采样方法和采样点
Swabbing method and location
本研究对3个独立的饲料生产设施(饲料厂1-3)进行评估和采样,并在每次拜访期间进行生物安全评估和审计(在ksuswine.org上公布)。每个饲料厂都有各自的生物安全挑战,要么是正常的操作程序,要么是必须在有限的设施内完成的任务。
Three separate feed manufacturing facilities (Sites 1-3) were evaluated and sampled for this study, with a biosecurity evaluation and audit (posted at ksuswine.org) performed during each visit. Each mill offered its own biosecurity challenges, either with the normal operating procedures or required tasks to be performed within facility limitations.
在4天的时间里,总共采集了573个样本,其中381个样本来自饲料原料或成品饲料,其余的192个样本是来自4个设施的环境样本。
A total of 573 samples were taken over the course of four days, with 381 of those samples consisting of feed ingredient or finished feed, and the remaining 192 samples environmental swabs, collected across the 4 sites.
饲料原料和成品饲料样品使用一次性塑料容器收集。每个原料/饲料最初收集10个独立样本。散装存储的产品是在传送过程中多次随机采样,袋装产品是从现场的10个不同的袋子中各取样品。每个样本都被独立分装和保存,以供各自分析,并从分析的10个样本中做一个混合样本进行分析。
Feed ingredient and finished feed samples were collected using single-use plastic tubs. For each separate item, 10 individual samples were collected initially. For bulk-storage products, samples were either drop-collected or grabbed at multiple times while being conveyed. For bagged products, samples were obtained from each of 10 different bags onsite. Each sample was kept separate for individual analysis, with an additional blended composite sample created from the 10 samples analyzed.
环境样品的采集使用了两种不同的采样方法。饲料厂内易于用拭子采样的区域用纱布拭子采样,采样方法是:取10cm×10cm的棉质纱布方巾,用5毫升pH为7.2的磷酸盐缓冲液(PBS)中浸湿,然后对面积约20cm×20cm的区域进行采样。对于不易进入的区域,如料仓或卡车拖车的内部,利用滚动油漆刷采样10。采样的地点根据每个设施的不同而有所不同,但在3个饲料生产设施(饲料厂1-3)中所选的采样点是类似的。每个拭子分别采集4个区域中的一个,包括饲料或原料的直接接触表面(区域1),近距离(1米内)非接触表面(区域2),非近距离(距离1米以上)非接触表面(区域3),和瞬态表面,例如可移动的工具、人员和非饲料或原料运输工具(区域4)。来自第四个设施(扩繁场)的试子样本是根据与猪的距离划分区域的。包括直接的饲料接触表面(区域5),栏内地板、栏位分隔板、饲喂器、饮水器等在内的直接的猪接触表面(区域6),以及包括员工通道、工作区、饲料存储区和风机等在内的非猪接触面(区域7)。
For the environmental samples, two different collection methods were used. Cotton gauze swabs were utilized for areas within the mill that had easy access for swab collection. The gauze swabs were collected by swabbing a surface area of approximately 20cm × 20cm with a 10cm × 10cm cotton gauze square soaked in 5 ml of phosphate buffered saline (PBS) with a pH of 7.2. For areas without easy access, such as the interior of storage bins or truck trailers, a paint roller was utilized, as described by Dee et al., 2014.10 Locations did vary based on each individual site, but within the 3 feed manufacturing facilities (Sites 1-3), similar locations were chosen. Each swab was assigned one of four zones, including direct feed or ingredient contact surfaces (Zone 1), close proximity (within 1m) non-contact surfaces (Zone 2), non-contact surfaces without close proximity (>1 m of separation) (Zone 3), and transient surfaces, such as moveable tools, employees, and non-feed or ingredient delivery vehicles (Zone 4). Swabs taken from the fourth facility, the multiplier farm, were assigned zones based on proximity to pigs. This included direct feed-contact surfaces (Zone 5), direct pig-contact surfaces (Zone 6) including pen flooring, pen walls, feeders, and waterers (pig contact), and non-pig contact surfaces (Zone 7) including employee walkways, work areas, feed storage, and fans (non-pig contact).
样本制备与分析
Sample preparation and analysis
饲料和原料样本是分别单独采集的,对于每个产品,从每个独立样本中取约25克样本并混合产生一个复合样本。所有产品样本在运输前均在4oC下储存。棉纱环境拭子样本在采样前先用5毫升的PBS溶液在圆锥管中对方形棉质纱布进行浸泡。采样后,再加入20毫升的PBS溶液。试子样本在运输前在4oC下储存。用滚动油漆刷采集的样本在采样后立即放在塑料自封袋中保存,每个油漆刷中加入200毫升pH为7.2的PBS溶液(以便运输),搅动,然后静置一小时。
Feed and ingredient samples were collected individual. For each product, a composite sample was created by dividing and blending approximately 25 g from each individual sample. All product samples were stored at 4˚C until shipped. Cotton gauze environmental swabs submitted for testing were initially prepared by adding 5 mL of PBS to a cotton gauze square in a conical tube prior to collection. After samples were collected, 20 ml of additional PBS were added. Swabs were kept at 4° C until shipped. The paint rollers used for sample collection were placed into large zipper-seal plastic bags immediately after use. To prepare them for shipment, 200 ml of 7.2 pH PBS was added to each roller. The sample was then agitated and allowed to set for 1 hour.
从每个样品中取出10毫升PBS溶液,在运输前在4oC下储存。取样后,样本用干冰保存并运送至爱荷华州兽医诊断实验室。样品在麦康凯琼脂上培养,然后鉴定每个样品中生长最多的三种细菌,并通过指定生长指数值进行报告。
10 mL of the PBS was removed from each sample and stored at 4˚C until shipped. After collection, samples were store and shipped on dry ice to the Iowa State Veterinary Diagnostic Laboratory. Samples were cultured on MacConkey agar, and the three types of bacteria with largest growth for each sample were identified and reported by assigning a growth index value.
统计分析
Statistical analysis
根据报告的生长情况,细菌生长结果的指标值分别为0、1、2、3或4。0表示阴性结果,1、2、3或4分别表示少量、低、中、高阳性结果。每个样品都有一个总体指数和,每个细菌都有一个生长指数值。用SAS GLIMMIX 程序(Version 9.4, SAS Institute Inc., Cary, NC)对数据进行分析,并用Tukey-Kramer法进行校正。
Bacterial growth results were assigned an index value of either 0, 1, 2, 3, or 4 based on reported growth, with 0 representing a negative result and values 1, 2, 3, or 4 a few, low, moderate, or high positive result, respectively. Growth values were reported as individual bacteria, with each sample receiving an overall index sum. The data were analyzed using the GLIMMIX procedure of SAS (Version 9.4, SAS Institute Inc., Cary, NC) with the Tukey-Kramer adjustment using the assigned location zones as the levels with the response variables of total growth (sum of index values) and presence of bacteria typical used to indicate fecal matter is present (fecal indicators).