VERTEBRATE-MOSQUITO RELATIONSHIPS m A MICHIGAN WATER QUALITY MANAGEMENT PROJECT Thesis. for the Degrea of M. S. NICHIGM‘ STATE UNIVERSIH - 30H]! ALBERT 911le 297.5% IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII“ ft mammal: gmewmmr “’m g; 4 I .I'e “:1 ‘ . I. {‘3 1} ‘f '9‘ Y ‘ or: 1 (j A"... {I ‘ .1, “*‘“~" ~th _ . _.___.__‘_"_l 7mmfisfl—7WLI". 717‘}: (“y-1:33.11- ' ‘ w-‘O— . ,—. J, I / .4 " , -_ 1 . / i ”no“ A .uo- _ - - . \" r, “ -' I _‘ . '1 _ ‘ ,.;' . .71!» ”5153‘ FEB 1 7 I999 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII"- '93 104, IIIIII 1 —_f-— IIII OJ ABSTRACT VERTEBRATE-MOSQUITO RELATIONSHIPS IN A MICHIGAN WATER QUALITY MANAGEMENT PROJECT BY John Albert Wildie This study was designed to obtain baseline data on California group virus activity in small indigenous mammals living in a sewage spray irrigation site prior to Spraying. The mammals were trapped in collapsible and non-collapsible live traps, anesthetized and blood samples taken by cardiac puncture. The sera were sent to the Michigan Department of Public Health, Virology Laboratory, and tested for hema- gglutination inhibition antibodies using the LaCrosse virus antigen and goose red blood cells. One hundred and sixty- two mammals, comprising 10 species, were trapped and 182 blood samples collected. Positive sera were obtained from 3 fox squirrels, Sciurus niger Linn.; 1 red squirrel, Tamiasciurus hudsonicus, Erxleben; and l chipmunk, Tamias striatus, Linn. An attempt was made to obtain data on the host feeding preferences of the mosquito species present in the spray irrigation area by using mammal-baited mosquito John Albert Wildie traps. Aedes triseriatus (Say), Aedes vexans (Meigen), Aedes sticticus (Meigen), Aedes fitchii—stimulans (Felt & Young), Culex pipiens Linn., Culex salinarius Coq., Aedes cinereus Meigen and Coquillettidia perturbans (Walk.) were collected but only in low numbers. There was low level California group virus activity in Sciuridae (squirrels and chipmunks) in the spray irri- gation area and at least one mosquito, Aedes triseriatus (Say), capable of transmitting the California encephalitis virus was in the study area. VERTEBRATE-MOSQUITO RELATIONSHIPS IN A MICHIGAN WATER QUALITY MANAGEMENT PROJECT BY John Albert Wildie A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1975 ACKNOWLEDGMENTS I wish to thank Dr. Harold D. Newson for his invaluable guidance in the preparation of this manuscript and for his support both financial and moral during the course of this study. I would like to extend my appreciation to Drs. Roland L. Fischer, James E. Bath, and Rollin H. Baker for serving on my guidance committee. I would like to thank Dr. Maurice Becker and the personnel at the Michigan Department of Public Health, Vir010gy Laboratory, for performing the necessary serolOgi- cal tests. I would like to extend appreciation to Drs. Howard Tanner, Thomas Bahn, and the personnel of the Water Quality Management Project for allowing me to conduct my research in the project site. I would like to extend a Special thanks to Dr. John N. Stuht, Rose Lake Research Station, for assisting me with the trapping and bleeding of mammals and for just being there when I needed advice. ii Finally a special note of appreciation to my wife, Suzanne, for her constant encouragement during my graduate studies. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . V LIST OF FIGURES O O O O O O O O O O O O 0 Vi INTRODUCTION 0 O O 0 O O O O O O O O O O 1 History of the California Encephalitis Virus . . 2 Effects of Water Management on Mosquito Production . . . . . . . . . . . . . 16 MATERIALS AND METHODS. . . . . . . . . . . 24 Description of Study Area. . . . . . . . . 24 Materials 0 O O O O O O O O O O O O O 27 MethOds O O O O C O O O O O O O O O O 3 5 RESULTS 0 O O O O O O O O O O O O O 0 38 DISCUSSION 0 O O O O O O O O O O O O O 45 SUMMARY 0 O O O O O O O O O O O O O O 50 LITERATURE CITED . . . . . . . . . . . . 52 APPENDICES Appendix A. Nembutal Dosages Based on Animal's Weight. . 59 B. HI Titer Results of Indigenous Small Mammals in a Water Quality Project Site, 1973-1974 0 o o o o o o o o o o o 60 C. Metric Conversion Table. . . . . . . . 64 iv LIST OF TABLES Known vertebrate hosts of mosquitoes occurring in MiChigan I O O O O O O O O O 0 Potential vectors of the California encephalitis virus group in Michigan. . . Potential Michigan animal reservoirs of California encephalitis viruses . . . . Potential mosquito—disease relationships in a sewage irrigation project in Belding, Michigan, 1972-1974 . . . . . . . . A survey of animals in the Michigan State University Water Quality Project site, 1973-19740 0 o o o o o o o o o o Serological survey of indigenous small mammals in the Michigan State University Water Quality Project site, 1973-1974 . . . . 1974 mosquito bait trap collection results at the Michigan State University Water Quality PrOjeCt Site. 0 O O O O O O O O 0 Host associations of fleas collected during 1973-1974 from Michigan State University Water Quality Project site . . . . . . Page 10 12 14 21 39 40 42 43 LIST OF FIGURES Figure Page 1. Map of study area . . . . . . . . . . 26 2. Collapsible live traps . . . . . . . . 30 3. Non-collapsible live traps . . . . . . . 3O 4. Mosquito bait trap with side collecting boxes detached . . . . . . . . . . 32 5. Mosquito bait trap opened to show location of caged animal . . . . . . . . . . 32 6. Mosquito bait trap with metal legs . . . . 34 7. A young raccoon anesthetized with Nembutal. . 34 vi INTRODUCTION During the early part of 1971 the Institute of Water Research of Michigan State University designed and initiated a water quality management project. The project was designed to determine the level of water purification that could be obtained by passing sewage effluent through a series of oxidation ponds. The water upon reaching the final pond was to be transported to a second portion of the project where spray irrigation distributed it to a variety of field crops, old-fields and woodland environments to determine whether dissolved water pollutants would be filtered out as the effluent passed through the soil and entered the ground water table. The study reported here was one of several conducted in the project area which included such diverse subjects as soil biology, ornithology, mammalogy and medical entomology. The medical entomology studies included a determination of the biting insects indigenous to the project site and the insect-borne disease potentials that existed in the project area before the start of any spray irrigation Operations. The human biting insect pOpulation studies were done by Tom Zorka, another graduate student, to determine the indigenous insect pOpulations that were in the area prior to the start of spray irrigation. The data obtained were to be used in the evaluation Of any changes that could occur in the course of future Spray Operations. Thus, the effects of the spraying Operation on the insect pOpulations produced remain to be evaluated at some future time. The part of the study reported here was divided into two parts: A serological survey to determine whether or not California encephalitis virus infections occurred in the indigenous small mammals and to determine the mammal host preferences of the mosquito Species present in the spray irrigation area. History of the California Encephalitis Group Virus ArthrOpod transmitted viruses or arboviruses have been known to cause central nervous system diseases in humans in the United States since the 19303. There are currently five epidemic or endemic forms of arboviral encephalitis of primary concern: Eastern equine (EEE), Western equine (WEE), Venezuelan equine (VEE), St. Louis (SLE), and California (CE) encelphalitis. The BBB, WEE, VEE, and SLE viruses are well documented in the literature and have received considerable study since they produce a relatively high mortality. CE virus infections, hoWever, produce a more benign disease and have become a leading cause of human arboviral encephalitis in the United States. The California encephalitis virus prototype was first isolated in 1943 and 1944 from Culex tarsalis Coq. and Aedes melanimon Dyar mosquitoes in Kern County, California, and was incriminated in three human cases of encephalitis that Occurred in 1945 in California's San Joaquin Valley (Henderson & Coleman, 1971; and Hammon & Reeves, 1945). This virus was not reported again until Thompson and co-workers isolated a virus with antigenic prOperties similar to the CE prototype. This was obtained from frozen portions of brain tissue Obtained from a 4-year- old girl who died Shortly after being hospitalized in LaCrosse, Wisconsin, in 1959 with a meningencephalitis diagnosed illness (Thompson 33 31., 1965). It took five years to develOp the techniques needed to isolate and identify this virus, subsequently named the LaCrosse Strain (CE group), after the location where the girl died. The importance of Thompson's work was not merely the isolation and identification of the LaCrosse virus, but the develop- ment of the techniques needed to detect this virus. Of the 519 confirmed or presumptive human encephalitis cases attributed to the CE group that were reported between 1945 and 1970, the majority occurred prior to 1964, but were not diagnosed until after 1964 when the techniques sen- sitive enough to detect the virus had been develOped (Sudia 3E 31., 1971; and Vianna 33 31., 1971). In 1964, the first reported CE epidemic occurred in Ripley County, Indiana, with 12 confirmed or presumptive cases in children under the age of 16 (Beadle, 1966). In the period between 1967 and 1969, the number of CE cases reported in the United States outnumbered the combined total of BBB, WEE, SLE, and VEE cases. Approximately 90 percent of these CE cases occurred in the north-central region of the country. The increasing number of reported human CE infections during this time is not necessarily an indication of the activity of the virus. Sudia 33 31. (1971) suggested that the large number of reported cases from certain areas (i.e., the north-central region) could be related to the high interest level of the investigatory groups looking for the virus in that area. For example, prior to 1968, there were no reported cases fromMichigan although numerous cases were reported from areas surrounding Michigan including Wisconsin, Ohio, Indiana, and Canada. Investigators in Michigan did not begin testing fOr CE until 1968, so it is not known whether human infections occurred there prior to that time. Human CE cases in the United States have been reported from 18 states with the majority occurring in Indiana, Ohio, Wisconsin, Minnesota, Iowa, and Michigan. Human CE infections have been reported as far south as North Carolina, indicating wide-spread occurrence of this virus. One difficulty in the diagnosis of this virus is that there are apparently eight to ten types or subtypes. Several authors have suggested that this variation results from the lack of mobility of the small mammals that are the vertebrate hosts of these subtypes which serves to isolate the virus in nature (Sudia 33 31., 1971; Johnson, 1970; Sather & Hammon, 1967; and Parkin, 1973). Johnson felt that these viruses are currently undergoing an evolutionary change which could explain the similiarities seen between the various strains in the CE group obtained from given locations at any one time. Of the types so far identified only four have been associated with human disease. These are CE prototype, LaCrosse subtype, Trivittatus subtype, and Jamestown Canyon subtype. The LaCrosse sub- type appears to be responsible for most of the known human cases that have been reported in the United States. Once man becomes infected with the virus, the sumptoms of the disease may or may not be expressed. Infections with the CE (LaCrosse subtype) generally product clinical symptoms in children under the age of 16, while adult infections typically are subclinical (Thompson 33 31., 1963; Thompson & Inhorn, 1967; and Johnson 33 31., 1968). The acute clinical symptoms seen in children are fever, headache, nausea, nuchal rigidity, convulsions and lethargy. Focal neurological signs that include paresis, paralysis and aphasia are also common. The typical illness usually runs its course in seven to ten days and is followed by complete recovery. However, there may be some post-encephalitic behavioral changes and impared scholastic abilities (Chun 33 31., 1968; and Matthews 33 31., 1968). The low mortality resulting from human CE infections IIIIIIIIIIII undoubtedly is why more research has not been conducted in many areas of the country. The natural transmission cycle of CE does not normally involve humans. It is maintained in nature in a mosquito-small mammal-mosquito cycle with man becoming involved when he intrudes on the woodland environment where the virus exists. The primary vector(s) Of the virus are mosquito Species Of the genus 33333. Small mammals serve as reservoirs of the virus and maintain a readily available source of the virus to infect other mosquitoes. The role that a particular mosquito Species plays in the trans- mission of the virus is partially based on its minimum field infection rate (MFIR), which is a reflection of the number of mosquitoes tested and the number of isolations obtained. 33333 species in general have higher MFIR'S than do non—33333_mosquitoes in the natural transmission cycle of the CE. This infection rate is a relative indication of the mosquito pOpulation infected and is not a direct relationship of the CE to human infection (Sudia 33 31., 1971). The role of the vertebrate host in the transmission of CE is that of maintenance Of a virus pool from which non-infected mosquitoes become infected. In this way a larger portion of the mosquito population can become infected and in turn infect more mammals. In nature this cycle would start as a small focus and keep building upon itself. Limiting factors that prevent or regulate this build-up include: (1) Not all vertebrates serve as reser— voirs; (2) not all mosquitoes serve as vectors; (3) environmental factors such as rain and snow that affect the pOpulation levels of suitable vector mosquitoes and; (4) the develOpment of immune mammal pOpulations as a result of the CE infections. In order for the vertebrate to serve as a reservoir the virus must be transmitted from the arthrOpod vector to the vertebrate host. There are two possible mechanisms by which the vertebrate host can be infected, either by mechanical or biological transmission Of the virus. CE viruses have been isolated from tabanoid Species including Hybomitra lasiOphthalma (Macq.) and ChgySOpS cincticornis (Walk.) incriminating them as possible vectors of CE in Wisconsin (Wright 33 31., 1970; and DeFoliart 33 31., 1969). This group Of insects may be responsible for the transmission Of the virus to the larger mammals such as deer, Odocoileus virginianus Zimmermann, and domestic animals with which these arthrOpOds are usually associated. The role these arthrOpOds play in CB transmission is still uncertain, for mechanical transmission of the virus via infected mouth parts is a possibility. Mechanical transmission is also possible in the primary vector (mosquitoes), but biological transmiSsion is considered more important. In this mechanism the virus multiplies within the tissues of the arthrOpod before it is transmitted to another vertebrate host. The detection of the virus in the insect tissues is a good indication that the insect serves as a vector Of the virus (Chernesky, 1967). Once infected the mosquito must maintain the virus at a level high enough to infect the vertebrate host and for long enough to enable the mosquito to feed on another susceptable host. Watts 33 31. (1972) were able to infect Aedes triseriatus (Say) mosquitoes by feeding them on 8- day-old hamsters which had been previously innoculated sub- cutaneously with the LaCrosse virus subtype. The mosquitoes were able to transmit the virus (after a prepatent period Of virus development of the virus of about 7 days) from day 7 to day 36 after feeding. This relatively long capability of a mosquito to transmit the virus is an important factor in determining the importance of the mosquito as a vector of the virus. Besides having a long lasting transmission ability, 33333 triseriatus (Say) is able to pass the virus to the eggs by transovarial transmission (Watts 33 31., 1973). Trans- mission of the LaCrosse virus by this method is a possible mechanism for the survival of this arbovirus during the winter season in the north-central United States (Watts 33 31., 1974). As a result of the transovarial trans- mission of the virus to the eggs, it could be possible to detect the virus in a particular location by testing larval and egg samples for the virus. Another criterion that the mosquito must exhibit before it can be incriminated as a potential vector is an association with the susceptable vertebrate host that serve as reservoirs of the virus. Much research has been con— ducted to determine these mosquito-host associations. Some mosquitoes such as Culex pipiens Linn. Show a preference for avian hosts and Aggpheles quadrimaculatus Say for mammalian hosts, while Culex salinarius Coq. and Coquillettidia perturbans (Walk.) feed freely on both types without showing any apparent preference (Murphy 33 31., 1967; Crans, 1963; Tempelis 33 31., 1967; Rempel 33 31., 1946; and Chamberlain 33 31., 1954). Although many mosquitoes show host preferences the availability of the host is an important factor in influencing the mosquitoes selection of its blood meal (Shemanchuk 33 31., 1963). Table 1 shows some of the mosquito-host associations of a variety of mosquito Species occurring in Michigan. Rodents, raccoons, rabbits and squirrels (including chipmunks) are the hosts most frequently used by these mosquitoes. Those mosquito species that utilize a wide range of hosts have a greater potential to serve as vectors of the CE virus than do those that only use a limited number Of hosts. In Table 1 Aedes canadensis (Theobald), Aedes cinereus Meigen, Aedes fitchii-stimulans (Felt & Young), Aedes sticticus (Meigen), Aedes triseriatus (Say), Aedes trivittatus (Coq.), Aedes vexans (Meigen), Culex pipiens Linn., Culex salinarius Coq. and Culiseta melanura (Coq.) were shown to 10 .ESmmomOIO .xosguwoozus .xcsxmum .xcsgdflnouo .Hmuuflswm vmmnmm .Hmmuflsvm xomumm .coooommuo mocom mo.am x x x x x x x x msumuufi>fluu moomm mm.mm x x x x x x x msumflummfiuu mopod mH.mH x x x mausnoauu mopom w©.mm x x x x x x x x x mSOAUOAOm mocwd mo.ma x x x x Houocsm mopom mo.ma x x x x x x x x mcmHSSHnmnflHnopHm mmpmm mm.ma x x mcdwosuoxm mopom mm.mv.am x x maammnov mopom ma x wHOOEEOO mmpmd mo.0m x x x x x x x x x msouocflo mopmm mo.om.hm x x x x x x x x x mflmcwpmamO mopod O 3 m U mm mm Udm ¢m m D m a on: mum a o v.0 u v.4 Mmumom Hmfiflcé mowommm ouflsvmoz .cmmeOHz cfl mcfluusooo mwouflsvmoe mo mumoz mumunouumb OBOGMII.H magma 11 use the widest variety of animal hosts for blood meal sources and all but Aedes sticticus (Meigen) and Culex salinarius Coq. have been incriminated as potential or proven vectors of one or more types of the CE (Table 2). Multiple feeding patterns of mosquitoes may be partially due to the host's activity. Edman and Downe (1964) found that those mosquitoes that had fed on multiple hosts had included a rodent as one of the hosts. The small rodent is able to brush away the mosquito feeding attempts. Once the mosquito is interrupted, it may move on to another host and possibly infecting several hosts before it receives a complete blood meal. The vertebrate hosts in acting as reservoirs Of the CE viruses may also serve to disperse the virus by their movements throughout the environment. This may be an important mechanism for dispersing the virus since some mosquitoes such as Aedes triseriatus (Say) have a limited flight range and usually do not venture more than a few hundred feet from their emergance site (Barr, 1958). There are probably other mosquito vectors that have greater flight capabilities but further research is needed to determine the mechanism of virus dissemination by the vertebrate's movement or mosquito diSpersion. A wide variety of mammal Species including rabbits, hares and squirrels, are known to have natural infections with California group viruses (Parkin 33 31., 1972; Hammon & Reeves, 1945; and Gresikova 33 31., 1964). Hares 12 Table 2.--Potential vectors of the California encephalitis virus group in Michigan.a Mosquito Species Subtypeb Aedes canadensis CAL,KEY,LAC,SH Aedes cinereus SH Aedes communis JC,LAC,SH,TVT Aedes dorsalis CAL,LAC Aedes fitchii-stimulans SH,JC Aedes tri33r13333 KEY,LAC Aedes trivittatus CAL,JC,LAC,TVT Aedes vexans CAL,JC,LAC,TVT,KEY,SH Angpheles 33nctipennis LAC Culex pipiens LAC,TVT Culiseta inornata CAL,JC,TVT,JS Culiseta melanura CAL Psorophora ferox CAL Coquillettidia perturbans TVT aSudia 3t; 31., 1971; Newhouse 313 31., 1963; Thompson 33 31., 1967; Thompson 33 31., 1972. bCAL-California encephalitis virus (prototype), JC-Jamestown Canyon virus, KEY-Keystone Virus, LAC- LaCrosse virus, SH-Snowshoe Hare virus, TVT-Trivittatus virus, JS—Jerry Slough virus. 13 appear to be associated with the Snowshoe Hare subtype, rabbits to the Keystone subtype and tree squirrels and chipmunks to the LaCrosse subtype (Burgdorfer 33 31., 1961; Newhouse 33 31., 1963; Bond 33 31., 1966; Moulton & Thompson, 1971; and Vianna 33 31., 1971). A number of other mammals tested in Michigan and elsewhere have had positive serological reactions to the hemagglutination inhibition test using the LaCrosse virus antigen, indi— cating that they have been infected with the virus (Table 3). The use of a single antigen test, however, may not be specific enough to detect only the antibodies of the virus strains suSpected (Parkin, 1973; and Sather & Hammon, 1967). A positive hemagglutiation inhibition test using the LaCrosse antigen is not positive proof that the LaCrosse virus was the etiologic agent in the infection, only that one of the CE subtypes probably was involved. Much is still unknown concerning the vertebrate— virus relationships and research is needed to determine which vertebrate hosts serve as reservoirs. Some Species may only serve as indicators of the presence of the virus and may not be able to produce a level of viremia high enough to reinfect the arthropod vector (Cook 33 31., 1965; and Issel 33 31., 1972a,b). Another factor affecting the expression of the virus in the host is the possible inter- action between the various CE types. Some of the types may be virulent and some non-virulent, and it may be possible to obtain an immunizing effect in the host in l4 .Aaoma ..mm mm ummHOOHSm “mnmauvnmfi .noxommv cmmHQOHE CH pmusudmo mameflcm Eonm Oocflmuno mumm A+v .msnfi> mcoumSOXIme .msufl> msumuuH>HHBIB>B .msufl> comcmu c3oummEmhIUO .mOHH> mumm mocmsochmm .msufl> mwmouuquumq .Ammmuououmv msufl> mwuflamnmoocm MHOHOMHHMOImO .Omcflmuoo coon w>m£ whom m>wuflmom coazz Eonm mmflommm Hmeflcd o m + + I + + + A+v + + + + I A+v l I I I + I I I A: +-+-+ I I I + - I I A: .. I I + A: -+ + -+ + -I + A: AmflHMHmsmumE magmawpflov EOmmOdo AxmcoE muoaumzv xosnopooz Am>H5m momao>v xom pom Imfluflzmme mfluflgmuzv xcsxm AmdomNEoummv mmsoz AHODOH comooumv coooomm Amscmflcflmufl> momaflooooov Homo AmOOMOHumEm msmuqv wumm wormBOcm Amscmpfluoam mommHH>HNWV aflonmm Hfimucouuoo Amcmao> mNEoomHmv mcflwam Amsmmcompsn mausmomeEmev 0mm Amflmcocflaoumo mausflomv ammo Auomflc msusflomv xom maouuasqm AmODMHHum mMHEmev xcsemflgo wmx B>B Uh mm U mmmfloomm HmEflcm .mmmsufi> mfluflamnmmoco MHCHOMHHOO mo mHHo>Hmmmu Hmeflcm SMOHSOHE HafiucouomII.m manme 312 IIIIIIIIII‘ 15 which the activity of the virulent type could be suppressed to a subclinical level by prior infections with the non- virulent (Henderson & Coleman, 1971). This could be a valuable tool in controlling those types able to produce human disease by introducing a non-virulent type into the mammal population to suppress the disease causing type. The detection of the first human CE infection in Michigan was in 1968 when the LaCrosse antigen was used to diagnose an illness of a 4-month-old boy in St. Johns (Clinton County) who became ill during the summer Of that year. Between 1968 and 1973, nine additional cases were confirmed using the hemagglutination inhibition test with the LaCrosse antigen. The data concerning human CE infections are still incomplete in Michigan and in many other states, partially due to the lack of interest or unawareness by physicians treating the children. There are probably many cases that are undetected (those that are subclinical) and undiagnosed (those termed "cause unknown" in which no blood sample was tested) (Gorton 33 31., 1975). The status of CE in Michigan will remain incomplete for some time because the Michigan Department Of Public Health (MDPH) lacks the facilities and resources needed to conduct the necessary research. The MDPH is currently attempting to increase the awareness of Michigan physicians as to the presence Of CE and have them submit blood samples from children with meningitis-encephalitis type illness. 16 This is important in determining the statewide occurrence of the virus in humans. There also must be concurrent research to determine the location of the natural trans- mission cycles of the virus in nature, the mammal-pathogen assocations and the extent of the mammal involvement in maintaining the virus in nature, and the evaluation of the vertebrate-mosquito relationships to determine which mosquito species might serve as vectors of vertebrate and human infections. Effects of Water Management on Mosquito ProductIOn "Water management" may have a bad connotation to segments of the general public. They envision the destruction of waterfowl and wildlife species and habitats. Consequently, mosquito control groups in favor of some form of water management for reducing mosquito breeding sites have been on the defensive when prOposing such programs (Brockway, 1960; and Springer, 1964). The use of prOper water management of marsh lands does not necessarily mean the destruction of wildlife but Often leads to better wild— life habitats. The successful use of a water management program is designed to reduce the number of favorable mosquito breeding sites (Rees, 1965). DuChanois and AlltOp (1957) showed that there was a relationship between the water table level and potential mosquito production. There appears to be a minimum level below which normal areas of standing water remain dry, thus producing no mosquitoes. 17 If imprOper water management is used, a water level could be produced to create abundant mosquito breeding Sites. The type of land use practice followed can also create con- ditions favorable for mosquito production. Hanson and Hanson (1970) evaluated a portion of rugged woodland that had been converted into a recreational area and found that these areas could produce potential health problems when humans intruded into areas with previously undetected natural enzootic virus transmission cycles. The type of water management used will to some extent determine the mosquito abundance and diversity (ChristOpher & Bowden, 1957). There are a wide variety of water management programs being used; including impound- ments for flood control, irrigation, hydroelectric power, recreational use and sewage treatment ponds. Irrigation is normally used in connection with farming practices to increase the crOp yield of a given area. The soil type present must be considered when using irrigation but this was not done in the Milk River Valley region of Montana and "lakes" were formed, due to the clay in the soil which created prolific mosquito production sites (Davis, 1959). Mosquito breeding sites created as a result of irrigation can be eliminated by the construction of a drainage system, such as ditches to remove excess water, or installing drain tiles in the field like those used by turf growers to eliminate standing water. These practices increase the 18 amount of arable land and decrease the number of potential mosquito breeding sites (Stivers, 1957). Besides irrigation, there is much interest in sewage lagoons as mosquito production sources. This is important as many sewage lagoons are constructed in close proximity to human habitation and recreational areas and are well within the flight ranges Of most mosquitoes. Smith (1969) noted potential dangers to humans in resort, camping and suburban areas and attempted to determine the relationship between the sewage lagoon and the mosquito Species produced. He found that mosquitoes of the genus Culex were the most common: including Culex pipiens Linn., Culex salinarius Coq., Culex tarsalis Coq. and Culex restuans Theobald. Smith indicated that there appeared to be an association between the low dissolved oxygen content due to the bacterial action and the attraction of ovi- positioning female members of this genus. The abundance and diversity of the Species present will determine whether or not a specific mosquito pOpu— lation will become a health problem. Where high diversity and low abundance occur, no great health problems are likely to develop. Where low species diversity and high abundance occur, the potential for develOpment of health problems is greater. Graham and Bradley (1969) found that a single species did not occur in abundance in those popu— lations having a relatively high diversity which is probably due to the competition between the species 19 present. The type of habitat available in the sewage lagoon will determine to some extent the diversity of the species present. Smith's work indicates that the genus Q3133_is the group that commonly utilizes this habitat and this low diversity could produce abundant mosquito pOpu- lations, thus creating potential health problems. In 1955 a severe annoyance develOped in the Chicago, Illinois, area due to Culex p1piens Linn. origi- nating in sewage lagoons. People were reported to have killed as many as 70 mosquitoes in their bedrooms in a single night (Wray, 1959). Larval samples taken in the lagoons with a standard one pint dipper contained up to 500 larvae per dip. Mosquitoes being produced under this situation were controlled chemically using a mixture Of DDT and fuel oil (Ibid.). Schober (1966) also noted high larval counts in sewage lagoons in Suffolk County, New York, with over 1200 larvae per dip. He attempted control measures by manipulation of the breeding habitat. It was known that mosquito survival was lower in water subjected to wave and wind action so a sprinkling system was set up to create artificial waves on the lagoons and within 24 hours after the sprinkling was started, no larvae, pupae or egg rafts were found. Upon stOpping the sprinkling, reinfestation occurred immediately. The wave action apparently prevented the females from ovipositing on the water surface. This Sprinkling system appears to be a good method of mosquito 20 control in sewage lagoons without using chemicals that might be potentially dangerous to the environment. The use of sewage lagoons in Michigan is not com- pletely new; however, until recently there has not been much research into the effects of these lagoons on mosquito production. In many cases sewage treatment is coupled with spray irrigation so the potential of mosquito production is increased by the creation of additional breeding sites outside the lagoon area (Newson, 1975). Sewage treatment ponds and spray irrigation projects are situations where mosquito control may be necessary because they can create breeding sites favorable for mosquitoes that may serve as potential vectors of diseases of man and his domestic animals (Table 4) (Newson, 1975). Two related studies are currently being conducted by investigators at Michigan State University. One of these, in Belding, Michigan, is to evaluate the insect production and insect-borne disease potentials resulting from spray irrigation using sewage effluent at this location. The other, south of the main campus of Michigan State University, is a water quality management project to determine the environmental effects of spray irrigation using sewage effluent. The Belding sewage project has been in Operation for several years but the spray irrigation portion was not begun until 1973. Prior to the spraying Operation Culex pipiens Linn. was the major species breeding in the oxidation ponds at Belding with sporadic 21 .mmop ou mm>uma nucHEHmn on» Omuuflamcmuu ouflswmoe may £0H£3 OH moapsum Hmucmeflummxo ma czoam Euozuummn mow mo uouom> cm>oumuo .mmflommm ouHOOmOE map Eoum Umcwmuoo mma mm>umH orb mo mompm o>fluoomcfl on» £OH£3 OH OHOSDHMOQ mow mo uouom> manflwmomIo .umos mumunounm> manmummomsm m on msufl> on» OouuHEmcmnp ouflsvmoe msu LOHLB OH mmflpsum Hmucmefluomxm >2 OBOnm mfluflamnmooco mo HOpoo> co>oumIn .mmfloomm ouwsvmoa may Eoum OODMHOmH coma mm: mOHH> may coats CA mflwfiamzmmocm mo Houom> manflmmomlm .Skma .comSsz I m ‘0 m (U U C) O C) 0 TI 0 M Q (U m Q m m mcmnusuumm OHUHDDOHHHSOOO Q MHSCMHQE M¥®mHHSU I mumcuocfl mummfiaso I mflscmmfiuocsm moaonmocm I mflumasomEfluomsw moamnmosd mcmxo> mopom M M WSHMHHQWHHfl m®©®4 m mcmufluuou xOHOO I mflammumu xoasu I mcmflmflm xmasu mHumEEH mflmflamsmwocm mammaflmoufio OHOHOMHHOO mfluflamnmmocm mflpflamnmoocm mmflommm ouflsvmoz mcflsgm cumumoz ocflsvm cumummm {.45aaumkmfi .cmmflnoflz .msfiwamm OH poomoum coflummwuufl mmmBom m CH mmflzmcoflumaou mmmomHOIouHSvaE Hmflpcmpomll.w magma 22 collections of a few Culex territans Walk., Anopheles quadrimaculatus Say and Anopheles punctipennis (Say). After the Spraying was started, the number and diversity of the mosquito Species using the spray areas and Oxidation ponds for breeding purposes dramatically increased. The irrigation spraying also created breeding sites in the spray area for the above species and three additional species: 33133 restuans Theobald, Culex salinarius Coq. and Culiseta inornata (Will.), with all of the species using the oxidation ponds as breeding sites (Newson, 1975). The semipermanent water sites created in the spray area were also used as breeding sites by Aedes vexans (Meigen), and to a lesser degree by Aedes triseriatus (Say), Coquillettidia perturbans (Walk.), Culex erraticus (Dyar & Knab), Culex tarsalis Coq., Culiseta impatians (Walk.) and Culiseta melanura (Coq.). In view of the wideSpread use of the spray area for breeding by these mosquitoes, there would seem to be a greater potential for health problems developing in those sewage treatment programs with associated Spray irrigation sites, as opposed to those with sewage oxidation ponds. The spray irrigation system apparently increases the diversity of mosquito species in the ponds and thus increases the potential of disease transmission since disease transmission is dependent on the abundance of the species present (Graham & Bradley, 1969). 23 The Michigan State University project was develOped to monitor the environmental effects of spray irrigation using sewage effluent. In 1973 the sewage oxidation ponds in the Michigan State University project were filled and in 1974 limited Spraying was conducted to determine if the spray system was working prOperly. A full spraying schedule is not expected until sometime in the future so a complete assessment of its effects on mosquito production and diversity and possible disease transmission will have to be deferred until some future time. MATERIALS AND METHODS Description of Study Area The study took place in the spray irrigation portion of a water quality management project develOped by the Institute of Water Research of Michigan State Univer— sity. The project is located in Ingham County on University prOperty about three miles south of the main campus and contains both field and woodland habitats. The woodland. portion of the irrigation site is a beech-maple-oak forest that contains several low areas in which water collects as result of spring rains and melting snow (map--Figure 1, locations A and B). These ponds were usually temporary and would dry up in the summer. An area Of mixed Oldfields and woodlands was trapped to determine the mammal Species present and the presence Of any CE activity. Mammal trapping was mainly concentrated in the woodland area because the natural transmission cycle of the CE (LaCrosse subtype) is known to occur in forested areas. Limited trapping was conducted in 1973 in the fallow fields east of the temporary pond "B" and along the road east Of the ditch. The area east of the pond proved to have few small mammals with only a few deer mice, Peromyscus maniculatus 24 III 25 Figure l. A map of the study area. Numbers 1 2 are the location of the mosquito-bait trzgd 3 So u on .................u u . 8» :13 00-315 Ilia-DH \\ Q ~ _ H ’33:. \\ lite |I|.|.o_| \\ m (fir-x 43m - m +( Ill..\ vascm 5 \ ‘ ‘ . ..e 0 C I" 3. «.8 3. an .9? 335..." we 2313300 on 039327395 26 "”nonocccc 00.. l 5.3.126 'I‘ D m . 0.000900009909000 OOOOOOOOOOOOOOOO UIII\II\|I‘Il‘\“‘\ Ll\\“|\‘\\\\l\\\11\\\\f\\\II “ I‘I 27 Wagner, being collected in 1973 so trapping was discontinued there early in the study. The area along the road had an abundant cottontail rabbit, Sylvilagus floridanus_Allen, pOpulation but trapping there had to be discontinued in 1974 due to construction in this area. Materials The mammals were collected in Sherman, Tomahawk, and Havahart live traps. The first two are collapsible while the third is non-collapsible. The Sherman traps (Figure 2A) were solid, sheet metal traps all of the same size (3"x3"x9")l and were used to sample the small rodents such as deer mice and meadow mice, Microtus. The commerical bait used consisted Of a mixture of grains and shelled corn mixed with mollasses and was called "horse crunch" or "colt feed." The Sherman traps were not effective and their use was discontinued early in the study. The Tomahawk traps used were of three sizes (Figures 2B, C, and D).‘ The smallest (5"x5"xl6")2 was very effective in collecting such rodents as chipmunks, Tamias striatus Linn.; fox squirrels, Sciurus niger Linn.; and very young raccoons, Procygn lotor Linn. The larger two sizes (6"x6"x24" and 9"x9"x32")3 were used to collect mammals larger than fox squirrels. The smaller mammals were able to escape through the opening l I I I For metric conver51on see Appendix C. 2 3 Ibid. Ibid. 28 between the door and the side Of the trap. The third trap type, Havahart, was a non-collapsible trap (Figure 3A and B). Due to its bulkiness and ineffective locking mechanism, it was not used after the first summer of collecting. The bait used in all of the Tomahawk and Havahart traps was dried ear corn. Three modified Villavaso and Steelman (1970) animal- baited traps were constructed to collect the mosquitoes attracted to the mammals occurring in the study area and thus determine the host preferences of these mosquitoes. The traps were 36"(L) x 34"(W) x 20"(H)1 and had two main components: (1) Two removeable side collecting boxes in which mosquitoes were collected (Figure 4); and (2) the center holding area (Figure 5) where the caged mammals were placed. A removeable screen barrier was placed in each collecting box to prevent the mosquitoes from feeding on the mammal used to attract the mosquitoes into the trap. The metal pan was used to prevent the mammal from scenting the trap with its wastes. It became necessary to attach metal legs on the traps in order to raise them Off the ground and decrease the number of Harvestmen (Arachnida: Phalangida) from entering the trap (Figure 6). A very low mosquito population existed in the study area in 1974 resulting in very small mosquito collections (usually Of l or 2'at a time). It was therefore felt that the Harvestmen, being 1 . . . For metric converSIOn see Appendix C. IIIIIIIII 29 Figure 2. Collapsible live traps. TIES 2135—; A. Sherman 3" x 3" x 9" B. Tomahawk 5" x 5" x 16" C. Tomahawk 6" x 6" x 24" D. Tomahawk 9" x 9" x 32” Figure 3. Non—collapsible live traps. Type Size A. Havahart 5" x 5" x 18" B. Havahart 6" x 6" x 30" - S .d ['1 ’ - 7 'lc' ’1 Figure 4. Figure 5. 31 Mosquito bait trap with side collecting boxes detached. .Mosquito bait trap opened to show location cxf caged mammal. 32 IIIIIIIII 3 33 Figure 6. Mosquito bait trap with metal legs. Figure 7. A young raccoon anesthetized with Nembutal. 34 35 scavangers, could affect the collection results so their access into the traps had to be minimized. The mosquito traps were all placed in the woodland area. Two of the traps were located in the north section (B) as it was in this section where the bulk of the mammals were trapped. All of the species trapped, except the deer mice, were used as bait in the mosquito traps. Because of the low mosquito populations present in 1974, it was often necessary to place several mammals of the same species in a trap to attract the mosquitoes. Methods The mammal traps were placed along three of the spray irrigation pipe lines at every fourth pipe joint to simplify the rechecking of the traps. Each trap was identified by a number-letter-number designation (e.g., l—A-l) used later in determining the ranges of the mammals trapped and to see if any areas were used more than others. The first number was the pipe line designation (numbering from west to east), the letter indicated the section of woods in which the trap was located, and the second number was the location of the trap along the pipe line (numbering away from the dirt road in either direction). A dirt road running west-east through the spray area separated the irrigation site into a north and south section, B and A, reSpectively. 36 A blood sample was taken by cardiac puncture and a numbered ear tag was affixed to each mammal trapped. A blood sample of at least 1 cc was needed to insure enough serum for the California encephalitis antibody test. Mammals such as Opossums, Didelphis marsupialis Linn.; cottontail rabbits; chipmunks; fox squirrels; red squirrels, Tamiasciurus hudsonicus Erxleben; and deer mice were anesthetized in the study area with chloroform and bled. Raccoons and woodchucks, Marmota monax Linn., were taken to the laboratory and partially anesthetized with chloroform and then given an injection (intramuscular-hip) of Nembutal (dosage determined by the mammal's weight, see Appendix A). When the mammal became semiconscious and could be handled safely (Figure 7), a blood sample was taken and a numbered ear tag affixed. After the mammal recovered from the Nembutal, it was released in the general area in which it had been trapped. The blood samples were transferred to test tubes (13 x 100mm) and were allowed to clot at room temperature for several hours after which the clotted samples were placed in a refrigerator (at 4 to 5°C) overnight to allow the clot to retract. The blood samples were then centri— fuged (at 1700 to 2000 rpms for 10 to 20 minutes) and the clear serum transferred with micro-pipettes into marked one-half dram vials and placed in a freezer (at -20 to -25°C). The samples were frozen until they could be tested for CEV antibodies at the Michigan Department of 37 Public Health, Virology Laboratory. The test used was a modified hemagglutination inhibition (HI) test using LaCrosse virus antigen in a suspension of goose red blood cells (Clarke & Casals, 1958). While the animals were anesthetized the fleas (Siphonaptera) on them were collected. This survey was conducted to determine the Species diversity associated with the animal Species present. RESULTS Ten Species of mammals were collected in the study area with chipmunks, fox squirrels, red squirrels, cotton— tail rabbits, raccoons, and Opossums making up approxi- mately 90 percent of the mammals trapped (Table 5). Two other Species: deer, Odocoileus virginianus Zimmermann and a starnose mole, Condylura cristata Linn. were observed in the study area but no blood samples were obtained from these animals, or from a trapped muskrat, Ondatra zibethica Linn.; and Skunk, Mephitis mephitis Schreber. Except for the rabbits and deer mice, all of the Species were collected in the woodland habitat. The majority of the rabbits trapped were taken from the field east of the ditch and were collected during the first summer. Table 6 Shows the results of the hemagglutination inhibition tests on the 187 blood samples obtained from the 162 mammals trapped. Approximately 30 to 40 percent of the fox squirrels and 40 to 50 percent of the chipmunks trapped were probably resamples but their correct identifi- cation was not possible because their ear tags were torn out thus conversion data for these was not possible. 38 39 Table 5.--A survey of animals in the Michigan State Uni- versity Water Quality Project Site, 1973-1974. Number Species Collected Chipmunk (Tamias striatus 28 Fox squirrel (Sciurus niger) 25 Red squirrel (Tamiasciurus hudsonicus) 20 Cottontail Rabbit (Sylvilagus floridanus)* 20 Raccoon (Progyon lotor) 30 Opossum (Didelphis marsupialis) 23 Woodchuck (Marmota monax) 8 Deer mouse (Peromyscus maniculatus)* 6 Muskrat (Ondratra zibethica) 1 Skunk (Mephitis mephitis) l Starnose mole (Condylura cristata) 1 (found dead) Deer (Odocoileus virginianus) ** *Taken in oldfield habitat, the other Species taken in woodlands. **A total of 5 were observed in study area. 40 Table 6.--Serological survey of indigenous small mammals in the Michigan State University Water Quality Project Site, 1973-1974. HI Titers to the LaCrosse subtype Animal SpeCIeS of California Encephalitis Virus* <1:10 1:10 1:20 1:40 Chipmunk 29 7 1 0 Fox Squirrel 19 21 2 1 Red Squirrel 16 3 l 0 Cottontail Rabbit 9 ll 0 0 Raccoon 21 9 0 0 Opossum 21 2 0 0 Woodchuck 6 2 0 0 Deer Mouse 5 l 0 0 Totals 126 56 4 1 *A titration : 1:20 is considered positive. 41 Positive sera were obtained from 3 fox squirrels, 1 red squirrel and l chipmunk, all of which were trapped in the woodland habitat. Three mosquito traps were constructed and used during the summer of 1974 to determine the host preferences of the mosquito Species present. Mosquito populations observed during the summer of 1974 were dramatically lower than those observed in 1973, resulting in only 60 mosquitoes of 8 different Species being attracted into these traps (Table 7): including Aedes cinereus Meigen, Aedes fitchii- stimulans (Felt & Young), Aedes sticticus (Meigen), Aedes triseriatus (Say), Aedes vexans (Meigen), Coquillettidia perturbans (Walk.), Culex pipiens Linn. and Culex salinarius Coq. In conjunction with the serological sampling of the small indigenous mammals, a survey of the fleas (Sip- honaptera) parasitizing these mammals was conducted (Table 8). Orchopeas howardii Baker made up 74.2 percent of the fleas collected. The remaining species consisted of Ctenophthalmus pseudagyrtes Baker, 17 percent; Cediopsylla Simplex (Baker), 5.5 percent; Opiscrotis bruneri (Baker), 0.9 percent; Orchopeas leucopus (Baker), 0.9 percent; Epitedia faceta (Rothschild), 0.5 percent; Megabothris asio (Baker), 0.5 percent; and Or0psylla arctomys (Baker), 0.5 percent. Birds frequently tripped the corn-baited mammal live traps used to sample the indigenous small mammals 42 + I I I I msflumcflHMm xmaso I I + + I mcmflmflm xmaso I I I I + mcmnusuumm mflofiuumaaflsmou I I I + + mcmxm> mooma I + + + + mauMHummHHu mopmm I I + + + msofluoflum mopmm I I + + + mcwHSEflumIfiflnouHm moved I I + + + msmumcflo mmomfi Edmmomo xosnooooz Hmuuflsqm com Hmuuwoqm xom cooomm mmflommm Hmaflcm mmflommm ouwdqmoz .muwm powmoum muflamso uwumz muwmnm> Ian: mumum savanna: may um muHSmmu cofluomaaoo many pawn ouflsqmoz whmHII.n magma 43 .uahnmm Hflmucouuooumam .mmsoz meQIzo .ESmmomOIo .coooomqumm .xcsemfi£UIU .Honufisvm pmmImm .kuuflsvm xomImmm o.ooH mam m ma mm mm am pm am mmflommm Hmaficm “we dance o.~a em ma mm mmuummmosumm mafiamsunmmcwuo m.o H a Oflmm mflunuonmmuz m.o m m Huwcdun mflumouoomflmo m.o H a mhsououm MHHMmmouo m.o N N mammusma mammonouo m.vn moa n mm NH mm vm fifipum3on mammonouo mwcflaasmmonoaaoo saasmm m.o a H muoUMM mflomuflmm MMUHHHMmmonowuummm xaaemm memEHm waahwmofiomu m.m NH NH mMGAOflHSm mHHEmm Hmuoe mo mmflommm moan so mdm 0 04m 0 mm mm mmflowmm mmam usmoumm mom mamuoe mmumom Hmfiflc¢ .muHm wommoum wuwamsa uwumz >uawum>flca wumum cmmfl£0flz Eoum vanIMSmH mafinso pmuomaaoo mmmam mo mSOHuwHUOmmm umozII.m mange 44 present. Seven Species of birds were trapped during the course of this study. Blood samples were obtained from three Species; including three grackles, Quisculus quiscula Vieill; two cardinals, Richmondena cardinal Linn.; and one purple finch, Carpodacus purpureus Gmel. No blood samples were obtained from the other four species trapped: including blue jays, Cyanocitta cristata Linn.; ruffed grouse, Bonasa umbellus Linn.; brown thrashers, Toxostoma rufum Linn.; and a rufous-sided towhee, Pipilo erythro- phthalmus Linn. DISCUSSION The movements of the forest mammals (see Table 5) were determined by a capture-recapture method. It was found that raccoons and Opossums showed the greatest degree of movement and diSpersion while chipmunks and fox squirrels Showed the least. Raccoons and Opossums did not have any location preferences but squirrels and chipmunks tended to stay in the general area where they were first trapped. The majority of the mammals trapped appeared to be juveniles which would seem to indicate a good reproducing mammal population in the study area. The number of juvenile raccoons and Opossums declined as the summer progressed. Whenever possible the mammals were re-bled monthly to determine the virus activity by monitoring antibody conversion. In order to confirm the presence of a virus infection in the animal a paired sera is required. Due to the high frequency of lost ear tags in the fox squirrels and chipmunks, it was not possible to determine which of them may have undergone serological conversion. Unfortu- nately, 4 of the 5 positive mammals were from this group. 45 46 The antibody titers of the mammals bled ranged from less than 1:10 to 1:40 with titers of 1:10 or less con- sidered negative and those of 1:20 or greater, positive. The reason for the relatively high percentage of reactions at the 1:10 dilution level is not known but may indicate the presence of heterologous CE antibodies. The infections in the mammals could involve strains of the CE other than the LaCrosse subtype that might cross react with the LaCrosse virus antigen used in the hemagglutination inhibition test. AS mentioned earlier, the use of a single antigen test may not be Specific enough to detect only one type of antibody. Sather and Hammon (1967) working in Wisconsin with antigenic patterns in the California encephalitis group found that there were apparent simi— liarities between the virus strains in the group, eSpecially in those strains obtained from the same area at the same time. They alSo found that cross reactions would occur between the Snowshoe Hare antibodies and the LaCrosse antigen. Similiar cross reactions between other strains of the CE group were Shown by Parkin (1973) with strains of the CE group found in domestic animals in Florida. This may explain in part the high proportion of 1:10 titers found in the animals tested in this study. The clarification of this will not be possible until the CE viruses present in this location have been isolated and typed. No isolations of the CE viruses have yet been reported from fleas but research in this area has been 47 very limited. Fleas are known to serve as vectors of such disease agents as bacteria (plague), Rickettsia (typhus) and viruses (Myxomatosis) (James & Harwood, 1969). Due to the close association fleas have with their hosts, it would seem possible that they could become infected with the CE virus while feeding on an infected host. Whether fleas have a role in the transmission of the CE viruses is not known, but its blood-feeding habits might enable it to reinfect its host, thus keeping the virus active so that the primary vectors (mosquitoes) could subsequently be infected. Sudia 33 31. (1971) indicate that the apparent mechanism by which the virus is disseminated in nature is by the movements of the vertebrate reservoirs. It is not known exactly how long the viremia remains high enough to infect the primary vectors. It is also not known what possible immune reactions might occur as a result of the CE infection, thus preventing the vertebrate host from being reinfected for a period of time. During this period the host might move to an area where non-infected mosquitoes are present. If the fleas then could transmit the virus, they (fleas) could reinfect the host when its immunity subsided. The number and abundance of possible vector species present in the study area will influence the virus trans- mission activity to the susceptable vertebrate hosts. Aedes cinereus Meigen, Aedes fitchii—stimulans (Felt & Young), Aedes triseriatus (Say), Aedes vexans (Meigen), 48 Coquillettidia perturbans (Walk.) and Culex pipiens were collected in the study area and have been incriminated elsewhere as either proven or potential vectors of one or more of the CE viruses (Tables 2 and 7). The abundance of these species is dependent upon the weather conditions (rain and snowfall, temperature, etc.) that make these Sites favorable for mosquito production. There were numerous fallen trees with holes; artificial containers such as tin cans, glass jars and rubber tires; and several temporary ponds (locations A and B on map) that could serve as potential breeding sites in the study area. Larvae of Aedes fitchii-stimulans (Felt & Young) were collected in the temporary ponds and a few Aedes triseriatus (Say) were taken from some of the tree holes sampled in the study area (Zorka, 1975). The adult mosquitoes collected during the summer of 1974 in mosquito bait traps gave an indication of the host preferences of these Species. Only five mammal Species attracted mosquitoes into the traps (Table 7) which, in part, may be due to the low mosquito populations occurring that year. These traps should be used again when the mosquito populations increase to obtain more complete host preference determinations. The host prefer- ences are necessary to determine what mosquito species may serve as vectors of the CE in the study area. Of the avian species occurring in the study area, blackbirds, cardinals, and grackles have been associated WWI) 3 49 with Eastern equine encephalitis (EEE) transmission else- where. Aedes triseriatus (Say), Aedes vexans (Meigen), and Coquillettidia perturbans (Walk.) have been incriminated as potential vectors of this virus (Williams 33 31., 1971). This would indicate that the potential for EEE transmission exists in the study area. The sera obtained from the birds sampled were not tested for EEE antibodies. Like CE, uncertainities will remain until virus isolations are done to determine which virus types are present in the bird pOpulationS in this location. The high mortality associated with the EEE infections could pose a greater threat to the humans living near this location than the CE, if the BBB is present. It is for this reason that further research is needed concerning arboviruses that may occur in the study area. The natural transmission cycle of the CE virus involves a mosquito—small mammal-mosquito cycle and man does not become involved until he intrudes on the habitat where this "natural" cycle occurs. These "natural" cycles usually occur in undisturbed woodland habitats where abundant vertebrate hosts and arthrOpod vectors exist. However, when man disturbs this habitat by developing campgrounds, hiking and nature trails, and suburban housing areas, he diSplaces the vertebrate hosts used as blood meal sources by the arthropod vectors. These vectors then use man as a readily available blood meal source and thus may infect him with the virus causing California encephalitis. SUMMARY Hemagglutination inhibition tests on indigenous small mammals collected in a sewage spray irrigation area on the Michigan State University campus indicated the presence of California encephalitis virus activity at this location. Of 182 serum samples obtained from 162 mammals, 5 were positive: 3 fox squirrels, 1 red squirrel and l chipmunk. Although the data iS limited, it suggests CE is maintained there in a mosquito-squirre1-mosquito cycle. Eight mosquito Species were collected in animal- baited mosquito traps: Aedes triseriatus (Say), Aedes cinereus (Meigen), Aedes fitchii-stimulans (Felt & Young), Aedes sticticus (Meigen), Aedes vexans (Meigen), Culex pipiens Linn., Culex salinarius Coq., Coquillettidia perturbans (Walk.). Of these Aedes triseriatus (Say), Aedes cinereus (Meigen), Aedes fitchii-stimulans (Felt & Young), Aedes vexans (Meigen), and Culex pipiens Linn. have been shown elsewhere to be capable of transmitting one or more of the CE group viruses. A relatively large bird population was observed in the Spray irrigation site, some of which may serve as 50 Sl reservoirs of the BBB virus. A number of mosquito species present in the Study area, including Aedes triseriatus (Say) and Aedes vexans (Meigen) are potential vectors of both BER and CE viruses (James & Harwood, 1969; Newson, 1975; and Sudia et 31., 1971). The potential for EEE transmission may exist in this location but additional studies will be required to determine whether or not the BBB virus is present. Of major concern to the develOperS of the water quality management project is the possible effects of the prOposed Spray irrigation system on mosquito production and transmission of insect-borne diseases. Since the potential for disease transmission may increase if the Spray irrigation procedures increase mosquito pOpulations and Species diversity in the Spray site, it will be important to closely monitor the biting insect pOpulations present and to continue research designed to assess the effects that the Spraying operations will have on both the natural zoonotic transmission cycles. LITERATURE CITED LITERATURE CITED Barr, A. R. 1958. The mosquitoes of Minnesota (Diptera:Culicidae:Culicine). Univ. Minn. Agr. Exp. Sta. Tech. Bull. 228. 154 pp. Beadle, L. D. 1966. 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Zorka, T. 1975. Personal communication. APPENDICES APPENDIX A NEMBUTAL DOSAGES BASED ON ANIMAL'S WEIGHT APPENDIX A NEMBUTAL DOSAGES BASED ON ANIMAL'S WEIGHT Raccoons Wt. (Lbs) Dosage (.75g/cc) Nembuta1* 1.0 .4cc 2-2.5 .5cc-.75cc 3-3.5 .6cc-l.lcc 4.0 l.0cc-l.25cc 5.5 1.3SCc 6.0 1.75cc 12-15.0 3.5cc 18.0 4.0cc Woodchuck Wt. (Lbs) Dosage (.7Sg/cc) Nembutal* 5-5.0 1.75cc-2.75cc 6.5 2.0cc 8.5 3.0cc 9.0 3.75cc 12.0 4.0cc *Due to the variance between individuals, the amount of Nembutal needed to sedate the animal also varied. 59 APPENDIX B HI TITER RESULTS OF INDIGENOUS SMALL MAMMALS IN A WATER QUALITY PROJECT SITE, 1973-1974 APPENDIX B HI TITER RESULTS OF INDIGENOUS SMALL MAMMALS IN A WATER QUALITY PROJECT SITE, 1973-1974 Fox Squirrel Chipmunk Tag No. Sex Trapped Result Tag No. Sex Trapped Result 44939 f 7—1-73 1:10 52441 m 7-1-73 44926 f 7-6-73 1:10 died f 7-1-73 44927 f 7-6-73 1:10 52442 f 7-4-73 52460 m 7-7-73 1:10 52450 m 7-4-73 52461 f 7-7-73 1:20 52441 m 7-5-73 1:10 52499 m 7-7-73 52430 f 7-6-73 1:10 52500 m 7-7-73 1:10 52435 m 7-6-73 52496 m 7-8-73 52475 f 7-13-73 died m 7-20-73 52469 m 7-14-73 52468 m 7-23-73 52473 m 7-14-73 52443 f 7-24-73 52450 m 7-21-73 1:20 52494 f 7-25-73 52487 f 7-22-73 52461 f 8-10-73 1:10 52487 f 7-24-73 5 f 8-10-73 52456 m 7-24-73 6 f 8-10-73 52492 f 7-24-73 52499 m 8-11-73 52444 m 7-24-73 13 m 8-21-73 52446 m 7-24-73 14 m 8-23-73 52435 m 8-10-73 15 m 8-25-73 1:10 7 f 8-10-73 16 m 8-25-73 8 m 8-10-73 52496 m 8-26-73 52430 f 8-11-73 101 f 6-23-74 1:10 died f 8-12-73 23 m 6-23-74 1:10 died m 8-12-73 1:10 147 m 6-25-74 1:10 52444 m 8-12-73 133 m 6-25—74 1:40 52492 f 8-20-73 1:10 29 f 7-9-74 52487 f 8-25-73 26 m 7-11-74 1:10 52435 m 8-25-73 38 f 7-12-74 1:10 untag m 6-19-74 1:10 110 m 7-12-74 1:20 79 f 6-25-74 52496 m 7-12-74 1:10 149 m 7-9-74 146 f 7-13-74 1:10 25 m 7-11-74 22 m 7-19-74 1:10 121 m 7-19-74 1:10 106 f 7-19-74 34 m 7-31-74 129 f 7-19-74 48 f 8-31-74 116 m 7-20-74 27 m 9-1-74 1:10 109 f 7-20-74 107 m 9-6-74 148 f 7-22-74 1:10 140 f 9-6-74 21 m 7-25-74 1:10 20 m 7-26-74 60 61 Fox Squirrel Chipmunk Tag No. Sex Trapped Result Tag No. Sex Trapped Result 101 f 8-2-74 1:10 38 f 8-22-74 1:10 29 f 8-24-74 1:10 119 m 9-6-74 Deleted titers were less than 1:10 (no reaction) Titers of 1:20 or greater are positive 62 13 Red Squirrel Cottontail Rabbit I! Tag No. Sex Trapped Result Tag No. Sex Trapped Result 52437 f 7-5-73 52451 u 7-1-73 1:10 died m 7-5-73 44915 u 7-20-73 1:10 52438 m 7-5-73 1:10 died u 7-20-73 1:10 52440 f 7-5-73 44914 u 7-20-73 1:10 died f 7-5-73 44908 u 7-21-73 1:10 52427 m 7-6-73 44907 u 7-22-73 died f 7-6-73 44906 u 7-22-73 1:10 52426 m 7-6-73 1:10 44911 m 7—23-73 1:10 52428 f 7-6-73 44912 m 7-23-73 52429 m 7-6-73 52453 m 7-23-73 52434 m 7-7-73 1:10 52458 f 7-23-73 1:10 52449 m 7-13-73 44922 f 7-24-73 52464 f 7-21-73 44918 f 7-24-73 1:10 died f 7-23-73 44924 m 7-25-73 52485 f 7-25-73 44913 f 7-25-73 52471 m 7-26-73 44920 f 7-26-73 12 f 8-20-73 5 f 8-20-73 1:10 43 m 6-25-74 8 f 8-24-73 1:10 132 m 7-22-74 1:20 64 f 8-23-74 105 f 7-27-74 70 f 8-31-74 Woodchuck Deer Mouse Tag No. Sex Trapped Result Tag No. Sex Trapped Result 44937 m 7-1-73 killed f 7-4-73 44931 m 7-6-73 killed f 7-15-73 44921 m 7-22-73 killed m 7-15-73 3 m 8-10-73 killed f 7-21-73 4 f 8-10-73 killed m 7-25-73 7 f 8-23-73 killed m 8-9-73 79 f 8-25-74 1:10 80 f 9-6-74 1:10 Deleted titers were less than 1:10 (no reaction) Titers of 1:20 or greater are positive u--sex unknown 63 Raccoon Opossum Tag No. Sex Trapped Result Tag No. Sex Trapped Result 44934 f 6-29-73 1:10 44929 m 7-6-73 44938 f 7-1-73 52452 f 7-7-73 1:10 44936 f 7-1-73 1:10 52498 m 7-8-73 44935 f 7-1-73 52497 f 7-8-73 44933 f 7-6-73 52432 f 7-13-73 44940 m 7-7-73 52465 m 7-15-73 44941 m 7-8-73 52447 m 7-20-73 44942 m 7-8-73 1:10 52433 f 7-21-73 44943 m 7-8-73 52448 f 7-23-73 44904 f 7-13-73 52439 f 7—24-73 44903 m 7-13-73 52495 m 7-25-73 44902 f 7-13-73 52490 m 7-25-73 44901 m 7-13-73 52482 f 7-25-73 44905 f 7-14-73 52463 f 7-26-73 44909 m 7-21-73 2 f 8-10-73 44910 f 7-21-73 18 f 8-26-73 44944 m 7-21-73 44 m 7-19-74 2 m 8-10-73 118 m 7-19-74 44938 f 6-20-74 128 f 7-22-74 63 f 6-23-74 1:10 46 f 8-22-74 1:10 30 f 6-27-74 1:10 47 f 8-22-74 31 f 6-27-74 134 m 9-1-74 32 m 6-27-74 127 m 9-7-74 69 m 6-27-74 1:10 71 f 6-27-74 1:10 54 m 6-30-74 49 f 7—10-74 77 f 7-30-74 1:10 78 m 7-30-74 27 f 9-8-74 Deleted titers were less than 1:10 (no reaction) APPENDIX C METRIC CONVERSION TABLE APPENDIX C METRIC CONVERSION TABLE English* Metric** 3in x 3in x 5in Sin 6in 6in X X X X Sin Sin Gin 6in 9in x 9in X X X X X 34in x 20in 9in 16in 18in 24in 30in 32in x 36in 7.62cm x 7.62cm x 22.86cm 12.7cm x 12.7cm x 40.64cm 12.7cm x 12.7cm x 45.72cm 15.24cm x 15.24cm x 60.96cm 15.24cm x 15.24 cm x 76.2cm 22.86cm x 22.86 cm x 81.28cm 86.36cm x 50.8cm x 91.44cm __ denotes inches. denotes centimeters. 64 HICH IIII)IIIIIIIIITIIIIIIIIQIIIZIIIIIIIITS 31293