 large swine herds from 2019 to 2020. The time to negative herd (TTNH) was 19, 28, 14, and 1 day from farm 1 to 4, respectively. Retention rates of pigs from farm 1 to farm 4 were 69.7%, 65%, 99.4%, and 99.72%, respectively. We anticipated that this innovative method would be a good alternative to the conventional stamping out method and greatly facilitate the control and eradication of ASFV in China and worldwide.\u003c/p\u003e","manuscriptTitle":"Development and application of Whole-herd-Sampling, qPCR-based- Testing, and Precision-Removal methods to Eliminate ASFV in Four Large Swine Herds in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-20 18:45:21","doi":"10.21203/rs.3.rs-4515313/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"922bf482-a95e-4b6f-bf46-a51719a64d61","owner":[],"postedDate":"June 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-17T11:08:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-20 18:45:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4515313","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4515313","identity":"rs-4515313","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}