High-dose dietary zinc oxide mitigates infection with transmissible gastroenteritis virus in piglets

Chai et al. BMC Veterinary Research 2014, 10:75 http://www.biomedcentral.com/1746-6148/10/75 RESEARCH ARTICLE Open Access High-dose dietary zinc oxide mitigates infection with transmissible gastroenteritis virus in piglets Weidong Chai1, Silke S Zakrzewski2, Dorothee Günzel2, Robert Pieper3, Zhenya Wang1, Sven Twardziok4, Pawel Janczyk5, Nikolaus Osterrieder1 and Michael Burwinkel1* Abstract Background: Zinc (Zn) supplementation has been shown to reduce the incidence of diarrhea and to protect animals from intestinal diseases, but the mechanisms of this protective effect against virus infection in vivo have not yet been elucidated. Transmissible gastroenteritis virus (TGEV) causes diarrhea in piglets with an age-dependent decrease of severity. Results: We used 60 weaned piglets that were divided into three groups to evaluate the effect of different Zn levels added to a conventional diet (50 mg Zn/kg diet, Znlow, control group). The other groups received the diet supplemented with ZnO at final concentrations of 150 mg Zn/kg diet (Znmed), or 2,500 mg/kg diet (Znhigh). Oral challenge infection with TGEV was performed when the pigs had been fed for 1 week with the respective diet. Half of the piglets of each group were sacrificed at day 1 and 18 after challenge infection. Fecal consistency was improved and body weights increased in the Znhigh group when compared to the other groups, but no direct effect of Zn concentrations in the diet on fecal TGEV shedding and mucosal immune responses was detectable. However, in the Znhigh group, we found a prevention of villus atrophy and decreased caspase-3-mediated apoptosis of jejunal epithelium. Furthermore, pigs receiving high Zn diet showed a down-regulation of interferon (IFN)-α, oligoadenylate synthetase (OAS), Zn transporter SLC39A4 (ZIP4), but up-regulation of metallothionein-1 (MT1), as well as the Zn transporters SLC30A1 (ZnT1) and SLC30A5 (ZnT5). In addition, forskolin-induced chloride secretion and epithelial resistance were controlled at a physiological level in the Znhigh but not the other groups. Finally, in the Znhigh group, we documented an earlier and higher systemic TGEV-specific serum antibody response. Conclusions: These results suggest that high dietary Zn could provide enhanced protection in the intestinal tract and stimulate the systemic humoral immune response against TGEV infection. Keywords: Zinc oxide, Coronavirus, Transmissible gastroenteritis virus, Cytokine, Morphometry, Electrophysiology, Zinc transporters Background Several in vitro studies have shown that zinc (Zn) has broad-spectrum antiviral activity against a variety of viruses, such as human immunodeficiency virus, transmissible gastroenteritis virus (TGEV), equine arteritis virus, and severe acute respiratory syndrome coronavirus [1-6]. Many potential mechanisms have been suggested to explain the potential beneficial effect of Zn against virus infections. For example, Zn mediates antiviral effects through the inhibition of nidovirus RNA-dependent * Correspondence: 1 Institut für Virologie, Freie Universität Berlin, Berlin, Germany Full list of author information is available at the end of the article RNA polymerases or other proteins essential for the different phases of the viral life cycle [5,6]. In addition, Zn participates in initiating and maintaining robust immune responses, in particular cytokine production and modulation of the activity of immune cells [7]. Zn induces the production of innate interferon (IFN)-α and also immune IFN-γ, and can potentiate the antiviral action of IFN-α, but not of IFN-γ [8]. Clearance of viral infections requires cytotoxic T lymphocytes, which are also highly dependent on the presence of Zn [7]. Antibody production during both the first and an immunological memory response is disturbed by Zn deficiency [9,10], indicating that Zn is necessary for optimal results following vaccination. © 2014 Chai et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chai et al. BMC Veterinary Research 2014, 10:75 http://www.biomedcentral.com/1746-6148/10/75 In swine nutrition, especially in the North American swine industry, high levels of Zn oxide (ZnO, 2,0003,000 ppm) are often added to the diet of weaned pigs, since such addition was shown to reduce non-specific post-weaning diarrhea and improve performance in this critical period of dietary change [11-13]. Diarrhea is caused by impaired intestinal epithelial barrier function, which most likely leads to malnutrition and decreased uptake of micronutrients, including Zn. It was shown that oral Zn supplementation with high doses was able to counteract this loss, improve intestinal mucosal integrity as well as absorption of water and electrolytes [12,14]. Furthermore, it leads to a faster regeneration of the gut epithelium [15]. However, because of environmental concerns, the maximum level of Zn allowed in pig diets was set up to 150 ppm in the European Union, irrespective of the Zn formulation [16]. Zn homeostasis is maintained in the body through a variety of transporters and Zn binding proteins [17]. High levels of dietary Zn provided as ZnO have been recently shown to outbalance Zn homeostasis with increased accumulation of Zn in various organs including the small intestine of piglets [18,19]. Since intestinal Zn uptake can also take place through passive diffusion, it is likely that very high dietary Zn levels would indirectly increase the intestinal barrier function as a protection mechanism of the epithelium. In addition, metallothionein that is induced by Zn accumulation in intestinal tissue may also protect the tissue from oxidative damage. Due to suboptimal immune functions, newborn as well as weaned piglets are particularly susceptible to infection by various pathogens, among them TGEV, which causes severe to mild gastroenteritis in piglets, depending on the age [20,21]. Our previous study [5] showed that high Zn levels markedly reduced TGEV titers as well as viral RNA and protein synthesis in vitro, but there is no report about antiviral effects of Zn supplementation in Page 2 of 10 pigs. The aim of this study was to close the knowledge gap and evaluate the antiviral potential and possible protection mechanisms of increased dietary Zn supplementation against TGEV infection in weaned piglets. Results High-dose Zn prevents diarrhea in piglets but does not affect other trace elements and virus shedding TGEV infection caused only mild symptoms and there was no difference in dehydration, anorexia, lethargy and body tempera (...truncated)