Selenium Depletion Reduces Vaccination Response in Horses

s / Journal of Equine Veterinary Science 31 (2011) 230-356 266 Selenium Depletion Reduces Vaccination Response in Horses M. Brummer, S. Hayes, S.M. McCown, A.A. Adams, D.W. Horohov, and L.M. Lawrence University of Kentucky, Lexington, KY, USA Introduction: Limited research has focused on the effect of Se on the equine immune system. Knight and Tyznik [1] found ponies fed a diet containing .22ppm Se had higher serum IgG levels and antibody titers in response to a sheep red blood cell vaccination than ponies fed a diet containing .02ppm Se. Those authors noted that a mechanism to explain the effect of Se on antibody production was not known, and even today the precise mechanism of action remains unknown. An immune challenge can stimulate the humoral and cell mediated components of the immune system. In other species Se status has been shown to affect the cell mediated immune response. Therefore the aim of our study was to examine the effect of low Se status on the ability of both the humoral and cell mediated components of the immune system to respond to a vaccine challenge. We hypothesized low Se status would alter immune responses at both a molecular and functional level in mature horses. Materials and Methods: Twenty eight horses, aged 6-24 yr, were used. The study included 28 wk of adaptation to diets prior to a 7 wk vaccine challenge. Horses were kept on pasture (<.06 ppm Se DM); when pasture availability declined, horses were fed hay from the same farm (<.05 ppm Se DM) and cracked corn (<.04 ppm Se DM). Horses were blocked by age and gender, and assigned to a low (LS) or adequate (AS) Se group. Horses were individually fed a mineral supplement, with (AS) or without (LS) sodium selenite, so that total Se intakes for AS and LS horses were 140% and 70% of the NRC recommendation [2], respectively. The AS group contained 7 horses and the LS group contained 21. More horses were allocated to the LS group in preparation for a later study. Blood samples, taken prior to and every 4 wks for 28 wk, were collected for analysis of whole blood Se (Michigan State University diagnostic laboratory) and glutathione peroxidase (GPx; Bioxytech GPx-340; OXIS Research, Portland, OR), GPx reported per mg hemoglobin (Hb). To ensure that the observed immune response was primary and not due to prior exposure, we used a novel antigen, keyhole limpet hemocyanin (KLH) for the vaccine challenge. Horses received 10mg KLH (IM) at the end of the 28wk feeding period, and another 5 mg dose 3 wks later. Serum KLH specific antibody levels were measured using an ELISA assay, developed in our laboratory, for 7 wks from initial vaccine. Blood collected in sodium heparin vacutainer tubes were used to isolate peripheral blood mononuclear cells (PBMC) before, 3 and 5 wks post vaccination. PBMC0s were used to examine the impact of Se status on in vitro cell-mediated immunity to KLH. Isolated PBMC0s were stimulated with media or KLH (200mg/mL). Stimulated PBMC0s were used for intracellular staining for interferon (IFN) g and tumor necrosis factor (TNF) a, and total RNAwas collected for the analysis of cytokine and transcription factors gene expression [3]. Lymphocyte proliferation was assessed using the 3H-thymidine incorporation method [4]. Data were analyzed as repeated measures using Proc Mix, SAS 9.2. with LS Means separation procedure. Data not normally distributed, were log transformed for data analysis. Data are reported as LS means. Results and Discussion: After 28 wks on treatment whole blood Sewas lower in LS (164.7ng/mL) than AS (211.1ng/mL; P< .05). GPx activity was also lower in LS (42.72mU/mg Hb) than AS (55.00mU/ mg Hb; P < .05). In response to vaccination, KLH-specific IgG levels increased over time in both groups, but AS responded quicker (time x treatment;P< .05)withhigherantibody levels at 3wkthanLS (P< .05). After the second vaccine, KLH antibody levels of the LS horses were comparable to those of AS horses. In contrast Knight and Tyznik [1] reported overall higher antibody response in ponies fed a high Se diet even after a second vaccine dose. Se status did not affect lymphoproliferation in response to KLH stimulation. Se status did not affect intracellular production of IFNg and TNFa following KLH stimulation. KLH-induced mRNA expression of IFNg, TNFa, interleukin (IL) -1, -2, -6, -8, -10 and -13 by PBMC was similar between groups. Transcription factor GATA-3 mRNA expression decreased from 3 to 5wk in both groups (P < .05) but did not differ between groups. Expression of the transcription factor T-bet was greater at 5 wks for AS (P < .05) than LS horses. Though T-bet induces an IFNg response, geared towards protection against viral and intracellular pathogens [5], we did not observe a related increase expression of this cytokine in PBMC from the AS horses. The AS horses responded faster to the initial vaccine by both producing greater amounts of KLH-specific antibodies and more effectively priming a cellular immune response, as evidenced by increased expression of Tbet. Initial vaccination of the LS horses appeared insufficient for stimulating the immune response as measured both by delayed antibody response and failure to upregulate T-bet expression in the PBMC cultures. This diminished response in the LS horses could be due to Se deficiency initially affecting the ability of the immune system to recognize the antigen or limiting the in vivo expansion of KLH-specific memory cells, hence a lag in antibody production and diminished cellular immune responses in vitro. Conclusion: Low Se status resulted in a delayed and reduced immune response to the vaccination. This could have implications with regards to immune responses to routine vaccination in horses kept in Se deficient areas of the U.S.