译者的话：<<

71°C处理10分钟和20°C处理7天同样可以灭活猪流行性腹泻病毒（PEDV），这些数据表明可利用温度和时间的方法来灭活病原微生物。这项温度/时间对物资消毒的方法的研究表明，在37°C（湿度< 20%） 条件下，沙门氏菌和猪繁殖与呼吸综合征病毒会在2天内失活，而在较低温度下可能需要更长的时间，长达7天或更久。 

过去的研究表明雾化和紫外消毒存在物资消毒不彻底的可能性，按温度/时间方法对物资的消毒不受物质的放置方式或组成限制，物资所有表面都基于时间和温度被同等处理。

正如这项研究所描述的，这种消毒方法减少了化学消毒剂的使用及其对人体健康安全的顾虑，易用且有效，因此在设计猪场生物安全时，该方法可以作为物资消毒的一种方案。

引言 Introduction<<

作者 Author:

J. Kordas¹; B. Blair¹; J. Lowe^1,2

¹伊利诺伊大学兽医学院

²伊利诺伊州劳氏咨询有限公司

¹College of Veterinary Medicine, University of Illinois, Urbana, Illinois

²Lowe Consulting Ltd., Mahomet, Illinois

引言 Introduction

设备和物资的入场会对养猪场构成疾病引入的风险。虽然已经确立了处理这类物品的准入的准则，但有关使用这些技术彻底消毒的效果仍然存在疑问。传统上，一般使用雾化消毒室和紫外线消毒室将个人物品和供应物品消毒后送入猪场。然而，最近的研究开始质疑这两种技术对农场常见病原体的影响，特别是在病毒隐藏在有机物或其他物资内的复杂情况下。

Entry of equipment and supplies pose threats of disease introduction to swine farms. Although practices have been established to handle the admittance of such items, questions regarding the efficacy of complete decontamination by use of these techniques still remain. The use of fogging rooms and UV chambers have traditionally been used to pass both personal and supply items into farms. However, recent research has begun to question the effect of both technologies on pathogens commonly found on farm, especially in complex situations where virus may be shielded by organic material or other supplies.

传统的雾化室对农场消毒的效果在最近的研究中开始受到质疑。虽然化学消毒剂对猪病原体有效是毫无疑问的，但在最近的一项研究中，由于消毒剂的重力积累以及被消毒物资表面的复杂性，使用标准工业雾化器的消毒仅显示出部分有效。¹ 这意味着雾化器不能将化学物质完全雾化，而是会产生冷凝物，使其保留液体的物理特性，抑制其完全渗透到物体内部的能力，从而存在消毒剂无法触及的区域。

The efficacy of traditional fogging chambers for decontamination on farms has begun to come into question in recent research. While it is not in question that chemical disinfectants are effective against swine pathogens, disinfection using standard industry foggers demonstrated only partial efficacy in one recent study due to gravitational accumulation of the disinfectant as well as the complexity of the surface to which it was applied.¹ The implication being that foggers do not fully aerosolize the chemicals but generate condensation that retains the physical properties of a liquid, inhibiting its ability to fully permeate the objects within the chamber and leaves areas untouched by the disinfectants.

此外，入场个人物品消毒的紫外线消毒方法在猪场的实践应用，引起了褒贬不一的评价。另一项研究表明，紫外消毒室的消毒可能是不彻底的，并不能完全灭活入场物资表面的病原体。一篇2019年发表的文章得出结论：操作条件对紫外消毒的效果有显著影响，完全使用紫外线对接种过沙门氏菌的物质进行消毒，由于背阴面的存在和不同的物品放置方式，在长达40分钟的时间内也没有将其灭活，进一步证明了这一观点。

Additionally, UV disinfection of personal item decontamination for entry onto swine farms has raised mixed reviews as to its practical application on farms. Another study suggests that UVC chamber decontamination may not be homogenous and do not fully inactivate surface pathogens on items entering facilities.² Furthering this idea, a 2019 publication concluded that operant conditions had significant impact on the efficacy of such chambers, due to shadowing potential and differing item placement, full decontamination using UVC on salmonella inoculated items was never achieved at times up to 40 minutes.³

此后，人们研究或提出了其他消毒方法，如熏蒸或时间/温度处理。已经研究了通过时间/温度处理对饲料成分和受污染的牲畜拖车中的病原体进行消毒。71°C处理10分钟和20°C处理7天同样可以灭活猪流行性腹泻病毒（PEDV）污染的粪便样品中的病毒。⁴此外，还发现某些饲料成分中病毒的半衰期既依赖于病毒又依赖于特定成分，其中豆粕和赖氨酸盐酸盐携带活病毒的时间最长可达26.6天。⁵虽然这些数据表明可利用温度和时间来灭活病原，但还没有关于将其用于农场物资消毒的研究。

Other methods of decontamination have since been investigated or proposed, such as fumigation or time/temperature treatment. Disinfection of pathogens by time/temperature treatment has been researched within feed ingredients and on contaminated livestock trailers. Temperatures of 71° C for 10 minutes as well as 20°C for 7 days were adequate in deactivating porcine epidemic diarrhea virus (PEDV) in contaminated fecal samples.⁴ Additionally, it was found that the half-lives of viruses within certain feed ingredients were dependent on both the virus and specific ingredient, with soybean meal, and lysine hydrochloride harboring viable virus the longest at up to 26.6 days.⁵ While this data is indicative of the possible use of temperature and time to neutralize infectious agents, no research exists on its use for the decontamination of supplies entering a farm.

材料和方法

Materials and methods

本研究的目的是探讨时间/温度处理对物体表面上沙门氏菌和猪呼吸和繁殖综合症病毒(PRRSV)的消毒作用。沙门氏菌和PRRSV被选为指示病原，是因为沙门氏菌对环境降解的抗性和PRRSV对养猪业的重要性。以脑心提取液(BHIB)作为各病原体的营养培养基，加入1头份大肠杆菌疫苗(Enterisol® Salmonella T/C)进行接种。在BHIB中（而不是使用无菌稀释剂）复苏PRRSV弱活苗(Ingelvac PRRSV MLV)。各取1mL接种的培养液，相当于1头份疫苗剂量，置于光滑的塑料表面。然后，所有表面都置于低湿度（< 20%）的3个不同温度的房间：4°C、18°C和37°C。每个处理组设置6个重复。受感染的表面每天拭子采样，PRRSV持续采样5天，沙门氏菌持续采样7天，共采集216个样本，以监测病原体降解。每天以可重复的、系统的方式使用棉签采样器采样，以确保对整个表面取样。  

The intent of this study was to investigate the use of time/temperature treatment on the decontamination of Salmonella and porcine respiratory and reproductive syndrome virus (PRRSV) on a surface. Salmonella and PRRSV were selected as indicator organisms for salmonella’s resistance to environmental degradation and PRRSV’s importance to the swine industry. Inoculation of brain heart infusion broth (BHIB), which served as a nutrient medium for each pathogen, was performed by adding one dose of Enterisol® Salmonella T/C. Ingelvac PRRSV MLV vaccine was reconstituted in the BHIB, rather than the provided sterile diluent. 1mL, the equivalent of one vaccine dose, of each inoculated solution was then placed on a smooth plastic surface. Once contaminated, all surfaces were placed in low humidity (< 20%) rooms at the three different temperatures: 4° C, 18° C, and 37°C. Six replicates of each treatment group were performed. Infected surfaces were swabbed each day for 5 days (PRRSV) or 7 days (salmonella) to monitor for pathogen degradation collecting a total of 216 samples. Surfaces were swabbed using a cotton tipped applicator in a repeatable, systematic fashion to ensure sampling of the entire surface each day.

采集后，将沙门氏菌样本拭子直接接种到沙门氏菌选择性XLD琼脂培养基，孵育24小时后进行菌落计数。直接接种的沙门氏菌疫苗作为阳性对照，而每一个相应的时间和温度处理的清洁的塑料表面作为该研究的阴性对照。将收集的PRRSV样品加入1ml无菌水中，储存在-20°C，直到进行进一步处理和细胞培养。

Once collected, salmonella samples were directly plated from the swabs onto salmonella selective XLD agar and incubated for 24 hours after which time colony counts were performed. Directly plated salmonella vaccine served as a positive control, while a clean plastic surface, subject to each time and temperature treatment served as negative controls for this portion of the study. PRRSV samples were collected, placed in 1ml of sterile water and stored at -20° C until further processing and cell culture could take place.

收集所有PRRSV样本后，各取500uL的样本在24孔板中感染MARC-145细胞，培养至近100%克隆率。罗斯威尔公园纪念研究所的完全培养基+ 5%胎牛血清被添加到每个孔中作为营养介质，以确保不会因缺乏营养而发生细胞损伤，样品在37°C和5%的二氧化碳的环境条件下孵育，在感染后第3天和第6天进行每个孔的细胞病变效应评估。在BHIB中复苏且不经过时间/温度处理的疫苗作为该研究的阳性对照，而相同时间/温度处理的清洁表面样本则作为阴性对照。阳性对照均有细胞病变，而阴性对照均无细胞结构改变。

Once all PRRSV samples were collected, 500uL of sample was used to infect MARC-145 cells cultured to near 100% confluency in 24-well plates. Complete Roswell Park Memorial Institute medium + 5% Fetal Bovine Serum was added as nutrient media to each well to ensure no cell damage would occur due to lack of nutrients, Samples were incubated at 37° C with 5% carbon dioxide and cytopathic effect of each well was assessed at 3 and 6 days post infection. Vaccine reconstituted in BHIB and not subject to time or temperature treatments served as the positive control for this portion of the study while clean surfaces, subject to the same time/temperature treatments as the samples served as the negative controls. All positive controls yielded cytopathic effect while all negative controls yielded no change in cellular structure. 

结果与讨论

Results and discussion<<

在37℃条件下，经过3天或更长的时间/温度处理，实现了感染塑料表面的沙门氏菌的完全消毒。一天的培养期后，菌落数量减少到最初的三分之一，然后在第3天下降到0。虽然在其他温度下对沙门氏菌的生长产生了影响，但在7天的研究期间并没有完成对所有样本的完全消毒。4℃时菌落计数保持一致，直到第7天菌落减少50%。在18°C下保存的所有样品的7天菌落计数都不低于最初接种的83%（表1），PRRSV与沙门氏菌相比是一种相当脆弱的病原体，在18°C条件下的第5天和37°C 条件下的第2天就被灭活（表1）。虽然4°C的条件下在第4天观察到PRRSV的部分失活，但在整个5天研究中没有达到完全的消毒。

Complete decontamination of salmonella on of surface was achieved by use of time/temperature treatment when subjected to 37° C for three or more days. Colony count was reduced to one third of the original inoculate after a single day incubation period then fell to 0 on day 3. While impacts were made to salmonella growth at other temperatures complete decontamination of all samples was not achieved during the 7 day study period. At 4° C colony count remained consistent until day 7 where a 50% reduction in colony growth was seen. All 7 day colony counts for the samples held at 18° C did not fall below 83% of the original inoculate (Table 1). Decontamination of PRRSV, a quite fragile pathogen as compared to salmonella, was attained by day 5 at 18°C and within 2 days at 37° C (Table 1). While some deactivation of PRRSV was observed at 4° C by day 4, complete decontamination was not accomplished throughout the five day study.

这项研究的结果表明，使用时间和温度或许可作为对进入农场物资的可行消毒方法。虽然并非所有物资，如疫苗，都应该暴露在最有效的高温下，但温度越低，暴露时间越长，其消毒效果可能会更好。此外，还有探索湿度对消毒过程的影响的项目。可以推断，较高的湿度可能会增加病原体降解所需的时间，因为孵化室中许多细菌物种生长的黄金标准是37°C时的高湿度。 收集这些额外的数据，以及扩大温度/时间处理的范围，可以制作出剂量反应曲线，以帮助对特定环境实施最有效的温度/时间处理。虽然进一步探讨温度/时间对不同表面和病原体（如疱疹病毒和流感病毒）的影响的研究仍有待完成，但这项研究表明，当暴露于 37°C 超出两天时，病原体将失活，而在较低温度下消毒可能需要更长的时间，长达7天或超出7天。  

The results of this study show that the use of time and temperature may be a viable option to decontaminate items upon entry onto farms. While not all items, such as vaccines, should be exposed to the most effective high temperatures, placement of an items at cooler temperatures for a longer period may prove efficacious. Additionally, there are plans for exploring the effect of humidity on the decontamination process. It is inferred that higher humidity levels may increase the time necessary for pathogen degradation, as the gold standard for growth of many bacterial species in an incubation chamber is high humidity at 37° C. Compiling these additional data points as well as expanding the range of temperature/time treatments may allow for the development of a dose response curve to aid in implementing the most effective temperature/time treatment for a specific environment. While further research exploring the effect of temperature/time on different surfaces and pathogens such as herpesvirus and influenza virus must still be completed, this research would suggest that when exposed to 37° C for greater than two days pathogens will become deactivated, while longer times, up to or exceeding seven days, may be necessary for decontamination at lower temperatures.

此外，像其他常用技术（如雾化）一样，按温度/时间对物体的处理不会被物体的方位或组成抑制。环境中的所有表面都基于时间和温度被同等处理。因为化学试剂的使用有限，这项的消毒方法减少了对人体健康安全的顾虑。正如这项研究所描述的，由于没有安全顾虑、易用且有效，因此在设计生物安全以消毒入场物资时，使用温度/时间处理的方法必须被考虑。

Additionally, treatment of objects by temperature/time is not inhibited by the orientation or composition of objects like other commonly used techniques, such as fogging. All surfaces within the environment are subject to equal treatment based on time and temperature. With limited chemical use, there are reduced human health safety concerns associated with this proposed method for decontamination. For its lack of safety concerns, ease of use, and efficacy as described by this research, the use of temperature/time exposure must be a consideration when designing biosecurity for the decontamination of items upon entry into a farm.

参考文献 References<<

1. Kettelkamp, E., Kordas, J., Blair, B., & Lowe, J. Impact of item orientation on the efficacy of supply decontamination with aerosolized chemical disinfectants. Proc AASV. 2019:11-114.

2. Rieland, K., Yang, M., & Torremorell, M. (2019). Evaluation of Efficacy of Ultraviolet Germicidal Chambers in Swine Farms. Swine Disease Eradication Center. Retrieved from https://vetmed.umn.edu/sites/vetmed.umn.edu/files/shmp_2018l19.48_evaluation_of_ultraviolet_germicidal_chambers-sciencepage.pdf.

3. Kordas, J., Kettelkamp, E., Blair, B., Lowe, J. Implications of operating conditions in UV light decontamination chambers for items entering swine facilities. Proc AASV 2019: 275-276.

4. Thomas, Paul R.; Karriker, Locke A.; Ramirez, Alejandro; Zhang, Jianqiang; Ellingson, Joshua S.; Crawford, Kimberly K.; Bates, Jessica L.; Hammen, Kristin J.; and Holtkamp, Derald J., “Evaluation of time and temperature sufficient to inactivate porcine epidemic diarrhea virus in swine feces on metal surfaces” (2015). Veterinary Diagnostic and Production Animal Medicine Publications. 120. https://lib.dr.iastate.edu/vdpam_pubs/120.

5. Dee, S. A., Bauermann, F. V., Niederwerder, M. C., Singrey, A., Clement, T., Lima, M. D., … Diel, D. G. (2019). Correction: Survival of viral pathogens in animal feed ingredients under transboundary shipping models. Plos One, 14(3). doi: 10.1371/journal.pone.0214529.