Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1975 The an tural history of trivittatus virus in central Iowa Ralph Thomas Alls Iowa State University
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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 75-25,312
ALLS, Ralph. Thoanas, 1940- IHE NAIURAL HISTORY OF IRIVnTAIUS VIRUS IN CENTRAL lOm,
Iowa State University, Ph.D., 1975 Entomology
Xerox University Microfilme, Ann Arbor, Michigan48106 The natural history of trivittatus virus in central Iowa
by
Ralph Thomas Alls
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Major: Entomology
Approved:
Signature was redacted for privacy.
Signature was redacted for privacy.
For the Major Department
Signature was redacted for privacy.
Iowa State University Ames, Iowa
1975 ii
TABLE OF CONTENTS
Page
INTRODUCTION 1
REVIEW OF LITERATURE 3
California Group Viruses 3
Mosquitoes and California Group Viruses 9
Vertebrates and California Group Viruses 13
MATERIALS AND METHODS 17
Description of the Study Area 17
Sampling of Mosquito Populations 18
Isolation of Viruses from Mosquitoes 19
California Virus Antibodies in Wildlife 21
Jamestown Canyon, Snowshoe Hare, and Trivittatus 22 Viruses in the Thirteen-lined Ground Squirrel
Infection of Aedes triseriatus with Snowshoe Hare 24 Virus from Viremic Thirteen-lined Ground Squirrels
Infection of Aedes trivittatus with Trivittatus 27 Virus from Viremic Cottontail Rabbits
RESULTS AND DISCUSSION 29
Sampling of Mosquito Populations 29
Isolation of Viruses from Mosquitoes 35
California Virus Antibodies in Wildlife 40
Jamestown Canyon, Snowshoe Hare, and Trivittatus 42 Viruses in the Thirteen-lined Ground Squirrel
Infection of Aedes triseriatus with Snowshoe Hare 45 Virus from Viremic Thirteen-lined Ground Squirrels iii
Page
Infection of Aedes trivittatus with Trivittatus 49 Virus from Viremic Cottontail Rabbits
CONCLUSIONS 53a
SUMMARY 54b
BIBLIOGRAPHY 55b
ACKNOWLEDGMENTS ^6
APPENDIX A. FORMULAE 9?
APPENDIX B. RECORDS OF MOSQUITOES TRAPPED IN DRY TOO ICE-BAITED CDC TRAPS FOR VIRUS ISOLATION ATTEMPTS
APPENDIX C. RECORDS OF SERUM SAMPLES FROM WILD MAMMALS 120 TESTED FOR THE PRESENCE OF ARBOVIRUS ANTIBODIES
APPENDIX D. FIGURES 123 IV
LIST OF TABLES
Page
Table 1. Summary of mosquito species collected by the New 30 Jersey light trap at the State Forest Nursery, Ames, Iowa, 1971
Table 2. Summary of mosquito species collected by the New 32 Jersey light trap at the State Forest Nursery, Ames, Iowa, 1972
Table 3. Summary of mosquito species collected by the New 34 Jersey light trap at the State Forest Nursery, Ames, Iowa, 1973
Table 4. Summary of mosquito species collected by the New 35 Jersey light trap at the State Forest Nursery, Ames, Iowa, 1971-1973
Table 5. Summary of mosquito species collected in CDC 37 traps at the State Forest Nursery, Ames, Iowa, and number of pools of each species processed for virus isolations, 1971-1973
Table 6. Isolations of trivittatus virus from Aedes 38 trivittatus at the State Forest Nursery, Ames, Iowa, 1971-1973, with field infection ratios
Table 7. Isolations of Flanders virus from Culex pipi ens 38 complex mosquitoes at the State Forest Nursery, Ames, Iowa, 1971-1973, with field infection ratios
Table 8. Virus titers in tissues of thirteen-lined ground 43 squirrels inoculated with 4.1 log,Q SMLDgQ of snowshoe hare virus
Table 9. Snowshoe hare virus titers in tissues of thirteen- 43 lined ground squirrel that died between 72 and 84 hours post-inoculation
Table 10. Viremias in thirteen-lined ground squirrels 46 inoculated with snowshoe hare virus
Table 11. Feeding percentages of Aedes triseriatus on 46 thirteen-lined ground squirrels inoculated with snowshoe hare virus
J V
Page
Table 12. Viremias in thirteen-lined ground squirrels 48 inoculated with 4.1 log,^ SMLDrn of showshoe hare virus
Table 13. Infection rates of Aedes trivittatus after 50 feeding on a viretnic (3.9 log.. SMLDcn/ml) cottontail rabbit VI
LIST OF FIGURES
Page
Fig. 1. Aerial photograph showing the location of the 125 State Forest Nursery near the intersection of U.S. Highways 30 and 69
Fig. 2. Detailed map of the study area, located at the 127 eastern edge of the State Forest Nursery, Ames, Iowa
Fig. 3. Seasonal distribution of all mosquito species, 129 Aedes trivittatus, and Aedes vexans collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1971
Fig. 4. Seasonal distribution of Culex pipiens complex 131 and Culex tarsalis col1ected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1971
Fig. 5. Seasonal distribution of all mosquito species, 133 Aedes trivittatus.and Aedes vexans col1ected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1972
Fig. 6. Seasonal distribution of Culex pi pi ens complex, 135 Culex tarsalis.and Culiseta inornata collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1972
Fig. 7. Seasonal distribution of all mosquito species, 137 Aedes trivittatus.and Aedes vexans collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1973
Fig. 8. Seasonal distribution of Culex pipiens complex. 139 Culex tarsal is.and Culiseta inornata collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1973
Fig. 9. Seasonal distribution of Aedes trivittatus 141 collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, 1971-1973 1
INTRODUCTION
Trivittatus virus (TVT) has been recognized as the virus most frequently isolated from mosquitoes in Iowa (Rowley et al. 1973).
One hundred seventy-eight isolations of TVT have been made in surveillance programs conducted in Iowa from 1955 to 1972 (Wong et al.
1970, 1973; Rowley et al. 1973; W. A. Rowley, Department of Entomology,
Iowa State University, Ames, Iowa, unpublished data). Trivittatus virus,
initially isolated from Aedes trivittatus in North Dakota in 1948
(Eklund 1957), was first isolated in Iowa in 1964 (U.S. Department of
Health, Education and Welfare 1965c). Although TVT has been recognized
for a considerable period of time, little was knov'n of its natural
history or ability to cause disease in humans.
Trivittatus virus was established as a member of the California
group of arthropod-borne viruses. The group consists of TVT, Melao
virus (MEL), and California encephalitis virus (CEV), which has seven
subtypes (Sudia et al. 1971b).
The realization that California group viruses cause serious disease
in humans came with the isolation of LaCrosse virus (LAC) from a
4-year-old girl who died of an encephalitis-like illness in Wisconsin
in 1960 (Thompson et al. 1955). Two fatal cases of California
encephalitis have also been reported from Ohio (Masterson et al. 1971).
In many states, surveillance programs for California encephalitis resulted
in a dramatic increase in the number of cases reported annually. An
average of 50 cases was reported yearly for the period 1964 to 1972 2
(U.S. Department of Health, Education and Welfare 1974b). The majority of California encephalitis cases have been attributed to LAC (Sudia et al. 1971b), although TVT has been found to occur throughout the north central states (Ohio, Indiana, Wisconsin, Minnesota, and Iowa) where most of these cases were reported (Sudia et al. 1971b). Monath et al. (1970)
provided serological evidence of TVT infections in residents of Minnesota.
A serological survey of selected premarital and prenatal sera collected
from Story County, Iowa residents indicated a 25.6% incidence of TVT
antibody in this group (Y. W. Wong, State Hygienic Laboratory of Iowa, University of Iowa, Iowa City, Iowa, personal communication).
Twenty-three isolations of TVT were made during August and September,
1969, from A. trivittatus collected south of Ames, Iowa. The public health
implications of a large number of virus-infected, potential disease
vectors prompted studies to evaluate the natural history of TVT in
central Iowa. Objectives of the studies were:(l) to determine the
Incidence of TVT infected A. trivittatus in this region, (2) to determine
the importance of other species of mosquitoes in the basic transmission
cycle of TVT, (3) to determine the natural hosts and potential reservoirs
of TVT, (4) to determine if A. trivittatus becomes infected with TVT
from feeding on viremic animals, and (5) to determine if A. trivittatus
are capable of transmitting TVT to susceptible hosts. 3
REVIEW OF LITERATURE
California Group Viruses
Classification
The World Health Organization (1967) defined arboviruses as
"...viruses which are maintained in nature principally, or to an important extent, through biological transmission between susceptible vertebrate hosts by hematophagous arthropods." Attempts to use the term, arbovirus, in both an epidemiological and a systematic context have been,in the main, confusing (Casals 1971).
The earliest attempt to systematize the large number of agents isolated from hematophagous arthropods was that of Casals (1957) who established the A, B, and C groups on the basis of serological relation ships. New isolates were assigned to existing serological groups or established as groups in themselves when other isolates were shown to be serologically related. Consequently, the CEV prototype isolated in 1943 (Mammon and Reeves 1952) became the nucleus of the California group when later isolates were shown to have a serological affinity to
CEV (Mammon and Sather 1966). When Whitman and Shope (1962) found that
California group viruses were related to Bunyamwera group viruses, the
Bunyamwera supergroup was formed which included a number of smaller groups (World Health Organization 1967).
As the serological classifications became more unwieldy and as the biochemical and biophysical properties of some of these agents became known, the designation "togavirus" was suggested to designate those viruses which 4 corresponded to the old serological groups A and B (Casals 1971). The
International Committee on Nomenclature of Viruses approved the family Toga- viridae, the genus Alphavirus (Group A), and remained undecided on Flavo-
virus or Flavivirus as the generic name for group B viruses (Wildy 1971).
Information concerning the biochemical, biophysical and replicative
properties of the Bunyamwera supergroup viruses as well as many other
viruses of similar morphology has been summarized by Murphy et al. (1973).
They propose that these viruses be established as a family, Bunyaviridae,
and those members showing a serological affinity (the Bunyamwera super
group) be included in the genus Bunyavirus with Bunyamwera virus as the
type virus.
Serology
California encephalitis virus was initially isolated from
dorsal is (later found to be A. melanimon) mosquitoes in 1943 and 1944
in the San Joaquin Valley of California (Mammon and Reeves 1952).
California encephalitis virus was determined to be serologically unrelated
to known arboviruses (Togaviruses); however, a serological affinity with
a virus isolated from A. trivittatus was shown (Mammon and Reeves 1952).
Trivittatus virus, as it came to be known, was isolated in 1948 in
North Dakota by Dr. Carl Eklund (Mammon et al. 1952).
During the 1950's several new isolates were added to the California
group. Melao virus was isolated from A. scapularis in Trinidad
in 1955 (Spence et al. 1952); Tahyna virus (TAH) was isolated from
A. caspius in Czechoslovakia in 1958 (Bardos and Danielova 1959); San
Angelo virus (SAN) was isolated from Anopheles pseudopunctipennis 5 pseudopunctipennis in Texas in 1958 (Grimes 1967); snowshoe hare virus
(SSH) was isolated from the blood of a snowshoe hare (Lepus americanus) in Montana in 1959 (Burgdorfer et al. 1961); and Lumbo virus (LUM) was isolated from A. pembaensis in Mozambique in 1959 (Kokernot et al. 1962).
Whitman and Shope (1962) showed that California group viruses (CEV,
TVT, and MEL) have some serological relationship to Bunyamwera group viruses through an intermediate virus, Guaroa virus. This work led to the establishment of the Bunyamwera supergroup consisting of the
Bunyamwera, Bwamba, group C, California, Capim, Guama, and Simbu groups after some members of these groups were shown to have antigenic cross- relationships (World Health Organization 1967).
Additional California group viruses were isolated during the early
1960's. LaCrosse virus was isolated from the brain of a 4-year-old girl who died of encephalitis in Wisconsin in 1960 (Thompson et al.
1965); Jamestown Canyon virus (JTC) was isolated from Culiseta inornata in Colorado in 1961 (LaMotte 1967); Jerry Slough virus [jtS) was isolated from Cu. inornata in California in 1963 (Reeves.1967b); and Keystone virus (KEY) was isolated from mixed pools of A. atlanticus and A. tormentor in Florida during 1964 (Bond et al. 1966).
Sather and Mammon (1967) used the complement-fixation test as the major tool in a detailed serological study of the California group. They identified 11 serological prototypes; nine (CEV, JES, JTC, KEY, LAC, MEL,
SAN SSH, and TVT) from the western hemisphere and two (LUM and TAH) from the eastern hemisphere. Subsequent workers have concluded that only three prototypes exist, TVT, MEL, and CEV, with seven subtypes of CEV. 6
These subtypes are JTC (=JES), KEY, LAC, SAN, South River (SOR) (isolated from crucians in New Jersey in 1960), SSH, and TAH (=LUM) (Sudia et al. 1971b).
In recent years the immunodiffusion-precipitin reaction has been used to confirm antigenic relationships within the California group.
Kurphy and Coleman (1967) demonstrated the usefulness of this technique in confirming the antigenic individuality of isolates that previously had been shown by other serological techniques. Calisher and Maness
(1970a) demonstrated similarities among CEV, JES, JTC, LAC, LUM, New
Jersey (SOR), SAN, SSH, and TAH by this technique. The immunodiffusion
precipitation test indicates that TVT and MEL are distinct from other
California group members.
Immunoelectrophoresis has been an additional tool in ascertaining antigenic relations within the California group, and has provided a slightly different picture. Trivittatus virus and KEY have been shown
to be unique in character with little demonstrable relationship to CEV,
JES, JTC, LAC, LUM, MEL, SAN, SSH, and TAH which are similar (Weilings
et al. 1971).
Biochemical and biophysical characteristics
The CEV prototype isolated by Hammon and Reeves (1952) was
determined to be 60-125 nm in diameter by collodion membrane filtration.
This virus was somewhat larger than other arthropod-borne viruses
isolated up to that time (Hammon et al. 1952).
Determination of virus size and structure was obscured by an
electron microscope study that indicated that MEL, Guaroa, Wyeomia, and 7
Cache Valley (CVV) viruses (the latter three Bunyamwera group virus) were similar to Venezuelan equine encephalitis virus, a group A virus with a size range of 50-60 nm (Bergold et al. 1969). Mammon and Sather (1966) cited an electron microscopy study that reported a virion also in the
50-60 nm size range for CEV.
Filtration studies using graded membrane series supported the idea of a virion in the 50-60 nm size range. Porterfield (1961)
reported TAH to be 32 nm in diameter and Whitney et al. (1969a) reported a virion of less than 50 nm for a New York isolate. Electron microscopy
with the New York isolate showed it to be 85 to 90 nm and
Whitney et al. (1969a) felt the discrepancy between filtration and
electron microscopy studies was attributable to flexibility of the
viral envelope.
Murphy et al. (1968a) examined five California viruses (CEV, KEY, LAC
SAN, and New York strain 65-8569) in infected mouse brain by electron
microscopy and found virions approximately 98 nm in diameter. The
same workers (Murphy et al. 1968b) also showed that three Bunyamwera
group viruses (Bunyamwera virus, Tensaw virus, and Maguari virus) had
similar morphology. This seemed to confirm the relationship between these
two groups of viruses that had been proposed by Whitman and Shope (1962)
on the basis of antigenic similarities. Electron microscopy
of several arboviruses confirmed the morphological similarity of what
are now considered to be Bunyamwera supergroup viruses (Holmes 1970).
Bonsdorff et al. (1969) studied unpurified preparations of Inkoo
virus (INK), a subtype of CEV isolated in Finland, with the electron 8 microscope and found virions about TOO nm with 15 nm spikes. Saikku et al. (1971b) isolated the ribonucleoprotein of INK and determined by electron microscopy that its morphology was similar to Rhabdoviruses and Uukuniemi virus, a Finnish Bunyamwera virus. All of these viruses exhibited helical symmetry with respect to the nucleoprotein as opposed to cubical symmetry for the Togaviruses. Further studies with INK
(Brummer-Korvenkontio et al. 1973) showed that these virions had a diameter of 100 nm with 15 nm spikes and demonstrated the presence of ribonucleic acid (RNA) by the use of tritiated uridine.
McLerran and Arlinghaus (1973) discovered that purified LAC infec
tious RNA was single-stranded and they isolated three polypeptides with molecular weights of 26,000, 45,000, and 85,000 daltons. The 85,000 d polypeptide is believed to be associated with the lipoprotein envelope and the 26,000 d polypeptide is probably associated with the nucleo- capsid. The 45,000 d polypeptide may be associated with the viral envelope but its role is undefined at this point. Bouloy et al.
(1974) found that the genome of LUM consists of three segmented single-
stranded RNA's. The total molecular weight of the RNA genome
(6.4 X 10^ d) was distributed in molecules of three size classes
one molecule of 2.9 X 10® d, one molecule of 2.0 X 10® d, and three
molecules of 0.5 X 10® d.
Replication
Murphy et al. (1968a) found that virus particles are assembled
at internal membrane surfaces and mature virions are formed by budding 9 into cisternae or vacuoles within the cell. A similar replication process occurs in the Bunyamwera group viruses studied by the same workers (Murphy et al. 1968b).
Lyons and Heyduk (1973) studied LAC infections in different host systems (mouse brain, Vero cells, and mosquito cells) by electron microscopy. Virus particles of about 95 nm were assembled in the region of internal cytomembranous structures, principally the Golgi complex.
Virus maturation occurred by budding of the virus into the lumen of cisternae and vesicles. A portion of the cell membrane is acquired in the process. Fewer cytopathic effects were noted in infected A. albopictus cells and persistence of the virus in mosquito cell cultures was explained on the basis of a steady-state interaction between a few lethally infected cells being replaced by sensitive cells. In mammalian cells and in mouse brain as the infection progressed the assembly sites became less focal due to the proliferation of Golgi membranes.
Lubiniecki and Henry (1974) noted that RNA synthesis by LAC occurred throughout the cytoplasm, in contrast with Eastern equine encephalitis and several group B Togaviruses (Japanese B encephalitis,
Dengue-2, and Powassan) where RNA synthesis occurred in the peripheral
cytoplasm and the perinuclear area of the cytoplasm, respectively.
Mosquitoes and California Group Viruses
Virus isolations from mosquitoes
CEV was the first arthropod-borne virus isolated from mosquitoes 10 before it was identified from another source (Mammon et al. 1952).
Only two subtypes (SSH and LAC) discussed in the preceeding section were originally isolated from sources other than mosquitoes. The literature concerning isolations of California group viruses is extensive and has been reviewed by Sudia et al. (1971b) who discuss the 1,353 isolations made from mosquitoes in North America from 1943 to 1970.
The three viruses most often isolated from mosquitoes, KEY, LAC and
TVT, have associations with particular vector species. Seventy- nine percent of the KEY isolations from mosquitoes are from A. atlanticus-tormentor. Forty-six percent of all LAG isolations are from
A. triseriatus. Eighty percent of the TVT isolates in Florida are from
A. infirmâtes while 86% are from A. trivittatus in the north central states (Sudia et al. 1971b).
In Iowa the virus most often isolated from mosquitoes is TVT.
From 1966 through 1972, 359 virus isolations were obtained from mosquitoes. Trivittatus virus was isolated from 178 mosquito pools;
156 of these were A. trivittatus. Trivittatus virus has also been isolated in Iowa from undetermined Aedes species, A. vexans, Coquillet- tidia perturbans, Culex pipiens, and inornata. LAC has been isolated from A. triseriatus on six occasions in Iowa. In addition, western equine encephalomyelitis (WEE), St. Louis encephalitis (SLE), CvV, Flanders
(FLN), and Turlock (TUR) are other viruses isolated from Iowa mosquitoes
(Wong et al. 1970, 1973; Rowley et al. 1973; Rowley, unpublished data). 11
Mosquito infection and transmission
Reeves and Mammon (1952) infected four species of mosquitoes with CEV in the laboratory. Aedes dorsal is, A. varipalpus, Ç. tarsal is,
and Cu. inornata were infected by feeding CEV virus suspensions in
defibrinated normal rabbit blood administered by soaked cotton pledgets.
Aedes dorsal is transmitted the virus to a rabbit 10 days after ingesting
the virus suspension.
Chernesky (1967) infected A. vexans by feeding defibrinated rabbit
blood, sugar, and CEV mixture and achieved virus transmission to rabbits
7 and 9 days after ingesting the virus suspension. Intrathoracic
injection of CEV in A. aegypti produced infected mosquitoes that
transmitted the virus to chickens after 4 days incubation.
Penny and Taylor (1969) infected A. infirmatus with KEY by the
virus,defibrinated-rabbit-blood,cotton-pledget method. Virus was
recovered from the abdomen of infected mosquitoes on days 1, 2, 3, and
5 and from the head, thorax, and abdomen on days 6, 7, and 8.
LaCrosse virus infection of A. triseriatus was accomplished by feeding
mosquitoes on viremic hamsters (Mesocricetus auratus) (Watts et al. 1972).
Infection rates of 33 to 100% occurred when mosquitoes fed on hamsters with
viremias ranging from 1.0 log^Q to 3.6 log-jg tissue culture 50% infective
dose (TCIDgg) per 0.1 ml. Infected mosquitoes transmitted the virus by
feeding on suckling mice as early as 7 days after feeding on the viremic
hamsters.
Aedes triseriatus has also been infected by feeding on viremic chip
munks (Tamias striatus) and gray squirrels (Sciurus carolinensis) 12
(Pantuwatana et al. 1972). Mosquitoes became infected after feeding on
chipmunks with viremias ranging from 2.5 log-jQ to 3.5 log^g suckling mouse
50% lethal doses (SMLDgg) per 0.02 ml and from feeding on gray squirrels with viremias ranging froir 3.1 log^g to 4.5 log^g SMLDgg/O.OE ml. Trans
mission of the virus to suckling mice was accomplished by 33% of the
infected mosquitoes which had fed on the chipmunk with 3.5 log^g
SMLDgg/0.02 ml viremia and by 20% of the infected mosquitoes which had fed
on the gray squirrel with a 3.1 iog-jQ SMLOgQ/0.02 ml viremia.
Watts et al. (1973a) tested nine species of mosquitoes, in addition to A. triseriatus, for their ability to become infected with LAC and transmit the virus. Mosquitoes were allowed to feed on a virus;defibri- nated-guinea-pig-blood mixture through a lamb skin membrane. Trans mission of the virus to suckling mice was accomplished by 70 to 77% of the
A. triseriatus infected by ingesting 4.4 log^g SMLDgg/O.GS ml of virus. At similar virus concentrations A. vexans, A. canadensis, A. aegypti, and
C'j. inornata transmitted the virus at rates of 27, 25-27, 10 and 33%, respectively. Other species were less efficient in transmission or failed to transmit the virus to suckling mice.
Transovarial transmission
Transovarial transmission of LAC in A. triseriatus was demonstrated
in the laboratory by Watts et al. (1973b). Mosquitoes ingested a virus
infected blood meal through a membrane and were maintained through
several ovarian cycles. Eggs, larvae, and adults produced by the
infected mosquitoes yielded LAC when inoculated into suckling mice.
Adult females (F^ generation) transmitted the virus to suckling mice 13 and chipmunks.
Pantuwatana et al. (1974) obtained LAC from field collected A. triseriatus larvae in Wisconsin during June and September 1972 and
Watts et al. (1974) isolated LAC from field collected larvae of A. triseriatus as well as male A. triseriatus reared from larvae collected in the field in Wisconsin during 1973. Female A. triseriatus reared from field collected larvae transmitted LAC to suckling mice. This additional evidence of transovarial transmission supported a hypothesis of probable overwintering of LAC via the egg stage (Watts et al. 1973b).
Vertebrates and California Group Viruses
Virus isolations from vertebrates
Mammon et al. (1952) in their original work with CEV demonstrated viremias in ground squirrels, Spermophilus (=Citellus) beecheyi, and domestic rabbits, Oryctolaqus sp., after subcutaneous inoculation of
CEV. Burgdorfer et al. (1351) isolated SSH from the blood of a showshoe
hare in Montana and SSH was subsequently isolated from snowshoe hares
in Alberta, Canada (Hoff et al. 1971). Domestic rabbits have been used
extensively as sentinel animals for the detection of California group
virus activity in Canada (McKiel et al. 1966), Florida (Jennings et al.
1968), Illinois (Kokernot et al. 1969a), Iowa (Wong et al. 1970), and
Wisconsin (Papadopoulos et al. 1970). In many cases, virus was isolated
from blood clots after serum samples showed rising antibody titers
indicative of infection with California group viruses. 14
Serological evidence of vertebrate infection
There are numerous serological studies on wildlife-California group virus associations. The literature was extensively reviewed by
Parkin et al. (1972). Most mammalian species, larger than mice, tested for California virus antibodies show serological evidence of infection.
Antibodies to California viruses have been found in high percentages of cotton rats (Sigmodon hispidus). ground squirrels (Spermophilus
(=Citenus) sp.), squirrels (Tamiasciurus and Sciurus sp.), chipmunks
(Tamias and Eutamias sp.), marmots (Marmota sp.), rabbits (Sylvilagus sp.), hares (Lepus sp.), deer (Odocoileus sp.), and moose (Alces alces).
Domestic mammalian species tested (dog, sheep, hog, donkey, horse, cow, and reindeer) have had California virus antibodies. Birds, reptiles, and amphibians show no serological evidence of involvement in California virus natural cycles.
The clinical disease in humans With the isolation of CEv in 1943 (Mammon and Reeves 1952), evidence was sought to determine if the virus caused disease in humans.
During the sunraer of 1945, three cases of encephalitis in Kern
County, California, were serologically shown to be caused by CEV.
In addition, a serological survey of residents of Kern County revealed
that approximately 11% of the population had neutralizing antibody to
CEV.
Aside from the isolation of new strains of California viruses
there was little concern over these viruses until the isolation of LAC 15 in Wisconsin in 1960 (Thompson et al. 1965). Public health officials and clinicians have attempted to determine the distribution and severity of California encephalitis since it was realized that
California viruses cause serious disease in humans.
The clinical disease is usually found in children between the ages of 5 and 9 and predominantly in the male (Henderson and Coleman
1971). Balfour et al. (1973c) describe two distinct clinical patterns.
The mild form consists of fever, headache, malaise, and gastro intestinal symptoms. After about 3 days, there is a temperature increase and patients experience lethargy and evidence of meningeal irritation. These symptoms abate gradually during a 7-to 8-day period with complete recovery.
The severe form begins with a fever and headache which is closely followed 12 to 24 hours later by the sudden onset of seizures. Most of these patients require supportive care and some have additional seizures after the initial, prolonged seizure activity. Patients are hospitalized for an average of 12 days and approximately 1/3 are discharged with abnormal neurologic findings, including ataxia, aggressive behavior, dysarthria, bilateral Babinski signs, and memory loss. Chun et al. (1968) also report a 29% incidence of neurological damage as demonstrated by abnormal electroencephalograms.
Clinical and psychological sequelae following California
encephalitis abate in time. Matthews et al. (1968) in a follow-up on
33 cases with an elapsed time period of from 1 to 6 years found similar
results between this group and a normal control group after examination 16 with a comprehensive psychological test battery.
Surveillance of California encephalitis
California encephalitis surveillance programs began in the mid-1960's.
Concommitant with the establishment of surveillance programs, there was a marked increase in the annual number of reported cases. A single case was reported in 1963. The number of reported cases increased to a high of 89 cases in 1970. Cases of California encephalitis have averaged 60 cases/year since 1964. The north central states of Ohio, Indiana,
Illinois- Wisconsin, Minnesota, and Iowa accounted for 504 of the 545 cases reported during 1963 to 1972 (U.S. Department of Health, Education and Welfare 1974b).
Serological surveys conducted in Indiana (Henderson and Coleman
1971), Ohio (Johnson 1970), and Minnesota (Monath et al. 1970b) revealed evidence of human exposure in 21, 16.1, and 20.3%, respectively, of the populations tested. Tnc Minnesota survey also turned up evidence of possible human infection with TVT as well as LAC. A survey conducted in
Iowa on prenatal and premarital serum samples disclosed a 25.6% incidence of TVT antibodies in residents of Story County (Y. W. Wong, personal communication). 17
MATERIALS AND METHODS
Description of the Study Area
Geography
The Iowa Conservation Commission State Forest Nursery is located southeast of the intersection of U.S. Highways 30 and 69 south of
Ames, Iowa. The study area is located on the eastern boundary of the
Nursery (Fig. 1).
The study area lies at the edge of the Skunk River flood plain and is characterized by a steep 25 foot drop in elevation from the upland into the study area. Several natural springs feed into a sluggish stream which flows into the Skunk River approximately one-half mile to the east. The stream is also fed at times by drainage from nearby fields, overflow from storm sewers, and overflow from a settling pond at a sewage treatment plant directly north of the study area
(Fig. 2). The State Nursery is west of the study area and a housing area is being developed to the south.
Vegetation
The woodlot located in the southeast corner of the study area
(Fig. 2) is practically a pure stand of ash (Fraxinus americana) with an understory of mulberry (Morus rubra). The wooded area bordering
the stream consists of mixed hardwoods including elm (Ulmus americana), oak (Quercus sp.), and maple (Acer sacchavinum) with an understory of mulberry and hackberry (Celtis occidental is). There are also several 18 stands of cotton wood (Populus deltiodes) and birch (Betula sp.).
The more open areas support a lush undergrowth of blackberry (Rubus allegheniensis), elderberry (Sambucus canadensis), and ragweed
(Ambrosia sp.).
A varied mammalian population is in residence in the study area including ground squirrels (Spermophilus tridecemlineatus tridecemlimeatus), chipmunks (Tamias striatus qriseus), fox squirrels
(Sciurus niqer ruflventer), cottontail rabbits (Sylvilagus floridanus mearnsii), jack-rabMts (Lepus townsendii campanius), raccoons (Procyon lotor hirtus), and oppossums (Didelphis marsupial is virginiana).
Mbsquitoes
During the warmer months of the year (May through October) there are often large mosquito populations in the study area. Battery-operated
CDC light trap and larval samplings made during 1969 and 1970, prior to the present study, revealed substantial populations of A. triseriatus,
A. trivittatus, A. vexans, An. punctipennis, £. pipiens, C^. san'narius, and C^. tarsal is.
Sampling of Mosquito Populations
A New Jersey light trap (Mulhern 1934) was operated at the study area during the summers of 1971, 1972, and 1973. The trap, located in the same place each year (Fig. 2), was turned on before sunset and off after sunrise by an electric timer. During 1971 the trap was in operation from June 2 through September 29. During 1972 and 1973 the 19 trap was set out May 1 and operated until October 31.
The trap catch was collected daily. Mosquitoes were sorted from other insects, identified to species and the number and sex recorded.
Each month was divided into three, 10-day periods (the last period of months having 31 days had an 11-day period) and each species count was totaled and averaged for the period. The average number of mosquitoes for each period was graphed on semi-logarithmic paper.
Isolation of Viruses from Mosquitoes
Mosquito trapping and identification
Battery-operated CDC light traps (Sudia and Chamberlain 1962), baited with dry ice (Newhouse et al. 1966) were employed to capture mosquitoes for attempts at isolation of virus. Several times each summer one to six traps were set out at various locations in the study area
(Fig. 2). Traps were operating at least 1 hour before darkness and picked up within 1 hour after sunup the following morning>
Insects other than mosquitoes were removed and the mosquitoes were frozen at -76°C. Mosquitoes were identified on a cold table (Sudia et al. 1965) and males and blood-fed females were discarded. Mosquitoes were pooled according to species and trap location, sealed in rubber- stoppered tubes, and returned to the -76*C freezer until virus isolations could be attempted. The July 1, 1971 collection was processed for virus isolation at Iowa State University; later samples were sent to the State Hygienic Laboratory of Iowa, Iowa City. 20
Virus isolations processed at Iowa State
Pooled mosquitoes were ground in glass tissue grinders using a
"cleaning fluid" (Appendix A) as a diluent. Mosquito suspensions were centrifuged at 1,000 X G for 15 minutes. Suckling mice 1 to 3 days old were inoculated intracerebrally (ic) with 0.02 ml of the supernatant using 1 cc disposable tuberculin syringes. Suckling mice were observed for signs of illness for 14 days. Sick or dead mice were removed and frozen at -76°C for passage.
The brains of mice which had died or showed signs of illness were removed, ground in "cleaning fluid", centrifuged and inoculated ic into l-to-3-day-old suckling mice. Sick or dead mice in this passage group
Were removed, frozen at -76°C and sent to the State Hygienic Laboratory for identification of the viral agent.
Virus isolations processed at the State Hygienic Laboratory of Iowa
Pooled mosquitoes were ground in a chilled mortar with Alundum
and 2 ml of 0.75% bovine albumin (BA) in phosphate buffered saline
(PBS) solution (Appendix A). Mosquito suspensions were centrifuged
at 1,000 X G for 20 minutes at 40°C. Suckling mice were inoculated ic
with 0.02 ml of the supernatant and the mice were observed for 14 days
for signs of illness. Mouse brains from sick or dead animals were
harvested and passed by ic inoculation in suckling mice. Isolates
were stored at -75°C for later identification. 21
Identification of arbovirus isolates
Arbovirus isolates were identified by the complement-fixation (LBCF) test (U.S. Department of Health, Education and Welfare 1965d).
Antigens were prepared by emulsifying tissue of infected mouse brain in PBS, pH 7.2, to obtain a 10% suspension. The supernatant obtained after centrifugation at 1,000 X G for 10 minutes was tested against known hyperimmune mouse ascitic fluids prepared by the method of
Tikasingh et al. (1966).
California Virus Antibodies in Wildlife
During the summer of 1973, sera from animals live-trapped in the Ames
area for studies of latent rabies virus were made available to determine
arbovirus antibody levels. The animals were captured in Sherman live-
traps^ under the direction of Dr. Richard E. Dierks for studies of latent
rabies viral infections in rodents (supported by NIH grant 5R22A111253-02).
The 78 sera consisting of 50 samples from S. tridecemlineatuSî 23
samples from T. striatus griseus and five samples from floridanus
mearnsii were sent to the State Hygienic Laboratory where they were tested
for the presence of neutralizing antibodies to WEE, SLE, LAC, TVT and JTC.
The neutralization test has been described by Finger (1974).
^H. B. Sherman, P.O. Box 683, Deland, Florida. 22
Jamestown Canyon, Snowshoe Hare, and Trivitattus Viruses
in the Thirteen-lined Ground Squirrel
Inoculation of ground squirrels
Juvenile thirteen-!ined ground squirrels (^. t. tridecemlineatus) were used to determine the susceptibility of the ground squirrel to
JTC, SSH, and TVT. Ground squirrels were captured by flooding their
burrows with water and netting the animals with an insect net as they
attempted to escape. Pregnant female squirrels were captured in early
May of 1973, and three litters of ground squirrels were born in captivity.
Twenty-one 4-month-old squirrels born in captivity, plus juvenile
squirrels captured at the Iowa State University golf course, constituted
test animals for these experiments. The exact age of the field-caught
squirrels was unknown, but because they were juveniles, their age was
judged to be 4 months or less.
Stock viruses used in this and subsequent experiments were obtained
from the State Hygienic Laboratory. Viruses were mixed with a BA in
PBS to give approximately 4.0 log^g SMLDgg per inoculum as determined
by previous titrations in mice. A portion of each inoculum was retained
for titration in mice to determine the exact dosage administered.
Eight squirrels were inoculated subcutaneously (sc) in the neck
with 4.3 log-jQ SMLDgg of JTC, eight were inoculated with 4.1 log-jQ SMLD^g
of SSH, and eight were inoculated with 4.5 log^g SMLDgg of TVT. One
uninoculated control animal was retained for each group for a total of
three control animals. The nine squirrels of each group were caged in
two cages, four in one cage and five in the other. 23
Handling of inoculated ground squirrels
Each 24-hour period after inoculation one infected squirrel from each group was sacrificed and tissues were removed for attempts at iso
lation of virus. Samples (0.1 ml) of whole blood, defibrinated plasma, red
blood cells and white blood cells were dispensed into vials containing
0,9 ml of BA and PBS. Solid tissues (spleen, liver, kidney, lung, lymph
nodes, brain, and brown fat) were placed in preweighed vials and reweighed with the tissue so that accurate titrations could be made at a later date.
All tissues were stored at -76°C until assays for virus could be completed. Squirrels were sacrificed daily for a period of 1 week.
One squirrel in the SSH group died during the night after the third
24-hour period. Tissues were removed the next morning as with sacrificed squirrels except that no plasma, red blood cell and white
blood cell samples could be taken. The squirrel was in poor condition and had no fatty tissue.
One squirrel from each group was retained until 24 days post-
inoculation. These animals were sacrificed and tissues taken in a
similar manner. The following day (25th day) the control animals were
sacrificed and tissues taken.
Virus isolation, titrations and identification
Blood samples and blood fractions from squirrels were inoculated
ic into suckling mice and the mice observed for 10 to 14 days for
mortality or signs of illness. Serial 10-fold dilutions were made from
samples that killed mice and dilutions 10"^ to 10"^ were inoculated ic
into suckling mice. The number of dead mice per dilution was recorded 24 over a period of 1 week and the titer of the virus was determined according to the method of Reed and Muench (1938).
Solid tissues were diluted by weight in BA and PBS to give a 10 ^ dilution, homogenized in ground glass tissue grinders, centrifuged and 0.02 ml of the supernatant inoculated ic into suckling mice. P^siFive samples were processed as above for determination of virus titer.
Virus isolates were identified by neutralization with specific antisera prepared in laboratory white rabbits. The neutralization test was carried out in suckling mice by the method described by Cunningham
(1966).
Infection of Aedes triseriatus with Snowshoe Hare Virus
from Viremic Thirteen-lined Ground Squirrels
Effect of dosage on viremia titers
Further studies were undertaken with SSH and ground squirrels to determine the effect of dosage on duration of viremias. Four ground squirrels were inoculated sc in the neck with 3.2 log^g SMLDgg of virus and two were inoculated with 4.2 log^^ SMLDgn of virus. Squirrels were divided into two groups of three squirrels, two inoculated with the lower dosage and the other inoculated with the higher dosage. An uninoculated control was included with the first group. Portions of the virus
inoculum were titered to determine the exact dosage used.
The first group of squirrels was bled 12 hours post-inoculation and at 24-hour intervals afterward up to and including 84 hours post-
inoculation. The second group was bled 24 hours post-inoculation and 25 at 24-hour intervals afterward up to and including 96 hours post- inoculation. Bleeding was accomplished by the orbital sinus method
(Sudia et al. 1970).
The 0.1 ml blood samples were dispensed into tubes containing
0.9 ml of BA and PBS and stored at -76°C until virus titrations could be attempted. Virus titrations and identifications were handled as in the previous experiment.
Model for California virus transmission
An attempt was made to determine if a laboratory model for
California virus transmission could be elucidated using ground squirrels,
SSH, and A. trivittatus.
Ground squirrels were inoculated sc in the neck with 4.1 log-jQ SMLDgg of SSH. Inoculations were made at 6-hour intervals so that when mosquitoes were allowed to feed, one squirrel would be 54 hours post-
inoculation, two squirrels would be 48 hours post-inoculation, one squirrel would be 42 hours post-inoculation and one squirrel would be
36 hours post-inoculation. One squirrel was kept as an uninoculated
control.
Squirrels were bled via the oroital sinus at 30, 24, 18, and 12
hours post-inoculation, respectively; the 0.1 ml samples were dispensed
into tubes containing 0.9 ml of a BA in PBS diluent and frozen at -76°C
for later virus assay. Squirrels were bled by the orbital sinus 24
hours later and the samples frozen as before. The squirrels were then
anesthetized by intraperitoneal injection of 0.15 ml of sodium 26 pentobarbital. Approximately. 50 female A. triseriatus were allowed to feed on each of the squirrels except the 36-hour post-inoculation squirrel which did not succumb to the anesthetic. The 10-day-old, unfed mosquitoes were contained in 1-pint ice cream cartons with a piece of nylon netting covering the top. Anesthetized squirrels were laid over the tops of the cartons so that the mosquitoes could feed on the exposed abdomens. Mosquitoes were allowed to feed for 30 minutes when another carton of mosquitoes replaced the first. At least
100 mosquitoes were exposed to each of the four squirrels.
Mosquitoes were anesthetized with ethyl ether and those that had not fed were removed and counted to determine feeding percentages. The blood-fed mosquitoes were placed in an incubator at 28°C and 80% relative humidity. They were checked daily for mortality and supplied with a fresh 0.3 M sucrose-soaked cotton pad. The squirrels were bled once more 24 hours later and the samples frozen at -76°C. Viruses were isolated and titered as in previous experiments.
Mosquitoes that had fed on squirrels with the highest titer viremias (one of the two exposed 48 hours post-inoculation and the one exposed 42 hours post-inoculation) were removed for virus assays to determine infection rates from day 10 to day 14 post-feeding.
Mosquitoes were sealed in rubber-stoppered tubes, frozen at -76°C and
sent to the State Hygienic Laboratory for assay. Mosquitoes were tested
individually for the presence of SSH virus by methods previously
described. 27
Infection of Aedes trivittatus with Trivittatus
Virus from Viremic Cottontail Rabbits
Cottontail rabbits (S.. floridanus mearnsii) were tested for their ability to produce viremias to TVT and provide infectious blood meals for A. trivittatus. Two male cottontail rabbits about 14 months of age were inoculated sc in the neck with 4.2 log^g SMLDgg of TVT. The animals were bled by the orbital sinus 24 hours later; two samples were taken from each rabbit.
During the 48-hour bleeding, one of the cottontails died. Samples of blood, lymph nodes, spleen, kidney, liver, lung and brain were removed and sent to the State Hygienic Laboratory for virus isolation attempts.
After bleeding, the remaining cottontail was anesthetized by intraperitoneal injection of 0.5 ml of of sodium pentobarbital. The abdominal area was shaved and wild-caught female A. trivittatus caged in 1-pint ice cream cartons were allowed to feed for approximately 20 minutes on the cottontail. The rabbit was bled again at 72 hours post-inoculation.
Mosquitoes exposed to the cottontail were anesthetized with ethyl ether and those that had not fed were removed. Blood-fed mosquitoes were placed in 1-pint ice cream cartons and provided an oviposition substrate. The mosquitoes were provided 0.3 M sucrose soaked cotton pads and checked daily for mortality. Mosquitoes were removed and frozen at -76°C for virus isolation attempts from day 10 to day 16
post-feeding. These and a group of 1,000 wild mosquitoes trapped the 28 night before the experiment were sent to the State Hygienic
Laboratory. Blood-fed mosquitoes were individually tested for the
presence of TVT and the wild-caught mosquitoes were tested in pools of
50 to determine the percentage of mosquitoes that were infected before feeding.
One group of rabbit blood samples was retained for virus isolation and titration by methods previously described; the other set of samples was sent to the State Hygienic Laboratory for isolation attempts. 29
RESULTS AND DISCUSSION
Sampling of Mosquito Populations
New Jersey light trap collections for the summer of 1971 are summarized in Table 1. Rainfall for the month of August totaled 0.48 of an inch, considerably less than the normal of 3.85 inches (U.S.
Department of Commerce 1971). This resulted in reduced populations of
A. trivittatus for the month of September. Table 1 shows that 60 A. trivittatus were collected in September versus a 3-year average
(1971-1973) of 574.
The data presented in Fig. 3 show two distinct population peaks for A. trivittatus in mid-June and mid-July. Aedes trivittatus populations began to decline in August. A slight leveling of the decline occurred in mid-August before population levels dropped pre cipitously by the end of the month. These data suggest that four broods of A. trivittatus were produced during 1971» each occurring during the middle portion of the months of June, July, August and September.
The seasonal distribution of C^. tarsal i s and C^. pi pi ens complex
{C_. pi pi ens, C^. restuans, and £. salinarius) mosquitoes for 1971 is presented in Fig. 4. Culex pipi ens complex mosquitoes increased in numbers throughout the season being less influenced by the drought, although a slight decline in the numbers of these species and £. tarsal is occurred during the latter part of August and early September.
Warmer than normal temperatures in May 1972 coupled with
increased amounts of precipitation in July (0.86 of an inch above the 30
Table 1. Summary of mosquito species collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, 1971
Species June July Aug. Sept. Total
Aedes triseriatus - 27 4 1 32 trivittatus 1817 4116 1981 60 7974 vexans 401 2317 1390 832 4940 Anopheles punctipennis 25 112 270 384 791 Coquillettidia perturbans 2 65 21 1 89 Culex erraticus - 1 1 pipiens complex* 227 1107 1590 2932 5856 tarsal is 51 378 445 124 998 Culiseta inornata 19 2 19 84 124 Psorophora ciliata - 3 1- 4 siqnipennis - 11 Uranotaenia sapphirina - 1 - 15 16 Unidentified 59 1-60
Total 2601 8129 5722 4434 20,886
^Includes £. pi pi ens, £. restuans, and £. salinarius. 31
normal), August (plus 1.35 inches), and September (plus 1.03 inches)
(U.S. Department of Commerce 1972) contributed to high numbers of
all mosquito species throughout the summer. The number of mosquitoes
trapped by the New Jersey light trap exceeded the number caught in
1971. Table 2 shows that the number of mosquitoes trapped during June
and July was high, despite the light trap being inoperative for 18
days because of electrical power failure and repairs at the packing
shed.
Aedes trivittatus appeared about 2 weeks earlier than in 1971 and
populations remained high into early October (Fig. 5). Four broods of
A. trivittatus occurred during the summer of 1972. Population peaks
occurred in late May, mid-June, mid-July and late August. There is a
suggestion of a small peak in late September.
Large numbers of Cu. inornata were collected in May and October
as a result of the extension of the light trap season. Culiseta
inornata, £. oioiens complex and C^. tarsal is seasonal distributions
are all included in Fig. 6. Culex pi pi ens complex numbers increased
throughout the season with the exception of a sharp decline in mid-
August which also occurred in C^. tarsal is populations. A possible
explanation for this decline may be that heavy rains during this period
caused increased runoff from breeding sites causing destruction of
some of the immature stages. Unusually low temperatures during this
period may have also contributed to a decline in numbers of these
species. 32
Table 2. Summary of mosquito species collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, 1972
Species May June* July^ Aug. Sept. Oct. Total
Aedes canadensis 4 4 dorsali s 2 1 3 niqromaculis 2 1 spenceri 1 1 triseriatus 1 1 trivittatus 768 1159 2969 2837 968 "92 8793 vexans 1429 2042 563 251 622 103 5010
Anopheles punctipennis 8 15 35 296 294 60 708 quadrimaculatus 6 6
Coquilletidia perturbans 14 14
Culex erraticus ^ 3 pi pi ens complex 283 351 5182 6275 6500 3341 21,932 tarsal is 20 73 162 186 102 24 567 territans 1 I
Culiseta inornata A AC 28 ?Q 50 556
Psorophora horrida
Uranotaenia sapphirina 10 11
Unidentified 27 224 92 11 354
Total 2985 3898 9021 9858 8535 3672 37,969
^Trap inoperative for last 7 days of month.
^Trap inoperative for first 11 days of month.
^Includes C^. pi pi ens, C. restuans and Z. salinarius. 33
May 1973 was cool and wet. Rainfall was 2.50 inches above the normal of 4.28 inches. June was dry; precipitation amounted to half of the normal 5.21 inches (U.S. Department of Commerce 1973). The onset of the breeding season for A. trivittatus was delayed by cool weather, and drought conditions caused populations to remain at low levels until August (Fig. 7). Aedes vexans populations were not influenced by the weather conditions, probably because of a greater tolerance to the cooler conditions in May.
Table 3 shows that larger populations of spring species, such as
A. sticticus and Cu. inornata, occurred during May. Fall populations of Cu^. inornata were also larger than the previous year. Culex tarsal is and £. pi pi ens complex populations developed normally over the course of the season (Fig. 8).
The seasonal distributions of A. trivittatus for the 3 years of the study were compared in Fig. 9. Peak populations usually occurred in mid-July with smaller peaks in rr.id-J'jr.s and late August. Finger
(1974) concluded from Malaise trap collections at the study area that there were five broods of A. trivittatus during the summer of 1973.
Light trap data presented in Fig. 9 indicate four population peaks during each season but sufficient males were not collected to clarify this
point. It seems probable that adult females from preceding broods obscure the emergence of an additional small mid-summer brood.
More than half of the mosquitoes collected by the light trap in
1972 and 1973 were C^. pi pi ens complex (Table 4). During the spring
C_. restuans is the predominant species of the complex. As the season 34
Table 3. Summary of mosquito species collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, 1973
Species May June July Aug. Sept. Oct. Total
Aedes cinereus - 1 - - - - 1 dorsal i s 2 - - - - - 2 niqromaculis 1 1 - 8 - - 10 sticticus 32 16 - - - 48 triseriatus - 11-- 2 trivittatus - 127 187 1087 595 72 2168 vexans 217 2183 1502 718 797 548 5965
Anopheles punctiDennis 5 54 91 314 293 115 872
Coquillettida perturbans - 5 34 3 - - 42
Culex erraticus - 1 - - - - 1 pi piens complex* 438 468 627 3004 4651 3180 12,368 tarsal is 190 312 211 122 62 7 904 terri tans 1 - - - - - 1
Culiseta i noma ta 778 171 2 - 47 148 1146
Orthopodomyia siqnifera - 1 - - 1
Psorophora cil iata - - - - 2 - 2
Uranotaenia sapphirina - - 12 4 18
Unidentified 21 2 1 4 28
Total 1664 3360 2658 5260 6552 4075 23,569
^Includes £. pi piens, restuans, and salinarius. 35
Table 4. Summary of mosquito species collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, 1971-1973
Species 1971 1972 1973 Total
Aedes canadensis - 4 - 4 cinereus - - 1 1 dorsal is - 3 2 5 niqromaculis - 3 10 13 spencen - 1 - 1 sticticus - - 48 48 triseriatus 32 1 2 35 trivittatus 7974 8793 2168 18,935 vexans 4940 5010 5965 15,915
Anopheles DunctiDennis 791 708 872 2371 quadrimaculatus - 6 - 6
CoQuillettidia perturbans 89 14 42 145
Culex erraticus 1 3 1 5 piPiens complex 5856 21,932 12,368 40,156 tarsal is 998 567 904 2469 terri tans - 1 1 2
Culi seta i noma ta 124 556 1146 1826
Orthopodomyia siqnifera - - 1 1
Psorophora ciliata 4 - 2 6 horrida - 2 - 2 siqmpenms 1 - - 1
Uranotaenia sapphirina 16 11 8 35
Unidentified 60 354 28 442
Total 20,886 37,969 23,569 82,424
^Includes C. pipiens, C. restuans. and C. salinarius. 36 progresses Ç.. pi pi ens and Ç^. salinarius become prevalent. The primary breeding site for these species is the small pond formed by the back-up of the stream as it passes through the culvert directly east of the packing shed (Fig. 2). The breeding potential of this body of water is enhanced by sewage runoff from a settling pond north of the study area (Fig. 2). Culex tarsal is larvae can also be found in this area, but in fewer numbers.
Isolation of Viruses from Mosquitoes
Battery-operated CDC light trap collections are summarized in
Table 5. Collection data for individual sampling dates are contained in Appendix B. Aedes trivittatus was the most common mosquito collected in CDC traps, representing 81.9% of the total catch. Culex pipiens accounted for 8.2% of the mosquitoes collected and 7.6% were A. vexans.
A total of 1457 pools of mosquitoes were processed for virus isolations. There were 960 pools of A. trivittatus, 259 pools of
C^. pi pi ens complex mosquitoes, and 96 pools of vexans. Viruses were isolated from 28 pools of mosquitoes. Twenty-five of the isolations were TVT from A. trivittatus (Table 6); the remaining three were Flanders virus from C^. pi pi ens complex mosquitoes (Table 7). Field infection ratios (FIR's), calculated by dividing the total number of mosquitoes tested by the number of virus isolations, are included in Tables 6 and 7.
Sixteen isolations of TVT were made from A. trivittatus collected
July 1, 1971. Isolations of California viruses had not been reported this early in the season in temperate regions. The presence of large 37
Table 5. Summary of mosquito species collected in CDC traps at the State Forest Nursery, Ames, Iowa, and number of pools of each species processed for virus isolations, 1971-1973
Species 197V 1972' 1973' Total
Aedes canadensis 1 (1) 1 (1) dorsal is 8 (1) 15 (1) niqromaculis 4 (2) 4 (2) spenceri 2 2 sticticus 364 (1) 365 (2) stimulans ! Ill 1 2 (1) triseriatus 3 (3) 6 (3) 16 (5) 25 (11) trivittatus 21,156(403) 44,774(460) 8926(97) 74,856(960) vexans 347 (15) 4695 (61) 1926(20) 6968 (96) species 32 (1) 32 (1)
punctipennis 228 (14) 131 (10) 95(12) 454 (36) CoQuillettidia perturbans no (7) 74 (3) 2 (1) 186 (11) Culex erratieus j 1 (1) 1 (1) pipi ens complex 3544 (89) 2565(111) 1465(59) 7574(259) tarsal is 63 (11) 126 (7) 150(14) 339 (32) Culi seta
1 f tw f f WW» 16 (6) 137 (11) 415(22) 558 (39) Psorophora horrida 3 (3) 2 (1) 5 (4) Uranotaenia saoDhirina 1 (1) 1 (1)
Total 25,468(549) 52,527(676) 13,403(233) 91,398(1457)
18 trap nights.
32 trap nights.
'22 trap nights.
Includes Ç. pi pi ens, C_. restuans and Ç. salinarius. 38
Table 6. Isolations of trivittatus virus from Aedes trivittatus at the State Forest Nursery, Ames, lowa, 1971-1973, with field infection ratios
Year July August September October
1971 16(1:168) 4(1:4436) 3(1:239) 0
1972 0 1(1 :7391) 0 1(1:1458)
1973 0 0 0 0
Table 7. Isolations of Flanders virus from Culex pipiens complex mosquitoes at the State Forest Nursery, Ames, Iowa, 1971-1973, with field infection ratios
Year July August September
1971 1(1:658) 0 1(1:803)
1972 0 1 (1 ;1167) 0
1973 0 0 0
^Includes £. pi piens, Ç. restuans, and Ç. salinarius. 39 numbers of infected first brood mosquitoes can best be explained by transovarial transmission of the virus from mosquito to mosquito and overwintering of the virus in the egg stage. Another possibility is
the presence of a spring vector species that provides a source of
viremic reservoirs for emerging A. trivittatus.
Large populations of Cu_. inornata occur in the study area in the
spring. Culiseta inornata is the only non-aedine species of mosquito
from which a large number of California virus isolations have been
made (Sudia et al. 1971b). In 1972 and 1973, CDC trap collections were
begun in May to determine if Cu_. inornata was an early spring vector
of TVT. Thirty-three pools of Cu. inornata, totaling 552
mosquitoes, were tested and found not to be infected with TVT.
There is no apparent pattern to the isolations of TVT from A.
trivittatus. Field infection ratios for isolations made in 1971 drop
from 1:168 mosquitoes in July to 1:4,436 in August and rise again to
1:239 ir. September (Table 6). The August 1972 FIR of 1:7391 is
comparable to the August 1971 FIR but no isolations were made from
22,310 A. trivittatus tested in July or from 192 A. trivittatus
collected in September of 1972. DeFoliart et al. (1972) also report
TVT in A. trivittatus at different times from year to year.
No isolations of TVT were made from mosquitoes during the summer of
1973. Sentinel rabbits did develop neutralizing antibodies to TVT,
indicating that the virus was present in mosquitoes in the study area
(Finger 1974). 40
Flanders virus is the second most-frequently-isolated virus in Iowa.
Eighty-four isolations have been made, primarily from pipiens complex mosquitoes and Ç. tarsalis (Wong et al. 1970, 1973; Rowley et al. 1973;
Rowley,unpublished data). Flanders virus has not attracted much attention because it has not been associated with disease in humans.
The isolation of Flanders virus from species of mosquitoes thought to be ornithophilic may be indicative of avian involvement.
California Virus Antibodies in Wildlife
Five of 78 wildlife sera tested for antibodies to arboviruses
were positive for California virus antibodies. None of the sera
neutralized WEE or SLE. A complete listing of animals tested is
contained in Appendix C.
None of the 50 ground squirrels were positive for antibodies to
JTC, LAC or TVT. Forty additional sera from captured ground squirrels
used in laboratory experiments were also negative. The thirteen-lined
ground squirrel has not been included in previous surveys for California
group virus activity and the current survey would tend to indicate
that the ground squirrel is not involved in the natural cycles of
JTC, LAC or TVT in Iowa.
Two of the 23 chipmunk sera tested were positive for LAC virus
antibodies. Both of the positive sera were from chipmunks trapped at
Ledges State Park, Boone County, and constituted the only samples from
that area. Moulton and Thompson (1971) suggested that the chipmunk
was the principal host of LAC virus. Pantuwatana et al. (1972) produced 41
LAC viremias in chipmunks and subsequently infected A. triseriatus by feeding them on viremic animals.
LaCrosse virus has been isolated in Iowa but only from the eastern third of the state, principally the northeast corner (Wong et al. 1973;
Rowley, unpublished data). Three cases of California encephalitis from Dallas and Polk Counties in 1970 (Wong et al. 1973) and the
presence of the two positive serum samples strongly suggest the existence of a LAC focus in central Iowa as well.
Three of five juvenile rabbits tested had neutralizing antibodies
to TVT. One serum also neutralized JTC in addition to TVT. More
significant than the incidence of neutralizing antibodies in cottontail
rabbits, is the finding that the two negative sera were taken before the
end of June and the three positive sera were collected after the first
week in July. The timing of the appearance of neutralizing antibodies
in cottontails closely approximates the appearance of neutralizing
antibodies in sentinel rabbits during the early summer of 1973
(Pinger 1974). This is an additional indication that TVT transmission
occurs shortly after the emergence of the first brood of A. trivittatus
in late spring.
No TVT antibodies were found in ground squirrels and chipmunks
captured in the same area where cottontails were positive. These data
indicate that sciurids probably are not involved in the natural cycle
of TVT. This is in agreement with Pantuwatana et al. (1972) who had
poor results with laboratory studies of TVT in chipmunks and gray
squirrels. 42
Jamestown Canyon, Snowshoe Hare and Trivittatus Viruses
in the Thirteen-!ined Ground Squirrel
Assays of SSH from tissues of ground squirrels are contained in Table 8. Virus was found in all tissue types examined except brain and brown fat. Viremias were present 24 and 48 hours post-inoculation. Highest virus titers occurred in spleen and lymph node tissues. Virus persisted in these tissues for 5 to 5 days. Liver, kidney, and lung tissues were also found to contain virus. Tissues from the control animal and the inoculated squirrel held for 24 days post-inoculation were found to be negative for SSH. Virus was also present in the tissues of the ground squirrel that died between day 3 and 4 of the experiment (Table 9). It was estimated that the squirrel had been dead between 2 and 12 hours before being discovered.
The lapse in time before tissue samples could be taken may have had an adverse effect on virus titers. Conversely, the virus found in the brain may have been a false positive due to the breakdown of the blood
brain barrier.
Tissues from squirrels inoculated with JTC were negative for the
presence of virus. In addition, only the squirrel sacrificed 24 hours
post-inoculation had TVT in trace amounts (less than 1 log^g
SMLDgQ/0.02 ml) from the plasma sample.
Studies conducted with hedgehogs (Erinaceus roumanicus) and TAH in
Czechoslovakia resulted in similar findings (Malkova et al. 1969a). Vire mias comparable in titer (1.5 log^g to 2.5 log^g SMLDgQ/0.01 ml) to those 43
Table 8. Virus titers in tissues of thirteen-!ined ground squirrels inoculated with 4.1 log-JQ SMLD^Q of showshoe hare virus
Days post-inoculation Tissue 1 2 3 4 5 6 24 Control
Whole blood 3.3a 4.6 Negb Neg Neg Neg Neg Neg Plasma 3.2 4.6 Neg Neg Neg Neg Neg Neg Red blood cells 3.1 3.9 Neg Neg Neg Neg Neg Neg White blood cells 3.4 4.9 Neg Neg Neg Neg Neg Neg Spleen 4.7 6.0 4.4 3.4 Neg T c Neg Neg Liver Neg T T Neg Neg Neg Neg Neg Lymph nodes 5.7 5.7 4.3 5.4 T Neg Neg Neg Kidney Neg 3.2 T Neg Neg Neg Neg Neg Lung 3.1 3.5 3.4 Neg Neg Neg Neg Neg Brain Neg Neg Neg Neg Neg Neg Neg Neg Brown fat Neg Neg Neg Neg Neg Neg Neg Neg
^Virus titer in log^Q SMLP^q/ihI .
'^Sample negative for virus.
^'Virus titer less than 1 log-jQSMLDgg/0.02 ml.
Table 9. Snowshoe hare virus titers in tissues of thirteen-!ined ground squirrel that died between 72 and 84 hours post-inoculation
Tissue Titer (log SMLDgg/ml)
Blood 3.3 Spleen 5.1 Liver 4.2 Lymph nodes 4.6 Kidney 3.5 Lung 4.2 Brain Ta
^Virus titer less than 1 log-,gSMLDgg/O.OZ ml. 44 obtained with SSH in ground squirrels were present in hedgehogs from 48 hours to 120 hours. Viremias remained detectable in hedgehogs until 8 days post-inoculation. Tahyna virus titers in spleen and lymph node tissues (2.5 log-jQ to 3.5 log-jQ SMLDgg/O.Ol ml) are also comparable to
SSH titers in similar ground squirrel tissues, although TAH persisted in hedgehog spleen and lymph nodes for 10 days. Tahyna virus was also found in liver, lung, and kidney tissues but not in brain tissue. The greater persistence of TAH In hedgehog tissues compared to SSH in ground squirrel tissues may be due to the titer of the TAH inoculum
(5.0 log-jQ SMLDgg). Possible differences in the pathogenesis of the two viruses must also be considered.
It has been assumed that California virus infections in natural rodent and lagomorph hosts are relatively benign (Hammon et al. 1952).
Burgdorfer et al. (1961) isolated SSH from an "...emaciated, rather sluggish snowshoe hare". Yuill et al. (1969) found evidence of higher mortality rates in smwshoe hares with neutralizing antibodies to arboviruses (WEE, SLE, CEV, and Silverwater virus). California virus infections in humans are thought to be mostly subclinical, although they can be severe (Balfour et al. 1973c) and, rarely, fatal
(Thompson et al. 1965). The thirteen-lined ground squirrel is obviously a good host for SSH and the evidence points to SSH as a contributing factor in the death of one of the experimental animals. 45
Infection of Aedes triseriatus with Snowshoe Hare Virus
from Viremic Thirteen-lined Ground Squirrels
Effect of dosage on viremia ti ters
Only one of the six squirrels inoculated with SSH produced a measurable viremia in this experiment. Table 10 shows that three of the four squirrels inoculated with 3.2 log^g SMLDgg of the virus developed trace (less than
1 log-jQ SMLDgQ/0.02 ml) viremias. The fourth squirrel had no detectable viremia. One of the two squirrels inoculated with 4.2 log-JQ SMLD^Q of SSH virus also developed only trace amounts of virus in the blood. A 3.2 log^g
SMLDgg dosage did not produce a measurable viremia but did initiate an infection in ground squirrels. In the previous experiment, a
4.1 log-iQ SMLDgg inoculum produced viremias in squirrels at 24 and 48
hours post-inoculation. The threshold dosage necessary to produce a sufficient viremia to infect mosquitoes must be greater than 3.2 log^g
SMLDgQ and less than 4.1 log-jQ SMLDgg. The titers of the 36 hour and
60 hour viremias in the squirrel that developed a measurable viremia
complement the virus titers obtained in the previous experiment at
24 and 48 hours post-inoculation. Differences in titer of the inocula (4.1 log^o vs 4.2 log^g SMLD^q) evidently had little influence on titer or
duration of SSH viremias. It remains to be determined whether
infective doses closer to the threshold dose capable of producing
viremias will have any influence on viremia titers or persistence.
Model for California virus transmission
Aedes triseriatus fed extremely well on the thirteen-lined ground 46
Table 10. Viremias in thirteen-lined ground squirrels inoculated with snowshoe hare virus
Animal Dosage Hours post-inoculation number (log^Q SMLDgg) 12 24 36 48 60 72 84 96
1 3.2 Neg Neg Neg Tb 2 3.2 Neg T Neg T 3 4.2 Neg 3.9c 3.3 Neg 4 3.2 Neg T Neg Neg 5 3.2 Neg Neg Neg Neg 6 4.2 Neg Neg T Neg C (uninoc.) Neg Neg Neg Neg
^Sample negative for virus.
^Virus titer less than 1 log-jg SMLDgg/O.OZ ml.
^Virus titer in log^g SMLDgg/ml.
Table 11. Feeding percentages of Aedes triseriatus on thirteen-lined ground squirrels inoculated with snowshoe hare virus
Animal First feeding (30 min.) Second feeding (30 min.) number No. exposed No. fed % No. exposed No. fed %
1 55 43 78.2 51 34 66.6 2 54 41 75.9 69 63 91.3 3 53 30 56.6 74 68 91.8 4 44 42 95.5 58 51 87.9
Total 206 156 75.7 252 216 85.7 47 squirrel. Feeding percentages are presented in Table 11. Viremias were present in three of four squirrels at the time mosquitoes fed. The viremia in the fourth squirrel did not occur until after the mosquitoes had fed, and the fifth squirrel did not develop a viremia. These data are presented in Table 12. The 102 mosquitoes that had fed on viremic squirrels were negative when tested for SSH.
Snowshoe hare virus was originally isolated from a snowshoe hare
(Burgdorfer et al. 1961) and has since been isolated from snowshoe hares on two occasions (Hoff et al. 1971; Feltz et al. 1972). Newhouse et al.
(1953; 1971) have found neutralizing antibodies to SSH in wild golden mantled ground squirrels (Spermophilus lateralis) and Columbian ground squirrels (S^. columbianus). McLean et al. (1973) reported neutralizing antibodies to SSH in 8% (1971) and 1% (1972) of arctic ground squirrels
(_S. undulatus). Laboratory inoculations of SSH resulted in viremias of up to 3.5 log-jQ SMLDgg/O.S ml in the golden mantled ground squirrel
(Newhouse et al. 1971). Snowshoe hare virus is evidently capable of producing infection in both lagomorphs and ground squirrels. Hammon et al. (1952) demonstrated a similar capability for the CEV prototype.
This suggests that some species of ground squirrels become infected and serve as reservoirs of SSH.
Natural vectors of SSH are probably A. communis in Canada
(McLean et al. 1973) and A. fitchii in Montana (Newhouse et al. 1971).
Snowshoe hare virus has not been isolated from A. triseriatus (Sudia et al. 1971b). The ability of A. triseriatus to become infected with
SSH is open to question. The Wisconsin B strain of A. triseriatus has Table 12. Viremias in thirteen-lined ground squirrels inoculated with 4.1 log-JQ SMLD^Q of snowshoe hare virus
Animal Hours post-inoculation number 12 18 24 30 " 36 42 48 54 60 66 72 78
1 Neg* Neg 3.2^
2 4.2 3.0
3 T 3.3 T
4 3.0 3.7 T
5 Neg Neg Neg
C Neg Neg Neg
^Sample negative for virus.
^Virus titer in log SMLDgg/ml.
^Virus titer less; than 1 log SMLDgjj/0.02 ml. 49
been shown to be less efficient than wild-caught mosquitoes in infection and transmission studies (Pantuwatana et al. 1972). It has been suggested that the inefficiency is associated with a genetic change
selected by colonization and laboratory rearing (Pantuwatana et al.
1972). It is suspected that a similar phenomenon exists with our
Wisconsin B strain and may be associated with the inability of A.
triseriatus to become infected with SSH after feeding on viremic
squirrels.
Infection of Aedes trivittatus with Trivittatus Virus
from Viremic Cottontail Rabbits
Two cottontail rabbits developed TVT viremias by 48 hours after
inoculation. One rabbit had a viremia titer of log 3.4 log-jg SMLDgg/ml
and the other rabbit had a titer of 3.9 log^q SMLDgg/ml. Trace viremias
were detected in both rabbits at 24 hours post-inoculation, and one
rabbit maintained a 3.7 lcg,„ SML.Dp„/ml viremia until the 72 hour bleeding; I u Du ^ Brain tissue taken from the dead rabbit was negative for TVT virus;
other tissue samples were positive.
A total of 196 A. trivittatus fed upon the cottontail 48 hours after
inoculation. Thirty-two of the blood fed mosquitoes were used for other
experiments and the remaining 164 were saved to determine infection rates.
The results of that study are presented in Table 13. Since field-caught
mosquitoes were used, field-collected mosquitoes were tested for the
presence of virus to determine the prefeeding infection rate. Five
of 20 pools containing 50 A. trivittatus each were positive for TVT 50 virus. The prefeeding infection ratio was calculated to be 1:200 mosquitoes. An attempt was made to transmit TVT by allowing A. trivittatus to feed on suckling mice. A total of 36 mosquitoes were provided 3-day-old suckling mice 15 and 16 days after they had fed on the viremic cottontail. None of the mosquitoes fed on the suckling mice.
Table 13. Infection rates of Aedes trivittatus after feeding on a viremic (3.9 lo9]o SMLDgg/ml) cottontail rabbit
Days No. mosquitoes No. mosquitoes % post-feeding tested positive infected
10 20 13 65
11 19 14 74
12 16 15 94
13 15 13 87
14 11 10 91
15 10 9 90
16 13 13 100
Total 104 87 84
Lagomorphs have been suspected as hosts for California viruses since Mammon et al. (1952) were able to produce CEV viremias in laboratory rabbits. Mammon and Reeves (1952) found serological evidence of CEV infection in 33% of cottontail rabbits. The SSH 51 subtype has been isolated from snowshoe hares on several occasions
(Burgdorfer et al. 1961; Hoff et al . 1971; Feltz et al. 1972) and SSH
neutralizing antibodies have been found in up to 65% of snowshoe hare
sera (McLean et al. 1971). Jennings et al. (1968) found that domestic
rabbits inoculated with KEY developed peak viremias of up to 2.4 log^g
SMLDgg/ml at 48 hours, and seven rabbits inoculated with TVT had
detectable viremias at 48 hours after inoculation. Finger (1974)
isolated TVT from the blood of a sentinel domestic rabbit.
Pantuwatana et al. (1972) inoculated six chipmunks with TVT. Two
of the chipmunks developed viremias at 48 hours but no viremias were
detectable at other sampling times. The cottontails used in this
experiment developed TVT viremias and maintained a high titer for 2
or more days. These results indicate that the cottontail rabbit is a
better host for TVT than either chipmunks or thirteen-lined ground
squirrels and must be considered as a prime candidate for the natural
host of TVT virus.
The results of the mosquito infection experiment compare favorably
with results obtained in other infection and transmission experiments
using live animal hosts. Watts et al. (1972) found 100% of A.
triseriatus infected with LAC 13 days after feeding on viremic hamsters.
Pantuwatana et al. (1972) found 67 and 80% of A. triseriatus
infected with LAC 15 days after feeding on viremic chipmunks and gray
squirrels. Table 13 shows that 100% of A. trivittatus were infected
with TVT 16 days after feeding on the viremic cottontail. Pinger (1974)
determined that 68% of the blood meals taken by A. trivittatus were 52 from cottontail rabbits. These results suggest a close relationship among A. trivittatus, the cottontail rabbit, and TVT virus. 53a
CONCLUSIONS
Trivittatus Virus and Aedes trivittatus
Trivittatus virus was isolated only from A. trivittatus. Over
16,000 mosquitoes of other species,collected in an area endemic for TVT, were tested and found not to be infected with TVT. Sudia et al. (1971b) reported isolations of TVT from 15 species of mosquitoes, but 86% of the
TVT isolations from the north central United States were from A. trivittatus. The high degree of specificity of TVT for A. trivittatus has been noted by Rowley et al. (1973) and DeFoliart et al. (1972).
Aedes trivittatus acquired TVT virus from feeding on viremic cottontail rabbits. The ability of A. trivittatus to transmit the virus to a susceptible host has not been determined.
There has been presumtive evidence of transovarial transmission of
TVT by A. trivittatus. Isolations of TVT have been made from first brood A. trivittatus on July 1= 1971 and June 19. 1974. Neutralizing antibodies to TVT began appearing in field collected sera of juvenile cottontail rabbits during early July 1973. Pinger (1974) isolated TVT from a sentinel rabbit shortly after the seasonal appearance of A.
trivittatus in 1973. Watts et al. (1973b) demonstrated transovarial
transmission of LAC in A. triseriatus, and LeDuc et al. (1975b) showed
transovarial transmission of KEY in A. atlanticus by isolation of the
virus from field collected larvae. Transovarial transmission of TVT
in A. trivittatus has not yet been found to occur. The idea that
TVT is transmitted transovarially in A. trivittatus and overwinters 53b via the egg stage has been the most attractive hypothesis to date.
Trivittatus Virus and Vertebrates
Cottontail rabbits developed high titer viremias after inoculation with TVT. Juvenile cottontail rabbits were found to develop neutralizing antibodies to TVT shortly after the seasonal appearance of A. trivittatus in 1973. Finger (1974) found a 45.5% incidence of neutralizing antibody in cottontail sera collected in 1973.
Thirteen-lined ground squirrels did not show evidence of infection after inoculation with TVT. Sera from field collected ground squirrels were negative for neutralizing antibodies to TVT. Eastern chipmunk sera also proved to be negative for TVT neutralizing antibodies.
Pantuwatana et al. (1972) found that the chipmunk and the gray squirrel were poor hosts for TVT. These data support a hypothesis that the eastern cottontail rabbit and not sciurids serve as hosts for TVT.
The Trivittatus Virus Natural Cycle
Under natural conditions the basic cycle of TVT is most likely a mosquito-to-mosquito cycle with the virus being passed transovarially in A. trivittatus. After overwintering in the egg stage, TVT is evidently trans mitted to cottontail rabbit populations by infected first brood Â. trivit
tatus. Cottontail rabbits may then serve as TVT reservoirs for uninfected
A. trivittatus. This may account for the high incidence of TVT in A.
trivittatus observed during the early summers of 1971 and 1974. Virus 54a transmission probably proceeds at low levels during mid-summer, but
FIR'S in late sunmer and early fall indicate the possibility of additional transmission activity. This activity may be associated with infection of susceptible juvenile cottontails from summer litters.
The cycle would be completed after infected A. trivittatus from this and preceeding broods deposited infected eggs which survived the winter and hatched the following spring.
Confirmation of this hypothetical cycle rests on proof of transovarial transmission of TVT in A. trivittatus and the ability of
A. trivittatus to transmit TVT. Additional information is needed on
TVT infections in cottontails and the seasonal occurrence of TVT in mosquitoes to lend credence to the proposed cycle. 54b
SUMMARY
Aedes trivittatus occurs in central Iowa from late May or early
June until late September or early October. New Jersey light trap collections indicate the presence of four broods of A. trivittatus during each season, with peak populations occurring in mid-July in two of the
three seasons. Populations of A. trivittatus are influenced by rainfall as indicated by reductions in numbers brought about by drought conditions in late summer of 1971 and early summer of 1973.
Trivittatus virus was isolated from 25 pools of field-collected
A. trivittatus during the 3-year study period. First brood mosquitoes accounted for 16 of the isolations on July 1, 1971. An isolation was made late in the season on October 4, 1972. Other mosquito species in the study area evidently are not involved in the TVT transmission cycle.
Three Flanders virus isolates were obtained from C_. pi pi ens complex mosquitoes. Serum samples from thirteen-lined ground squirrels
(^. t. tridecemlineatus) and eastern chipmunks (T. striatus qriseus),
collected during the sunmer of 1973, were negative for neutralizing
antibodies to TVT and JTC. Three of five cottontail rabbits (^. floridanus
mearnsii) collected in the same area had TVT neutralizing antibodies. Two
rabbits collected before June 27 were negative and three collected after
July 10 were positive. The only two chipmunk samples taken from Ledges
State Park were positive for neutralizing antibodies to LAC.
Thirteen-lined ground squirrels developed viremias ranging from
3.0 log^Q to 4.6 log^g SMLDgg/ml after inoculation with SSH virus. Virus 55a was also recovered from spleen, liver, lymph nodes, kidney and lung tissues of infected squirrels. Virus was isolated from brain and other tissues of one squirrel that died during the experiment. Aedes triseriatus that fed on viremic squirrels did not become infected with SSH virus. Ground squirrels inoculated with TVT and JTC failed to produce any evidence of viral replication.
Cottontail rabbits became infected with TVT and 48 hours after inoculation developed viremias ranging from 3.4 log-jQ to 3.9 log^g
SMLDgg/ml. Virus was also recovered from samples of spleen, liver,
kidney and lung tissues. Trivittatus virus infections developed in A.
trivittatus after feeding on a viremic cottontail. After 16 days of
incubation, 100% of the mosquitoes were infected with TVT. Attempts to
transmit TVT to suckling mice using infected A. trivittatus were
unsuccessful. Difficulties were encountered in inducing the infected
mosquitoes to feed on suckling mice.
The results of these studies indicate that sciurids such as
thirteen-lined ground squirrels and eastern chipmunks are not involved
in the natural cycle of TVT. The cottontail rabbit develops TVT
viremias that remain at a high titer for 2 or more days and A.
trivittatus can become infected by feeding on viremic cottontails. 55b
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Wellings, F. M. 1968. Epidemiological-virological study of the California encephalitis virus groups in Tampa Bay area of Florida, 1963=1968. Ph.D. Thesis, Univ. Pitt. 140 p. (Diss. Abstr, B,30:1778).
Wellings, F. M., G. E. Sather and W. McD. Hammon. 1970. A type- specific immunodiffusion technique for the California encephalitis virus group. J. Immunol. 105:1194-1200.
Wellings, F. M., G. E. Sather and W. McD. Hammon. 1971. Immuno- electrophoretic studies of the California encephalitis virus group. J. Immunol. 107:252-259. 94
Wellings, F. M., A. L. Lewis and L. V. Pierce. 1972. Agents encountered during arboviral ecological studies: Tampa Bay area, Florida, 1963 to 1970. Amer. J. Trop. Med. Hyg. 21:201-213.
Whitman, L. and R. E. Shope. 1962. The California complex of arthropod-borne viruses and its relationship to the Bunyamwera group through Guaroa virus. Amer. J. Trop. Med. Hyg. 11:691-696.
Whitman, L., R. C. Wall is and E. A. Leventhal. 1968. California group arbovirus from Aedes abserratus (Felt and Young) in Simsbury, Connecticut. Amer. J. Tr'op. Med. Hyg. 17:449-450.
Whitney, E. and R. Deibel. 1971. Propagation of arboviruses in Singh's Aedes cell lines. III. Growth studies of California Encephalitis Virus in two Aedes mosquito cell line cultures. Curr. Top. Microbiol. Immunol. 55:138-139.
Whitney, E., H. Jamnback, R. G. Means, A. P. Roz and G. A. Rayner. 1969a. California virus in New York State. Isolation and characterization of California encephalitis virus complex from Aedes cinereus. Amer. J. Trop. Med. Hyg. 18:123-131.
Whitney, E., A. P. Roz, G. A. Rayner and R. Deibel. 1969b. Serologic survey for arbovirus activity in deer sera from nine counties in New York State. Bull. Wildl. Dis. Ass. 5:392.
Wilcocks, C. and P. E. C. Manson-Bahr. 1972. Diseases due to viruses: Arbovirus diseases. Pages 355-383 in C. Wilcocks and P.E.C. Manson-Bahr. Manson's Tropical Diseases. Seventeenth edition. Williams and Wilkins, Co., Baltimore. 1164 p.
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Yunker, C. E. and J. Cory. 1968. Infection of Grace's Antheraea cells with arboviruses. Amer. J. Trop. Med. Hyg. 17:889-893.
Zarate, M. L., R. H. Geiger, R. E. Shope and W. F. Scherer. 1968. Intergroup antigenic relationships among arbovirus manifested by a Mexican strain of Patois virus and viruses of the Bunyamwera, C, California, Capim and Guama groups. Amer. 0, Epidemiol. 88: 273-286. 96
ACKNOWLEDGMENTS
I would like to express my appreciation to Dr. Wayne A. Rowley for his help and guidance with my research and his critical comments during the preparation of this manuscript. To the other members of my committee. Dr. Paul A. Dahm, Dr. C. John Mare, Dr. Loyd Y. Quinn and Dr. Michael K. Petersen, I would like to express a sincere thanks for advice and encouragement during various phases of my research.
A special thank you is due to Mr. Yau Wai Wong, Mr. Donald C.
Dorsey and Mr. James P. Brinker of the State Hygienic Laboratory of
Iowa for invaluable assistance and advice. I would also like to acknowledge Mr. Jerry Grebasch for his cheerful cooperation in allowing me to pursue my research at the Nursery.
I would like to thank my fellow graduate students Dr. Robert R.
Pinger, Jr. and especially Mr. Bruce G. Johnson, Jr. and Mr. Wayne
N. Andrews who gave invaluable assistance at crucial periods in my research.
We always seem to save the best for last. I would like to thank my wife Merry Ellen for encouragement, understanding and help with my
research project and the preparation of this manuscript. 97
APPENDIX A. FORMULAE 98
Cleaning Fluid
Penicillin 100,000 lu
Streptomycin 100,000yugm
Fungizone 2,000 jugm
Distilled water to 1,000 ml 99
Phosphate Buffered Saline
Solution A. 0.1 M monobasic sodium phosphate
NaHgPO^'HgO 13.8 gm
NaCl 8.77 gm
Distilled water to 1000 ml
Solution B. 0.1 M dibasic sodium phosphate
NagHPO^ 14.2 gm
NaCl 8.77 gm
Distilled water to 1000 ml
Antibiotic solution lOOX
Streptomycin 1.0 gm
Penicillin 1,000,000 lu
Sterile distilled 10 ml water to
0.1 M Bovi ne Albumi n-Phosphate Buffered Saline
pH 7.6 to 7.8
Solution A 89.7 ml
Solution B 8.3 ml
Bovi ne Albumin 1.0 gm
Antibiotic solution 1.0 ml (optional) 100
APPENDIX B. RECORDS OF MOSQUITOES TRAPPED IN DRY
ICE-BAITED CDC TRAPS FOR VIRUS ISOLATION ATTEMPTS 101
Collection of July 1, 1971
Mosquito species Trap site^ Species 1 2 3 4 5 5 total
Aedes
trivittatus 910 357 83 600 482 261 2693
vexans 3 - - 13 17 14 47
Anopheles
punctipennis 1 1 1 11 32 46
Coquillettidia
perturbans 7 3 - 5 56 18 89
Culex
pipiens complex 354 106 4 53 121 20 658
tarsal is 1 1 - - 4 1 7
Culiseta
inornata - 1 - - - - 1
Trap site total 1276 469 87 672 691 346 3541
Vrap site numbers for this and subsequent collections refer to locations in Fig. 2. 102
Collection of August 3, 1971
Mosquito species Trap site Species 1 3 4 5 6 total
Aedes
triseriatiis 1 - - 1
trivittatus 4088 2526 3190 691 7250 17,745
vexans - 8 5 16 29
Anopheles
punctipennis 2 27 12 5 3 49
Coquillettidia
perturbans 117 1 1 20
Culex
pipiens complex 402 397 1 22 1 06 446 1 473
tarsal is 2 13 4 23 3 45
Trap site total 4495 2988 3334 842 7703 19,362 103
Collection of August 31, 1971 (September)
Mosquito species Trap site Species 1 2 3 5 6 total
Aedes
triseriatus - 1 - - - 1
trivittatus 268 170 73 26 116 653
vexans 4 6 7 1 7 25
Anopheles
punctipennis 19 12 48 7 22 108
Coquillettidia
perturbans - 1 - - 1
Culex
Pipi ens complex 263 254 180 62 44 803
tarsal is - 1 8 1 10
Quii seta
i no mat a 3 2 1 - - 6
Uranotaeni a
sapphirina 1 _ _ _ _ 1
Trap site total 558 445 311 104 190 1608 104
Collection of September, 1971
Mosquito species Trap 1 Trap 1 Species 9-21 9-28 total
Aedes
triseriatus 1 1
trivittatus 62 3 65
vexans 224 22 246
Anopheles
punctipennis 23 2 25
Culex
pipiens complex 363 247 610
tarsal is 1 - 1
Culi seta
i nornata 7 2 9
Trap site total 680 277 957 105
Collection of May 3, 1972
Mosquito species Trap site Species 2 3 4 5 6 total
Culex
pipiens complex 2-1 216
Culiseta
inornata 3 1 2 8 1 15
Trap site total 5 1 3 10 2 21 106
Collection of May 18, 1972
Mosquito species Trap site Species 1 2 3 4 5 6 total
Aedes
dorsal 'i s 1 - - - - 7 8
sticticus 1 - - - - - 1
trivittatus - - 1 2 - 1 4
vexans 41 30 12 39 6 24 152
Anopheles
punctipennis 1 - - - - 1
Culex
pi piens complex 10 8 5 7 2 32
Culiseta
inornata 29 6 8 15 - 13 71
Trap site totals 82 45 26 63 6 47 269 107
Collection of June 1, 1972
Mosquito species Trap site Species 1 2 3 4 5 6 total
Aedes
canadensis ______i
niqromaculis ______2
stimulans ______1
trivittatus 4331 3379 253 1648 2447 568 12,626
vexans 586 350 172 222 615 205 2150
Anopheles
punctipennis ______20^
Culex
pipi ens complex 104 68 74 12 48 40 346
tarsal is - - - - 31^
Culiseta
inornata - - - - 27 ^
Trap site totals 5021 3797 499 1882 3110 813 15,204
Hrap site not recorded. 108
Collection of July 7, 1972
Mosquito species Trap site Species 1 4 6 total
Aedes
triseriatus
trivittatus 5126 9561 7623 22,310
vexans 54 133 128 315
Anopheles
punctipennis 29
Coquillettidia
perturbans 49'
Culex
pipiens complex 382 285 174 841
tarsal is 65
Culi seta
i nornata 8'
Psorophora
horrida
Trap site totals 5562 9979 7925 23,621
1 Trap site not recorded. 109
Collection of August 9, 1972
Mosquito species Trap site Species 6 total
Aedes
triseriatus
trivittatus 2069 524 4798 7391
vexans 29 741 773
Anopheles
punctipennis 54
Coquillettidia
perturbans 25
Culex
pipiens complex 972 8 187 1167 1 tarsal is 22
Culiseta
inornata
Trap site totals 3070 535 5726 9434
Hrap site not recorded. no
Collection of September 19, 1972
Mosquito species Trap site Species 14 6 total
Aedes
triseriatus 11- 2
trivittatus 18 35 139 192
vexans 113 52 440 605
Anopheles
punctipennis 6 8 7 21
Culex
erraticus 1 1
pi piens complex 118 11 20 149
Psorophora
horrida - 1 1
Traps site totals 257 108 606 971 Ill
Collection of October 4, 1972
Mosquito species Trap site Species 6 total
Aedes
niqromaculis 1 1
trivittatus 153 824 480 1457
vexans 11 29 62 102
Culex
pi piens complex
Psorophora
horrida
Trap site totals 165 858 545 1568 112
Collection of October 26, 1972
Mosquito species Trap site _ Species 6 total
Aedes
trivittatus
vexans
Culex
pipi ens complex
Trap site totals 113
Collection of May 18, 1973
Mosquito species Trap site Speci es 6 total
Aedes
dorsal is 3 7
stimulans 1 1
spenceri - - 2 - 2
sticticus 135 174 40 12 361
vexans 391 587 171 98 1247
Anopheles
punctipennis
Culex
pipiens complex 8 18 14 1 41
tarsal is 59 16 21 34 130
Culi seta
i noma ta 115 162 45 83 405
Trap site totals 710 963 295 228 2196 114
Collection of July 3, 1973
Mosquito species Trap site Species 1 2 total
Aedes
triseriatus 7 - 7
trivittatus 281 155 436
vexans 270 149 419
species 32 - 32
Anopheles
punctipennis 10 3 13
Coqui11ettidia
perturbans 1 î 2
Cul ex
pipiens complex 10 16 26
tarsal is 3 2 5 115
Collection of July 12, 1973
Mosquito species Trap site Species 1 total
Aedes
sticticus 3 3
trivittatus 133 133
vexans 52 52
Anopheles
punctipennis 3 3
Culex
pi piens complex 16 16
tarsal is 4 4
Trap site totals 211 211 116
Collection of August 15, 1973
Mosquito species Trap site Species gl total
Aedes
triseriatus
trivittatus 656 1324 1253 3233
vexans 3 5 6 14
Anopheles
punctipennis
Culex
pipi ens complex 147 97 95 339
tarsali s
Trap site totals 813 1427 1358 3598
Hwo traps in area. 117
Collection of August 16, 1973
Mosquito species Trap site Species 1 4 6 total
Aedes
tri seri atus 3 14
trivittatus 813 887 1269 2969
vexans 27 34 27 88
Anopheles
punctipennis 16 16 13 45
Culex
pi pi ens complex 354 101 225 680
tarsal is 1 6 7
Culiseta
i noma ta - 11
Psorophora
horrida 2 - 2
Trap site totals 1216 1038 1542 3796 118
Collection of September 10, 1973
Mosquito species Trap site Species i total 1
Aedes
trivattatus 323 323
vexans 8 8
Anopheles
punctipennis 17 17
Culex
pipiens complex 137 137
tarsal is 2 2
Culiseta
inornata 1 1
Trap site totals 488 488
1 Two traps in area. 119
Collection of September 11, 1973
Mosquito species —^sHe species 1 4 6 total
Aedes
triseriatus 2 - - 2
trivittatus 500 524 608 1632
vexans 13 20 65 98
Anopheles
punctipennis 6 11 8
Culex
pipi ens complex 119 4 103 226
tarsal is - - -
Culiseta
i noma ta 7 1 - 8
Trap site totals 647 550 777 1974
Vwo traps in area. 120
APPENDIX C. RECORDS OF SERUM SAMPLES FROM WILD MAMMALS
TESTED FOR THE PRESENCE OF ARBOVIRUS ANTIBODIES
Symbols used with the following data are:
I Species
GS Spermophilus tridecemlineatus tridecemlineatus
CM Tamias striatus griseus
CT S.ylvilaqus floridanus mearnsii
Result
I m L.MV Neutralizing antibodies to LaCrcsse virus
JTC Neutralizing antibodies to Jamestown Canyon virus
TVT Neutralizing antibodies to trivittatus virus 121
No. Species Sex Location Sample Resuit date
1 6S M St. For. Nur. 8-9-72 Neg 2 GS M Lakeside Lab. 4-5-73 Neg 3 GS F Lakeside Lab. 4-5-73 Neg 4 GS F Lakeside Lab. 4-16-73 Neg 5 GS F South Ames 4-27-73 Neg
6 GS M South Ames 4-27-73 Neg 7 GS F South Ames 4-27-73 Neg 8 GS F South Ames 5-1-73 Neg 9 GS F South Ames 5-3-73 Neg 10 GS F South Ames 5-7-73 Neg n GS F South Ames 5-10-73 Neg 12 GS M South Ames 5-10-73 Neg 13 GS M South Ames 5-10-73 Neg 14 GS M South Ames 6-11-73 Neg 15 GS M South Ames 6-11-73 Neg
16 CM F Ledges St. Pk. 6-21-73 LAC,JTC,TVT 17 CM M Ledges St. Pk. 6-21-73 LAC 18 CM M South Ames 6-21-73 Neg 19 CM F South Ames 6-22-73 Neg 20 CM F South Ames 6-26-73 Neg
21 CM M South Ames 6-26-73 Neg 22 CM M South Ames 6-26-73 Neg 23 CT F South Ames 6-22-73 Neg 24 rx c Cmufh âmoc 6-27-73 Neg 25 GS F South Ames 6-28-73 Neg
26 CM M South Ames 6-28-73 Neg 27 CM F South Ames 6-28-73 Neg 28 CM M South Ames 6-28-73 Neg 29 CM F South Ames 6-28-73 Neg 30 GS F South Ames 6-28-73 Neg
31 CM M South Ames 6-28-73 Neg 32 CM F South Ames 6-29-73 Neg 33 CM M South Ames 6-29-73 Neg 34 GS M South Ames 6-29-73 Neg 35 CM M South Ames 7-2-73 Neg
36 CM M South Ames 7-2-73 Neg 37 CM M South Ames 7-3-73 Neg 38 CM M South Ames 7-3-73 Neg 39 CT South Ames 7-10-73 TVT 40 CT South Ames 7-21-73 TVT 122
No. Species Sex Location Sample Resuit date
41 GS F South Ames 7-5-73 Neg 42 CM M South Ames 7-9-73 Neg 43 CM F South Ames 7-9-73 Neg 44 CM M South Ames 7-9-73 Neg 45 CM F South Ames 7-12-73 Neg
46 GS F South Ames 7-19-73 Neg 47 CM F South Ames 7-19-73 Neg 48 GS F South Ames 7-19-73 Neg 49 GS F South Ames 7-19-73 Neg 50 GS F South Ames 7-19-73 Neg
51 GS M South Ames 7-19-73 Neg 52 GS F South Ames 7-23-73 Neg 53 GS F South Ames 7-23-73 Neg 54 GS F South Ames 7-23-73 Neg 55 GS F South Ames 7-23-73 Neg
56 GS M South Ames 7-23-73 Neg 57 GS M ÎSU Campus 7-26-73 Neg 58 GS F South Ames 7-26-73 Neg 59 GS M South Ames 7-26-73 Neg 60 GS M ISU Campus 7-26-73 Neg
61 CT West Ames 7-19-73 JTCJVT 62 GS F South Ames 7-28-73 Neg 63 GS F ISU Campus 7-28-73 Neg 04* GS F OW un rviicd 8-2-73 Neg 65 GS F South Ames 8-2-73 Neg
66 GS F South Ames 8-2-73 Neg 67 GS M South Ames 8-2-73 Neg 68 GS M South Ames 8-2-73 Neg 69 GS M South Ames 8-2-73 Neg 70 GS M South Ames 8-2-73 Neg
71 GS r South Ames 8-2-73 Neg 72 GS M South Ames 8-2-73 Neg 73 GS M South Ames 8-8-73 Neg 74 GS F South Ames 8-8-73 Neg 75 GS M South Ames 8-8-73 Neg
76 GS M South Ames 8-8-73 Neg 77 GS F South Ames 8-8-73 Neg 78 GS M South Ames 8-8-73 Neg 123
APPENDIX D. FIGURES Fig. 1. Aerial photograph showing the location of the State Forest Nursery near the intersection of U.S. Highways 30 (east-west) and 69 (north-south)
The study area is framed in black. 125 Fig. 2. Detailed map of the study area, located at the eastern edge of the State Forest Nursery, Ames, Iowa
C Culverts CS Coniferous stands DS Deciduous stands L New Jersey light trap M Malaise trap PL Property 1i ne PS Packing shed R Road S Stream SP Settlement pond I -u uuv I lyiiL UI apd 127 Fig. 3. Seasonal distribution of all mosquito species, Aedes trivittatus, and Aedes vexans collected by the New Jersey li()ht trap at the State Forest Nursery, Ames, Iowa, during 1971 A All mosquito species 500 • — Aedes trivittatus Q Aedes yexami
200
100
50 -a
20 -a
10
10 20 31 10 20 :)0 10 20 31 10 20 31 10 20 30 10 20 31 May June July August September October Fig. 4. Seasonal distribution of Culex pi piens complex (including Ç. pipiens. Ç. restuans, and £. salineirius) and Culex tarsalis collected by the New Jersey light trap at the State Forest Nursery, Ames., Iowa, during 1971 • Culex pipiens complex 500 - O Culex tarsalis
200 -
100 -
50 -
I I \ /
1 I .1 I 1 ^ J 1 1 _i I I I I I III I il 10 20 31 10 20 30 10 20 31 10 20 31 10 20 30 10 20 31 May June July August September October Fig. 5. Seasonal distribution of all mosquito species, Aedes trivittatus. and Aedes vexans collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1972 A — All mosquito species 500 - • — Aedes trivittatus Q_ —Aedes vexans f.... >.. No data
A 200 .
"U0) 4J a 10 H I 5 - J. 20 30 10 20 31 10 20 30 10 20 31 June August September October l-ig. 6. Seasonal distribution of Culex pi pi ens complex (including C_. pi pi ens. Ç. restuans, and C. salinariu:;). Culex tarsal is. and Culiseta inornata collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1972 Culex piplens complex 500 - Culex tarsalls A - Culiseta inornata No data 200 - 100 - 50 - 20 - 10 - o—o 10 20 May June July August September October Fig. 7. Seasonal distribution of all mosquito species, Aedes trivittatus, and Aedes vexans collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1973 A All mosquito species ID ' — Aedes trivittatus 500- 0 Aedes vexans 200- A-.-A 100- 50- 20- 10- 10 20 31 10 20 30 10 20 31 10 . 20 31 10 20 30 10 20 31 May June July August September October Fig. 8. Seasonal distribution of Culex uipiens complex (including £. pipiens. Ç. restuans. and Ç. salinarius). Culex tarsal is, and Culiseta inornata collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, during 1973 Culex piplens complex 500. O — — Culex tarsaliîi ^ Cullseta inorriata 200 - 100 10 20 31 10 20 30 10 20 31 10 20 31 10 20 30 10 20 31 May June July August September October Fig. 9. Seasonal distribution of Aedes trivittatus collected by the New Jersey light trap at the State Forest Nursery, Ames, Iowa, 1971-1973 500- No data 200. 100- 50- 20- 10. 10 20 31 10 20 30 10 20 31 10 20 31 10 20 30 10 20 31 June July August September October