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Swine influenza virus (SIV) causes severe suffering at the animal level and significant economic losses in the swine industry worldwide. Since epithelial cells in pig trachea contain both human and avian type receptors (α 2, 6- and α 2, 3-linked sialic acid, respectively), pigs are supposed to be the “mixing vessels” for a wide range of influenza A viruses and as the potential source for new human-avian influenza A virus reassortants. Therefore the control of swine influenza viruses plays an important role both from the animal health and from the public health point of view. To address this question, two studies were performed in porcine model - In vitro and in vivo.
Concerning the in vitro study, we assessed the inhibitory potential of the probiotic Enterococcus faecium NCIMB10415 on the replication of two porcine strains of influenza virus (H1N1 and H3N2 strain) in a continuous porcine macrophage cell line (3D4/21) derived from lung macrophages and in the continuous epithelial cell line, MDBK cells.
Cell cultures were treated with E. faecium at the non-toxic concentration of 1x106 CFU/ml in growth medium for up to 90 min before, during and after SIV infection.
After further incubation of cultures in probiotic-free growth medium, cell viability and virus propagation were determined at 48 h or 96 h post infection in 3d4/21 and MDBK cells, respectively. The results obtained reveal an almost complete recovery of viability of SIV infected cells and an inhibition of virus multiplication by up to four log units in the E. faecium treated cells. In both 3D4- and MDBK-cells a 60 min treatment with E. faecium stimulated NO release which is in line with published evidence for an antiviral function of NO. Furthermore, E. faecium caused a modified cellular expression of selected mediators of defense in 3D4-cells: while the expression of TNF-α, TLR-3 and IL-6 were decreased in the SIV-infected and probiotic treated cells, Il-10 was found to be increased.
Since we obtained experimental evidence for the direct adsorptive trapping of SIV through E. faecium, at the cellular level, this probiotic microorganism inhibits influenza viruses by at least two mechanisms, direct physical interaction and strengthening of innate defense.
Concerning the in vivo study, we tested if probiotic Enterococcus faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements could provide beneficial effects on SIV vaccination and infection in piglets. Seventy-two weaned piglets were fed three different diets containing either E. faecium or high (2,500 ppm) or normal levels of Zn oxide (natural Zinc content: 50-80 ppm, control). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine.
All piglets were then infected intranasally with H3N2 SIV. Clinically, significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Zn and E. faecium groups gained weight, while those in the control group lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Zn+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Compared to the control group, significantly higher CD4+CD8+ and CD8+ (cytotoxic T lymphocytes, CTL) were detected in the Zn and E. faecium groups at various time points after infection as determined by flow cytometry.
Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology.