Protective Effects of Cell-Free Supernatant and Live Lactic Acid Bacteria Isolated from Thai Pigs Against a Pandemic Strain of Porcine Epidemic Diarrhea Virus

Abstract

Porcine epidemic diarrhea virus (PEDV) is a coronavirus which causes severe diarrhea and fatal dehydration in piglets. In general, probiotic supplements could enhance recovery and protect piglets against enteric pathogens. Seven local lactic acid bacteria (LAB), (Ent. faecium 79N and 40N, Lact. plantarum 22F, 25F and 31F, Ped. acidilactici 72N and Ped. pentosaceus 77F) from pig feces were well-characterized as high potential probiotics. Cell-free supernatants (CFS) and live LAB were evaluated for antiviral activities by co-incubation on Vero cells and challenged with a pandemic strain of PEDV isolated from pigs in Thailand. Cell survival and viral inhibition were determined by cytopathic effect (CPE) reduction assay and confirmed by immunofluorescence. At 1:16, CFS dilution (pH 6.3-6.8) showed no cytotoxicity in Vero cells and was therefore used as the dilution for antiviral assays. The diluted CFS of all Lact. plantarum showed the antiviral effect against PEDV; however, the same antiviral effect could not be observed in Ent. faecium and Pediococcus strains. In competitive experiment, only live Lact. plantarum 25F and Ped. pentosaceus 77F showed CPE reduction in the viral infected cells to <50% observed field area. This study concluded that the CFS of all tested lactobacilli, and live Lact. plantarum (22F and 25F) and Pediococcus strains 72N and 77F could reduce infectivity of the pandemic strain of PEDV from pigs in Thailand on the target Vero cells.

Keywords: Antiviral activity; Cell-free supernatants; Lactic acid bacteria; Porcine epidemic diarrhea virus; Probiotics.

Fig. 1

a Adhesion ability of seven studied LAB strains on Vero cell monolayers. The observed magnification fold is ×100. The arrows indicate adhering bacterial cells. b Number of bacterial cells adhered per Vero cell. The results are expressed as mean ± standard deviation. The different letters indicate statistically significant differences between LAB strains (P < 0.05)

Fig. 2

The cytotoxicity evaluation by neutral red assay on Vero cell monolayers after exposure to undiluted and serially twofold dilutions (1:2 to 1:64) of CFS after day 4 of incubation

Fig. 3

Antiviral activity of LAB against pandemic strain of PEDV on infected Vero cells. The CPE reduction was observed under a microscope at ×100. a No infection. b–d shows the CPE scores as + (<50% of observed field area), ++ (50–75% of observed field area), and +++ (>75% of observed field area). The reduction of the fluorescent signals of the infected cells were observed under a fluorescent microscope as e no infection; f–g shows the reduction signals scored as + (<50% of observed field area), ++ (50–75% of observed field area), and +++ (>75% of observed field area). The circles indicate the infectivity area