Serological Survey of Mosquito-Borne Arboviruses in Wild Birds from Important Migratory Hotspots in Romania.

In the context of climate change, globalization, and enhanced human traveling, arboviruses continue to represent a threat to public health. West Nile and Usutu viruses are mosquito-borne viruses belonging to the Flaviviridae family (Flavivirus genus) and members of the Japanese encephalitis virus serocomplex. Included in the Togaviridae family (Alphavirus genus), the Sindbis virus is also vectored by mosquitoes. In the present study, we aimed to analyze the presence of antibodies concerning the abovementioned viruses in migratory and resident birds in the South-Eastern region of Romania, as avian hosts represent the main reservoir for human infection. Blood samples were collected from wild birds between May 2018 and October 2019 in nine locations from three counties. All the samples were serologically tested by ELISA and a serum neutralization test. Overall, a seroprevalence of 8.72% was registered for the West Nile virus, 2.71% for the Usutu virus, and 0% for the Sindbis virus. To our best knowledge, this is the first large-scale comprehensive study to assess the West Nile virus seropositivity in wild birds and the first serological confirmation of the Usutu virus in wild birds in Romania. Moreover, this is the only follow-up study reviewing the current seroprevalence of the Sindbis virus in Romania since 1975.

Potential mosquito vector attraction to- and feeding preferences for pigs in Romanian backyard farms.

Introduction: Mosquitoes either biologically or mechanically transmit various vector-borne pathogens affecting pigs. Mosquito species display a wide variety of host preference, as well as host attraction and behaviours. Mosquito species attraction rates to- and feeding rates on pigs or other potential hosts, as well as the seasonal abundance of the mosquito species affects their pathogen transmission potential. Methods: We caught mosquitoes in experimental cages containing pigs situated in Romanian backyard farms. The host species of blood meals were identified with PCR and sequencing. Results: High feeding preferences for pigs were observed in Aedes vexans (90%), Anopheles maculipennis (80%) and Culiseta annulata (72.7%). However, due to a high abundance in the traps, Culex pipiens/torrentium were responsible for 37.9% of all mosquito bites on pigs in the Romanian backyards, despite low feeding rates on pigs in the cages (18.6%). We also found that other predominantly ornithophilic mosquito species, as well as mosquitoes that are already carrying a blood meal from a different (mammalian) host, were attracted to backyard pigs or their enclosure. Discussion: These results indicate that viraemic blood carrying, for instance, African swine fever virus, West-Nile virus or Japanese encephalitis virus could be introduced to these backyard pig farms and therefore cause an infection, either through subsequent feeding, via ingestion by the pig or by environmental contamination.

Seasonal dynamics of Phlebotomus neglectus (Diptera: Psychodidae) in cave microhabitats in Romania and the rediscovery of Sergentomyia minuta (Rondani, 1843) after 50 years.

BACKGROUND: In a countrywide study aiming to update the knowledge on diversity of sand fly species in Romania, a sand fly population was observed in an isolated system of cave microhabitats. The caves are located in the protected area of Canaraua Fetii, Dobrogea region, southeastern Romania. The highest sand fly diversity was recorded in this area between 1968 and 1970. This work presents a study conducted to estimate the seasonal variation of the sand fly species in correlation with the particular environmental factors of the isolated system of cave microhabitats. METHODS: Sand flies were collected between May and October 2020 from one trapping site of interest in Canaraua Fetii. The trapping site consisted of a cave entrance. CDC miniature light traps and sticky traps were used to collect insects from the exterior walls of the cave entrance. Species identification of collected sand flies was done using morphological keys. Statistical analysis of the trapping and climatic data was performed. RESULTS: From all collected sand flies, 99.7% (818/822) were Phlebotomus neglectus, 0.1% (1/822) Ph. balcanicus and 0.2% (2/822) Sergentomyia minuta. Sand fly activity was first observed on 2 July and last on 24 September. A monomodal abundance trend was present, with the peak activity between 16 and 17 July. The analysis of the climatic data showed correlations between the total number of captured sand flies and both average temperature and average relative humidity. The total number of collected specimens was statistically higher when CDC miniature light traps were used compared to sticky traps. The number of females on the sticky traps was significantly higher than the number of males on the same trap type. Compared with the sticky traps, significantly more males were collected by CDC miniature light traps. This is the first record of Se. minuta in Romania after 50 years of no records (despite the trapping effort of the last 5 years in the country). Also, Ph. sergenti, previously present in this location, was not found. CONCLUSIONS: In the investigated natural habitat, the diversity of the sand fly species appears to have changed, with the predominance of Ph. neglectus instead of Ph. balcanicus and Se. minuta (recorded as the two predominant species in 1968-1970). A monomodal abundance trend was observed as in other regions of the country. The sand fly activity in this particular cave microhabitat appears to be longer than in other regions in Romania. Longer sand fly activity increases the zoonotic risk of various pathogenic species' transmission, with an impact on public health, as sand flies are important insect vectors.

Evidence of West Nile Virus (WNV) Circulation in Wild Birds and WNV RNA Negativity in Mosquitoes of the Danube Delta Biosphere Reserve, Romania, 2016.

West Nile virus (WNV) is a zoonotic flavivirus whose transmission cycle in nature includes wild birds as amplifying hosts and ornithophilic mosquito vectors. Bridge vectors can transmit WNV to mammal species potentially causing West Nile Fever. Wild bird migration is a mode of WNV introduction into new areas. The Danube Delta Biosphere Reserve (DDBR) is a major stopover of wild birds migrating between Europe and Africa. The aim of this study was to investigate the presence of WNV in the DDBR during the 2016 transmission season in wild birds and mosquitoes. Blood from 68 wild birds (nine different species) trapped at four different locations was analyzed by competitive ELISA and Virus Neutralization Test (VNT), revealing positive results in 8/68 (11.8%) of the wild birds by ELISA of which six samples (three from juvenile birds) were confirmed seropositive by VNT. Mosquitoes (n = 6523, 5 genera) were trapped with CDC Mini Light traps at two locations and in one location resting mosquitoes were caught. The presence of WNV RNA was tested in 134 pools by reverse transcription quantitative PCR (RT-qPCR). None of the pools was positive for WNV-specific RNA. Based on the obtained results, WNV was circulating in the DDBR during 2016.

Species diversity, host preference and arbovirus detection of Culicoides (Diptera: Ceratopogonidae) in south-eastern Serbia.

BACKGROUND: Culicoides (Diptera: Ceratopogonidae) is a genus of small biting midges (also known as "no-see ums") that currently includes 1368 described species. They are proven or suspected vectors for important pathogens affecting animals such as bluetongue virus (BTV) and Schmallenberg virus (SBV). Currently little information is available on the species of Culicoides present in Serbia. Thus, the aim of this study was to examine species diversity, host preference and the presence of BTV and SBV RNA in Culicoides from the Stara Planina Nature Park in south-eastern Serbia. RESULTS: In total 19,887 individual Culicoides were collected during three nights of trapping at two farm sites and pooled into six groups (Obsoletus group, Pulicaris group, "Others" group and further each group according to the blood-feeding status to freshly engorged and non-engorged). Species identification was done on subsamples of 592 individual Culicoides specimens by morphological and molecular methods (MALDI-TOF mass spectrometry and PCR/sequencing). At least 22 Culicoides species were detected. Four animal species (cow, sheep, goat and common blackbird) as well as humans were identified as hosts of Culicoides biting midges. The screening of 8291 Culicoides specimens in 99 pools for the presence of BTV and SBV RNA by reverse-transcription quantitative PCR were negative. CONCLUSIONS: The biodiversity of Culicoides species in the natural reserve Stara Planina was high with at least 22 species present. The presence of C. imicola Kieffer was not recorded in this area. Culicoides showed opportunistic feeding behaviour as determined by host preference. The absence of SBV and BTV viral RNA correlates with the absence of clinical disease in the field during the time of sampling. These data are the direct outcome of a training programme within the Institutional Partnership Project "AMSAR: Arbovirus monitoring, research and surveillance-capacity building on mosquitoes and biting midges" funded by the programme SCOPES of the Swiss National Science Foundation.

The complete sequence of a West Nile virus lineage 2 strain detected in a Hyalomma marginatum marginatum tick collected from a song thrush (Turdus philomelos) in eastern Romania in 2013 revealed closest genetic relationship to strain Volgograd 2007.

In this study the first complete sequence of the West Nile virus (WNV) lineage 2 strain currently circulating in Romania was determined. The virus was detected in a Hyalomma marginatum marginatum tick collected from a juvenile song thrush (Turdus philomelos) in the Romanian Danube Delta close to the city of Tulcea, end of August 2013. Our finding emphasizes the role of ticks in introduction and maintenance of WNV infections. Sequence analyses revealed close genetic relationship of the Romanian WNV strain to strain Reb_Volgograd_07_H, which was isolated from human brain tissue during an outbreak of West Nile neuroinvasive disease (WNND) in Russia in 2007. In 2010 the Eastern European lineage 2 WNV caused an outbreak of human WNND in Romania. Partial sequences from subsequent years demonstrated that this WNV strain became endemic in Eastern Europe and has been causing outbreaks of varying sizes in southern Russia since 2007 and in Romania since 2010.

European mustelids occupying pristine wetlands in the Danube Delta are infected with Trichinella likely derived from domesticated swine.

Abstract We analyzed 32 specimens from nine species of Mustelidae for Trichinella; six infections from two Trichinella species were observed from three host species. This provides documentation of Trichinella in Mustela erminea and Martes foina in Romania and Trichinella spiralis in a mustelid host from Europe. Trichinella spiralis continues to be a public challenge characterized by a wide host range and geographical distribution ( Pozio 2007 ). During the past 20 yr, Romania has had the most reported human cases of trichinellosis in the world ( Blaga et al. 2007 ). Transmission occurs among domesticated swine, rats, and wild mammals that feed by scavenging or predation ( Pozio 2000 ). Trichinella transmission to humans may occur by consumption of meat of livestock infected after exposure to wildlife ( Pozio et al. 2009 ).

First report of Borrelia burgdorferi sensu lato in two threatened carnivores: the marbled polecat, Vormela peregusna and the European mink, Mustela lutreola (Mammalia: Mustelidae).

BACKGROUND: Lyme disease is a widespread cosmopolitan zoonosis caused by species belonging to the genus Borrelia. It is transmitted from animal reservoir hosts to humans through hard - ticks of genus Ixodes which are vectors of the disease. CASE PRESENTATION: Borrelia burgdorferi sensu lato infection was identified in a marbled polecat, Vormela peregusna, and two European minks, Mustela lutreola, from Romania, by PCR. RFLP revealed the presence of a single genospecies, Borrelia burgdorferi sensu stricto. CONCLUSIONS: This is the first report of the Lyme disease spirochetes in the two mentioned hosts.