Monitoring Survivability and Infectivity of Porcine Epidemic Diarrhea Virus (PEDv) in the Infected On-Farm Earthen Manure Storages (EMS)

Abstract

In recent years, porcine epidemic diarrhea virus (PEDv) has caused major epidemics, which has been a burden to North America's swine industry. Low infectious dose and high viability in the environment are major challenges in eradication of this virus. To further understand the viability of PEDv in the infected manure, we longitudinally monitored survivability and infectivity of PEDv in two open earthen manure storages (EMS; previously referred to as lagoon) from two different infected swine farms identified in the province of Manitoba, Canada. Our study revealed that PEDv could survive up to 9 months in the infected EMS after the initial outbreak in the farm. The viral load varied among different layers of the EMS with an average of 1.1 × 10(5) copies/ml of EMS, independent of EMS temperature and pH. In both studied EMS, the evidence of viral replication was observed through increased viral load in the later weeks of the samplings while there was no new influx of infected manure into the EMS, which was suggestive of presence of potential alternative hosts for PEDv within the EMS. Decreasing infectivity of virus over time irrespective of increased viral load suggested the possibility of PEDv evolution within the EMS and perhaps in the new host that negatively impacted virus infectivity. Viral load in the top layer of the EMS was low and mostly non-infective suggesting that environmental factors, such as UV and sunlight, could diminish the replicability and infectivity of the virus. Thus, frequent agitation of the EMS that could expose virus to UV and sunlight might be a potential strategy for reduction of PEDv load and infectivity in the infected EMS.

Keywords: Swine; earthen manure storages (EMS); infectivity; porcine epidemic diarrhea virus (PEDv); survivability.

FIGURE 1

Schematic diagram of the study design indicating porcine epidemic diarrhea virus (PEDv) outbreak and sampling schedule for monitoring survivability and infectivity of the virus in two on-farm Manitoba earthen manure storages (EMS).

FIGURE 2

The layout of sampled EMS in this study. (A) The layout of farm-1 EMS indicating the locations of 12 sampling points and pH/temperature data loggers. (B) Three sampling depths at farm-1 EMS. (C) The layout of farm-2 EMS indicating the locations of 16 sampling points and pH/temperature data loggers. (D) Two sampling depths at farm-2 EMS.

FIGURE 3

Standard curves for the duplex real-time RT-PCR assay. (A) Synthetic DNA standard curve for virulent PEDv strain. (B) Synthetic DNA standard curve for variant-INDEL PEDv strain.

FIGURE 4

Temperature and pH dynamics in the studied EMS. The temperature and pH records for farm-1 EMS (A,B) and for farm-2 EMS (C,D) during fall sampling. The temperature and pH records for farm-2 EMS (E,F) during spring/summer sampling.

FIGURE 5

Survivability of PEDv in two infected on-farm EMS. Using real-time RT-PCR targeted to S gene, the survivability of PEDv over time was tested based on the detectable viral RNA copies number in 1 ml of EMS at each sampling time point during fall 2014 sampling for farm-1 EMS (A), and from fall 2014 to summer 2015 sampling for farm-2 EMS (B). The bar shows average RNA copy number of PEDv in the respective layer of EMS. The error bars show the standard deviation based on 12 replicates per layer of the EMS in farm-1 and 16 replicates in farm-2. Each biological sample was analyzed in triplicate using real-time RT-PCR. ^∗∗∗P < 0.001 and ^∗∗∗∗P < 0.0001.