Mosquito surveillance is a major component of the surveillance and control of mosquito-borne arboviruses. Many principles and techniques are equally applicable to operations planned around different diseases. Where mosquito surveillance programmes already exist for other mosquito-borne diseases it may be practical to adapt these programmes to address West Nile virus surveillance needs. Modifications may be required depending on factors such as the climate of the area (e.g. sampling of larvae is not applicable during the winter in temperate climates, as mosquitoes are not active and breeding at that time), the main mosquitoes involved in transmission of a particular virus and the importance of the disease.

Surveillance of mosquitoes has several purposes related to gathering data about the disease and also for control efforts. There are three major aspects of mosquito surveillance: surveillance of larvae, surveillance of adults and surveillance for virus in mosquitoes: "The primary purpose of mosquito surveillance is to determine the species composition, abundance, and geographic distribution of mosquito species within each county by collection of larval and adult mosquitoes. Adult mosquito specimens that are submitted for virus testing can also provide evidence of infection status." (D72)

• Collection of mosquitoes, identification of species and pooling for virus detection should be conducted at a local level. However use of a central laboratory for virus detection has been recommended to ensure consistency and quality control. (P32.1.w19)

The goals of mosquito surveillance are to (D147):

• Use data on mosquito populations and virus infection rates to assess the threat of human disease;
• Identify geographic areas at high-risk of disease;
• Assess the need for and timing of intervention (management) events;
• Identify larval habitats for targeted control;
• Monitor the effectiveness of this type of surveillance, and improve prevention and control measures;
• Develop a better understanding of transmission cycles and potential vector species.

(D67, D73, D147, P32.1.w23, P32.1.w27)

Timing and level of surveillance:

The appropriate level of surveillance of mosquitoes will vary across the USA depending on the period of the year during which mosquitoes are active in a local area. The level of surveillance considered appropriate in different areas is likely to change as a greater understanding of the ecology of WN virus in North America develops and as the virus spreads westwards across the continent. Availability of financial and personnel resources are also likely to affect mosquito-based surveillance programs. (D67)

The transmission season of WN virus is based on the activity of the insect vectors, thus the period of surveillance varies depending on the vector season in a given geographic area; in some areas a long period of surveillance is required during the year. (D147)

• Northeastern and Midwestern USA: Active ecological surveillance should begin in early spring and continue through the fall (autumn) until mosquito activity ceases because of cold weather. Surveillance in urban and suburban areas should be emphasized. (D147)
  • During 2001-2002 onset of human illness onset in the northeastern states occurred as early as early July and as late as mid-November; in the same years cases in birds occurred as early as the first week of April and as late as the second week of December. (D147)
• Southern USA: Active ecological surveillance should be conducted year round in these areas. (D67, D147)
  • N.B. in the Southern states, WN virus circulates throughout the year, with activity detected from January to December. In 2001-2004, onset of human illness was reported from as early as April to as late as mid-December; equine and avian infections were reported in all months of the year in these geographical areas. (D147, P39.3.w1, J84.10.w5, J84.11.w4)
• Western USA:  Ecological surveillance should be encouraged beginning in early spring and continuing through the fall until mosquito activity ceases due to cold weather. (D147)
  • In 2002, WNV activity was first reported among humans and animals in Rocky Mountain states and among animals in Pacific coast states. These events occurred relatively late in the year (mid-August). (D147)
• Western Hemisphere outside the USA: Surveillance in other countries of the Western Hemisphere should be encouraged. (D67)
  • In Canada, in 2002 a WNV epidemic occurred in Ontario and Quebec provinces and an equine/avian epizootic occurred extending from the maritime provinces to Saskatchewan. By 2005, WV virus had been detected in Alberta, Manitoba, New Brunswick, Nova Scotia, Ontario, Quebec and Saskatchewan. (B526.21.w21, D147)
  • Development of surveillance systems capable of detecting WNV activity should be encouraged in the Caribbean and Central and South America. WNV surveillance should be integrated with dengue surveillance in these areas, and with yellow fever surveillance in areas where urban or peri-urban transmission of this virus occurs. (D147)
    • Wild bird and sentinel chicken surveillance has been recommended as part of surveillance for WNV in the Caribbean and in South America (Brazil). (J408.40.w1, W705.May08.w1)
    • By 2005, WV virus had been detected in equines and/or birds in Mexico, the Caribbean (the Bahamas, the Cayman Islands, Cuba, the Dominican Republic, Guadeloupe, Jamaica, Puerto Rico, Trinidad), Central America (Belize, Guatemala, El Salvador), and South America (Colombia); in 2006 it was detected in Venezuela and caused fatal illness in equines in Argentina; samples taken from birds in Argentina were first positive in 2005. (B526.21.w21, J84.12.w3, J84.13.w1, J84.14.w1, J270.45.w1, J491.19.w1)
• Southern Africa: transmission of WNV increases in the early months of the year, following heavy rainfall in spring and summer. (J84.11.w4)

Additional factors may affect decisions regarding surveillance effort:

• Human population density, landscape features such as catch basins, landfills, wetlands and forests as well as historic occurrence of virus should be used in designing sampling strategies. (P32.1.w19)
• Data from dead wild bird surveillance (dead bird density plotted geographically) may be used to assist in directing mosquito surveillance efforts. (P39.3.w12)

It has been suggested by the Centers for Disease Control (CDC) that for any mosquito control district a Minimum Entomological Surveillance Program would include (D67):

• Obtaining basic literature and expertise on mosquito identification, biology and surveillance;
• Developing contacts with established regional mosquito surveillance programs, and local and national mosquito associations;
• Development of a surveillance program for mosquito larvae, mosquito adults and detection of WN virus within mosquitoes;
• Establishment of a database and analysis of data on regular basis to evaluate disease risk, and to direct and evaluate control efforts. This would include the establishment of electronic data bases using GIS format to provide a spatial appreciation of anomalies in population measurements;
• Sharing of results with cooperating public health agencies and other mosquito control districts.
• (D67)

(D67)

The CDC Epidemic/Epizootic West Nile Virus in the United States: Guidelines for Surveillance, Prevention and Control 3rd Revision (D147) suggest the following minimal requirements of an entomological surveillance programme for West Nile virus:

The following example of an action plan for mosquito surveillance has been taken directly from the New York State West Nile Virus Response Plan - Guidance Document (D72): [Text copied directly]

• Guidelines for Arbovirus Surveillance Programs in the United States

• Epidemic/Epizootic West Nile Virus in the United States: Revised Guidelines for Surveillance, Prevention, and Control

• Hazard Assessment of the Organophosphates

• New York State West Nile Virus Response Plan - Guidance Document

• Health Canada - Municipal Mosquito Control Guidelines

• Data from dead wild bird surveillance (dead bird density plotted geographically) may be used to assist in directing mosquito larva surveillance efforts. (P39.3.w12)

Larval surveillance should include the location, mapping, characterization of aquatic habitats (mosquito breeding habitats), including use of GIS; the collection and identification of immature forms from larval habitats; and the determination of control options. Responsible control programs target vector and pest populations for control and avoid disturbing habitats which support benign species. (D67, D72)

• "Larval mosquitoes are collected by taking dip samples from a variety of habitats to identify species present in the area and to identify mosquito sources. Thorough mapping of larval habitats will facilitate larval control or source reduction activities. In addition, where larval management is not feasible, quantitative estimates of larval densities will permit anticipation of new adult emergences. Minimally, the number of larvae collected per dip and location where collected should be recorded to provide a basis for tracking larval production and association of larval density with resulting adult mosquito population density." (D147)

Identification of habitats:

Mosquitoes may breed anywhere that standing water is available. Natural and large-scale man-made habitats include temporary flooded areas, tidal (brackish) or fresh water wetlands, lakes, ponds and rivers, tree holes, water-holding plants, flooded depressions, stream edges where quiet water pools exist, municipal sewage or wastewater treatment plants, flooded basements of (abandoned) buildings etc. Examples of smaller man-made potential breeding habitats include household articles and discarded trash such as tires, drums, pails, garbage cans, plant pots, swimming pools, the tops of swimming pool covers, bottles, discarded automobiles and household appliances. (D70, D72)

Experienced personnel may be able to identify probable habitats where mosquito breeding occurs in a given area by means of a rapid reconnaissance survey. Detailed inspection is required to determine specific breeding sites at which larval sampling stations may be established. (D70)

Collection of larvae:

Different habitats such as ponds, tree holes and artificial containers must be searched for larvae of different species. (D70)

• Knowledge of the appropriate breeding habitats of the different mosquito species is important if the correct habitats are to be surveyed. 
• An understanding of larval behaviour is also required in order to collect larvae successfully, for example avoiding excessive water disturbance or the casting of a shadow over surface-dwelling larvae as this may cause them to dive to the bottom of the water. (D70)
• Sampling must be repeated every one to two weeks throughout the mosquito breeding season; areas free of larvae at one collection time may contain large numbers of larvae at a different point in the breeding season. (D70)
• Further information is provided in: Collection and Counting of Mosquito Larvae for West Nile Virus Control (Techniques)

Identification of larvae:

Correct identification of mosquito larvae is a specialist task and is best undertaken by appropriately trained persons; most established programs have a team of trained inspectors to collect larval specimens on a regular basis from known larval habitats, and perform systematic surveillance for new sources; properly trained mosquito identification specialists can separate mosquito nuisance and vector species. (D67, D70)

Record keeping:

Accurate and precise record keeping is essential. Records must identify the date, the site of inspection/collection, a description of the site (habitat type), the identity of the person undertaking the collection, the number of dips undertaken, and the number of larvae found per dip and in total for the site. (D70)

• "Minimally, the number of larvae collected 
• Examples of record forms for larval mosquito surveys are available, e.g. D73 -Appendix A.

• Collection and Counting of Mosquito Larvae for West Nile Virus Control (Techniques)
• Flaviviridae- West Nile Virus (Viral Type)
  • West Nile Virus - Intermediate Hosts and Vector Species (Viral Reports)
• Chemical Control for West Nile Virus
• Biological Control for West Nile Virus
• Habitat Management for Control of West Nile Virus
• Wild Bird and other Animal Surveillance for West Nile Virus

• "Adult mosquitoes are collected using a variety of trapping techniques and are used to identify the mosquito species and primary vector species present in an area and the relative density of those species. When coupled with virus detection protocols, mosquito collections can be screened for the presence of virus and provide a quantifiable index of WNV activity. Adequate sampling requires trapping regularly at representative sites throughout a community, and rapid testing of collections of sufficient size to detect low infection rates in the vector population. Minimally, adult mosquito density (number collected per trap night) and infection rate (number of individual mosquitoes estimated containing WNV per 1,000 specimens tested) should be recorded for each area to provide a basis for tracking mosquito density and virus incidence." (D147)
• Data from dead wild bird surveillance (dead bird density plotted geographically) may be used to assist in directing adult mosquito surveillance efforts. (P39.3.w12)

It is advantageous for a mosquito surveillance programme to have the ability to utilise both fixed trap sites and a flexible trapping system:

• A network of fixed trap sites is needed in order to develop a database allowing temporal and spatial evaluation of changes in mosquito population size. It is important to recognise that it may take an entire transmission season to identify the best fixed sites in an area and to establish the fixed network of trap sites. 
• A flexible trapping system will maximize the likelihood of obtaining virus isolates, for example, by moving traps to sites near suspect human cases or sites of crow deaths. Flexible trapping is also necessary if mosquito counts are used to evaluate targeted control efforts. 

(D67)

CDC Epidemic/Epizootic West Nile Virus in the United States: Revised Guidelines for Surveillance, Prevention and Control (D67) give the following guidelines for surveillance of adult mosquitoes: [Text copied directly]

Gravid traps are often used to monitor the ovipositing segment of populations of Culex pipiens complex - Northern house mosquito and Culex restuans - White dotted mosquito; CDC light traps baited with CO2, readily collect host-seeking Culex tarsalis in areas where this species is common, resting boxes are often used for collecting the bird-feeding mosquito Culiseta melanura, and pigeon-baited traps may be used to collect host-seeking Culex sp. mosquitoes which amplify WN virus. Collection of a representative sample of day-active species such as Aedes albopictus - Asian tiger mosquito may be more difficult: CDC light traps baited with CO2, used during the day, or Fay traps or traps using a counterflow geometry may be useful for these. (D147)

Use of mosquito surveillance information for control programs:

Information from mosquito surveillance efforts can be helpful both in determining when to conduct mosquito control and in monitoring the effectiveness of control activities. While it is not necessary to test all collected mosquitoes for the presence of WN virus, those that are tested can provide valuable information regarding spraying decisions. 

• It is important to realise that surveillance efforts to detect virus in mosquitoes are much more difficult to conduct than are similar efforts to detect virus in birds. Mosquito trapping is however the best way to determine the number and species of vector mosquitoes in an area and gathering this information may be more important than testing mosquitoes for WN virus. 
• It is important also to recognise that systematic mosquito trapping, requires specially trained staff and is time intensive. For localities which do not have such capacity, there are other potential sources of information on mosquito activity, for example visual inspection by staff of the area where a positive bird was found, or around human population centres for habitats likely to be conducive to mosquito breeding, and personal observations by staff of mosquito activity. 

(D72)

• Flaviviridae- West Nile Virus (Viral Type)
  • West Nile Virus - Intermediate Hosts and Vector Species (Viral Reports)
• Use of Light Traps for the Collection of Adult Mosquitoes (Techniques)
• Use of Oviposition Traps for the Collection of Adult Mosquitoes (Techniques)
• Chemical Control for West Nile Virus
• Biological Control for West Nile Virus
• Wild Bird and other Animal Surveillance for West Nile Virus

• "When coupled with virus detection protocols, mosquito collections can be screened for the presence of virus and provide a quantifiable index of WNV activity. Adequate sampling requires trapping regularly at representative sites throughout a community, and rapid testing of collections of sufficient size to detect low infection rates in the vector population. Minimally, adult mosquito density (number collected per trap night) and infection rate (number of individual mosquitoes estimated containing WNV per 1,000 specimens tested) should be recorded for each area to provide a basis for tracking mosquito density and virus incidence." (D147)
• Rapid detection and reporting of virus-positive samples are important for implementation of control measures. (P32.1.w19) 
  • Rapid testing can be used to help target applications of pesticides to areas where infected mosquitoes are present. (P32.1.w8)

It is important to determine the infection rates of mosquitoes by species. It is appropriate to focus initially on Culex spp. mosquitoes to provide first indication of WN virus presence. Once virus has been detected in Culex mosquitoes, the protocol should be changed: pool and test all potential vector species with emphasis on incriminated or suspected species. (D67)

• Following collection mosquitoes may be anaesthetised with triethylamine, the species identified and the mosquitoes sorted into pools of the same species, then frozen for transport to a central laboratory for virus detection/isolation. (P32.1.w19)
• Use of a central laboratory is recommended for virus detection to ensure consistency and quality control of results. (P32.1.w19)
• It is suggested that for mosquitoes collected at low temperatures (e.g. overwintering mosquitoes), holding at a higher temperature such as 26°C for a two to three days before testing may increase the rate of virus detection and isolation. (J110.38.w2)

Within New York State, the following prioritisation has been determined (D72): [Text copied directly]

Virus testing:

• RT-PCR of mosquito pools has proven to be a reliable method for use in surveillance. (D67, D147)
• Well-characterised antigen-capture ELISAs are now available for detection of WNV (and St. Louis encephalitis virus, SLE) in mosquito pools. (D147)
• Further information on different methods of detection of virus, viral RNA or viral genome is provided in: West Nile Virus - Detection and Identification Techniques (Viral Reports)

Laboratory case definitions: 

Laboratory definitions for mosquito infection from CDC Epidemic/Epizootic West Nile Virus in the United States: Guidelines for Surveillance, Prevention and Control 3rd Revision (D147) [Text copied directly]: 

(D147)

Additional notes on interpretation of findings:

• Preferably, the proportion of the mosquito population carrying WN virus should be expressed as the infection rate (IR): the estimated number of infected individual mosquitoes per 1,000 specimens tested [see: Minimum infection rate]. This is a more useful index of virus prevalence than simply the number of WN virus-positive mosquito pools collected during a defined time period at a given location. (D147)
• It is suggested that if WN virus is recovered from the legs of a mosquito then the infection may be considered to be disseminated, whereas if it is recovered only in the body it may be that the infection is nondisseminated, restricted only to the midgut. (J110.38.w2)
• It should be noted that although the detection of virus in overwintering mosquitoes confirms its presence, absence of detection of the virus in overwintering mosquitoes does not confirm absence of the virus since the prevalence of infection is probably low. (P32.1.w19)

Record keeping and protocols for sending specimens:

It is important to ensure that mosquito specimens for WN virus testing are correctly and completely labelled and are shipped in the correct containers and at the correct temperatures. Precise instructions should be sought from the laboratory to which the mosquitoes are to be sent in advance of the start of mosquito collection operations. Examples of appropriate forms and shipping instructions can be found in New York State West Nile Virus Response Plan - Guidance Document (D72) Appendix D and CDC Epidemic/Epizootic West Nile Virus in the United States: Guidelines for Surveillance, Prevention and Control 3rd Revision (D147) Appendix A.

• CDC requests that mosquitoes are shipped on dry ice. (D147- Appendix A)
• Details of requirements for samples sent to CDC are provided in: West Nile Virus - Detection and Identification Techniques (Viral Reports) - Sample Collection & Shipping

Potential Problems and Erroneous Results:

• Erroneous data may arise due to:
  • Misidentification of the mosquito species.
  • Accidental placement of a correctly-identified mosquito into the wrong holding dish.
  • The wrong species name being written on the data sheet or into a computer record.
  • Cross contamination between mosquito pools, either when originally sorted or in the laboratory where processing of specimens occurs: "A single leg from a WN virus-infected mosquito can contain up to 10,000 plaque-forming units of virus." This means that even minor cross-contamination (a single leg) could result in a separate mosquito pool being wrongly identified as infected.
  • Cross contamination by means of infected instruments (e.g. forceps contaminated with haemolymph from an infected mosquito).
  • Virus being contained within blood recently ingested by an engorged mosquito, rather than the mosquito actually being infected.

  (J214.267.w1)

Recent Experience:

• USA (New York City) 2001. study in 2001, testing mosquitoes (and birds) at three locations where single dead WN-virus positive birds had been found early in the transmission season in 2000, found WN virus-positive mosquitoes, mainly Culex pipiens (Culex pipiens complex - Northern and Southern house mosquitoes) / Culex restuans (Culex restuans - White dotted mosquito), at all three sites. (J91.67.w1)

• A study of the identification of 45 blood-fed field collected Culex spp. mosquitoes from New York City, checking morphological identification against molecular identification, found that although Culex salinarius - Unbanded saltmarsh mosquito mosquitoes were correctly distinguished from Culex pipiens and Culex restuans, 3/23 mosquitoes identified morphologically as Culex pipens (Culex pipiens complex - Northern and Southern house mosquitoes) were actually Culex restuans - White dotted mosquito. It was considered that there was a need for an improvement in the quality of mosquito identification. (P39.2.w3)

• In 2001 in the USA in 16/359 counties (4.5%) the earliest detection of WN virus activity was by detection of virus in mosquito pools. (P39.3.w7)

• Totals of WNV-positive mosquito pools reported to CDC were 515 in 2000 and 905 in 2001. (P39.3.w7)

• In New York City in 2001 mosquitoes provided the best means of early detection of WN virus transmission. (P39.3.w7)

• In Illinois in 2001 20/81 Culex spp. pools collected and tested in Cook County were positive; all the positive pools were collected 15th August to 12th October. The results were followed closely by positive dead birds in the same area. (P39.3.w20)

• During 2002 1.5 million mosquitoes were tested. A total of 6,033 WNV-positive pools of mosquitoes were detected and virus was detected in 29 species. The earliest WNV-positive in mosquitoes was in Culex restuans - White dotted mosquito from Monmouth County, New Jersey, collected 22 May 2002 while the latest positive was found in Culex quinquefasciatus - Southern house mosquito in Catham county, Georgia, 12 November 2002. [Data as of 21 Jan 2003]. (P39.4.w1)

• During 2003 a total of 7,725 mosquito pools positive for WN virus were reported from 38 states, the district of Columbia and New York City. [Data to 25 November 2003](N7.52.w6)

• In Chicago in 2002 positive mosquito pools were found concurrently with human cases. (P39.4.w6)

NOTE: Data on surveillance for WN virus in mosquitoes in the USA to 2007 is available summarised in map form. See: Map0001 - Spread of West Nile Virus in the USA (2000-2007) - Mosquitoes maps

• Flaviviridae- West Nile Virus (Viral Type)
  • West Nile Virus - Intermediate Hosts and Vector Species (Viral Reports)
  • West Nile Virus - Detection and Identification Techniques (Viral Reports)

There is some data to show that WN virus may overwinter in mosquitoes in the USA, although the level of importance of such overwintering to the maintenance of infection in an area is not yet known. 

• USA, 2000. "Virus isolation data suggest the 1999 outbreak was transmitted by Culex species mosquitoes, principally Culex pipiens. Overwintering mosquitoes of this species collected in January and February 2000 were found to be positive for WN virus". (J84.7.w33)
• USA (New York City) January to February 2000. WN virus RNA detected by TaqMan RT-PCR assay from three pools of mosquitoes (two pools confirmed by PCR as Culex pipiens) and virus was isolated from one pool, indicating overwintering of WN virus within infected, hibernating Culex pipiens mosquitoes. (N7.49.w1, N7.49.w2, J84.7.w26)

It is clear that more information is needed regarding the role that overwintering mosquitoes play in the maintenance of WN virus between transmission seasons (D147).

• Flaviviridae- West Nile Virus (Viral Type)
  • West Nile Virus - Intermediate Hosts and Vector Species (Viral Reports)
  • West Nile Virus - Detection and Identification Techniques (Viral Reports)
• West Nile Virus Disease (Viral Disease)
  • West Nile Virus Disease - Time Course-Persistence of Disease in a Susceptible Population (Disease Reports)