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Drcnunnn, 1986
IorNrrrrcarroN
oF AEDEs Celrprsrnrs
529
IDENTIFICATION OF AEDES CAMPESTRISFROM NEW MEXICO:
WITH NOTES ON THE ISOLATION OF WESTERN EQUINE
ENCEPHALITIS AND OTHER ARBOVIRUSES'
H.CALISHER3
c. L.cRABBSbStt[?:%k"1t]!fl,CHARLES
GARY
G.CLARK,2
Department of Arboviral Entomology, Disease Assessment Divisi"on, U.S. Army Medical Research Institute
of Infectious Diseases,Fort Detrick, Frederick, MD 21701-501I
ABSTRACT. An arbovirus survey was conducted during August 1985 at White Sands Missile Range in
southcentral New Mexico following a suspected arboviral disease epizootic among feral horses. A total of
20,566 mosquitoes (18,505 females and 2,061 males) and 8,900 biting gnats were collected and assayed for
virus. Femalt mosquitoes were principally Aedes campestru (54.8Vo), Aedns dorsalis (30.4Vo) and Culex tarsalis
(13.27o). Arboviruses were not isolated from biting gnats, but mosquitoes yielded a total of 37 viral isolates,
including western equine encephalitis (WEE) (18,1,California serogroup (15), Cache Valey (l), and Hart Park
(l) viruses in additioir to 2, as yet unidentified, rhabdoviruses. Isolatesbf WEE virus were from 9 pools of Aa.
campestris,6 of Cx. tarsalis and 3 of Ae. dorsalis. California serogroup viruses, including 2 subtypes, were
obtained from 7 pools of females and I pool of males of Ae. campestrisand from 4 pools of Ae. d.orsalis.Cache
Valley and Hart Park viruses were isolated from single pools of ,4e. dorsalis and Cx. tarsalis, respectively, and
the rhabdoviruses were obtained from,4e. campestrisand Psorophora signipennis.
Sacramento Mountains. Elevation in the area
of greatest mortality is approximately 380 m
In late July and early August 1985, heavy
above sea level. The principal water sources are
mortality occurred in feral horses at the White
Malpais Spring, a freshwater spring (pH 7.!,
Sands Missile Range (White Sands), New
and the highly alkaline Salt Creek (pH 8.0-8.6)
Mexico. Twenty-nine (78%) of the 37 carcasses which originates in the mountains north of the
were found near salt marshes just south of
study area and terminates in alkali flats to the
Malpais Spring in Otero County (33"17'N,
south. The area is characterized by desert
106'18'W) and near Salt Creek approximately
grass/shrub vegetation. Salt cedar (Tamarix
7 km west in Sierra County (Fig. l). Mortality
spp.) is the principal plant along the waterways,
was limited to a moderately cohesive herd
with alkali sacaton (Sporobolw airoides), mescomposed of about 200 of the estimated 750
quire (ProsopisjuliJlora), and chamiza (Atriplex
ferai horses in the 3 herds at White Sands.
canescens) between Salt Creek and Malpais
Large numbers of mosquitoes were encounSpring (Neher and Bailey 1976).
tered by veterinarians soon after the first
This area experiences a rainy season from
horses were found dead. As a result of these
to September, usually providing about half
July
observations, and with extensive equine mortalthe annual precipitation. Tularosa, 30 km
ity of unclear etiology, an entomologic/virologic
southeast of Malpais Spring, the closest Nasurvey was undertaken to identify mosquitoes
tional Weather Service recording site, has an
present in this locality and to determine if these average annual precipitation of
26.2 cm (U.S.
'Ihe
mosquitoes were infected with a virus that
Department of Commerce 1984).
average
might have caused the equine deaths.
daily maximum temperature duringJune, July
and August ranges from 35.0 to 36.1"C.
METHODS AND MATERIALS
Because of the moderately high elevation, the
average daily minimum temperature for these
Sruov srrr. The survey was conducted in the
months is 14.4 to 17.8'C.
Tularosa Basin, a flat valley in southcentral
Cor-r-scrroN Mnrnoos. On August 15-17,
New Mexico bordered on the west by the San
1985, Army miniature solid-state light raps,
Andres Mountains and on the east by the
supplemented with dry ice as a CO2 source,
were suspended at I to 2 m in salt cedar trees
I The views of the authors do not purport to
along Salt Creek and adjacent to Malpais
Spring by 1930 hr. The following morning,
reflectthe positionsof the Departmentof the Army or
the Departmnt of Defense.
collection containers from the traps were
z Present address: CDC-San
Juan Labs, G.P.O. gathered by 0900 hr. Containers from each site
Box 4532, SanJuan, PR 00936.
were placed in coolers with dry ice for 5 min to
3 Divisionof Vector-BorneViral Diseases,
Centers
immobilize the insects. The contents were then
for DiseaseConttol, P.O. Box 2087,Fort Collins,CO
transferred to 50-ml plastic, screw-cap centri80522-2087.
' Departmentof BiologicalSciences,Universityof
fuge tubes, sealed, labeled with site and date,
Notre Dame.IN 46556.
and retained on dry ice to preserve any virus
INTRODUCTION
I
530
R^M
JounNer- oF THE Anrrnrceru Mosqurro CoNrnol AssocrerroN
A
l-sruoY^aEA
I*{
\ ,
Vol. 2, No. 4
prepared against the following viruses:
Jamestown_ Canyon (California Jerogroup),
Flanders (Hart Park serogroup),
furlock
(Turlock serogroup), St. Louis encephalitis
(arbovrrusserogroup B = flaviviruses),west_
ern equine encephalitis (WEE) (arbovirus
s^erggloupA = alphaviruses),and a grouping
fluid for Bunyamweraserogroupuiruies. itepresentative California ICALy group virusLs
were subtyp.d
- 'al.
U a serum dlluti6n-plaque
reduction neutralization test (Lindsey et
1976).Viral stockswere prepared from supernatanr fluids from virui-inhcted Vero cells.
Titers of the stock viral preparations were
determined by plaque assayiin-Vero cellsand
subsequentneutralization tests performed bv
using_infection-immune
hamsteriera prepared
with JamestownCanyon, California eircefutratitis (CE).and SanAngelo(SA)viruses(KaraLatsos
and Mathews1980).
Fig. l. Location of horse mortality in southcentral
New Mexico, 1985.
RESULTS
that the insects might contain until returned to
the U.S. Army Medical Research Institute of
Infectious Diseasesat Fort Detrick. Frederick.
Maryland. Mosquito larvae were collected with
a standard dipper from a 3 m x 3 m isolated
ground pool and from numerous water-filled
hoofprints near Malpais Spring. The larvae
were allowed to emerge to adults for species
identification and viral assay.
Leeonerony Mrruoos. Specimens from the
light trap collections and' laboratory-reared
mosquitoes were placed on a chill table (4.C),
and bloodsucking Diptera were separared from
other arthropods. Mosquitoes were identified
to,spe,cies under a stereomicroscope with the
aid of a key produced by Darsis and Ward
(1981) and placed in pools of up ro 50
according to sex. site of collection, aate and
presence or absence of visible blood.
Ceratopogon idae (Culicoil"es spp.) were placed
il p"otr of 100 without regard to speCies or
bloodfed status. Procedures for inilial viral
assay were described previously (Clark et al.
1985).
Arthropod suspensions also were inoculated
intracranially into each of a group of eight 2- to
4-day-old suckling mice and observed for 14
days for evidence of morbidity or mortality.
Suspensions of brains from sick or dead miie
were tested by complement fixarion (CF) by the
LBCFbo method modified for microtiter (Casev
1965); crude alkaline-extracted,infected suckling mouse brain was clarified and used as
antigen (Calisher and Maness lg75). For the
viral identification tests, we used hyperimmune
mouse ascitic fluids (Tikasingh et al. 1960)
A total of 20,566 mosquitoeswere collected,
pooled, and assayedfor virus (Table l). The
principaf speciescollected were Aedescampestris
!/ar and Kryb, Aedcsdorsalis (Meigen) and
CulextarsalisCoquillett. At Malpais Spring, Ae.
campestris
and Cx. tarsaliswere founci in Equal
numbers, whereas over 5 times as many-Ae.
campestris
as Cx. tarsaliswere collected near Salt
Creek.
Males represented 10.0% of the total mosquito_collection(Table l). At Malpais Spring,
24.07oof the Cx. tarsaliswere malei, comparel
with 3.4% at Salt Creek. Aedesmales,predomi'Ja\'itly Ae. campestris,comprised 23.4% of alt
Aedescollectedat Malpais Spring and l0.4Voof
the Aedesmosquiroesfrom Salt Creek. While
sorting the mosquitoes,Ae. campeslris
pairs were
frequently found ia copula.
All laboratory-reared mosquitoes (2b) collected from hoofprints at Malpais Spring
were
Cx. tarsalis. Adults reared from immature
mosquitoes collected from the larger ground
pool at this location were all Ae. campeitris(17
females and 5 males). Numerous Ae. campestris
maleswere also present in the low vegetation in
and around this pool. When disturbed, they
wo,uld fly to a height of approximately 30 cm
before settling back to the vegetarion.
The rainfall data recorded at Tularosa for
June I through August 18 are presented in
Fig. 2. Only 0,2 cm of rain was recorded in
May, which was below normal. However, the
1.7cm and 4.8 cm of rainfall in lune and "The
lulv.
respectively,were normal for iularosa.
most significant climatic events recorded during the 2 weeks,prior to the initial observarion
of dead horses were the 2.3- and 1.2-cm
DrcrMsnn. 1986
Table
531
IneurrrrcerroN oF AEDESCenpnsrnrs
l. Mosquitoes collecteh for viral assay at White Sands Missile Range, New Mexico,
August 16-18, 1985.
Collection area
Malpais Spring
Species
Ae. cam,pestrxs
Ae. dorsalis
Ae. sollicitans
Ae. aexans
Aedasspp. (males)
An. pseudopunctipennis
Cx. erythrothorax
Cx. tarsalis
Cx. tarsalis (males)
Cs.inornata
Ps.confinnis
Ps.signipennis
Total
No. females
650 (36.8)*
374 (2r.2)
8 (0.5)
32 (1.8)
249
l5 (0.8)
34 (l.e)
649 (36.8)
9nq
I (0.r)
2 (0.1)
0
.) c)<).
r,765
Salt Creek
9,495(56.7)
5,243 (3r.3)
32 (0.2)
58 (0.3)
l,539
34 (0.2)
4 (<0.1)
1 , 8 0 0( 1 0 . 8 )
64
0
71 (0.4)
3 (<0.1)
18,343
16,740
Total
(54.8)
10,145
5,617(30.4)
40 (0.2)
e0 (0.5)
I,788
4e (0.3)
38 (0.2)
2,44e(r3.2)
97.
l (<0.1)
73 (0.4)
3 (<0.1)
20,566
18,505
* No. collected (% of females at this site).
rainfalls of July 15 and 27, respectively.The
temperaturefor the l0 days following the first
precipitationinJuly ranged from 13.3 to 36.1"
(i = 24.6'C).
Thirty-six virus isolations were obtained
from 18,505 female mosquitoes collected at
White Sands during 3 nights of light-trapping
(Table 2). Following initial virus isoltion, all
isolates were reisolated in Vero cells and
sucklingmice. Eighteenisolateswere identified
as WEE virus. Nine of these were from .4e.
carnpestris;
however, of the 3 principal species,
Cx. tarsalis had the highest minimum field
infection rate (MFIR).
Fourteen CAL serogroup virus isolateswere
obtained from pools of female mosquitoes, 4
from Ae. dorsalisand ll frorn Ae. campestris
(Table 2). Another CAL virus isolate, not
shown in Table 2, was from a pool of 50 male
Aedesspp. This pool of males as well as one of
female Ae. campestriswere identified as CE
virus, while one pool of Ae. dorsalisyielded a
rioJtcttotttroo
ot
roaQUtro
co|-ltc?rox
2.5
I
2.O
1.5
E
3
J
J
t.o
l!
-
o.5
G
o
JUNE
JULY
AUGUST
Fig. 2. Relationship of rainfall and horse mortality at White Sands Missile Range, New Mexico,
June-August, 1985.
532
Jounr'rel oF THE Au*rceN
Mosqurro coNrnor
AssocrerroN
Vor-. 2, No. 4
Table 2' viral isolations from female mosquitoes_collectedat White Sands
Missile Range, New Mexico,
August l6-lB, 1985.
Virus*
CAL
8p.
Ae. eampestrk
Ae. dorsalis
Cx. tarsalis
Ps. signipennis
Total
0
l (0.2)
0
0
WEE
l0 11.0;*r'
4 (0.7)
0
0
t4
0
0
l (0.4)
U
I
I
x CV : Cache Valley, CAL
gp = California serogroup, Hp :
encephalitis
x* No. virus
vir
isolates (Minimum field infection rate/1,000).
subtype of SA virus. Single strains of Cache
Valley (CV) and Hart Park viruses were
isolated from Ae. dorsalis and Cx. tarsalis,
resDectivelv.
fwo additional viral isolates have not been
specifically identified. However, elecrron microscopic examination revealed morphologic characteristics similar to viruses in thJ familv
Rhabdoviridae. Preliminary serologic results
indicate that these rwo are identicil to each
other, yet different from the 6 rhabdoviruses
known from this region of the USA.
Virus was not obtained from the 22 ad.ultAe.
campestris reared from immatures. Likewise,
virus was not detecred in Culicoides(89 pools)
collected on August 16 near the lava flowin the
Malpais Spring area.
The product of the WEE virus MFIR and the
percentage of the 3 principal species in the
collection yielded a "population infection index" (Table 3). In this analysis, the overall
index value in both areas for Ae. campestriswas
3.2 times as grear as Ae. dorsalis
and 1.5 as large
as Cx. tarsalis.
DISCUSSION
The presence of substantial numbers of Cx.
tarsalis and, Ae. dorsalis in our collections was not
e (0.9)
3 (0.5)
6 (2.4)
0
l8
Rhabdoviridae
Total
9n
8
7
I
I
0
0
I
2
JO
Hart park, WEE = *.rt..*qui.,.
unexpected, based on the reported distribution
of these 2 species(Darsie and Ward lgSl). Aedes
campestris has been reported from northern
New Mexico (Sudia et al. 1967) and from
southwesternTexas (McGregor and Eads 1943),
but not from southcentral New Mexico (Darsi,e
and Ward l98l). Initially regarded as a
univoltine_species of late spring and early
summer (Rees 1943), Ae. campestriswas suggested by Chapman (1966) to-be multivoltirie
but with only one generation per year because
of climatologic and ecologic'faciors. Apparently, midsummer rainfall and elevated imbient temperatures caused the emergence of this
species prior to the occurrence of horse deaths
at White Sands. The portrayal of the habitat,
mating and anthropophilic behavior of this
species and concentration of Ae. campestri;at an
emergence site were similar to those reported
by Knab (Carpenter and Lacasse1955).
Culex tarsalis is regarded as the principal
vector of WEE virus in the western USA
(reviewed by Hess and Hayes 1967). The MFIR
that we observed for this specieswas similar to
the 2.3/1,000 found in 1983 in Kern Countv.
California. by Reisen t I984). However. our
findings are rhe first reported isolations of
WEE virus from Ae. cambestrisin this countrv.
Table 3. Western.equine encephalitis virus isolations from and population infection index of three
mosquito species collected at white Sands Missle Range, New Mexico, August 16-lg, Iggb.
Collection area
Species
Malpais Spring
Ae. campestris
0/650
Ae. dorsalts
o/374
Cx. tarsalh
(1.5)
1/649
Salt Creek
9/9,495(0.9)*
51.0*,k
3t5,243(0.6)
18.8
5 / l r , 8 0 0( 2 . 8 )
30.2
Both areas
(0.9)
9/10,145
49.3
3 / 5 , 6 1 7( 0 . 5 )
r5.2
6t 2,44s(2.4)
31.7
* No. viral isolates/no.mosquitoesassayed(minimum field infection rate/l,000)
xx Population infection index :
collection.
(minimum
field infection rate/1,000) x species, percenrage of fernale
Dacerrasrt, 1986
IorNuucarIoN
or Arnrs Cerr.rptsrnrs
JJJ
The postmortem conditions of the horses at
White Sands precluded collection of usable
diagnostic samples required to ascertain the
etiology of the sudden die-off. While WEE
virus was the only recognized equine pathogen
that we isolated, it was not possible to unequivada.
ocally link the isolation of this virus from
The infrequent isolation of WEE virus from
mosquitoes to the deaths of the horses.
Ae. carnpestris may reflect the relatively small
numbers that have been assayed for virus (i.e., Transmission of WEE virus in this locality by
Aedes mosquitoes, involving the principal spe5 , 7 2 6 - l v e r s e n e t a l . 1 9 7 9 14 , 0 8 7 - M c l i n t o c k
et al. 1970; cies that we collected, is a tenable hypothesis
et al. 1966; 2,338-Mclintock
and should be pursued bv further field, as well
et al. 19731 less than 1,000974-Burton
as laboratory studies. These results warrant
Shemanchuk and Morgante 1968; l8-Crane
"few"-Sudia
laboratory studies to define the ability of this
et al. 1967; Hammon
et al. 1977;
species to l) become infected with WEE virus
et al. l94l). No Ae. campestriswere found by
after feeding on sources with viremias equivaHayes et al. (1976) among 44,444 mosquitoes
lent to those developed by naturally infected
collected during 1972 in 8 New Mexico
vertebrates and 2) to successfully transmit this
counties, not including Otero and Sierra
virus to other susceptible vertebrate hosts.
counties.
The most abundant mammal observed durAlthough infection rates are not synonymous
ing ground and aerial surveillance at White
with transmission rates (Reeveset al. l96l), our
"population infection index" was similar to one
Sands was the black-tailed jackrabbit (kpus
californicus). A natural transmission cycle involvcalculated for St. Louis encephalitis virus and
ing this hare, WEE virus, and Aed,esmelanimon
birds bv Lord et al. (197$. This index clearlv
Dyar has been described from northern Calisuggested that A?. campestriswas potentially the
fornia (Hardy and Bruen 1974). Recently,
most important WEE virus vector based on
Reisen (1984) reported an MFIR of 1.9/1,000
biomass and field infection rate. Although we
for Ae. tnelanimon in Kern County, a rate twice
collected more Ae. campestris near Salt Creek
that we found in Ae. campestris.Aedesmelanimon
than near Malpais Spring, the l0-mile [6 km]
flight range for this species (Rees 1943) easily is reported only from northwestern New
encompassesthe latter area, where the greatest Mexico (Darsie and Ward l98l) and was not
found in our collections. However, in Hale
number of dead horses was observed. The
County, Texas, Bowers et al. (1969) obtained 5
relatively high percentage of males in light trap
"fresh"
collections, the
condition of the speci- WEE virus isolates from L. califurnictu in 1965
mens, and the frequency of pairs found en and found that 48 (86%) of 56 had hemagglutination-inhibiting antibody to WEE virus.
copula, implied that there was a recent emerThe ecological situation described here is an
gence of Ae. campestrisat this locality.
The possibility of transovarial transmission
example of a unique focus that could provide
the basis for important field studies on the
of WEE virus in this remote location should be
ecology of WEE virus in southwestern United
explored. The MFIR in females without visible
blood and the large percentage of males in the
States. In this small, well-defined area with
minimal human activity, we found WEE virus,
collection, but relatively small number of males
a recognized epizootic (Cx. tarsalis) and potenassayed,reinforce the need for this study.
The isolation of CE virus from a pool of Ae. tial maintenance (Ae. campestris)vector of WEE
virus, and a possible vertebrate host (L.
campestrismales was suggestive of tiansovarial
californicus\.Because of restricted accessto this
transmission of this virus. Previously, Crane et
al. (1977) reported transovarial transmission of
area and the presence of an endangered
CE virus tn Ae. dorsalisat Blue Lake, Utah; they
species, the White Sands pupfish (Cyprinodon
isolated CE virus from 2 pools of Ae. dorsalis, tularosa), the current land-use policy and
one of males and one of females, reared from
regulations against use of insecticides strongly
field-collected larvae.
preclude significant man-made changes in this
Although rarely isolated, SA virus has been
desert habitat in the near future.
obtained from Ps. signipennis collected at Las
Cruces, New Mexico (Tesh 1980). The only
ACKNOWLEDGMENTS
prior report of an SA virus isolation from the
genus Aedes was from a single mixed pool of
We thank Dr. Lelia T. Gaines, Director of
Aedesatlanticus-infirmatus(Bartnett et al. 1967). Health Services, and her staff at White Sands
The role of these 2 Aedes species in the
and Dr. William L. Lumpkin, Deputy Commaintenance of these CAL serogroup viruses mander for Veterinary Services,and his staff at
in this area requires further definition.
William Beaumont Army Medical Center in El
In the mid-1960s,Mclintock et al. (1970)
reported 3 WEE virus isolates, and J. O.
Iversen (personal communication, 1985) obtained a singleWEE virus isolatein 1976 from
collected in Saskatchewan,Can4e. cantpestris
534
oF THEAurnrcen Mosqurro CoNrnor Assocrerron
Jounr.rer-
Paso, Texas, for their superb logistic support.
The field assisranceof Mr. Max"Canestoip of
White Sands and Dr. DeWayne G. Tavlor and
SpecialistT. R. Otin of the U.S. Army'Medical
Research Institute of Infectious disease is
appreciated. The assistance of Ms. Daisan
Taylor and Captain Dario Montelongo at
White Sands is also acknowledged. A"t the
Centers for Disease Control, i'ort Collins.
Colorado, Ms. S. Jones provided enrhusiastic
technical assistance. Dr. Lewis T. Nielserr
confirmed the identifiction of the Ae. cambestrk
and Dr. Douglas M. Warts provided nitpfut
assistance with the manuscript.
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