Urban greening has benefits for both human and environmental health. However, urban greening might also have negative effects as the abundance of wild rats, which can host and spread a great diversity of zoonotic pathogens, increases with urban greenness. Studies on the effect of urban greening on rat-borne zoonotic pathogens are currently unavailable. Therefore, we investigated how urban greenness is associated with rat-borne zoonotic pathogen prevalence and diversity, and translated this to human disease hazard. We screened 412 wild rats (Rattus norvegicus and Rattus rattus) from three cities in the Netherlands for 18 different zoonotic pathogens: Bartonella spp., Leptospira spp., Borrelia spp., Rickettsia spp., Anaplasma phagocytophilum, Neoehrlichia mikurensis, Spiroplasma spp., Streptobacillus moniliformis, Coxiella burnetii, Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL)/AmpC-producing Escherichia coli, rat hepatitis E virus (ratHEV), Seoul orthohantavirus, Cowpox virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Toxoplasma gondii and Babesia spp. We modelled the relationships between pathogen prevalence and diversity and urban greenness. We detected 13 different zoonotic pathogens. Rats from greener urban areas had a significantly higher prevalence of Bartonella spp. and Borrelia spp., and a significantly lower prevalence of ESBL/AmpC-producing E. coli and ratHEV. Rat age was positively correlated with pathogen diversity while greenness was not related to pathogen diversity. Additionally, Bartonella spp. occurrence was positively correlated with that of Leptospira spp., Borrelia spp. and Rickettsia spp., and Borrelia spp. occurrence was also positively correlated with that of Rickettsia spp. Our results show an increased rat-borne zoonotic disease hazard in greener urban areas, which for most pathogens was driven by the increase in rat abundance rather than pathogen prevalence. This highlights the importance of keeping rat densities low and investigating the effects of urban greening on the exposure to zoonotic pathogens in order to make informed decisions and to take appropriate countermeasures preventing zoonotic diseases.

Seoul virus (SEOV) is a zoonotic orthohantavirus carried by black and brown rats, and can cause hemorrhagic fever with renal syndrome in humans. Human cases of SEOV virus infection have most recently been reported in the USA, United Kingdom, France and the Netherlands and were primarily associated with contact with pet rats and feeder rats. Infection of rats results in an asymptomatic but persistent infection. Little is known about the cell tropism of SEOV in its reservoir and most available data is based on experimental infection studies in which rats were inoculated via a route which does not recapitulate virus transmission in nature. Here we report the histopathological analysis of SEOV cell tropism in key target organs following natural infection of a cohort of feeder rats, comprising 19 adults and 11 juveniles. All adult rats in this study were positive for SEOV specific antibodies and viral RNA in their tissues. One juvenile rat was seropositive, but negative in the rRT-PCR. Of the 19 adult rats of which subsequently additional organs were tested, SEOV RNA was detected in all lungs, followed by kidney (79%) and liver (74%). Histopathologic changes associated with SEOV infection were primarily found in the liver, consistent with a pathological diagnosis of a mild hepatitis. In conclusion, natural SEOV infection results in mild inflammation of the liver in the absence of clinical disease.

Seoul orthohantavirus (SEOV) is a rat-associated zoonotic pathogen with an almost worldwide distribution. In 2019, the first autochthonous human case of SEOV-induced hemorrhagic fever with renal syndrome was reported in Germany, and a pet rat was identified as the source of the zoonotic infection. To further investigate the SEOV reservoir, additional rats from the patient and another owner, all of which were purchased from the same vendor, were tested. SEOV RNA and anti-SEOV antibodies were found in both of the patient’s rats and in two of the three rats belonging to the other owner. The complete coding sequences of the small (S), medium (M), and large (L) segments obtained from one rat per owner exhibited a high sequence similarity to SEOV strains of breeder rat or human origin from the Netherlands, France, the USA, and Great Britain. Serological screening of 490 rats from breeding facilities and 563 wild rats from Germany (2007–2020) as well as 594 wild rats from the Netherlands (2013–2021) revealed 1 and 6 seropositive individuals, respectively. However, SEOV RNA was not detected in any of these animals. Increased surveillance of pet, breeder, and wild rats is needed to identify the origin of the SEOV strain in Europe and to develop measures to prevent transmission to the human population.

Two different methods for preventing the binding of cross-reacting antibodies to the genus-reactive chlamydial lipopolysaccharide (LPS) were used to improve the specificity of an enzyme immunoassay for the determination of antibodies to Chlamydia trachomatis. Coated elementary bodies were treated with either sodium periodate, to oxidize the antigenic sites of the LPS, or Triton X-100, to extract the LPS. By using these new enzyme immunoassays, the standard enzyme immunoassay, and the whole inclusion fluorescence (WIF) assay, antibodies to C. trachomatis were determined in sera from different groups of patients and controls. Paired serum samples from patients with culture-proven urogenital C. trachomatis infections showed similar responses in all three assays. Paired serum samples from patients with Chlamydia psittaci infections showed similar responses in the WIF assay and the standard enzyme immunoassay, whereas significantly reduced titers were obtained in the enzyme immunoassays with treated antigen, especially in the convalescent-phase serum samples. Serum samples from patients with symptoms suggestive of infection with C. trachomatis, pregnant women, and blood donors were evaluated by all three types of assays. Eighty percent of the significant reductions in immunoglobulin G (IgG), IgA, and IgM titers were observed in sera with WIF assay titers in the lower classes (IgG, 1: < or = 256; IgA, 1: < or = 32; IgM, 1: < or = 16). From these results we conclude that oxidation of the antigen by sodium periodate is a simple and effective method of producing an enzyme immunoassay with enhanced specificity that could be useful for diagnostic purposes and seroepidemiological studies.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Seoul virus (SEOV) is a zoonotic orthohantavirus carried by rats. In humans, SEOV can cause hemorrhagic fever with renal syndrome. Recent human SEOV cases described in the USA, United Kingdom, France and the Netherlands were associated with contact with pet or feeder rats. The prevalence of SEOV in these types of rats is unknown. We collected 175 pet and feeder rats (Rattus norvegicus) from private owners, ratteries and commercial breeders/traders in the Netherlands. Lung tissue of the rats was tested using a SEOV real-time RT-qPCR and heart fluid was tested for the presence of antibodies against SEOV. In all three investigated groups, RT-qPCR-positive rats were found: in 1/29 rats from private owners (3.6%), 2/56 rats from ratteries (3.4%) and 11/90 rats from commercial breeders (12.2%). The seroprevalence was largely similar to the prevalence calculated from RT-qPCR-positive rats. The SEOV sequences found were highly similar to sequences previously found in domesticated rats in Europe. In conclusion, SEOV is spread throughout different populations of domesticated rats.