Detection of African Swine Fever Virus in Feed and Feed Mill Environment Following Extended Storage

Table 1

Proportion of qPCR positive and interactive means of cycle threshold (Ct) value and log₁₀ genomic copies/g of feed samples for ASFV DNA survival after experimental inoculation of swine feed and subsequent feed batch sequencing.

Item	Batch of feed^†
	2	3	4	5	6
Proportion PCR positive
Day 1	40/40	20/20	19/20	19/20	17/20
Day 3	6/6	4/6	3/6	3/6	1/6
Day 7	6/6	5/6	1/6	2/6	2/6
Day 14	6/6	6/6	4/6	5/6	3/6
Day 28	6/6	6/6	3/6	3/6	0/6
Day 60	6/6	6/6	3/6	1/6	2/6
Day 90	6/6	6/6	2/6	2/6	1/6
Day 180	6/6	5/6	2/6	2/6	0/6
Cycle threshold^‡
Day 1	33.0^a	37.5^b,c,d,e	39.5^e,f,g,h,i	39.3^e,f,g,h,i	40.1^e,f,g,h,i
Day 3	31.7^a,b	39.5^c,d,e,f,g,h,i	42.4^e,f,g,h,i	41.4^e,f,g,h,i	43.8^g,h,i
Day 7	31.6^a	37.8^a,b,c,d,e,f,g,h	44.3^h,i	42.2^e,f,g,h,i	43.5^f,g,h,i
Day 14	31.8^a,b	36.9^a,b,c,d,e,f,g	40.6^e,f,g,h,i	39.6^d,e,f,g,h,i	41.4^e,f,g,h,i
Day 28	31.3^a	36.5^a,b,c,d,e,f	42.7^e,f,g,h,i	42.5^e,f,g,h,i	45.0^i
Day 60	32.4^a,b,c	37.8^a,b,c,d,e,f,g,h	42.3^e,f,g,h,i	44.3^h,i	43.2^e,f,g,h,i
Day 90	32.6^a,b,c,d	36.2^a,b,c,d,e	43.0^e,f,g,h,i	43.6^f,g,h,i	43.6^f,g,h,i
Day 180	32.0^a,b	39.7^d,e,f,g,h,i	43.5^f,g,h,i	41.5^e,f,g,h,i	45.0^i
Log10 genomic copies/g^§
Day 1	4.7^i	3.6^f,g,h,i	3.1^d,e,f,g,h	3.1^c,d,e,f,g,h	2.8^b,c,d,e,f,g,h
Day 3	5.0^h,i	2.5^a,b,c,d,e,f,g,h,i	1.5^a,b,c,d,e,f	1.7^a,b,c,d,e,f,g	0.6^a,b,c
Day 7	5.0^h,i	3.2^b,c,d,e,f,g,h,i	0.5^a,b	1.3^a,b,c,d,e,f	0.9^a,b,c,d,e
Day 14	4.9^h,i	3.7^b,c,d,e,f,g,h,i	2.2^a,b,c,d,e,f,g,h	2.7^a,b,c,d,e,f,g,h,i	1.7^a,b,c,d,e,f,g
Day 28	5.1^h,i	3.8^b,c,d,e,f,g,h,i	1.4^a,b,c,d,e,f	1.5^a,b,c,d,e,f	0.00^a
Day 60	4.8^g,h,i	3.5^e,f,g,h,i	1.5^a,b,c,d,e,f	0.5^a,b	1.0^a,b,c,d,e
Day 90	4.7^g,h,i	3.8^b,c,d,e,f,g,h,i	1.1^a,b,c,d,e	0.9^a,b,c,d,e	0.6^a,b,c,d
Day 180	4.9^h,i	2.7^e,f,g,h,i	0.9^a,b,c,d,e	1.4^a,b,c,d,e,f	0.0^a

Note: ^†Swine gestation feed was inoculated with African swine fever virus (ASFV) for a final concentration of 5.6 × 104 TCID50/gram inoculated feed (Batch 2) following an initial priming of the feed manufacturing equipment with ASFV free feed. Four subsequent batches of initially ASFV-free feed were then manufactured (Batches 3–6). On Days 1, 3, 7, 14, 28, 60, 90, and 180 after manufacture following room temperature storage, three samples were mixed with approximately 35 mL of phosphate buffered solution, incubated for 2 hr at room temperature then centrifuged at 1,000 × g for 3 min. Samples were then analyzed using qRT-PCR for detection of the gene encoding for the p72 protein. Analysis of Day 1 feed samples have been reported by Elijah et al. [12] and are included in the current analysis of ASFV detection over time. ^‡Samples that had no detectable ASFV DNA were assigned a Ct value of 45.0. Batch × day: . SEM for Batch 2, Day 1 = 0.64; SEM for Batches 3–6, Day 1 = 0.69; All other SEM = 1.27. ^§Log₁₀ genomic copies/g of feed. Batch × day, . SEM for Batch 2, Day 1 = 0.27; SEM for Batches 3–6, Day 1 = 0.30; All other SEM = 0.56. ^a–iMeans within item lacking common superscript differ () using Tukey multiple comparison adjustment.