Revealing new tick-borne encephalitis foci by screening antibodies in sheep milk

Climate changes have increased the tick-distribution in Sweden, and the prevalence of ticks has been predicted to increase towards the northern parts of the country, increasing the risk of tick-borne zoonosis in new regions. Tick-borne encephalitis (TBE) is the most important viral tick-borne zoonosis in Sweden as well as in Europe. TBE virus (TBEV) infection often leads to severe CNS disease, including encephalitis and severe myelitis, which may lead to paralysis and respiratory failure in humans. TBEV and antibodies against TBEV are excreted in milk of goats, sheep and cattle and the virus can be ingested orally by consumption of non-pasteurized dairy products. Since the prevalence of TBEV in the tick population is low there is a need for new and robust surveillance techniques identifying new risk areas of TBEV at early stages. In this study we have developed a novel strategy for identifying new TBEV foci. We have collected raw milk and colostrum samples from sheep and goats in Örebro County, Sweden. The milk samples were analyzed for the presence of TBEV antibodies by ELISA, and validated by an in-house Western Blot assay where milk samples were used as primary antibody to detect purified TBEV E-protein. By monitoring TBEV antibodies in milk we have found three novel foci in the Örebro County which also overlap with the plausible place of infection of registered human TBE cases reported during 2009-2018. Furthermore, the stability of TBEV in milk and raw milk was studied at different temperatures. Our data indicates that keeping unpasteurized milk at 4 ˚C will preserve the infectivity of TBEV for several days. Ticks have also been collected from areas with TBEV positive milk. We aim to extract total RNA from the sampled ticks, followed by TBEV detection by nested-PCR and next-generation sequencing. Here we present a novel technique to reveal risk areas of TBE in Sweden, which is robust, reliable, and non-invasive and can accordingly be used to map TBEV “hotspots”