Victor A. Brugman

Epidemiological perspectives on West Nile virus surveillance in wild birds in Great Britain.

West Nile virus (WNV) is a zoonotic arthropod-borne pathogen with continued geographical expansion in Europe. We present and evaluate data on the temporal, spatial and bird species focus of the WNV surveillance programme in dead wild birds in Great Britain (2002-2009). During this period all bird samples tested negative for WNV. Eighty-two per cent of the 2072 submissions occurred during the peak period of vector activity with 53% tested during April-July before human and equine infection would be expected. Samples were received from every county, but there was significant geographical clustering (nearest neighbour index=0·23, P<0·001). Over 240 species were represented, with surveillance more likely to detect WNV in resident bird species (92% of submissions) than migrants (8%). Evidence indicates that widespread avian mortality is not generally a reported feature of WNV in Europe and hence additional activities other than dead bird surveillance may maximize the ability to detect WNV circulation before the onset of human and equine infections.

Blood-feeding patterns of native mosquitoes and insights into their potential role as pathogen vectors in the Thames estuary region of the United Kingdom

BackgroundThe range of vertebrate hosts on which species of mosquito blood-feed is an important parameter for identifying potential vectors and in assessing the risk of incursion and establishment of vector-borne pathogens. In the United Kingdom, studies of mosquito host range have collected relatively few specimens and used techniques that could only broadly identify host species. This study conducted intensive collection and analysis of mosquitoes from a grazing marsh environment in southeast England. This site provides extensive wetland habitat for resident and migratory birds and has abundant human nuisance biting mosquitoes. The aim was to identify the blood-feeding patterns of mosquito species present at the site which could contribute to the transmission of pathogens.MethodsTwice-weekly collections of mosquitoes were made from Elmley Nature Reserve, Kent, between June and October 2014. Mosquitoes were collected using resting boxes, by aspiration from man-made structures and using a Mosquito Magnet Pro baited with 1-octen-3-ol. Blood-fed specimens were classified according to the degree of blood meal digestion using the Sella scale and vertebrate origin determined using sequencing of a fragment of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes that were morphologically cryptic were identified to species level using multiplex PCR and sequencing methods.ResultsA total of 20,666 mosquitoes of 11 species were collected, and 2,159 (10.4%) were blood-fed (Sella scale II-VI); of these 1,341 blood-fed specimens were selected for blood meal analysis. Vertebrate origin was successfully identified in 964 specimens (72%). Collections of blood-fed individuals were dominated by Anopheles maculipennis complex (73.5%), Culiseta annulata (21.2%) and Culex pipiens form pipiens (10.4%). Nineteen vertebrate hosts comprising five mammals and 14 birds were identified as hosts for mosquitoes, including two migratory bird species. Feeding on birds by Culex modestus and Anopheles atroparvus populations in England was demonstrated.ConclusionsThis study expands the vertebrate host range of mosquitoes in the Thames estuary region of the UK. Feeding on both resident and migratory bird species by potential arbovirus vectors including Cx. pipiens f. pipiens and Cx. modestus indicates the potential for enzootic transmission of an introduced arbovirus between migratory and local bird species by native mosquito species.

How often do mosquitoes bite humans in southern England?: a standardised summer trial at four sites reveals spatial, temporal and site-related variation in biting rates

BackgroundThis field-based study examined the abundance and species complement of mosquitoes (Diptera: Culicidae) attracted to humans at four sites in the United Kingdom (UK). The study used a systematic approach to directly measure feeding by mosquitoes on humans at multiple sites and using multiple volunteers. Quantifying how frequently humans are bitten in the field by mosquitoes is a fundamental parameter in assessing arthropod-borne virus transmission.MethodsHuman landing catches were conducted using a standardised protocol by multiple volunteers at four rural sites between July and August 2013. Collections commenced two hours prior to sunset and lasted for a total of four hours. To reduce bias occurring due to collection point or to the individual attractiveness of the volunteer to mosquitoes, each collection was divided into eight collection periods, with volunteers rotated by randomised Latin square design between four sampling points per site. While the aim was to collect mosquitoes prior to feeding, the source of blood meals from any engorged specimens was also identified by DNA barcoding.ResultsThree of the four sites yielded human-biting mosquito populations for a total of 915 mosquitoes of fifteen species/species groups. Mosquito species composition and biting rates differed significantly between sites, with individual volunteers collecting between 0 and 89 mosquitoes (over 200 per hour) of up to six species per collection period. Coquillettidia richiardii (Ficalbi, 1889) was responsible for the highest recorded biting rates at any one site, reaching 161 bites per hour, whilst maximum biting rates of 55 bites per hour were recorded for Culex modestus (Ficalbi, 1889). Human-biting by Culex pipiens (L., 1758) form pipiens was also observed at two sites, but at much lower rates when compared to other species.ConclusionsSeveral mosquito species are responsible for human nuisance biting pressure in southern England, although human exposure to biting may be largely limited to evening outdoor activities. This study indicates Cx. modestus can be a major human-biting species in the UK whilst Cx. pipiens f. pipiens may show greater opportunistic human-biting than indicated by earlier studies.

Quantification of within- and between-farm dispersal of Culicoides biting midges using an immunomarking technique

Summary Culicoides biting midges (Diptera, Ceratopogonidae) are vectors of arboviruses that cause significant economic and welfare impact. Local‐scale spread of Culicoides‐borne arboviruses is largely determined by the between‐farm movement of infected Culicoides. Study of the dispersal behaviour of Culicoides by capture–mark–recapture (CMR) is problematic due to the likelihood of mortality and changes in behaviour upon capture caused by the small size and fragility of these insects, evidenced by low recapture rates. To counter the problem of using CMR with Culicoides, this study utilised an ovalbumin immunomarking technique to quantify the within‐ and between‐farm dispersal of Culicoides in southern England. Both within‐ and between‐farm dispersal of Culicoides was observed. Of the 9058 Culicoides collected over 22 nights of trapping, 600 ovalbumin‐positive Culicoides, of 12 species including those implicated as arbovirus vectors, were collected with a maximum dispersal distance of 3125 m. This study provides the first species‐level data on the between‐farm dispersal of potential bluetongue, Schmallenberg and African horse sickness virus vectors in northern Europe. High‐resolution meteorological data determined upwind and downwind flight by Culicoides had occurred. Cumulative collection and meteorological data suggest 15·6% of flights over 1 km were upwind of the treatment area and 84·4% downwind. Synthesis and applications. The use of immunomarking eliminates the potential adverse effects on survival and behaviour of insect collection prior to marking, substantially improving the resolution and accuracy of estimates of the dispersal potential of small and delicate vector species such as Culicoides. Using this technique, quantification of the range of Culicoides dispersal with regard to meteorological conditions including wind direction will enable improved, data‐driven modelling of the spread of Culicoides‐borne arboviruses and will inform policy response to incursions and outbreaks.

Detection of Theileria orientalis in mosquito blood meals in the United Kingdom

Theileria spp. are tick-borne protozoan parasites that infect a wide range of wild and domestic animals. In this study, the utility of xenosurveillance of blood-fed specimens of Culiseta annulata for detecting the presence of piroplasms in livestock was investigated. Blood-fed mosquitoes were collected at Elmley National Nature Reserve, Kent, United Kingdom. All specimens were morphologically identified, and DNA barcoding was used to confirm the morphological identification. Both the vertebrate host species and Theileria genome was detected within the bloodmeal by real-time PCR. Sequencing was used to confirm the identity of all amplicons. In total, 105 blood-fed mosquitoes morphologically identified as Cs. annulata were collected. DNA barcoding revealed that 102 specimens were Cs. annulata (99%), while a single specimen was identified as Anopheles messeae. Two specimens could not be identified molecularly due to PCR amplification failure. Blood meal analysis revealed that Cs. annulata fed almost exclusively on cattle at the collection site (n=100). The application of a pan-piroplasm PCR detected 16 positive samples (15.2%) and sequence analysis of the amplicons demonstrated that the piroplasms present in the blood meal belonged to the Theileria orientalis group. This study demonstrates how xenosurveillance can be applied to detecting pathogens in livestock and confirms the presence of Theileria species in livestock from the United Kingdom.

Molecular approaches for blood meal analysis and species identification of mosquitoes (Insecta: Diptera: Culicidae) in rural locations in southern England, United Kingdom

Thirty-four species of Culicidae are present in the UK, of which 15 have been implicated as potential vectors of arthropod-borne viruses such as West Nile virus. Identification of mosquito feeding preferences is paramount to the understanding of vector-host-pathogen interactions which, in turn, would assist in the control of disease outbreaks. Results are presented on the application of DNA barcoding for vertebrate species identification in blood-fed female mosquitoes in rural locations. Blood-fed females (n = 134) were collected in southern England from rural sites and identified based on morphological criteria. Blood meals from 59 specimens (44%) were identified as feeding on eight hosts: European rabbit, cow, human, barn swallow, dog, great tit, magpie and blackbird. Analysis of the cytochrome c oxidase subunit I mtDNA barcoding region and the internal transcribed spacer 2 rDNA region of the specimens morphologically identified as Anopheles maculipennis s.l. revealed the presence of An. atroparvus and An. messeae. A similar analysis of specimens morphologically identified as Culex pipiens/Cx. torrentium showed all specimens to be Cx. pipiens (typical form). This study demonstrates the importance of using molecular techniques to support species-level identification in blood-fed mosquitoes to maximize the information obtained in studies investigating host feeding patterns.

The Role of Culex pipiens L. (Diptera: Culicidae) in Virus Transmission in Europe.

Over the past three decades, a range of mosquito-borne viruses that threaten public and veterinary health have emerged or re-emerged in Europe. Mosquito surveillance activities have highlighted the Culex pipiens species complex as being critical for the maintenance of a number of these viruses. This species complex contains morphologically similar forms that exhibit variation in phenotypes that can influence the probability of virus transmission. Critical amongst these is the choice of host on which to feed, with different forms showing different feeding preferences. This influences the ability of the mosquito to vector viruses and facilitate transmission of viruses to humans and domestic animals. Biases towards blood-feeding on avian or mammalian hosts have been demonstrated for different Cx. pipiens ecoforms and emerging evidence of hybrid populations across Europe adds another level of complexity to virus transmission. A range of molecular methods based on DNA have been developed to enable discrimination between morphologically indistinguishable forms, although this remains an active area of research. This review provides a comprehensive overview of developments in the understanding of the ecology, behaviour and genetics of Cx. pipiens in Europe, and how this influences arbovirus transmission.

Detection of malaria sporozoites expelled during mosquito sugar feeding

Malaria is a severe disease of global importance transmitted by mosquitoes of the genus Anopheles. The ability to rapidly detect the presence of infectious mosquitoes able to transmit malaria is of vital importance for surveillance, control and elimination efforts. Current methods principally rely on large-scale mosquito collections followed by labour-intensive salivary gland dissections or enzyme-linked immunosorbent (ELISA) methods to detect sporozoites. Using forced salivation, we demonstrate here that Anopheles mosquitoes infected with Plasmodium expel sporozoites during sugar feeding. Expelled sporozoites can be detected on two sugar-soaked substrates, cotton wool and Whatman FTA cards, and sporozoite DNA is detectable using real-time PCR. These results demonstrate a simple and rapid methodology for detecting the presence of infectious mosquitoes with sporozoites and highlight potential laboratory applications for investigating mosquito-malaria interactions. Our results indicate that FTA cards could be used as a simple, effective and economical tool in enhancing field surveillance activities for malaria.

13. Semiochemical tools for a new generation of livestock pest control

Arthropod-borne diseases and ectoparasite infestation of livestock remain a serious veterinary and economic problem worldwide. Furthermore, nuisance biting by haematophagous arthropods can result in a significant disruption to livestock behaviour and production. Existing tools for managing pest arthropods have so far not been successful in providing elimination or effective control of many pest arthropod species. The rapid emergence and re-emergence of pathogens and the on-going burden of those currently circulating mean that the need to develop new tools and interventions for pest management is urgent. A vast array of host-derived, arthropod-derived and environmental semiochemicals regulate the behaviour of pest arthropods and, therefore, many of these present promising targets for control interventions. This chapter details the principles of semiochemical-based approaches to arthropod surveillance and control and the major arthropod behavioural targets (host-seeking, sugar-feeding and oviposition/larviposition behaviour) that need to be understood to enable successful interventions to take place. The chapter then reviews current progress towards semiochemical-based approaches for the surveillance and control of major arthropod pests: tsetse flies (Diptera: Glossinidae), mosquitoes (Diptera: Culicidae), biting midges (Diptera: Ceratopogonidae), sand flies (Diptera: Phlebotominae), black flies (Diptera: Simuliidae), horse flies (Diptera: Tabanidae), horn flies and stable flies (Diptera: Muscidae), and ticks (Ixodida: Ixodoidea).