Using improved technology for filter paper-based blood collection to survey wild Sika deer for antibodies to hepatitis E virus

Abstract

Recent reports from Japan implicated wild Sika deer (Cervus nippon) in the zoonotic transmission of hepatitis E to humans. Seroprevalence studies were performed to determine if imported feral populations of Sika deer in Maryland and Virginia posed a similar risk of transmitting hepatitis E virus (HEV). Hunters collected blood on filter paper discs from freshly killed deer. The discs were desiccated and delivered to a collection point. The dried filters were weighed to estimate the amount of blood absorbed and were eluted and collected in one tube via a novel extraction system. The procedure was quantified and validated with negative and positive serum and blood samples obtained from domestic Sika deer before and after immunization with HEV recombinant capsid protein, respectively. None of the 155 tested samples contained antibody to HEV, suggesting that Sika deer in these populations, unlike those in Japan, do not pose a significant zoonotic threat for hepatitis E. However, the new method developed for collecting and eluting the samples should prove useful for field studies of many other pathogens.

Figure 1

Ultra Free-Cl centrifugal filter device. A. Each sample filter was incubated with gelatin solution in the inner compartment of the filter device. B. Eluate after extraction and centrifugation.

Figure 2

Serial dilution of pre-immunization and post-immunization samples from immunized Sika deer. Two filters, inoculated with 50 μL and 400 μL of deer blood, respectively and a serum sample were diluted and tested for deer IgG anti-hepatitis E virus in an ELISA as described in the text.

Figure 3

Optical density based on total deer IgG. Eluates from four filters with different amounts of deer blood were tested at dilutions of 1:1 000 for total deer IgG by ELISA. The amount of total deer IgG was determined by extrapolating from known concentrations of human IgG standards run in parallel on a (8–16%) SDS gel.

Figure 4

The distribution of total deer IgG in the Sika deer sample eluates based on optical density. Each eluate was tested for total deer IgG at a 1:1 000 dilution by ELISA (n = 165). Ten samples with OD values that were less than 0.700 were removed from the study.