Arbovirus Field Surveillance Techniques

The following information is presented to introduce the basic procedures of field surveillance. Completion of proper training and adherence to procedures as listed under the guidelines is necessary for participation in the Arbovirus Surveillance Program.

Pathogenic mosquito-borne encephalitis viruses affect the well-being of humans in Texas and are a continuing public health threat. Although most attention is given to this infectious group of diseases when explosive outbreaks occur, scattered cases are reported each year.

• Purposes
• Materials
• Preparation of Materials (Supplied by the Laboratory)
• Methods
• Quality Control (Live, Adult Mosquito Shipments)

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1. Collection of vector mosquitoes to be shipped to the Laboratory for identification and arboviral isolation studies.
2. Determination of vector mosquito breeding sites.
3. Training of field investigators in techniques of surveillance.

1. Adult mosquitoes

1. Mosquito collection traps
2. Mechanical aspirators
3. Flashlight
4. Labeled mosquito shipping boxes
5. Mosquito collection cartons
6. Newspaper or other packing material
7. 1 quart plastic container filled with water and frozen prior to sending mosquitoes, or polar packs

2. Larval mosquitoes

1. Dipper, 8 oz., long handle (4 feet)
2. Vials, 6 dram with screw caps (or other vials with secure lids)
3. Disposable bulb pipet ("eye dropper")
4. Alcohol, 70% isopropyl
5. Mailing containers
6. Mosquito submission forms

Preparation of Materials:
(Supplied by the Laboratory)

1. Mosquito shipping boxes

   These boxes consist of an outer and inner box, which are separated by fiber insulation. A return-addressed label is attached to the mosquito shipping box. One inside and one outside box flap has the following information written with a bold, permanent marker: "MOSQUITO SURVEILLANCE." A box may be used repeatedly. Plastic containers with ice or polar packs along with newspaper are placed in each box.

2. Mosquito Collection Cartons

   These cartons are made from pint, plastic containers with lids especially prepared for proper airflow. A corked hole is located on the side of the container. A mosquito submission form must accompany each container. Attach it to the container with a rubber band.

3. Larval collecting kits

   These kits include: small vials with secure leak-proof lids and mosquito submission forms.

1. An overall perspective of the area to be surveyed is possible by consulting location (county, city, area) maps, discussing mosquito problems and cases of encephalitis with local officials (sanitarians, mosquito control personnel, Zoonosis Control investigators), and reviewing any records pertaining to mosquito activities in that area. Areas where mosquitoes are most likely to pose problems are circled on the map and/or recorded on a referral list (preferably in convenient order of investigation).
2. A map and/or referral list is carried with field investigators when they begin fieldwork. Materials for live adult mosquito collections are collection traps, insulated mosquito shipping boxes, mosquito collection cartons with attached mosquito report forms, aspirators, flashlights, and frozen containers that are kept in the shipping boxes. If larval collections are to be conducted, vials, dippers, bulb pipets, alcohol, and mosquito submission forms are also taken.

   Note: Whenever both larval and adult collections are to be conducted, specimens in both stages, if present, should be taken from the same address. Use the proper collection containers for the type of stage. Adults should never be placed in alcohol or other fluid as this will alter important taxonomic characteristics and will not be processed.

3. Each area marked on the map or list should be investigated. Although the amount of time spent at each site will vary, one should allow sufficient time for sites separated by a considerable distance. Random surveying of sites in scheduled areas may prove productive.
4. Permission from the property owner or other authorized person must be secured prior to surveying any site. It is usually most practical to seek such permission at the time of arrival at that site. Sometimes, due to long travel distances, permission may be sought by telephone prior to leaving headquarters.
5. Suitable mosquito collecting locations at a site may be found by:

1. Questioning a resident or property owner about recent mosquito activity.
2. Observing any attempts to feed by daytime biting mosquitoes.
3. Examining any fairly open structures that have areas or corners somewhat protected from sun and wind; some frequently productive collecting locations are garages, chicken houses, barns, stables, under bridges, and large diameter storm drains. Any structure housing chickens and/or livestock is particularly suitable because many species of mosquitoes are attracted to chickens and will readily feed on them. Diurnal or evening biters are most often found at these locations. Therefore, surveying at sites not listed but where these animals are noticed can be productive.

6. Daytime biters that are encountered while surveying may be removed from the person (such as from the pants leg) by using an aspirator. Several specimens may be collected at a time (making certain that either suction is maintained or the open end of rigid tubing is covered to prevent specimens from escaping) and then blown into the mosquito collection containers. The mosquitoes are introduced into the containers by removing the cork, blowing the mosquitoes through the hole, and rapidly reinserting the cork to prevent loss of specimens.
7. Mosquitoes found in structures are removed from their resting sites (webbing, boards, tires, walls, etc.) and placed in the cartons. Because most specimens found in this type of habitat are frequently in the darkest and most protected corners or containers, a flashlight is necessary to locate individual specimens.
8. Separate cartons should be used for different collecting locations at a site. For instance, mosquitoes collected from the pants leg while surveying the grass at a site should be in a different carton than those collected from a chicken coop at the same site. Of course, different sites will also require separate cartons. Collect about 50 mosquitoes at a site; then enter all collection information on the mosquito submission from that accompanies the carton. Please use a ballpoint pen. Attach the form to the carton with a rubber band.

   Note: The above method is most often used; however, if precise information pertaining to location and site is not important to the investigator, mosquitoes may be combined in the same cartons and labeled as desired (such as county, city, block, or code number).

9. Collected specimens are placed into a cooler that also contains several frozen containers. This will keep the live specimens cool and relatively inactive during the remainder of the survey.
10. Following surveillance activities, the collected mosquitoes are returned to headquarters. Plastic containers with frozen water or polar packs are placed into the shipping box with the collecting cartons.

    IMPORTANT: Several moistened paper towels or moistened newspaper should be added to the inner box to keep humidity at a high level. Avoid saturating the paper because excess water in the cartons will damage the mosquitoes.

11. The prepared shipping box is then labeled for proper transit to the DSHS Laboratory. Shipment by bus has proven most efficient for the majority of program participants.
12. Examine standing water to collect larval mosquitoes. The following types of water are most often productive:

1. Roadside ditches and other drainage water
2. Artificial containers (such as birdbaths, discarded or stacked tires, vases, watering troughs, barrels)
3. Ponds, stock tanks, creeks. The amount of larvae present will, in large part, depend on the amount of aquatic predators that may be present.
4. Other areas. Almost any water that stands for several days may be productive. It can be worthwhile checking such habitats as tree holes, livestock tracks (following rain), and clogged rain gutters.

13. To determine the presence of larvae, take a sample of water using a dipper. If larvae are observed, remove them from the dipper with a bulb pipet and place them in a vial. Fill in the mosquito submission form with a ballpoint pen and attach it to the vial with a rubber band.

    Note: A relative amount of immature mosquitoes at various sites may be estimated by taking a count of larvae and pupae taken in each dipper sampling. If this information is desired, a record should be kept of each dip or average of several dips per site.

14. If larvae are mature (4th instar), alcohol should be placed in the vial to preserve specimens until they can be identified. Alcohol should be composed of at least 50 percent (estimated) of the liquid volume; therefore, it may be necessary to drain off some of the collected water. The loss of specimens that may occur while draining the water can be avoided by placing a small piece of cheesecloth, filter paper, or paper toweling over the mouth of the vial when pouring off water or by using a bulb pipet to remove water. Immature specimens (1st through 3rd instar) are quite small and may lack taxonomic characteristics true to the mature larval stages of species. Therefore, these specimens can be left in the collection water for a day or two until they develop into mature larvae prior to submission in alcohol.
15. Vials with mosquito larvae for identification may be sent to the Laboratory in one of two ways:

1. In the adult mosquito shipping box with adult mosquitoes
2. In any suitably-sized mailing tube with enough packing to prevent breakage of the vials. These tubes are available upon request

Quality Control:
(Live, Adult Mosquito Shipments)

1. Handling

   The most critical part of submitting satisfactory specimens is in shipping techniques.

1. Temperatures

   Sustained high temperatures are very detrimental to mosquitoes. Plastic containers with frozen water or polar packs should be used during both collection and submission stages.

2. Humidity

   This is another important aspect that can be easily overlooked. High humidity is necessary and can be achieved by placing several moistened paper towels or moistened newspaper into the inner box. Avoid saturating the paper because excess water in the cartons will damage the mosquitoes.

3. Proper labeling

   Collection data should be written on forms. Lack of proper information can decrease the value of arboviral isolation studies, particularly if arbovirus positive mosquito specimens have been collected.

4. Holding specimens

   Although variable, the natural life span of a typical adult, female mosquito is quite short (about a month). In captivity, mortality increases significantly after two or three days. Therefore, specimens being sent for arboviral isolation studies should be sent as soon after collection as possible—preferably the same day as collected. It is also detrimental to unnecessarily handle the specimens, such as by conducting "pre-identifications." All mosquito specimens submitted are identified and, therefore, such handling by the submitter does not assist the laboratory process but often increases the mortality rate of the mosquitoes during transit.

5. Shipping arrangements

   It is always preferable to arrange a general shipping schedule with the laboratory in advance of the season's surveillance program. Shipments late in the week may not be received until Saturday. Because mosquito processing is not routinely conducted on a weekend, a delay may occur that could damage the quality of the specimens. State holidays should also be taken into consideration. Whenever an emergency situation (such as an outbreak) develops, please call the DSHS Arbovirus/Entomology Laboratory to arrange for special pick-up and/or processing of such specimens.

6. Contamination

   Mosquitoes for arboviral isolation studies are quite susceptible to pesticides; contact should be avoided or damage to the shipment will occur. This should be kept in mind when storing equipment and supplies.

2. Sampling methods

1. Sampling patterns

   It is most productive to routinely sample all areas of concern. Whether a city, county, or other designated area, a schedule for routine sampling should be established. Few programs are comprehensive enough to cover all areas at the same time; therefore, quadrants or some other subdividing of the area may be necessary. Each subdivision of the area can then be surveyed at regular intervals.

2. Pool size

   Although a total of one mosquito can be tested for encephalitis viruses, an ideal pool for arbovirus testing consists of about 50 mosquitoes. Having too many mosquitoes in a carton will stress them and can compromise the identification process.

3. Time of day

   The time of day that collections are made can by very significant. Although daytime biters can be collected at almost any time during the daylight hours, evening biters may present a problem if survey times are not properly scheduled. Collection of evening biters, which includes Culex quinquefasciatus, the primary vector of West Nile and St. Louis encephalitis, is usually made in protected areas where these mosquitoes rest during the day. Generally, collection traps are placed in the late afternoon and checked early the next morning.

4. Habitats

   The primary vectors of West Nile, St. Louis encephalitis, and western equine encephalitis are frequently found in resting shelters during the day. Potential vectors of eastern equine encephalitis include some daytime biters that may be difficult to find in such places. These factors are important when surveying for particular types of encephalitis vectors.

5. Light trap for collecting adult mosquitoes

   The CDC miniature light trap is productive for surveillance of some vector species but counterproductive for others. Scheduling for setting out light traps and picking up collected specimens may present some problems. An important drawback of this method is the lack of proper species collected when surveying for West Nile and St. Louis encephalitis viruses; the primary vector in Texas Cx. quinquefasciatus, is only weakly attracted to light. The attachment of carbon dioxide in the form of dry ice will increase the yield of the primary vector, but the results can still be very unsatisfactory. However, light traps can be effective tools when surveying storm sewers, collecting potential vectors of La Crosse encephalitis, eastern or western equine encephalitis, or determining the presence of some mosquito species that are seldom collected by other methods.

6. Gravid trap for collecting adult mosquitoes

   The gravid trap provides a more effective and economical sampling system for female Culex mosquitoes as they come to oviposit. It is therefore selective for females that have already taken at least one blood meal and the chance of isolating an arbovirus is greatly increased.