engleski

SARS-CoV-2 investigation in wildlife and environment

Almost two years of coexistence with COVID-19 are behind us and it is very much clear that disease is likely to become endemic. To make it manageable, the knowledge of all aspects of the disease and its causative agent is of paramount importance. Human-to-animal contact, as people encroach on animal habitats has shown in the past to cause spillover events of numerous pathogens from animals to humans, and vice versa. When it comes to SARS-CoV-2, it has been proven that humans sporadically can transmit the virus to other animals or even cause an epidemic wave within some species such as outbreak of infection in farm minks. Due to the number of infected people, possible animal susceptibility, high viral recombination rates of coronaviruses and recent reports of environmental, especially wastewater contamination, reverse spillovers of SARS-CoV-2 can be expected. Therefore, the aim of this study was to investigate a possible transmission of SARS-CoV-2 to free ranging and captive wildlife as well as the environment after the first infection wave and during the second one. We tested the environmental samples which were represented by wastewaters (n=21 ; nine wastewater treatment plants from seven Croatian counties ; from 1st to 3rd treatment stage), surface waters (n=30 ; two nature parks and one city lake) and bivalves from the Adriatic Sea (n=77). Regarding the wildlife species, we tested samples of wild boar (n=153), red fox (n=204) and jackal (n=65), for the presence of SARS CoV-2 RNA and anti-SARS CoV-2 antibodies. We also tested yellow-legged gulls (n=111) since they feed on garbage pins nearby highly populated cities. To fulfil the epidemiological data, we tested samples of Zoo animals (n=32) that were consecutively in contact with SARS CoV-2 positive humans. Finally, samples from one bat colony (n=38) located relatively close to the human settlement were tested as well. The presence of SARS-CoV-2 was examined by applying real-time RT-PCR directed towards different genome targets ; the presence of anti-SARS CoV-2 antibodies were examined by two commercially available serological assays (ELISA and VNT). Eight out of nine wastewater plants were positive in influent wastewaters on at least one genome target. Most effluent wastewaters from 2nd and 3rd treatment stage plants were SARS-CoV-2 negative (81.8%), as well as all surface waters and bivalves. However, no wildlife samples were either serologically or virologically positive, regardless of the locality and distance from human settlements. Samples of wild animals from the Zagreb Zoo, which are in daily contact with people, including those proven to be positive for COVID-19, were also negative. Nevertheless, 58% of the bats tested ELISA positive, and neutralising antibodies were detected in 29% of bats. Guano samples (26%) tested positive with SARS-CoV-2 E-gene-Sarbeco real-time RT-PCR protocol. The detected coronavirus sequences were classified within both alphaCoVs and betaCoVs. The discovered betaCoV sequence was phylogenetically clustered with other SARS-CoV-2-like viruses, but not with the original pandemic virus. In conclusion, no evidence of spillover of SARS CoV-2 was found in tested wildlife species regardless of their habitat, however, the SARS CoV-2 pandemic strain derived from wastewater and the SARS CoV-2-like strain detected in bats indicate that a possibility of virus transmission is present, and monitoring must be carried out.

SARS-CoV-2, gulls, zoo animals, wildlife, bats, virus-neutralization test, ELISA, real-time RT-PCR

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