Belgian Anopheles plumbeus Mosquitoes Are Competent for Japanese Encephalitis Virus and Readily Feed on Pigs, Suggesting a High Vectorial Capacity.

Anopheles plumbeus, a day-active mosquito known to feed aggressively on humans, was reported as a nuisance species near an abandoned pigsty in Belgium. Since Japanese encephalitis virus (JEV) is an emerging zoonotic flavivirus which uses pigs as amplification hosts, we investigated (1) whether An. plumbeus would feed on pigs and (2) its vector competence for JEV, to investigate whether this species could be a potential vector. Three- to seven-day-old F0-generation adult mosquitoes, emerged from field-collected larvae, were fed on a JEV genotype 3 Nakayama strain spiked blood meal. Blood-fed mosquitoes were subsequently incubated for 14 days at two temperature conditions: a constant 25 °C and a 25/15 °C day/night temperature gradient. Our results show that An. plumbeus is a competent vector for JEV at the 25 °C condition and this with an infection rate of 34.1%, a dissemination rate of 67.7% and a transmission rate of 14.3%. The vector competence showed to be influenced by temperature, with a significantly lower dissemination rate (16.7%) and no transmission when implementing the temperature gradient. Moreover, we demonstrated that An. plumbeus readily feeds on pigs when the opportunity occurs. Therefore, our results suggest that Belgian An. plumbeus mosquitoes may play an important role in the transmission of JEV upon an introduction into our region if temperatures increase with climate change.

Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by Stomoxys calcitrans.

Lumpy skin disease virus (LSDV) is a vector-transmitted capripox virus that causes disease in cattle. Stomoxys calcitrans flies are considered to be important vectors as they are able to transmit viruses from cattle with the typical LSDV skin nodules to naive cattle. No conclusive data are, however, available concerning the role of subclinically or preclinically infected cattle in virus transmission. Therefore, an in vivo transmission study with 13 donors, experimentally inoculated with LSDV, and 13 naïve acceptor bulls was performed whereby S. calcitrans flies were fed on either subclinical- or preclinical-infected donor animals. Transmission of LSDV from subclinical donors showing proof of productive virus replication but without formation of skin nodules was demonstrated in two out of five acceptor animals, while no transmission was seen from preclinical donors that developed nodules after Stomoxys calcitrans flies had fed. Interestingly, one of the acceptor animals which became infected developed a subclinical form of the disease. Our results show that subclinical animals can contribute to virus transmission. Therefore, stamping out only clinically diseased LSDV-infected cattle could be insufficient to completely halt the spread and control of the disease.

Relevant Day/Night Temperatures Simulating Belgian Summer Conditions Reduce Japanese Encephalitis Virus Dissemination and Transmission in Belgian Field-Collected Culex pipiens Mosquitoes.

Japanese encephalitis virus (JEV), a zoonotic mosquito-borne Flavivirus, can be considered an emerging infectious disease. Therefore, vector competence studies with indigenous mosquitoes from regions where JEV is not yet endemic are of great importance. In our study, we compared the vector competence of Culex pipiens mosquitoes emerged from Belgian field-caught larvae under two different temperature conditions: a constant 25 °C and a 25/15 °C day/night temperature gradient representing typical summer temperatures in Belgium. Three- to seven-day-old F0-generation mosquitoes were fed on a JEV genotype 3 Nakayama strain spiked blood-meal and incubated for 14 days at the two aforementioned temperature conditions. Similar infection rates of 36.8% and 35.2% were found in both conditions. The observed dissemination rate in the gradient condition was, however, significantly lower compared to the constant temperature condition (8% versus 53.6%, respectively). JEV was detected by RT-qPCR in the saliva of 13.3% of dissemination positive mosquitoes in the 25 °C condition, and this transmission was confirmed by virus isolation in 1 out of 2 RT-qPCR positive samples. No JEV transmission to saliva was detected in the gradient condition. These results suggest that JEV transmission by Culex pipiens mosquitoes upon an accidental introduction in our region is unlikely under current climatic conditions. This could change in the future when temperatures increase due to climate change.

Comparison of Serological Methods for Tick-Borne Encephalitis Virus-Specific Antibody Detection in Wild Boar and Sheep: Impact of the Screening Approach on the Estimated Seroprevalence.

Tick-borne encephalitis virus (TBEV) is a flavivirus transmitted by ticks. Serological screenings in animals are performed to estimate the prevalence and distribution of TBEV. Most screenings consist of a primary screening by ELISA, followed by confirmation of positive samples by plaque reduction neutralization tests (PRNTs). In this study, 406 wild boar sera were tested with 2 regularly used commercial ELISAs for flavivirus screening in animals (Immunozym FSME (TBEV) IgG All Species (Progen) and ID Screen West Nile Competition (Innovative Diagnostics)) and PRNTs for TBEV and USUTU virus. The results showed that the Immunozym and IDScreen ELISAs had low relative sensitivities of 23% and 20%, respectively, compared to the PRNT results. The relative specificities were 88% and 84% due to cross reactions with USUTU virus-specific antibodies. The minimal TBEV prevalence in our sample set was 8.6% when determined by PRNT. When the screening approach of ELISA testing followed by PRNT confirmation was applied, a TBEV seroprevalence of only 2.0% and 1.7% was found. The suboptimal performance of the ELISAs was confirmed by testing sera collected from experimentally TBEV-infected sheep. While the PRNT detected TBEV specific antibodies in 94% of samples collected between 7 and 18 days post-infection, the ELISAs classified only 50% and 31% of the samples as positive. Both routinely used ELISAs for TBEV antibody screening in animal sera were shown to have a low sensitivity, potentially leading to an underestimation of the true prevalence, and furthermore cross-react with other flavivirus antibodies.

Tick-Borne Encephalitis Virus Prevalence in Sheep, Wild Boar and Ticks in Belgium.

Tick-borne encephalitis virus (TBEV) is the most important tick-borne zoonotic virus in Europe. In Belgium, antibodies to TBEV have already been detected in wildlife and domestic animals, but up-to-date prevalence data for TBEV are lacking, and no studies have assessed its seroprevalence in sheep. Serum samples of 480 sheep from all over Belgium and 831 wild boar hunted in Flanders (northern Belgium) were therefore screened for TBEV antibodies by ELISA and plaque reduction neutralization test (PRNT), respectively. The specificity of positive samples was assessed by PRNTs for TBEV and the Louping Ill, West Nile, and Usutu viruses. TBEV seroprevalence was 0.42% (2/480, CI 95%: 0.11-1.51) in sheep and 9.27% (77/831, CI 95%: 7.48-11.43) in wild boar. TBEV seroprevalence in wild boar from the province of Flemish Brabant was significantly higher (22.38%, 15/67) compared to Limburg (7.74%, 34/439) and Antwerp (8.61%, 28/325). Oud-Heverlee was the hunting area harboring the highest TBEV seroprevalence (33.33%, 11/33). In an attempt to obtain a Belgian TBEV isolate, 1983 ticks collected in areas showing the highest TBEV seroprevalence in wild boars were tested by real-time qPCR. No TBEV-RNA-positive tick was detected. The results of this study suggest an increase in TBEV prevalence over the last decade and highlight the need for One-Health surveillance in Belgium.

Japanese Encephalitis Virus Interaction with Mosquitoes: A Review of Vector Competence, Vector Capacity and Mosquito Immunity.

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus and a major cause of human viral encephalitis in Asia. We provide an overview of the knowledge on vector competence, vector capacity, and immunity of mosquitoes in relation to JEV. JEV has so far been detected in more than 30 mosquito species. This does not necessarily mean that these species contribute to JEV transmission under field conditions. Therefore, vector capacity, which considers vector competence, as well as environmental, behavioral, cellular, and biochemical variables, needs to be taken into account. Currently, 17 species can be considered as confirmed vectors for JEV and 10 other species as potential vectors. Culex tritaeniorhynchus and Culex annulirostris are considered primary JEV vectors in endemic regions. Culex pipiens and Aedes japonicus could be considered as potentially important vectors in the case of JEV introduction in new regions. Vector competence is determined by various factors, including vector immunity. The available knowledge on physical and physiological barriers, molecular pathways, antimicrobial peptides, and microbiome is discussed in detail. This review highlights that much remains to be studied about vector immunity against JEV in order to identify novel strategies to reduce JEV transmission by mosquitoes.

Temperature and food sources influence subadult development and blood-feeding response of Culicoides obsoletus (sensu lato) under laboratory conditions.

BACKGROUND: Culicoides obsoletus (s.l.) is the most abundant Culicoides species in northern Europe and an important vector of bluetongue virus and Schmallenberg virus. Nevertheless, information on its subadult life stages remains scarce and no laboratory-reared colony exists. METHODS: C. obsoletus (s.l.) adults were collected in Belgium and transferred to the laboratory in an attempt to establish a laboratory-reared colony. C. obsoletus (s.l.) were reared from eggs to adults at different temperatures (28 °C, 24 °C, 20/16 °C) and under different food regimes. RESULTS: The most suitable temperature for rearing seemed to be 24 °C for most developmental parameters, but resulted in a biased 3:1 male/female sex ratio. The latter could be optimized to a 1:1 sex ratio when a 20/16 °C day/night temperature gradient was applied, but rearing at these low temperature conditions resulted in significantly lower egg hatching and pupation rates and a longer subadult development time. Independent of the rearing temperature, adding dung as an additional food source during larval development resulted in a significantly higher adult emergence rate and a decrease in subadult development time. Furthermore, blood-feeding rates of field-collected C. obsoletus (s.l.) were compared for different blood sources and feeding systems. The overall blood-feeding success was low and only successful with cotton pledgets (2.7% blood-fed midges) and through a membrane system with chicken skin (3.5% blood-fed midges). Higher feeding rates were obtained on cattle blood compared to sheep blood. CONCLUSIONS: These results will help us to determine the necessary conditions to rear a viable laboratory colony of this important vector species, although further optimization is still required.

Longitudinal monitoring of Culicoides in Belgium between 2007 and 2011: local variation in population dynamics parameters warrant cautious use of monitoring data.

BACKGROUND: Several European countries suffered important economic losses during the past decade due to the emergence of bluetongue and Schmallenberg viruses. Both are viruses of veterinary importance and are spread by Culicoides spp. This triggered many European countries to start Culicoides population monitoring. Recently a one year monitoring study at 16 sites in Belgium revealed that important variation existed in Culicoides abundance and species diversity between collection sites. In order to analyze whether this variation is consistent over years, a detailed analysis of monitoring data collected at seven locations in Belgium between 2007 and 2011 was performed in this study. At all locations, biting midges were collected with OVI black light traps set-up in close proximity to livestock. RESULTS: In total, 42 different Culicoides species were morphologically identified. Species of the subgenus Avaritia represented 83% of all collected midges. Nevertheless, important differences in species composition were found between sites. Furthermore, statistical differences between sites were found for the total and maximum annual abundance, showing that a consistent higher or lower number of Culicoides could be collected depending on the selected collection site. Yearly, up to 16 and 30-fold differences in total and maximum annual abundances between sites, respectively, were found. Also the month in which most Culicoides were collected varied greatly between years, both at local (from May to October) and country level [May (2008), June (2010), July (2009), August (2011), October (2007)]. Finally, the average vector-free period over all sites and years was 173 days and could roughly be defined between November and the end of April. Interestingly, important yearly variations of up to two months in the duration of the vector-free period were found between the studied collection sites. In contrast to the abundance parameters, no specific sites could however be identified where monitoring consistently showed shorter or longer vector-free periods. CONCLUSIONS: In conclusion, our results show that the selection of collection sites for Culicoides monitoring, even in a small country such as Belgium, strongly influences abundance parameters and that yearly variation in seasonality occurs. This emphasizes that care should be taken when using such parameters in risk assessments for transmission of Culicoides-borne diseases and that more clear and strict guidelines for Culicoides monitoring should be considered when monitoring data are used for legislative purposes.

Unchanged Schmallenberg virus seroprevalence in the Belgian sheep population after the vector season of 2014 and 2015 despite evidence of virus circulation.

Schmallenberg virus (SBV) emerged in North-Western Europe in 2011 and induces congenital defects in ruminants. Many epidemiological studies were undertaken to study the spread of the virus during the first two years after its emergence, but little data is available on the current antibody protection rate against SBV. A cross-sectional seroprevalence study was therefore carried out in the Belgian sheep population and showed that the total seroprevalence against SBV was 26% (CI95%: 21-32) at the end of the vector season of 2015, being significantly lower than the seroprevalence of 84% detected after the outbreak in 2011. Nevertheless, 63% (CI95%: 51-73) of the Belgian sheep flocks still had a certain level of protection against SBV. Despite the fact that PCR detection of SBV in aborted calves in April 2016 evidenced that SBV had circulated in 2015, no change in seroprevalence between 2014 and 2015 was found in the Belgian sheep population.

Increased detection of Aedes albopictus in Belgium: no overwintering yet, but an intervention strategy is still lacking.

In 2013 and 2014, routine surveillance for invasive mosquito species was implemented in Belgium at 13 potential points of entry. Following the introduction of Aedes (Stegomyia) albopictus (Skuse 1895) to Belgium via a used tyre import company (Vrasene, Province of East Flanders) in July 2013, one female and 17 larvae were collected outdoors during a period of intensive surveillance in summer and autumn 2013, but no control measures were implemented. Although climatic conditions were suitable during the winter of 2013-2014, this reproducing population did not overwinter. Lack of genetic variation, incomplete diapause adaptation and egg desiccation due to long dry periods during diapause or competition with endemic species are possible reasons. More studies on the diapause/longevity of Ae. albopictus eggs in northern temperate climatic conditions and on the competition with endemic species in western and central Europe are warranted to assess the potential for this invasive mosquito to overwinter. Furthermore, following the detection of four Ae. albopictus larvae in a shipment of lucky bamboo at the port of Antwerp in August 2014, one female, one male, 11 pupae and six larvae were collected at the destined lucky bamboo company (Lochristi, Province of East Flanders) in autumn 2014. In this case, immediate control measures were successfully implemented at the nursery. Because of increasing threats and the absence of an invasive mosquito species control policy in Belgium, the need for a permanent vector surveillance and control plan has never been so high.

Implementation of surveillance of invasive mosquitoes in Belgium according to the ECDC guidelines.

BACKGROUND: In 2012, the new guidelines for the surveillance of IMS in Europe, produced by the European Centre for Disease Prevention and Control (ECDC), were tested in Belgium. This study aimed at (1) testing the usefulness and applicability in the field of the ECDC guidelines for the surveillance of IMS in Europe and (2) surveying IMS throughout Belgium. METHODS: First, the scenarios, which Belgium is facing, were identified according to the ECDC guidelines. Second, the surveillance strategy and the methods were identified based on the guidelines and adjusted to the Belgium context. Two areas colonised by IMS and 20 potential points of entry (PoE) were selected. Mosquito Magnet Liberty Plus (CO2-baited) traps (23) and oviposition traps (147) were set-up, and larval sampling was performed monthly or bi-monthly from July till October 2012. Finally, the costs and workload of the surveillance activities were compared to the estimates provided by the ECDC guidelines. RESULTS: Surveillance at 20 potential PoE (complying with scenario 1) revealed that no new IMS were established in Belgium. Surveillance at two sites colonised by IMS (scenario 2) indicated that although control measures have drastically reduced the Ae. j. japonicus population this species is still present. Furthermore, Ae. koreicus is permanently established. For both scenarios, the problems encountered are discussed and recommendations are given. In addition, the actual workload was lower than the estimated workload, while the actual costs were higher than the estimated ones. CONCLUSIONS: The ECDC guidelines are helpful, applicable and efficient to implement surveillance of IMS in Belgium. Recommendations were customised to the local context (political demands, salary and investment costs, and existing expertise). The workload and costs related to the preparatory phase (i.e., planning, contacts with the PoE, writing a protocol) were found to be missing in the cost evaluation suggested in the guidelines. Updates on the occurrence of IMS in Belgium and the related risk for disease agents they can transmit will only be available once a structured and permanent surveillance system is implemented.