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Mosquito Pooling: Getting a Return on your Investment

As Florida mosquito control programs approach the busiest time of the year, it is more important now than ever to review arbovirus surveillance programs and protocols. In the last issue of Buzz Words, Dr. Walter Tabachnick discussed the potential for a major outbreak of West Nile in Florida - the "calm before the storm" (Buzz Words, Volume 3, Issue 1; Jan/Feb 2003). Working smart is the key to efficient and effective arbovirus surveillance and disease prevention. And working smart means review, discussion and planning for surveillance programs now, rather than in the middle of an epidemic.

The collection and combining of mosquitoes for virus testing, commonly referred to as "mosquito pooling", is a part of many arbovirus surveillance programs throughout the U. S. Mosquitoes are trapped, identified to species, and tested, or frozen for later testing, in pools of 1--100 mosquitoes. The mosquito pools are ground into one mass and tested by RT-PCR or cell culture assay. The lab conducting the testing provides the client with a list of positive and negative pools. What is a mosquito control district to do with this list? How will it be used to plan control operations?

There are several questions to be asked prior to incorporating mosquito pooling into a surveillance program, as well as after it has been initiated.

  1. Where will mosquitoes be collected?
  2. What species will be targeted in the collections?
  3. When will mosquitoes be collected?
  4. When will the pools be submitted for testing?
  5. How will the mosquitoes be tested (what test will be used?)?
  6. What does a "positive" pool mean?
  7. What is the threshold for positive pools to activate or modify control measures?
  8. What species will be important if positive?
  9. What is the turn around time for processing the mosquito pools and getting results?
  10. What is the overall goal of the mosquito pool testing?

There has been a lot of discussion to address these questions in the past 2 years by Florida medical entomologists. I urge you to read Dr. Don Shroyer's article in Wing Beats, "The collection and processing of mosquitoes for arbovirus assay: some fundamental considerations" (Wing Beats, Summer 2001). Dr. Shroyer discusses the meaning of a "positive pool." The four possibilities for mosquitoes included in any positive pool are:

  1. Uninfected mosquitoes that harbor residual virus from a previous blood meal;
  2. Mosquitoes with dead-end infections--meaning they can not transmit the virus;
  3. Mosquitoes with young infections--the mosquito can possibly transmit the virus, but has not completed the extrinsic incubation period; and
  4. A fully competent vector able to transmit the virus.

Mosquito pool results do not distinguish these four possibilities. The results that you receive cannot indicate more than "positive" or "negative". And at most, a positive pool means that one mosquito in the pool was "positive".

Dr. Walter Tabachnick pointed out in "West Nile virus detection: The details are important" (Buzz Words, Vol. 2, Issue 2, Mar-Apr 2002) that PCR can detect viral remnants in a mosquito that fed on a viremic host long after the blood meal has been digested. PCR cannot answer the question of infected vs. infectious mosquitoes. Additionally, some mosquitoes contain live virus but they are not able to TRANSMIT. The important question is can the mosquito transmit the virus? Dr. Tabachnick's myth #3 in "West Nile in North America: Sorting through four years of myths" (Buzz Words, Vol. 2, Issue 5, Sep/Oct 2002) is another reminder that a majority of the WN positive identified species play little or no epidemiological role in sustaining virus amplification and pose little risk to humans. In a previous article, "Mosquito Vector Competence Tests for West Nile Virus: What do they mean for Florida?" (Buzz Words, Vol. 1, Issue 4, July/Aug 2001), he discussed the value of vector competence and reminded us that we have to look beyond the "positive" mosquito to evaluate the importance of potential arbovirus vectors in Florida. For example, other important traits would be host preference, longevity, extrinsic incubation time, environmental effects on the virus, and species abundance.

In Rutledge et al., "West Nile virus infection rates in Culex nigripalpus (Diptera: Culicidae) do not reflect transmission rates in Florida" (J. Med. Ent., In press) we reported infection rates in mosquitoes of 1.0-7.5 per 1000 mosquitoes and transmission rates of 0.8-1.0 per 1000 over a four night period during epidemic transmission in Jefferson County, Florida during 2001. During the study, we collected 12,000 mosquitoes, primarily of 3-4 targeted Culex species, over a four night period. This effort was very labor intensive, but we had an objective that made use of the surveillance data we had at the time. Compare this effort to a few thousand mosquitoes collected over an entire mosquito season during a non-epidemic period: what are the chances of obtaining information that is useful in control operations? Often, mosquito pooling can address research questions, but is it useful for operations?

An increase in sentinel chicken programs in Florida, and an increase in numbers of flocks in existing programs, combined with an increase in sampling frequency and programs that now run all year long, is going to place a huge burden on the labs that test for arboviruses. The labs that process these samples test the sentinel animal serum first as it has proven to be the most valuable information for control efforts. If mosquito pooling is in your plan, reconsider your sampling effort. It is not efficient or effective to ship a box of mosquitoes to be tested if you have no real objective in mind prior to making the collections. What is the potential for realtime, or close enough to real-time, data that will have meaning for your control program with mosquito pooling?

For those programs that continue to use mosquito testing as part of your surveillance program, be smart about it. Consider the questions asked above and determine what the real goal of the testing is--don't just sample every mosquito out there without consideration of why you are doing it. For the benefit of the testing labs and for a greater return on your investment of time and dollars, remember the following--some very important rules for smart collection and testing of mosquito pools:

DO DON'T
Identify mosquitoes to species Include more than one species in a pool
Remove blood-fed females from the pool Include more than 100 mosquitoes in a pool
Separate males from females Include male and female mosquitoes in the same pool
Maintain a cold chain Forget to identify the mosquito species
Target and prioritize your collections Include mosquito pooling in surveillance without meaningful objectives

C. R. Rutledge, Associate Professor
Florida Medical Entomology Laboratory,
University of Florida/IFAS