A Method to Increase Efficiency in Testing Pooled Field-Collected Mosquitoes

Abstract

Testing field-caught mosquito collections can result in thousands of pools, and testing pools of 50 mosquitoes each can be both time consuming and cost prohibitive. Consequently, we have developed an alternative approach to testing mosquito pools for arboviruses, utilizing a superpool strategy. When mosquito samples are processed for extraction of viral RNA and subsequent virus testing via quantitative real-time polymerase chain reaction, each pool is tested individually. Using the method described here, 0.025 ml from each of 10 pools is combined into a superpool for RNA extraction and testing. When a virus-positive superpool sample is found, each of the original 10 pools that constitute this sample is tested individually in order to find the specific positive sample. By retesting the original samples after the initial superpool screen, we are still able to obtain reliable estimates for minimum infection rates or maximum likelihood estimations. To test this principle, we created controlled mosquito pools of known titer and subjected them to our superpool process. We were able to detect our entire range of laboratory-created pools as being West Nile virus (WNV) positive. In 2005, field surveillance efforts from our laboratory resulted in over 4,000 mosquito pools tested, with 8 resulting WNV-positive samples. We found that all of these field samples were detected as WNV positive using the superpool method and contained calculated virus titers from < 0.1 to 4.1 log10 plaque-forming units/ml WNV, indicating that the limit of superpool detection of WNV is below this point. These results reveal that the superpool method could be accurately used to detect WNV in field-collected specimens.

Full text

SCIENTIFIC NOTE
A METHOD TO INCREASE EFFICIENCY IN TESTING POOLED
FIELD-COLLECTED MOSQUITOES
DANIEL M. CHISENHALL,
1,2
CHRISTOPHER J. VITEK,
1
STEPHANIE L. RICHARDS
1
AND
CHRISTOPHER N. MORES
1,2,3
ABSTRACT. Testing field-caught mosquito collections can result in thousands of pools, and testing pools
of 50 mosquitoes each can be both time consuming and cost prohibitive. Consequently, we have developed
an alternative approach to testing mosquito pools for arboviruses, utilizing a superpool strategy. When
mosquito samples are processed for extraction of viral RNA and subsequent virus testing via quantitative
real-time polymerase chain reaction, each pool is tested individually. Using the method described here,
0.025 ml from each of 10 pools is combined into a superpool for RNA extraction and testing. When a virus-
positive superpool sample is found, each of the original 10 pools that constitute this sample is tested
individually in order to find the specific positive sample. By retesting the original samples after the initial
superpool screen, we are still able to obtain reliable estimates for minimum infection rates or maximum
likelihood estimations. To test this principle, we created controlled mosquito pools of known titer and
subjected them to our superpool process. We were able to detect our entire range of laboratory-created pools
as being West Nile virus (WNV) positive. In 2005, field surveillance efforts from our laboratory resulted in
over 4,000 mosquito pools tested, with 8 resulting WNV-positive samples. We found that all of these field
samples were detected as WNV positive using the superpool method and contained calculated virus titers
from ,0.1 to 4.1 log
10
plaque-forming units/ml WNV, indicating that the limit of superpool detection of
WNV is below this point. These results reveal that the superpool method could be accurately used to detect
WNV in field-collected specimens.
KEY WORDS West Nile virus, mosquito pool, quantitative reverse transcriptase polymerase chain
reaction, arbovirus testing, surveillance
Arthropod-borne encephalitis viruses are an
ongoing health risk to humans in the United
States. In Florida, 3 encephalitis viruses are
commonly found in the resident mosquito pop-
ulation: Eastern equine encephalitis (EEE), St.
Louis encephalitis (SLE), and West Nile virus
(WNV). A variety of surveillance methods are
employed in Florida for early detection of
potential outbreaks, including passive monitoring
of dead birds and horses, as well as monitoring
human cases and seroconversions in sentinel
chicken flocks (Blackmore et al. 2003, Godsey
et al. 2005). Testing mosquito pools is another
method employed during routine surveillance,
outbreaks, and in field-based experiments
(Shroyer 1991, Vitek et al. 2008).
The generalized procedure for pooling mosqui-
toes for virus testing involves sorting samples by
species and dividing the collection into pools of
up to 50 individual mosquitoes (Lee et al. 2002,
Condotta et al. 2004, Farajollahi et al. 2005,
Lampman et al. 2006). Mosquito pools have been
used previously for detection of other arboviruses
(Chiang and Reeves 1962, Dow et al. 1964, Le
1981, Shroyer 1991) and, since the discovery of
WNV in the United States in 1999, mosquito
pooling has been utilized throughout the country
for WNV detection (Lanciotti et al. 2000,
Lanciotti and Kerst 2001).
It is possible to obtain reliable estimates for
minimum infection rates or maximum likelihood
estimations (Bernard and Kramer 2001, Condotta
et al. 2004) utilizing pools of approximately 50
mosquitoes. However, increasing the numbers of
mosquitoes tested per pool may lead to inhibition
of the growth of certain viruses in cell culture–
based assays (Hubbard et al. 1989). When testing
field-caught specimens, mosquito collections can
result in thousands of pools, and testing pools of
50 mosquitoes each can be both time consuming
and costly. Additionally, quantitative reverse
transcriptase–polymerase chain reaction (qRT-
PCR) assays may be more beneficial for surveil-
lance despite its higher costs because these assays
outperform conventional RT-PCR due to its
speed, lower contamination risk, ability for
quantification, and higher sensitivity (Vijgen et
al. 2005). In our laboratory, the estimated cost of
testing a single pool using standard RT-PCR is
roughly $3.00/pool, while qRT-PCR costs ap-
proximately $8.00/pool; however, costs can rise
steeply depending on the reagents and methods
employed. In 2005, field surveillance efforts from
2
Present address: Louisiana State University, School
of Veterinary Medicine, Department of Pathobiological
Sciences, Skip Bertman Drive, Baton Rouge, LA 70803.
3
To whom correspondence should be addressed.
1
University of Florida, Florida Medical Entomology
Laboratory, 200 9th Street SE, Vero Beach, FL 32962.
Journal of the American Mosquito Control Association, 24(2):311–314, 2008
Copyright
E
2008 by The American Mosquito Control Association, Inc.
311

our laboratory resulted in over 4,000 mosquito
pools tested, with 8 resulting WNV-positive
samples (Vitek et al. 2008). The cost of testing
these field samples for WNV would be approxi-
mately $32,000 using standard qRT-PCR meth-
ods as described by Lanciotti et al. (2000).
Consequently, we have developed an alterna-
tive approach to testing mosquito pools for
arboviruses, utilizing a superpool strategy. By
combining aliquots of 10 mosquito pools into 1
superpool prior to viral RNA extraction, this
method decreases the costs associated with
arbovirus testing of mosquito pools by a factor
of approximately 10. This reduced cost is
especially important when dealing with large
sample numbers. Mosquitoes are sorted and
pooled using standard protocols. When mosquito
samples are processed for extraction of viral
RNA and subsequent virus testing via qRT-PCR,
we draw and then combine 0.025 ml from each of
10 pools, instead of drawing 0.25 ml from each
pool. Then, each 0.25-ml sample is added to
0.25 ml of extraction buffer prior to extraction of
viral RNA. In addition to the cost benefits, this
allows us to test 10 times as many samples at
once. When a virus-positive superpool sample is
found, each of the 10 pools that constitute this
sample is tested individually in order to find the
specific positive sample.
To test this methodology using laboratory-
infected mosquitoes, 7-day-old female Culex
quinquefasciatus Say from a colony established
from Alachua County in Gainesville, FL, in 1995
(generation .F
40
) were utilized and maintained
at 28uC under a 14:10 (L:D) cycle simulating a
long day photoperiod. We used WNV strain WN-
FL03p2-3 (Doumbouya 2007), originally isolated
from a pool of Cx. nigripalpus Theobald in Indian
River County, FL, in 2003, passaged 5 times in
African green monkey kidney cells and once in
baby hamster kidney cells. Female Cx. quinque-
fasciatus were starved for 48 h and then allowed
to feed on artificial membrane feeders containing
citrated bovine blood warmed at 35uC for 15 min
with 6.8 60.03 log
10
plaque-forming units
(PFU)/ml WNV. After feeding, mosquitoes were
immobilized with cold and fully engorged spec-
imens transferred to 1-liter cardboard cages with
mesh screening. Mosquitoes were transferred to
incubators, held for 13 days at 25uC, and
provided 20%sucrose ad libitum. After 13 days,
surviving mosquitoes were removed from each
cage, killed by freezing, and their legs removed.
Mosquito bodies were triturated in 0.9 ml BA-1
diluent (Lanciotti et al. 2000) and stored at
280uC for later processing. Prior to virus assays,
2 4.5-mm zinc-plated beads (BB-caliber air gun
shot) were added to each mosquito sample.
Samples were homogenized at 25 Hz for 3 min
(TissueLyser; Qiagen, Inc., Valencia, CA) and
centrifuged at 4uC and 3,148 3gfor 4 min.
In order to determine the sensitivity for the
superpool method to detect a single positive pool
from 9 potentially negative mosquito pools, we
used the body of a single laboratory-infected
colony mosquito to create a known positive pool
to simulate the lowest possible number positive in
a wild-caught pool. As a point of reference, we
used our standard pool testing procedure: 0.25 ml
of a laboratory-infected positive pool containing
a single mosquito of known titer was combined
with 0.25 ml of extraction buffer. The superpool
procedure was tested over a range of titers using 3
known positive laboratory-infected mosquitoes
that were chosen based upon their previously
determined WNV titers. Mosquito samples were
of high (7.4 log
10
PFU/ml WNV), medium (5.6
log
10
PFU/ml WNV), and low (2.3 log
10
PFU/ml
WNV) titers. We added 0.025 ml of 1 of these
positive pools to 0.225 ml of a negative pool, and
0.25 ml of extraction buffer. Negative pools
contained 50 virus-negative Cx. nigripalpus col-
lected from the field in 2005 (Vitek et al. 2008).
A standard was created by adding 0.25 ml of
WNV stock (8.0 log
10
PFU/ml WNV) to 0.25 ml
of extraction buffer as a reference sample for the
calculation of viral titers, and a negative control
consisting of 0.5 ml of extraction buffer were
processed along with the experimental samples to
detect any cross-contamination.
To further evaluate the superpool method, 8
WNV-positive mosquito pools collected in 2005
(Vitek et al. 2008) were processed as described
above. Each superpool consisted of 0.025 ml of a
field-collected positive pool, combined with
0.025 ml from each of 9 negative field-collected
mosquito pools, and 0.25 ml of extraction buffer.
Positive standards and negative controls were
created and samples were processed as previously
described.
All samples were extracted with the MagNA
Pure LC System and Total Nucleic Acid Isolation
Kit (Roche, Mannheim, Germany), and eluted in
0.05 ml of elution buffer prior to qRT-PCR
testing.
The amount of viral RNA in each sample was
determined using the LightCyclerH480 system
(Roche) and Superscript III One-Step qRT-PCR
kit (Invitrogen, Carlsbad, CA) carried out as
described previously for WNV (Lanciotti et al.
2000, Lanciotti and Kerst 2001). Samples were
amplified using the following operation guide-
lines: 48uC for 30 min, 95uC for 2 min, 45 cycles
of alternating temperatures of 95uC for 10 sec
and 60uC for 15 sec, followed by 50uC for 30 sec.
Standard curves were created based on data
acquired from 10-fold serial dilutions of WNV
stocks of known concentration and used to
extrapolate concentrations of experimental sam-
ples (Bustin 2000).
We were able to detect the entire set of
laboratory-created mosquito pools using the
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standard viral RNA extraction and qRT-PCR
methods. Under standard pool testing conditions,
high-, medium-, and low-titered pools were
detected and calculated to contain 7.4, 5.6, and
2.3 log
10
PFU/ml WNV, respectively. In the
superpool method, the high-, medium-, and low-
titered pools were detected with calculated
concentrations of 5.3, 3.0, and 1.8 log
10
PFU/ml
WNV, respectively. The positive control derived
from WNV stock was detected at 9.1 log
10
PFU/
ml WNV and negative controls were not detected
as positive for WNV. This shows that our limit of
detection for the superpool method is below our
low-titered mosquito pool.
All of the field samples were detected as WNV
positive using the superpool method and con-
tained calculated virus titers from ,0.1 to 4.1
log
10
PFU/ml WNV (Table 1). The positive
control contained 8.9 log
10
PFU/ml WNV and
the negative controls were not detected as positive
for WNV. Since the lowest titer from field-
collected samples was detected using the super-
pool method and calculated to contain ,1.0 log
10
PFU/ml WNV, this indicates that the limit of
superpool detection of WNV is below this point.
These results reveal that the superpool method
would be able to detect WNV in field-collected
specimens under these conditions.
Compared to standard qRT-PCR methodolo-
gy, our superpool method allows 10 times the
number of mosquito pools to be tested simulta-
neously at roughly one-tenth of the total cost.
With the potential benefit of saving both time and
money, this method is currently being used to test
samples collected in Florida during the 2006 field
season. In 2006, we collected 289,663 mosquitoes,
divided into 6,476 pools. Using the superpool
method will cost us approximately $5,200 com-
pared to $51,700 for testing these samples via the
standard pooling method, saving $46,500 while
also allowing us to test the samples in less time.
The cost to test these pools by nonquantitative
RT-PCR would be approximately $19,400, while
the superpool method would save $14,200. It
would also provide the added benefits of qRT-
PCR compared to RT-PCR, such as increased
sensitivity, increased processing speed, lower
contamination risk, and the ability to quantify
viral loads (Vijgen et al. 2005).
Admittedly, there was a 2- to 3-fold reduction
in calculated WNV titer via the superpool method
in the laboratory-created samples when compared
to our standard pooling method. Approximately
1 log
10
PFU WNV of the observed reduction can
be explained by the reduction of the amount of
sample added. The remaining decrease in sensi-
tivity could be due to an inhibitory effect on the
qRT-PCR, resulting from the increase in co-
extracted mosquito nucleic acids, a decrease in
the efficiency of the reaction by limiting the
amount of starting RNA template, as has been
shown in experimental amplification of phage
DNA (Kainz 2000), or by some other means we
have yet to discover. It is unlikely that this
reduction was created by differences in sample
preparation as both methods were run at the
same time with each sample experiencing identi-
cal preparation, i.e., same extraction protocol,
same number of freeze–thaw cycles, same general
laboratory conditions. The impact of this de-
crease in titer-calculation accuracy when com-
pared to the standard method is offset by the
benefits gained from the streamlined superpool
approach. The calculated titers using the super-
pool method would remain considerably more
accurate than relative quantification using RT-
PCR.
The minimum infection rates (MIRs) and
maximum likelihood estimations (MLEs) are still
able to be calculated after a superpool assay.
When a positive superpool is identified, it is
broken down into its component sub-pools, and
each of those pools is individually tested via qRT-
PCR analysis. This enables a much more reliable
MIR or MLE estimate, based on pool sizes of 50
mosquitoes or fewer and not the superpool size of
approximately 500 mosquitoes. It is important to
note that during epizootics or epidemics in focal
zones, it may be worthwhile to utilize standard
procedures of individual testing of mosquito
pools as more superpool samples will be positive
for virus. Therefore, the number of component
pools tested will also increase, offsetting the cost
and time benefits of the superpool method.
Further research is underway to determine the
absolute sensitivity of the superpool method, the
reasons for the decreased calculated titers ob-
served, and the ability of multiplex assays to use
the superpool method. The multiplex assay for
detecting WNV, SLE, and EEE using the super-
pool method would be of particular interest to
researchers in Florida and other states where
mosquitoes are abundant and multiple arbovi-
ruses are endemic. Such a test would save
considerable time and money, thereby streamlin-
ing mosquito control efforts and protecting
public health.
Table 1. Field-collected mosquito samples positive for
West Nile virus (WNV), as tested by
superpool methodology.
1
Sample no. WNV titer (log
10
PFU/ml)
351 3.3
493 4.1
510 3.6
522 0.1
558 4.0
967 3.9
1,102 ,0.1
2,186 3.4
1
PFU, plaque-forming units.
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This research described in this publication was
made possible by funding from the Florida
Department of Agriculture and Consumer Ser-
vices. We would like to thank Chelsea Smartt and
Kendra Pesko for their critical reading of this
manuscript.
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