"- N“R'”qv\«~‘0009uo~o«;§cg.mwfi_._ _‘ _. V: -I’vw‘. AN EPIDEMIOLOGICAL STUDY OF MOSQUITO - BORN-E CALIFORNIA EN’CEPHA-LITIS IN A SOUTHERN MICHIGAN SUBDIVISION Thesis for the Degree of M. S MICHIGAN STATE UNIVERSITY. RALPH}. GORTON 1973 \mfi'fi?’ "L" a LIBRAIL 1’ a... Michigan S ta ta UniVCtSity "III \\\\\\\\\ III IIIIIII IIIII 3\1293 1073 ..-._— ‘L ' ”NEINO ‘IIIAG & SIIIIS’ aunx emnmv ms 1 L'BRARY BINDE autumn. Ill: 7,; “9V 2 41399 ABSTRACT AN EPIDEMIOLOGICAL STUDY OF MOSQUITO - BORNE CALIFORNIA ENCEPHALITIS IN A SOUTHERN MICHIGAN SUBDIVISION by Ralph J. Gorton The presence of California Group arthropod - borne viruses, and the epidemiological conditions which may main- tain these viruses in nature, were investigated in a woodlot - subdivision community in Shiawassee County, Michigan during the late summer and early fall of 1971. Three clinical cases of encephalitis caused by infec- tions with California Group viruses have occurred in children living in this community; two in 1970 and one in 1971. A serological survey of 30 residents (ages 1 - 39 years) indi- cated the presence of hemagglutination inhibition antibody titers (La Crosse antigen) of E 1:20 in 23.5% of the suscep- tible - age group tested (i.e., children under 16 years of age). One of these children had a history of severe encephalitis - like disease in 1967 and a convalescent serum titer (u years later) of 1:80, which was considered presump- tive evidence of infection with California encephalitis virus. Epidemiological conditions in the community include a large number of susceptible - age children that play in the Ralph J. Gorton woodlots and are exposed to biting insects, 56 species of vertebrates which potentially may serve as mosquito and/or virus hosts, and at least 9 species of man - biting mosqui- toes, several of which are proven or suspected vectors of California Group viruses in other North Central states. Of 312 mosquitoes collected at human bait, 60% were Aedes triseriatus, 17% Ag. vexans, 11% Coquillettidia per- turbans, 5% Anopheles quadrimaculatus, M% 53. fitchii, 1% Ag. canadensis, < 1% Ag. stimulans, and-< 1% 52. page: tipennis. Culex pipiens was abundant in the woodlot in immature stages, but was not collected at human bait. Only Aedes triseriatus, a proven vector of La Crosse viral sub- type in Wisconsin, exhibited a biting pattern which coin- cided with the daytime play activities of subdivision children. AN EPIDEMIOLOGICAL STUDY OF MOSQUITO - BORNE CALIFORNIA ENCEPHALITIS IN A SOUTHERN MICHIGAN SUBDIVISION by Ralph J. Gorton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1973 ACKNOWLEDGEMENTS I wish to thank Dr. Harold D. Newson for his invaluable guidance during the course of the study and in the prepara- tion of the manuscript. To Drs. Gordon E. Guyer, Ronald E. Monroe, and Donald W. Twohy, I extend my appreciation for serving on my guidance committee. Special thanks also go to Dr. Gordon B. Guyer, whose financial assistance made this study possible; Drs. Maurice Becker and Richard Bail of the Michigan Department of Public Health for performing the virological tests; and my wife, Marion Yvette, for her constant encouragement during my graduate studies. ii TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . INTRODUCTION. . . . . . . . . . . . . . . . . . HISTORY OF THE CALIFORNIA GROUP ARBOVIRUSES . . METHODS . . . . . . . . . . . . . . . . . . . . Description of the Study Area. . . Epidemiological Procedures . . . . Vertebrate Survey Procedures . . Mosquito Survey Procedures . . . . RESULTS AND DISCUSSION. . . . . . . . . . . . . Clinical Cases of California Encephalitis. Human Serological Survey . . . . . . . . . Mosquito Fauna . . . . . . . . . . . . . . Vertebrate Fauna . . . . . . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . . . . . . LITERATURE CITED. 0 O O O O O O O O I O O O O APPENDICES O O O O O O O O O O O O O O O O O O O I. Average monthly temperatures for Owosso, Michigan, 1967 - 1971 (May - September). . . . . II. Daily temperatures for Owosso, Michigan, May - SEPtembeP, 19710 0 o o o 0 o o 0 III. Rainfall recorded for Owosso, Michigan, 1967 - 1971 (May - September). . . . . IV. Daily rainfall recorded for Owosso, Michigan, May - September, 1971. o o o o o o o 0 iii Page vi 17 17 23 2n 25 27 28 31 u1 su so an 70 70 71 72 73 TABLE OF CONTENTS--continued CONTENTS V. Birds recorded in the Owosso study area, based on 38 field observation days (April 29 - September 19, 1971). iv Page 7% LIST OF TABLES TABLE 1. 2. A chronological summary of the California Group arboviruses of the world . . . . . . . . Confirmed and presumptive human cases of California encephalitis in the North Central United States (excluding Michigan), 1995 - 1970. . . . . . . . . . . . . . . . . . Results of a serological survey for hemagglutination inhibition antibody to California Group arbovirus (La Crosse antigen) in the Owosso woodlot - subdivision residents. . . . . . . . . . . . . . . . . . Frequency of eight species of mosquitoes collected at human bait between July 20 - September 7, 1971, in the Owosso woodlot - subdivision. . . . . . . . . . . . . The relative abundance of immature Culex pipiens in the Owosso woodlot - subdivision between JUly 28 - SGPtembeP 7, 1971. o o o o o o o o o The mosquito Species of the Owosso woodlot - subdivision and the virus subtypes isolated from them in North America, 1993 - 1970 . . . . . . A list of the mammals, reptiles and amphibians observed in the Owosso woodlot - subdivision between April 29 - September 1a, 1971. . . . . Most frequently observed birds in the Owosso woodlot - subdivision between April 29 - September 1”, 1971 (based on data in Appendix V). . . . . . . . . . . . . . . . . . Page 11 33 H3 H8 51 5,5 57 LIST OF FIGURES FIGURE 1. 2. Topography of the Owosso, Michigan study area and the region surrounding it. . . . . ._ Map of the 1971 woodlot - subdivision study area, Owosso, Michigan. . . . . . . . . . . . The geographical distribution of the homes of the confirmed cases of California encephali- tis and the families involved in the serological survey, Owosso, Michigan. . . . . Seasonal distribution of California encephalitis and encephalitis - like illness- es in children admitted to Memorial Hospital, Owosso, Michigan (according to dates of onset, 1967 - 1971) compared with seasonal distribution of 239 cases reported in the literature. . . . . . . . . . . . . . . . . . . Time of biting of the major pest species of mosquitoes in the Owosso study area, based on #3 twenty minute collections at human bait, July 20 - September 7, 1971 . . . . . . vi Page 17 21 37 H0. H5 INTRODUCTION Within the last decade there has been a tremendous increase in the number of known arthropod - borne animal viruses (arboviruses) throughout the world.' The American Committee on Arthropod - borne Viruses (1970) lists 2H3 known or presumed arboviruses, but in only about half of these are the vectors known. The others are designated as arboviruses on the basis of biochemical properties and on their ability to multiply in both experimentally infec- ted arthropod and vertebrate tissues (Mattingly, 1969). According to James and Harwood (1969, page 206), "Arthropod - borne viruses are causal agents of some of the most serious and widespread diseases of man and animals. They are by far the most varied and numerous pathogens transmitted by arthropods..." Classified as arboviruses are such well known diseases as dengue, Japanese B encephalitis, urban yellow fever, and Venezuelan equine encephalitis, all of which can occur in epidemic proportions with occasional high mortality to man. In the United States, arboviruses have been associated with human central nervous system disease since the 1930's, when Eastern equine encephalitis, Western equine encephalitis and St. Louis encephalitis viruses were shown to cause encephalitis in man (Reeves and Hammon, 1962). These viruses are maintained in essentially mosquito-bird cycles and have been responsible for numerous human epidemics and epizootics in horses and pheasants (James and Harwood, 1969). A fourth arbovirus causing human illness in the United States and Canada is California encephalitis virus, which, unlike the equine and St. Louis encephalitis viruses, seems to be a mammalian virus, transmitted primarily by mosquitoes of the genus Agdgs (Mattingly, 1969; Thompson, 1969; Sudia g: 31., 1971). As presently constituted, there are eight sero- typically distinct California encephalitis viruses known to occur in North America, which are called, collectively, the California Group arboviruses. In 196M the first epidemic of encephalitis caused by the California Group viruses was reported in North America, and produced 12 cases of central nervous system disease in young children in Indiana (Marshall, 1965). Since 1969 the number of confirmed and presumptive human cases due to this group of viruses has risen to more than 520, primarily in the North Central and Eastern United States (Sudia st 31., 1971). In less than a decade this arbovirus, which causes illness primarily in children under the age of 15, has be- come an important late summer - early fall health problem in the North Central United States (Thompson and Inhorn, 1967; Hanson and Hanson, 1970) and a number of institutions have initiated research programs in an effort to study the epidemiological and ecological aspects of the natural disease cycle. The first evidence of California Group virus in Michigan was reported by Newhouse g: 31. (1963), who found neutraliz- ing antibodies in 8 of 9 snowshoe hares (Lepus americanus) collected at Shingleton (Alger County) in the Upper Penin- sula. During the summers 1968 - 1970, the Michigan Depart- ment of Public Health Laboratory (MDPH) began selectively testing blood samples submitted by private physicians for routine virus diagnosis in an attempt to determine if Cali- fornia Group viruses were involved in human central nervous system diseases occurring in Michigan. During this period the MDPH selected and tested 25% serum samples which met the following criteria: (1) Patients had illnesses of menigitic and/or encephalitic nature but their sera were negative in tests with routine antigens such as mumps, herpes, measles, poliomyelitis and the equine encephalitides; (2) patients were not more than 15 years of age; and (3) the onset of disease occurred between July 1 and October 1. The paired serum samples were subjected to hemagglutination inhibition tests using the La Crosse subtype antigen obtained from the Center for Disease Control in Atlanta, Georgia. The re- sults of the tests were as follows (Becker and Bail, 1972): Titer increase of 2-fold or greater - 6 Titer increase of l-fold - 3 Initial elevated titer of 1-20 or more - 2 Negative - 293 ll (H.6%) Total with California virus antibodies Although the number of sera with California Group virus antibody was minimal, Newson (1971) noted in an unpublished report that . . . "no special effort was made to obtain human sera from patients with central nervous system disease and the samples tested were only those routinely submitted to the State Public Health Department Laboratory. Test re- sults reported on human sera, therefore, will in no way be indicative of the total number of human California encephali- tis virus infections that may have occurred in Michigan during this three year period." An important result of these MDPH tests was the detection of the state's first known clinical illness resulting from an infection with a California Group virus. During the summer of 1969, a four month old child living in St. Johns, Clinton County, was hospitalized with a clinical illness later shown, through serological tests, to have been caused by California encephalitis virus. Epidemiological evidence indicates that the virus must have been contracted in that community. In the summer of 1970, the MDPH tested sera of small mammals collected in the St. Johns City Park and found hemagglutination inhibition anti- bodies for California Group viruses in 2 of 12 Fox Squirrels (Sciurus niger) and l of 5 raccoons (Progyon lotor) (Becker and Bail, 1972). During the summer of 1970, in a community approximately 20 miles east of St. Johns, two more clinical human cases of encephalitis were detected. In late August, two young boys from the same neighborhood in Owosso, Shiawassee County, were hospitalized with high fevers, headaches and convul- sions. Both of these cases were also confirmed by MDPH officials as central nervous system diseases caused by Cali- fornia Group viral infections. In view of the occurrence of these first clinical cases in Michigan and the increasing health problem created by these viruses in neighboring states, a preliminary epidem- iological investigation was conducted near the residence of the two Owosso clinical cases with the objectives of: (1) Assessing the extent of human California encephalitis group virus infections in that area; (2) determining the vertebrates in the area which might serve as potential mosquito and/or virus hosts; (3) ascertaining the composi- tion of pest mosquito species during the late summer and early fall; and (M) elucidating the epidemiological impor- tance of the man — biting mosquitoes in the study area as potential vectors of California Group viruses. It is hoped that this study will provide some of the basic data needed for future arbovirus research in the community of Owosso and in the State of Michigan. HISTORY OF THE CALIFORNIA GROUP ARBOVIRUSES In 19u3 and 1999 an unknown virus was isolated from two species of mosquitoes, Aedes melanimon Dyar and Culex tarsalis Coquillett, in Kern County, California. In a series of pub- lications (Hammon and Reeves, 1952; Hammon st 31., 1952; and Reeves and Hammon, 1952) the unknown viral agent was named California encephalitis virus and was successfully transmitted in the laboratory to a rabbit by infected Aedes melanimon. In addition, serological evidence implicated the newly found virus as the causative agent of three human cases of encephalitis in the San Joaquin Valley of California during 1995 (Hammon and Reeves, 1952). After the initial isolation, the virus was not reported again in California for nearly two decades, even though virus isolations were attempted for over 500,000 hematophagous arthropods during that period (Reeves and Hammon, 1962). It was assumed that some unknown ecological changes had resulted in the disappearance of California encephalitis virus from Kern County, although antibodies were found in humans and small mammals in 1963 (Gresikova 33 31., 1969). During this unexplained 20 year absence in California, other laboratories in the United States began to isolate viruses that were antigenically related to, but distinct from, the 1993 - nu California isolate. In 19MB, Trivittatus virus was isolated from Aedes trivittatus (Coquillett) in North Dakota by Dr. C.M. Eklund (Hammon gt gt., 1952). A decade later San Angelo virus was isolated from Angpheles pseudopunctipennis (Say) in Texas (Taylor, 1967) and for the next six years, new antigenically related viruses were isolated almost yearly in North America. During this period related viruses also were being discovered in Trinidad, Czechoslovakia and Africa. Table 1 lists the California Group viruses by the year and locality of their initial isolation. The column "Mosquito Species" lists the number of species from which the virus subtypes have been isolated, and is based on the reported isolations by all researchers in North America from 19MB to 1970 (compiled by Sudia gt §_1_., 1971). The two most recent and comprehensive reviews of this group of arboviruses (Henderson and Coleman, 1971; Sudia gt gt., 1971) indicate that the classification is still in a state of flux. A tentative classification has been established by the Subcommittee on Immunological Relation- ships Among Cataloged Arboviruses of the American Committee on Arthropod-borne Viruses (reviewed by Sudia gt gt., 1971). This places three complexes in the California Group of arboviruses: California, Trivittatus and Melao. The latter two complexes presently contain only one virus type (the original isolated agents), while the California complex now has eight subtypes. In this classification, Lumbo is .naufib saunas mo unadunb 4 A .u:u«> nomddo nebunuluh mo uauuuce < u .353 tmflww. 133 loan suds @3303" soon can 2—53.. on» no? loam 3.32.. no weal—z.— .onaoxiz” .naunuuabaualfl .IH:HON«H¢0I0I neuaushou OH coma ..H¢ uo anon Ivuuonm Icaouundduo manic coma «couches o I somd .uoahua aunu0mwddo nuunuonq duouwdao meme vanuaoam muuunv 0 Ha Roma .aoaln: and wusuom ovuuoaoo quaduoaa uuoaaaso acmu nomuuo unbuuolun o d 32 . add Mm 833.: 5382:. A33 13.5 8x 83 835 3 o m :3 . .flm mm. «:38 .683 82 3338 Sausage 83 “95. 58m 6 Ansuduwuuau unmoav ma HomH .amu;mm.uouuovmusn «sauna: and: «cannonm mmmH can: oosusoam u . $3 rflm mm. ”0533. 333.3: 383.33 .83 $2 “.6455 u mama I .a>oaowaun can movunm uwx¢>oauosuouo mawmuuo uuvo< mmma unmask o canaunwuuaamdvaonn n 33 .318. mass .m modunmofi 3.3 359.. an... o I NGmH ..mm:mw ouaomm vuvaawua manuaamdoo moved nmma oaaoz z ma Nan ..mmwmm.cosanm uuoxoa :uuoz mnuauua>auu mmvo< mama mauuuuw>wuy a n Nnma .nmm.wm coaam: casu0uwaau coaqaodoa mowo< mama ammuOuoum o moaoumm acquaaomH aowuuaomn coauaaouH cuaammoz vouao unauauouqq mo muaauoog mo mausom no use» ammunsw «nodaloo vauos ozu mo momauw>onua anouu owauomfidno msu mo muqaaau HouawOHoaouno < .H manna considered a variant of Tahyna virus and Jerry Slough a variant of Jamestown Canyon. Henderson and Coleman (1971) present an excellent detailed review of the antigenic struc- ture and classification, the physical and biochemical prop- erties, and 12.11322 and 12.2112 studies that have been made of these viruses. After the three initial human cases of encephalitis in California in l9u5, additional evidence of human clinical infections with California Group viruses in the United States did not appear until the early 1960's. In 1962, Thompson gt gt. (1963) recorded the first known evidence of widespread human infections in an area other than California. They found neutralizing antibodies in 51 of luu wildlife conserva- tion workers tested in Wisconsin. Twenty cases of Cali- fornia Group encephalitis, diagnosed from a retrospective study of 351 patients with central nervous system disease, occurred in Wisconsin from 1960 through 196% (Thompson and Evans, 1965). During August and September of 196%, the first epidemic of California Group virus occurred in the United States in southeastern Indiana (Marshall, 1965). The epidemic included twelve serologically confirmed cases, all associated with children under 16 years of age. In the same year cases of California Group encephalitis in children were also reported in Ohio (32 cases), Wisconsin (4 cases) and North Carolina (1 case) (Beadle, 1966). The first documented human fatality due to these viruses occurred in La Crosse, Wisconsin in 1960. In 10 September of that year a 9-year-old girl died with meningoencephalitis, but it was not until 196”, when virus isolation procedures were conducted on frozen brain and spinal cord tissues, that a California Group viral agent (now called the La Crosse subtype) was isolated (Thompson gt 3;., 1965). As diagnostic laboratories throughout the United States began to include California virus tests in their routine surveillance of central nervous system illnesses, the number of reported cases began to increase, particu- larly in the North Central United States. Sudia gt gt. (1971) indicates that 519 confirmed and presumptive cases were diagnosed in the United States during the period 19u5 - 1970. Of these, H66 (90%) were reported from the North Central region. The distribution of these cases, shown in Table 2, emphasizes the increasing incidence of infections with this arbovirus in states bordering Michigan. Infections in man may be even more widespread than the reported cases indicate since extensive serological surveys have revealed the presence of antibodies in human popula- tions in the absence of reported clinical disease, in many parts of North America. A recent review of the existing data from these surveys (Henderson and Coleman, 1971) points out several important correlations: Antibodies are most prevalent in rural populations and in certain subgroups within these populations (e.g., wildlife .AvovsHouH you «HuoamoHv waHvaoa moQGUV oungIoo uoz 11 .22 tum .ulo 33m Sue” was mm as no an em mm mm NH aHauoa NOH on ow HH o NH «H e wH cHuaoouHa me~ mm on ms nu an mm an I OHgo mm c 0H m m MH H N H auouoanz mm «H o H OH I o I I aaoH em I I H N N n ~H I naquaH N I I I H H I I I aHoaHHHH poan momH oomH momH oomH momH comH nomHInemH mHuuoa uuaom ma woman no unnauz ouuum .23 I 32 483:3: wflBHoxoo 3:3 “.32: 33:8 fiuoz any :H uHuHHanmooao «HauOMHHao mo manna amass o>Huqa=muun can vuauHmaoo .N oHnaa 12 conservation workers, Seminole and Miccosukee Indians in Flordia, and Wisconsin cranberry workers); and virus activity seems to be endemic in certain localities, as shown by an increase in prevalence of antibody in older age groups. Clinical studies (Thompson and Evans, 1965; Cramblett gt gt., 1966; Young, 1966; Thompson and Inhorn, 1967; Chun gt 31" 1968; and Johnson gt_gt., 1968) indicate that ill- nesses produced by infections with California Group viruses are most prevalent in children under the age of 15, par- ticularly in the 5 - 9 age group, and occur predominantly during the months of July, August and September. Infections in adults are generally subclinical, although Vianna gt_gt. (1971) report a case of California encephalitis in a 33-year- old male in southeastern New York. Patients typically experi- ence a 5 to 10 day febrile period, headache, vomiting or nausea, a lethargic feeling and, in severe cases, convul- sions and coma. The initial clinical diagnosis in such cases is often aseptic meningitis or meningo-encephalitis (Chun gt gt,, 1968). There are few studies investigating the subsequent effects of acute central nervous system disease caused by the California Group viruses in children, but it appears that major sequelae are uncommon (Chun gt gt., 1968; Matthews gt gt., 1968). With the exception of the original three cases in l9u5, associated with California encephalitis prototype virus 13 infections, most of the virus isolates from human cases have been the La Crosse subtype. Recent studies have indicated the possibility of human disease related to in- fections with Trivittatus subtype in Minnesota (Monath gt gt., 1970) and Jamestown Canyon in Wisconsin (Sudia gt gt., 1971). Human deaths from clinical disease are extremely rare; only three deaths have thus far been re- ported in North America (Thompson gt gt., 1965; Parson, 1967). The viruses in this group are transmitted to humans and other susceptible vertebrates by the bite of hema- tophagous arthropods. In North America, California Group viruses have been isolated from two species of Tabanidae (Diptera), two Species of ticks (Acarina: Ixodidae) and at least 38 species of mosquitoes (Culicidae). Wright gt gt. (1970), who succeeded in isOlating La Crosse subtype from three specimens of the horse fly Hybomitra lasiophthalma (Macq.), speculated as to whether these tabanids were mechanically or biologically infected. None of the speci- mens pooled together for testing had contained full or even partial blood meals, and it may be that these were true in- fections (i.e., multiplication of the virus within the in- sect's tissue) and not residual ingested virus. DeFoliart gt gt. (1969) isolated Jamestown Canyon subtype from both E. lasiophthalma and the deer fly Chgysgps cincticornis (Walk.). California Group viruses have been found in ticks in only one instance. In western Montana, Newhouse gt gt. in (1963) obtained two isolations from the wood tick Derma- centor andersoni Stiles (collected from a chipmunk and a golden-mantled ground squirrel) and one isolation from a rabbit tick Haemaphysalis leporispalustris (Packard) (collected from a snowshoe hare). Based on preliminary laboratory experiments conducted on other specimens of these two Species which indicated that all traces of the virus were lost within #8 hours after infectious feedings, Newhouse felt that ticks are of a very minimal, if any, importance in the maintenance of California viruses in nature. This is substantiated by Burgdorfer gt gt. (1961), who were unable to isolate virus from a pool of 131 Q. andersoni which had been collected from a viremic snowshoe hare in western Montana. Their attempts to infect other species of ticks, Ornithodoros parkeri Cooley and Q. turicata Duges (Argasidae) proved unsuccessful. McLean gt gt. (1972) failed to isolate virus from H. leporigpalustris collected in the Yukon. The majority of the virus isolations have been from mosquitoes, primarily those of the genus ggggg. Sudia E£.§£' (1971) report that California Group viruses have been isolated in North America from 38 species of mosqui— toes: 21 species of Aedes; 5 Species of Psorophora; u species of Anopheles; u species of Culex; 3 species of Culiseta; and Coquillettidia pgrturbans (Walker). These data clearly establish that mosquitoes are the major vec- tors of California Group viruses, but the Significance of 15 tabanids and ticks in the transmission cycle presently is uncertain. It is generally thought that these viruses are harbored in small mammals such as hares, rodents and raccoons, and that they are transmitted by mosquito vec- tors. Humans do not appear to be involved in the natural cycle, but may become infected when their activities en- croach upon the natural mosquito - wild mammal cycle. In order for mosquito - born viruses to be maintained in nature, a set of complex interactions must take place. A blood -_feeding female mosquito must be attracted to, and ingest blood from, a vertebrate in which the virus is circulating in the peripheral blood stream in sufficiently high quantities to infect the mosquito. Obviously, the relationship between the vertebrate and the virus must be such that the virus does not kill its host before an infec- tive level of viremia develops. The virus, once ingested by the mosquito, must then be capable of multiplying within the tissues of the insect. It is at this point that many mosquito species are eliminated as true vectors of a par— ticular virus, for the virus may be unable to multipy to an infectious level. Therefore, only unengorged mosquitoes should be used when conducting virus isolation studies in order to differentiate between actual vectors and those species which may harbor the virus for a short period while the blood is being digested. Once an arthropod vector is infected with the virus, it remains so for life (Reeves and Hammon, 1952; Reeves, 1965). 16 It is essential for a vector to acquire at least two blood meals during its adult life if transmission of the virus is to occur. The virus must first be acquired from a viremic vertebrate and then, after a proper incubation period in which the virus multiplies and infects the mosquito's tissues, be introduced into a susceptible ver- tebrate at a subsequent feeding. This multiple feeding must occur in a significant proportion of a vector popula- tion before the virus can be transmitted and maintained in the environment. In this manner a natural cycle of mosquito to wild vertebrates to mosquito can exist in an area. Man or his domesticated animals normally become infected as a result of their encroachment upon this natural cycle. The viremia in vertebrates is of relatively short duration so the maintenance of arboviruses becomes more com- plex in temperate regions since mosquito populations are not active throughout the year and some type of over - wintering mechanism is required. No over — wintering mechanism has as yet been substantiated for the California Group viruses (Rosicky, 1969), although an annual introduction by migrating birds might be possible, since neutralizing antibodies for the Tahyna subtype have been reported from a number of migratory bird species in Europe (Bardos gt gt., 1960). METHODS Description of the Study Area Epidemiological studies were conducted in a 70 acre woodlot - subdivision complex located 500 yards west of the residence of the two children who had contracted infections with California Group virus during the late summer of 1970. This study area is situated on the eastern edge of the city of Owosso, Michigan (1970 population: 18,500) and slightly northwest of Corunna (1970 population: 3,000). The Shiawas- see River flows in an east-west direction one half mile south of the study area, and a small airport lies between the river and the woodlot. Figure 1 shows the basic topography of the region, which is primarily residential-business south and west of the study area and rural-agricultural north and east of the study area. Details of the woodlot - subdivision are shown in Figure 2. Construction of the subdivision disturbed little of the woodlot, and except for the paved roadways and the modest lawns around the houses, no woods were cleared. At the time of this study there were ten homes in the sub- division, with three more being built (Figure 2). Children living in the subdivision played in all parts of the woodlot, but particularly in the northwest corner where they had 17 18 .HH MGHUGSOQQSm GOHmms may paw mmpm mpsym ammwgowx .Ommo3o may mo >5amnmom09 .H mssmflm . 000000000000000 . . 0 0 0 0 0 0 ooooooo . . . . ....u.".u.u...u.u.u.3.3anuuu“nun”nunnunuuuuuuunnuuunuunuunun”unuuuunu." u.u...u.n.u.n.u.n.n........ . . . 00H0H0H.H.u.u0w.w.40u J0u0u0u0n0r0 ..0 .0 ..0 0000000000000000000000000000000000000000000000000000000000000000000000000000 0.0... O I O 0 O I O O O O O O C O O I O O O I O O I OOOOCOOOOOOOOCOOO .00... ...... I O ....... ....... 0000000000000 ...... O ........... OOOOOOOOOO OOOOOOOOO ........ ......... H mfismwm meOHm>mUcS "mmmmm Umpmnmcz 19 .......... run”. UMOL Um>mm ................. 000000000000000000 I. O ...... OOOOOOOOOOOOOOOOOOOOO mQMUCSOQ mono mosvm 993nm wommmzmwnm \/\I FOHUooz mSOSpHooQ UHmHm c900 “I... 0 0 . ”0H0? .I...‘ 0 0 0 3.”. 00.00. .0000. 0.0.1.... IIIIIIIIIIIIIIIIIIIIIIII 00000000000000 0 . n n 0.000.000. 00000000000 .0000. 000000000000000000 0 . 0 0 0 0 0 . 0 0 0 . 0 0000000000000000 0 0. 000000 . 0.00.0000 .00.0000.0 00.000.00.0..0.000000000000... .0 0. .0 0 0 I I I I .“.0.0 0.0. .H... 0H0. 0H0 0 0 . 0 00 0 .00 0 00 O DI. I I I .00 .. O”. O 0 0 O - . mmsm HMHpcmonmm b mafia m\H _ l ,. 2O .cmmfinowz .Ommozo .mmpm >p3pm COHmH>HoASm I pOHUooz HmmH mzy mo as: .m opsmfim 21 m mpzwfim mmpHm GOHPomHHoo Hm>hmq opHm cowpomHHoo mcflpfim mmflog GGQB HOHoooz mSOSpHoma amp ammgpm gowns mamascmz pmop pm>mm COHPOSh¥mCOO hmflfifi Omfiom monmUHmmm A AA‘A ‘A—‘A -‘AA.A‘ A v A... "7 "WV va 'fiv‘v‘v‘v" -- AAA“ A.‘ “A‘A vi" fr '7 22 constructed an elaborate 25-feet high tree-house. In the western half of the woods a number of trails had been made by children riding bicycles and motorscooters, and these trails were frequently used by local and non-subdivision children. The 50 acre woodlot proper was a Beech-Maple-Oak-Hickory association. Shagbark Hickory (Qgtzg gggtg) predominated in the eastern half of the woodlot and the undergrowth there was not as thick as in the central and western portions. Two shallow stream beds, running east and south, contained snow-melt water in early spring, but were generally dry by mid-May. The only permanent aquatic habitat found in the woodlot was a one-meter wide, man-made ditch extending #00 feet into the woods, and was fed by rains, ground water and sewage runoff. A large amount of leaf litter and other organic matter lined the bottom of the ditch and extensive algal growths covered this substrate. During the latter half of June, 1971, a large portion of this ditch dried up and subsequent rains in July created conditions favorable for the larvae of Culex pipiens Linneaus. Weather data for the Owosso area (see Appendix I - IV) were recorded by the United States Department of Commerce at a weather station located at the Owosso Wastewater Plant, 3 miles west northwest of the study area. Information re- corded at this station was obtained from the Environmental Data Service in East Lansing, Michigan. 23 Epidemiological Procedures Preliminary data concerning encephalitis - like i11- nesses in children living in the study area were obtained through personal interviews with the parents of the children. Information such as the age and sex of the child, areas in which the child frequently played, date of onset of the ill- ness, activities for several weeks prior to onset, descrip- tion of the symptoms observed at onset, the child's play- mates, and the name of the family physician was obtained in this manner. Details of the clinical symptomology were ob- tained from patients' personal folders at Memorial Hospital in Owosso. Records of encephalitis - like illnesses in other Owosso area residents were also obtained by screening the diagnostic records at the hospital. The serological survey for antibody to California Group viruses was conducted on October 18, 1971. Thirty residents of the subdivision being studied were bled (1 serum sample per person) by a physician from the Michigan Department of Public Health (MDPH). Blood samples were collected in disposable vacutainers, allowed to clot, and transported to the MDPH Laboratory in Lansing for hema- gglutination inhibition tests, utilizing goose red blood cells and La Crosse subtype antigen (obtained from the Center for Disease Control in Atlanta, Georgia). Sera with California encephalitis virus antibody titers of 1:20 29 or greater were considered positive and indicative of prior infection with this agent (Bail, 1971). Vertebrate Survey Procedures Field observations were made of the vertebrates in the study area in an effort to establish the spectrum of potential sources of mosquito blood meals, and thus the available hosts for California Group viruses. Tally Sheets which listed common species were used for recording observa- tions. Mammals, reptiles and amphibians present were noted while conducting mosquito studies in the woodlot and sur- rounding areas. Several small, nocturnal species of rodents were found drowned in the steep - sided portion of a ditch leading into the woods. Road killed animals near the study area also were identified. In early fall, 20 live traps ("Have-a-Heart") were operated in the woodlot for several days in an attempt to obtain vertebrate serum samples, but with little success. To avoid repetitious observations of birds due to nesting pairs and singing males, only the presence of a species, rather than the number of individuals, was noted in order to obtain the frequency of Species in the study area. 25 Mosquito Survey Procedures Human - bait biting collections were made while Sitting on a camp stool near the center of the study area (Figure 2). During each collection period an equal amount of skin was exposed. Both trouser legs were rolled above the knees and the left shirt sleeve was rolled above the elbow. By plac- ing the left hand on top of the head, mosquitoes which would normally land on the face, and be difficult to catch, would land on the back of the hand and arm, and by slowly lowering the arm they could be seen and captured. A chloroform kill- ing tube equipped with a paper cone in the mouth to prevent mosquitoes from tumbling out on subsequent captures was manipulated with the right hand. For collecting after dark, a flashlight with red cellophane Shading the lens was used to locate landing mosquitoes. When possible, collections were conducted for at least one hour. The longest consecutive collection was 3 hours, 20 minutes. The catches were segregated at 20-minute inter- vals by transferring the contents of the killing tube to prelabeled field tins containing tissue paper. Several drops of chloroform were pipetted onto the tissue to ensure the death of all mosquitoes. The field tins were transported to the laboratory where the mosquitoes were mounted on card points, labeled and identified to species according to keys in Carpenter and LaCasse (1955). 26 In an effort to supplement data provided by the biting study, collections of immature mosquitoes were made at various times throughout the summer. A standard one pint, white enamel dipper was used to sample ditches and mud pud- dles in the study area. The contents of the dipper were poured through an Open plastic tube with cheese cloth cover- ing one end, and the larvae and pupae remaining in the cloth were transferred to labeled shell vials containing 70% ethyl alcohol. A basting pipette was used to sample certain tree holes. RESULTS AND DISCUSSION Because of the lack of epidemiological studies in the state, the natural transmission cycle of California Group viruses in Michigan and the subtype(s) involved are unknown. Most of the knowledge concerning this arbovirus has been accumulated in other North Central states where environmen- tal conditions, vertebrate fauna and mosquito populations may vary considerably from those of Michigan. The vectors and vertebrate hosts of the viruses in Michigan will remain unknown until state-wide virus isolation studies are com- pleted. The true incidence of human California encephalitis in Michigan will also remain unknown until a comprehensive effort is made to obtain and test acute and convalescent serum samples from suspect patients throughout the state. This study is an attempt to gain at least a partial under- standing of the epidemiological conditions that exist in a Michigan community where human California encephalitis is known to have occurred. With this understanding, it may be possible to more accurately evaluate the feasibility of pursuing in-depth, state-wide studies of this arbovirus. 27 28 Clinical Cases of California Encephalitis The two cases of California encephalitis in Owosso which prompted this investigation occurred in the late summer of 1970. These cases were particularly Significant in that they were the second and third human cases reported for the State of Michigan, the first case having occurred in St. Johns in 1969. The histories of the two Owosso cases, based on hospital records and interviews with the parents, were as follows: One case involved a 10-year old boy (T.T.), who was admitted to Memorial Hospital in Owosso on August 31, 1970 with a severe headache, abdominal pains, and a sore throat. He experienced headaches for two days prior to admission, and two hours after being brought to the hospital went into convulsions and remained comatose for a time. His temperature peaked at 103.5°F. and began to decline after the seizures. His Speech remained garbled for several days, but then improved steadily and he was dis- charged on September 9, 1970. The hemagglutination inhibi- tion (HI) antibody titer to La Crosse antigen was 1:1280 in his acute-phase serum and 1:320 in convalescent serum tested on December H, 1970. Follow-up examinations of T.T. by a physician revealed no abnormal developments and his recovery was apparently complete and uneventful. The other case of California encephalitis involved a l3-year old boy (R.T.) who was admitted to the hospital on September 3, 1970. 29 Five days previous to this, he had developed headaches, fever and vomiting. Upon admission he was incoherent and having seizures, with a peak temperature of 105°F. The patient was fed intravenously for three days and according to hospital reports, "thrashes about wildly in bed with somewhat sunken eyes. Does not recognize people or faces, and restraints and pains seem to make him wilder." About the third day after admission his sensorium cleared, and he was released four days later. The HI antibody titer in his acute serum was 1:u0, whereas the titer of the con- valescent serum tested on September 25, 1970, was 1:6u0. Several aspects of these case histories seem to be of special epidemiological interest. T.T. and R.T. were unre- lated playmates, living next door to one another in a reSi- dential area 3/8 mile east of the woodlot - subdivision. Both boys spent a considerable amount of time together in this woodlot during July and August, 1970 and were frequently exposed to biting insects while playing there. They had traveled with their families to other parts of Michigan in the week previous to the onset of symptoms, but the families did not visit the same areas and the parents reported that the boys had not spent any appreciable amount of time out- doors during the trips because of heavy rains. Thus, while there is the possibility that the boys were bitten by in- fected mosquitoes in separate areas of the state, it seems likely that they became infected with the virus while play- ing together near their homes. 30 In addition to these two confirmed California encephalitis illnesses in 1970, there is also evidence that California virus transmission may have occurred near the same area in 1971. In January, 1972, the Michigan De- partment of Public Health reported the results of serological tests made on the serum of an Owosso child who had had a central nervous system disease during the early fall of 1971. The patient, a 2-year old girl (C.H.), had been ad- mitted to Memorial Hospital on September 9, 1971. She had experience a fever of 101°F. for two days prior to ad- mission. At the hospital her fever rose to 10u°F. and She had seizures Similar to those of R.T. and T.T. She was released ten days later and recovered uneventfully. Her HI antibody titer to La Crosse antigen, 1:80 in her acute- phase serum and 1:320 in convalescent serum, indicates an infection with California Group viruses. The home address of C.H. was obtained from hospital records. She lived one mile east of the residences of the two 1970 cases in a 96-house subdivision with a Beech-Maple-Oak woodlot along the northern boundary (see Figure 3). The home of C.H. was situated at the edge of this woodlot, and her mother stated in an interview that C.H., and many other children in the subdivision, played in this woodlot throughout the summer of 1971. Bicycles, swings, and toys were seen in many of the yards in this subdivision, indicating that many families had one or more young children. The 31 close proximity of this subdivision to the study area and to other deciduous woodlots, and the presence of a large number of children in a susceptible age group, would make this an important area for future epidemiological studies. Human Serological Survey As a result of interviews with subdivision residents of the study site throughout the spring and summer of 1971, it was learned that three children in the immediate area had previously been hospitalized for illnesses which, based upon descriptions by the parents, appear to have been of encephalitis-like nature. A girl (M.G.) who had lived in the subdivision since 1965 was hospitalized on September 28, 1967, (age 6 when hospitalized) with a headache, sore neck and high fever. She suffered convulsions, was unable to talk coherently for several days, and reacted violently to both hospital personnel and members of her family. She was more alert by October 5, but complained of a constant head- ache. Her recovery was uneventful and she apparently suf- fered no ill effects. A boy (B.B.) who had lived in the subdivision since early 1967 was also hospitalized in September of 1967 with Similar symptoms, although no con- vulsions occurred (age 9 when hospitalized). These two children lived within 700 feet of one another and played frequently in the woodlot around their homes. Neither child 32 had traveled to other parts of Michigan for several weeks prior to the onset of symptoms. In late August of 1969, a third child, a boy (M.M.) who had lived in the subdivision since 1965, suffered from headache, high fever and vomiting. These symptoms began on August 26 and he was admitted to the hospital on August 31, 1969 with a diagnosis of meningi- tis. During his hospitalization (age 9 at the time) he was sleepy and irritable but made-an uneventful recovery. Two weeks prior to the onset of symptoms he had returned from a summer camp in northern Michigan where he spent several weeks. According to records at Memorial Hospital in Owosso, where the three children had been admitted for central ner- vous system disorders, there had been no diagnostic tests for arboviruses conducted at the time of illness. It was important, therefore, to determine if these three children had had any previous experience with the California Group viruses. Results of the serological survey, which included mem- bers from 8 of the 10 subdivision families, are presented in Table 3. Of the 30 individuals tested, four children (13.3%) exhibited significantly high hemagglutination inhibi- tion (HI) antibodies (i.e., titer a 1:20) to La Crosse anti- gen, which indicates that they had had previous exposure to California Group viruses. More importantly, when only the number of individuals (17) in the age group most susceptible 33 Table 3. Results of a serological survey for hemagglutination inhibition antibody to California Group arbovirus (La Crosse antigen) in the Owosso woodlot - subdivision residents. Age Group Sex Number* ‘ Number Titer Subject (years) Tested --I Positive Level Code M 0 0 - 4 F 2 0 M 6 1 1:20 R.G. 5 - 9 F 5 1 1:80 R.R. M 3 O 10 - 14 F 2 2 1:20 L.R. 1:80 M.G. M 2 0 15 - 19 F 1 0 M 0 20 - 29 F 1 0 M 5 0 30 - 39 F 5 0 Total: 30 4 (13.32) * Date of serum collection was October 18, 1971. 31+ to California virus infections (i.e., children under 15 years of age) is considered, the percentage of positive reactions is 23.5% (9/17). Antibodies were not found in any individual over lu years of age. One of the four sero- positive individuals was M.G., a 10-year old girl whose 1967 encephalitis-like illness was described above. Be- cause no acute-phase serum had been obtained for subsequent comparative testing, and because no California Group virus isolation was attempted at the time of her illness, it is impossible to definitely attribute her encephalitis to this virus. However, her antibody titer was 1:80, which is presumptive evidence of a past infection with this arbovirus. Sera from the other two subdivision children with encephalitis-like diseaSe histories (B.B. and M.M.) exhibited titers of 1:10 and < 1:10, respectively. These levels cannot be considered indicative of previous Cal- ifornia virus infections. In addition to the high California virus antibody titer of M.G., significant HI antibody levels were present in three other children: R.G., a 9-year old boy who had lived in the subdivision since 1967 had an HI titer of 1:20; L.R., a 12-year old girl who had lived there since 1966 had an HI titer of 1:20; and R.R., an 8-year old Sis- ter of L.R. had a titer of 1:80. None of these three children have a record of any clinical central nervous system disease, although R.R., whose high titer strongly 35 indicates an infection with this arbovirus, had experienced a non-encephalitic illness during the late summer of 1970. The geographical distribution of the homes of the confirmed clinical cases of encephalitis and the seroposi- tive individuals, as shown in Figure 3, indicates the close association with deciduous woodlots where exposure to biting insects is likely. The number of susceptible individuals living in this narrow strip was augmented by visiting play- mates and children who lived within bicycling distance. On numerous occasions during the summer, non-subdivision chil- dren were seen walking, bicycling or riding motor scooters on the trails in the study woods. Many local children utilized this woodlot for extended daytime play activities, as was evident by their elaborate tree—house, underground huts and well-worn trails. The large number of children playing in this area is an important factor which would tend to increase the potential for transmission of Cali- fornia encephalitis virus. In an effort to locate possible encephalitis cases in other Owosso localities, the diagnostic records at Owosso Memorial Hospital were screened for the months of June - November, 1965 - 1971. All "suspect-diagnoses" of aseptic meningitis, meningo - encephalitis, or encephalitis of probable viral etiology, which are often the initial diagnoses of what are actually found to be infections with California Group viruses (Cramblett gt gt., 1966), were 36 .cmwwonz .OmmOSO .>o>n:m HmonOHonom may SH pm>Ho>cH mmHHHEmm opp paw meHHmnmmocm MHSQOMHHMU mo mommo meLHmcoo exp mo meoc mzp mo cowysnflhpmfln HMOHAQMpmoow mnB .m madman m onsmflm mmmcHHH mxHHImeHHM£mmoco mo mpoumwn npwa .Aomquv pnMOHMHcmHmGH Ho>oH mooowwc< Aomquv vamoHMchHm Ho: Hm>oH moonwun< wmoGHHH oxHHImHHHHmnmoono 0: van .AomuHmV >GOAHHQM “CMOHmwcmHm meHHwnmooao MHCAOMHHMU mo mmmo o>wumadmonm .0604 mHvHHmnmmocw MHcpomemu.Ho ommo ooanwmaoo 37 » was: a: . HbmH RIC} I IIIII 38 noted and the corresponding patients' folders were then checked for age, sex, date of admission to the hospital, and general symptoms of the illness. In addition to the six patients whose illnesses were already known and have been described earlier (patients R.T., T.T., C.H., B.B., M.G., and M.M.), seven other patients were found to have had central nervous system diseases of "suspect-diagnosis" during the months of June - November, 1967 - 1971. Two of these patients were over 30 years of age, and are omitted from the discussion since infections with California viruses are extremely uncommon in age groups above 20 years (Henderson and Coleman, 1971). Figure M compares the seasonal distribution (1967 - 1971) of both California encephalitis cases and encephalitis- 1ike illnesses of unknown origin in the Owosso area to the distribution dates of 239 cases of California encephalitis reported from North America, 1963 - 1968 (Henderson and Coleman, 1971). Although the two early November illnesses of "suspect-diagnosis" are questionable as being California Group virus induced diseases, the two mid-September (1971) illnesses, as well as the one in late August (1970), would perhaps indicate more extensive virus activity, since the latter three cases occurred at times when other cases of confirmed California encephalitis were reported in Owosso. According to addresses obtained from the patients' folders, the "suspect-diagnosis" children lived in Owosso, with the exception of the mid-September 2-year old who lived in 39 .mnzpmnopHH any cw omppommn mommo :mm mo SOHvsnwhvao HMCOmmom LPHS ownedeoo AHmmH I mmmH .Pomco mo mopmo ow wafiohooomv cmmHnowz .Omwo3o .Hmwwmmom HMHLOEmz ov omvPHEpm cosprno CH mommoCHHH mxHH I meHHmnmooco one meHHmnmooco MHQLOMHHMU mo coapznfippmflo Hmcommom .: madmflm HO : oCCMHm .HHmmH .CMEmHoo UCm COmCmpComv mmmH I mmmH .HomCo mo mwvmo C30Cx Csz MOHCoE< CHCoz CH mHHHHmnmmOCo MHCCOMHHMU mo mommo :mm mo COHHCAHpHmHU HMCOmmmm « .mammh CH HCoHHmm may mo own oCH onoHUCH mHonahm 0H onC mmmmzaz CHwHCo CsoCMCC mo ommmmHU mxHH I mHHHHMCaoOCm AV .mUCoooC OHHmOCMMHU Hmeamon Cwsonnp mHCo poHMUOH wmmmoCHHH onoHUCH mmqwzHpm§moCm G meHHmnamOCo MHCCOMHHMQ mo mmmo pwECHMCoo C .mmCm mpswm mCH CH mCH>HH CwCoHHCo oCm 0mmozo CH mommo HMOHCHHU CzoCx wwmoHoCH mmqomHo _ mmmzmsoz _ mmmoauo _ mmmzmaamm _ $83 _ 52. L _ P p . C —o_H—~_3_ms_om_sm_§_NN—NHT moo... .53 AV mH .mmmH 2 Q S 3” GE .33 m 4 .23 ~40“ 91 Ovid, Michigan, eight miles west of Owosso. In any future arbovirus study in the Owosso community, a detailed sero- logical follow—up of these "suspect-diagnosis" patients would be necessary, since these five illnesses indicate the possibility of virus activity extending beyond the immediate study area. Mosquito Fauna The primary objective of the mosquito survey was to ascertain which human-biting Species were active during the late summer and early fall, and might be regarded as po- tential vectors of California Group viruses in the study area. All collections from human bait were made at a single point in the woodlot, approximately 900 feet from the home of the 1967 presumptive case of California encephali- tis and 1/2 mile from the homes of the two 1970 confirmed cases. Residents of the woodlot - subdivision stated that the density of mosquitoes throughout the late Spring and summer of 1971 was abnormally low. They reported that in some years mosquitoes were unbearable in the subdivision and made backyard picnics, parties and other prolonged outdoor activities extremely unpleasant. The low density of mosqui- toes during 1971 may have been associated with the unusually low amount of rainfall in May, June and August, which would lower the number of available breeding sites. The monthly H2 rainfall for May, June and August was 2.21, 2.H1 and 1.2a inches below a 30-year norm, respectively (see Appendix III for comparisons with 1967 - 1970). Human-bait biting col- lections conducted between July 20 and September 7, 1971, yielded only 312 female mosquitoes, including 5 species of Aedes, 2 species of Anopheles, and Coqgillettidia perturbans (Walker). Table u indicates the frequency of collections for each of the 8 species during 56 20-minute collection periods. Aedes triseriatus (Say), the most widely distributed treehole- breeding mosquito in North America, constituted 60% of the total catch at human bait. The biting patterns for the three major pest species encountered in the study area between 12 noon and 9 p.m. are shown in Figure 5. This period corresponds to the time when many children in the subdivision were actively playing in the woodlot. Adult residents were also engaged in out- door activities during this period and several families had frequent late afternoon and early evening barbeques and lawn parties for large groups of people throughout the summer. Female mosquitoes seeking blood meals during this period of time could come in contact with many human hosts in the subdivision and, as discussed earlier, visiting friends and playmates of the residents could increase the number of available human hosts. AS shown in Figure 5, Aedes triseriatus was attracted to, and fed upon, human bait throughout the afternoon and early evening, but terminated its feeding soon after sunset. H3 Table 4. Frequency of eight species of mosquitoes collected at human bait between July 20 - September 7, 1971, in the Owosso woodlot - subdivision. Species Number I of Total Caught Catch ggggg_triseriatus 188 60 92.4.9.9. 1222.113 54 17 Coquillettidia perturbans 35 ll Anopheles guadrimaculatus l7 5 Egg fitchii 13 4 £9.23. canadensis 3 l _A_g_dg_s_ stimulans 1 <1 Anonheles punctigennis 1 <1 Total 312 1001 nu .HmmH .m CmnEonmw I on mHCb .pHmn CMECC Hm mCOHHowHHoo mHSCHEImHCoSH m: Co comma .momm mpspm 0mm030 may CH mmOHHCdmoE mo meommm «mom Comma on“ mo wCHHHn mo mEHB .m oCCmHm #5 m opsmHm mQOHmmm mDOmIH 2H mZHB Zm_z< mlm mlh hIw mlm ml: :Im mIN NIH HINH - b b pl 5 h b b O . x — szzm x .5 \\\\\\\\\ 1H “ C /..\\\\\\\\Q IN 0 X rm 0 O O I: 0 1m 1w 15 m mCmnCCHCom .m X C C m mmew> .fl 0 / mCHMHCmmHCH .fl 0 O OH HOIVO Hlflle-OZ 83d SHOIIHOSON JO HHHNHN NVHN NB Feeding occurred most frequently between 5 p.m. and 7 p.m., and dropped off considerably after sunset. These data agree with fi. triseriatus studies by Loor and DeFoliart (1970), who also noted that biting was very rare after 8 p.m. The latest collection of this species was made be- tween 10:10 and 10:30 p.m. on August 17, when a single fe- male was caught at human bait in the tree house at a height of 25 feet. Aedes vexans Meigen and Coquillettidia pertur- gggg rarely appeared at human bait during the day but became more numerous from one hour before sunset until 9 p.m., when most collecting was terminated. A single fi. vexans was collected between 10:10 and 10:30 p.m. on August 17 in the tree house. The other five species attracted to human bait were Anopheles punctipennis (Say), fig. quadrimaculatus Say, Aedes fitchii (Felt and Young), fig. stimulans (Walker), and fig. canadensis (Theob.), but they were collected too infrequently to determine any biting pattern. A survey of summer breeding sites in the study area resulted in the collection of immature stages of Aedes triseriatus, Anopheles punctipennis and Culex pipiens Linn. Despite the frequency of adult fig. triseriatus in the woodlot, only 17 larvae were collected during the study. All 17 were first instar larvae collected on July 2, 1971 in a small depression in the trunk of a partially uprooted maple tree. On the previous day the area had received 0.62 inches of rain, the first substantial rainfall since June 13 (see Appendix IV), which most likely stimulated the hatching of 47 the eggs. No other tree holes which could be sampled were found to contain water long enough to allow mosquito larval development. Larvae of Anopheles punctipennis were found in a mud puddle in the northeastern corner of the nub- division. A total of 39 larvae, taken in a series of dips made on September 15 and 18, were reared to adults in the laboratory to confirm the identification. Several smaller puddles were checked but no other immatures were found. Adult fig. quadrimaculatus were collected in the woodlot but larvae of this species could not be found at any breeding site in the study area. Immature stages of Culex pipiens were collected in several habitats in the area: A child's plastic swimming pool containing leaf litter; a discarded tar bucket; the flooded basements of two houses under con- struction; and the man-made ditch described earlier which extended MOO feet into the woodlot. This ditch was dry earlier in the summer of 1971 but contained rather high densities of Q. pipiens larvae in mid—July after rains earlier in the month (Appendix IV) provided sufficient water levels for their development. This drying - flooding con- dition removed most of the algae which grew heavily in the ditch in May and June. A total of 2283 larvae and pupae of 9. pipiens were collected from the ditch from July 28 - September 7, 1971 (Table 5). Three sites, designated A, B, and C and approximately 200 feet apart (Figure 2), were sampled periodically. Each larval collection consisted of ”8 Table 5. The relative abundance of immature Culex pipiens in the Owosso woodlot - subdivision between July 28 - September 7, 1971. Date Collecting water Pupae Station Temp. ?C I and II III and IV A 17.0 66 34 3 JUL 28 B 17.0 120 124 43 C 17.0 9 26 1 A 21.0 42 40 4 AUG 2 B 19.5 24 48 2 C 20.0 4 3 3 A 18.0 30 66 4 AUG 4 B 18.5 27 49 8 C 18.5 14 34 O A dry - - - AUG 10 B 23.5 16 4 0 C 22.0 16 10 3 A 20.0 8 2 0 AUG 12 B 19.5 35 15 0 C 22.0 11 10 1 A 21.0 5 l 0 AUG 16 B 21.0 31 l 4 C 20.0 46 56 4 A 22.5 0 3 0 AUG 19 B 23.0 4 4 0 C 23.0 7 O 0 A 21.0 39 18 1 AUG 25 B 21.0 49 39 0 C 20.5 2 30 0 A 18.0 27 111 2 AUG 31 B - - - - C - .. _ - A 24.0 10 4O 0 SEP 7 B 22.5 15 71 l C 22.0 211 334 158 Total: 868 1173 242 Ave/Dip: 16.1 21.7 4.5 Total number of immatures collected: 2283 Average number of immatures/Dip: 49 two dips taken near the center of the ditch at each Site. Recorded water temperatures were taken 1 inch below the surface. The effects of dry periods of weather on available larval habitats are reflected in the data for August 10 and 19 (Table 5). On August 10, after 12 days of essen- tially no rain, site A was completely dry and the water at site B was only several inches deep. Similarly, the few larvae at the three sampling sites on August 19 followed 7 rainless days. Despite these periods of partial drying there were always some portions of the ditch contain- ing sufficient water to which the larval population could move. Immature Culex pipiens, averaging 92.3 per dip, was the only mosquito Species taken from the ditch. No females of this species were taken at human bait although it is known to feed on man (Carpenter and LaCasse, 1955). A total of 9 species of mosquitoes were obtained in biting and larval collections in the Owosso woodlot-subdivison during the summer and early fall of 1971. Since these Spe- cies are known to feed on man, and are the ones that are most likely to contact children in the subdivision, it is important to consider their role as potential vectors of California Group virus in this area. The most comprehensive review of the species of mosquitoes which may be involved in the transmission of California Group viruses is that of Sudia gt gt. (1971). They have reviewed all of the published 50 literature concerning virus isolations from North American mosquitoes between 1943 - 1970, as well as much unpublished data from laboratories active in arbovirus research. Table 6 lists the 9 species of mosquitoes present in the Owosso study area and summarizes the published accounts of the isolations of California Group viruses from these Species. As shown in this table, there are several species of mosquitoes which could serve as potential vectors in the study area. Certainly the most suspect mosquito is figggg - triseriatus, which is the,proven vector of La Crosse virus in the North Central United States. According to Sudia gt gt. (1971), this is the only species which has fulfilled the four basic criteria considered necessary to prove vector status, namely: (1) The virus must be isolated from mosqui- toes collected in the field; (2) there must be field evidence to confirm an association of the infected mosquito species with a vertebrate population in which the infection is occurring; (3) the mosquito must experimentally be infec- ted by feeding upon a viremic vertebrate; and (H) the mosquito must be capable, in laboratory experiments, of transmitting the virus to a susceptible vertebrate host by bite after a period of extrinsic incubation of the virus. Aedes triseriatus, which comprised 60% of the total number of mosquitoes collected at human bait in the study area, was a persistent feeder throughout the afternoon and early evening and was likely to be encountered by 51 Table 6. The mosquito species of the Owosso woodlot - subdivision and the virus subtypes isolated from them in North America, 1943 - 1970,a Virus Number of Species Subtypeb Isolation. Breakdown by States AnOpheles punctipennis LAC* 1 Minnesota Anopheles quadrimaculatus 0 Coquillettidia pgtturbans TVT* 1 Iowa UNT 1 Florida Aedes fitchii SSH 5 Montana Aedes stimulans JC* 3 Wisconsin SSH 1 Alberta, Canada Aedes canadensis KEY 2 Maryland LAC 16 2-Georgia; 14-0hio SSH 2 lc-British Columbia; 1-Massachusetts UNT 6 léMassschusetts; l-New York; 4-Ohio Aedes triseriatus KEY 1 Louisiana LAC 34 4-Iowa; SAMinnesota; 8-0hio; l7-Wisconsin UNT 1 Ohio Aedes vexans 'CE* 2 New Mexico JC 1 Wisconsin KEY 2 léMaryland; l-Misaissippi LAC 6 l-New Mexico; 4-Ohio; l-Wisconsin ssu 2 1c-British Columbia; l-Wisconsin TVT 13 Iowa UNT 15 13-Alaska; l-California; 1-Utah Culex pipiens LAC ld Wisconsin TVT 1 Iowa 8Based on the data of Sudia.gtugt. (1971). CE—California encephalitis; JC-Jamestown Canyon; KEY-Keystone; LAC-La Crosse; SSH-Snowshoe Hare; TVT-Trivittatus; UNT-untyped. cFrom mixed pool of Aedes vexans - canadensis. dFrom mixed pool of Culex pipiens - restuans (data from Thompson gt gt., 19721 *Subtype implicated in human disease. 52 children playing in the woodlot. No other mosquito species collected during the late summer and early fall exhibited a biting pattern which would seem to coincide with the daytime outdoor activities of local children (Figure 5). The second most frequent mosquito species collected at human bait in the study area was Aedes vexans, but its num- bers were low and may have been well below the densities of other years. As shown in Table 6, Six subtypes of California Group viruses have been isolated from fi. vexans in North America, including those subtypes known to cause human ill- ness, but this species has not as yet met the four vector criteria for any of the California Group viruses. The biting pattern of Aedes vexans in the study area was such that any outdoor activity on the part of subdivision residents after 5 or 6 p.m. would bring them in contact with this species. Several children slept infrequently in the tree house during warm summer nights and may have encountered Aedes vexans in this manner. Arbovirus studies in Ohio suggest that Aedes canadensis may also be a vector of La Crosse subtype, since 14 isola- tions have been made from this Species in that state (Table 6). In the present study, fi. canadensis was col- lected only three times at human bait during the late summer — early fall season, although larvae and pupae were found in several snow pools in the woodlot during late April and early May, 1971. According to Carpenter and LaCasse 53 (1955), the adults of this species generally emerge in April, May and early June and the females, which appear to be long - lived, persist in small numbers in woodlands un- til late summer. The feeding habits of this Species are varied. It is attracted to, and feeds readily upon, turtles, birds, deer, raccoon, rabbits, and other small mammals (Means, 1968; Wright and DeFoliart, 1970). Because of this wide spectrum of vertebrate hosts and the demonstrated association with La Crosse virus in Ohio, it is possible that fi. canadensis in Michigan could serve to amplify the virus in indigenous mammal pOpulationS during the Spring and early summer and produce a greater number of viremic mammals later in the season. In the woodlot - subdivision small numbers of five other culicids were collected from human bait: Angpheles punctipennis (1/312); fig. quadrimaculatus (17/312); Coquillettidia perturbans (35/312); Aedes fitchii (13/312); and fig. stimulans (1/312). As indicated in Table 6, none of these species have as yet been consistently associated with any of the subtypes of the California Group, although DeFoliart (1972) feels that Aedes stimulans is one of the vectors in the typical transmission cycle of Jamestown Canyon subtype in Wisconsin. Culex pipiens, which was not collected at human bait but was nevertheless abundant in the study area in the immature stages, has a very Slight association with 5H California Group viruses and could be tentatively ruled out as a vector in the Owosso area (Henderson and Coleman, 1971). Studies of the feeding habits of Q. pipiens indicate that it is primarily a bird feeder, but does occasionally feed on mammals (Tempelis and Washino, 1967; and Wright and DeFoliart, 1970). This preference for avian hosts may ex- clude g. pipiens from the small mammal - mosquito cycle which typifies the California Group viruses but could pos- sibly involve it with transmission of these viruses to cer- tain avian species. The possibility of birds serving as hosts for California viruses is discussed in the next section. Vertebrate Fauna The species of mammals, reptiles and amphibians which were observed in and around the study area are listed in Table 7. Live trapping attempts in the woodlot to obtain blood samples for serological tests had limited success; only two Peromyscus and one Eastern Cottontail (Sylvilggus floridanus) were trapped and one of the mice died before being bled. The absence of chipmunks and red squirrels throughout the summer was particularly noticeable, since the woodlot appeared to be a suitable habitat for both of these species. Forty one species of birds were observed in the woodlot - subdivision from April 29 to 55 Table 7. A list of the mammals, reptiles and amphibians observed in the Owosso woodlot - subdivision between April 29 - September 14, 1971. Species Number Observed MAMMALIA* Order Insectivora Starnose Mole (Cond lura cristata) Shorttail Shrew (Blarina brevicauda) Order Carnivore Raccoon (Proczon lotor) Domestic Dog (Canis familiaris) Domestic Cat (Felis domesticus) Order Rodentia Woodchuck (Marmota monax) Eastern Fox Squirrel (Sciurus niger) White-footed Mouse (Peromyscus leucopus) Jumping Mouse (Zapus or Napaeozgpus) Order Lagomorpha Eastern Cottontail (Sylvilagus floridanus) Order Artiodactyla Whitetail Deer (Odocoileus virginianus) REPTILIA** Garter Snake (Thamnophis sirtalis) AMPHIBIA** American Toad (Bufo americanus) Spring Peeper (Hyla crucifer) Green Frog (Rana clamitans melanota) 1 (dead) 1 (dead) 3+tracks in cornfield. 5 seen daily 1 2 (1 dead) no actual count (1 or 2 seen almost daily) 3 (2 trapped) ' 1 (dead) 6 (l trapped) 2+tracks in cornfields usually seen daily heard infrequently 1 or 2 seen daily * Taxonomy follows Burt (1957) ** Taxonomy follows Conant (1958) 56 September 14, 1971, and these are listed in Appendix V. Table 8 lists the 13 species which were most frequently observed during the study. These frequencies are based on the number of days a particular species of bird was seen or heard during 38 days of field observation. As an indication of the prevalence of tree hole situations in the woodlot and therefore breeding Sites for certain species of mosquitoes, 7 of these 13 frequent species utilize tree holes as nesting sites. Of the 11 species of mammals recorded in the study area (Table 7), 6 have been reported in the literature as having antibody for California Group viruses: Raccoon (Newhouse gt gt., 1964; Bond gt gt., 1966; and Whitney gt gt., 1969); Woodchuck (Whitney gt gt., 1969); Fox Squirrel (Moulton and Thompson, 1971); Perogyscus (Gresikova gt gt., 1969); Cottontail (Whitney gt gt., 1969; Moulton and Thompson, 1971) and Whitetail Deer (Cook gt gt., 1965; Emmons, 1968; and Whitney gt gt., 1969). Tests for California virus antibodies in sera from two mammals trap- ped in the study area (1 Peromyscus, l Cottontail) were negative. Serological surveys of vertebrates in other areas of the state have yielded evidence of California Group virus infections in a number of mammals and birds. Serum samples from various species of mammals and birds have been collected in other areas of Michigan by personnel of the Rose Lake Wildlife Pathology Laboratory 57 Table 8. Most frequently observed birds in the Owosso woodlot - subdivision between April 29 - September 14, 1971 (based on data in Appendix V). Species 0bservation* Rank Frequency Blue Jay 742 1 Tufted Titmouse** 712 2 Red-headed Woodpecker** 612 3 Red-tailed Hawk 532 4 White-breasted Nuthatch** 532 4 Robin 47% 5 Yellowbshafted Flicker** 452 6 Red-eyed Vireo 402 7 Crested Flycatcher** 342 8 Common Crackle 34X 8 Black-capped Chickadee** 29% 9 Starling** 29% 9 Cardinal 292 9 * Number of days observed/38 field observation days ** Indicates the species nests in tree holes 58 and tested for H1 antibody to La Crosse antigen by the Michigan Department of Public Health. Sera were considered positive if they exhibited a titer of 1:20 or greater. The results of these tests (Becker and Bail, 1972) were as fol- lows (number positive/total tested for each species): Snowshoe Hare (27/160); Opossum (Didelphis marsgpialis) (0/6); Raccoon (1/8); Fox Squirrel (2/12); Chipmunk (Tamias striatus) (5/12); Woodchuck (0/1); Cottontail (0/55); Striped Skunk (Mephitis mephitis) (0/1); Thirteen-lined Ground Squirrel (Citellus tridecimlineatus) (0/1); Peromyscus Sp. (0/2); Whitetail Deer (3/132); Blue Jay (Cyanocitta cristata) (3/21); Woodcock (Philohela minor) (0/2); Cowbird (Molothrus ater) (0/1); Robin (Turdus mtgratorius) (0/1); and Ring- necked Pheasant (Phasianus colchicus) (2/2). These data suggest that a wide spectrum of vertebrates may play a role in the natural transmission cycle of California Group viruses in Michigan although further studies are needed to ascertain the specific hosts of the virus. Of particular interest are the finding of seropositive birds, which are not considered likely hosts of these viruses. The three positive Blue Jays, which were collected during May, 1972, on High Island in Lake Michigan, had titer levels of 1:20, 1:40 and 1:80, which would strongly indicate previous infection with California Group viruses. Blue Jays were the most frequently observed birds in the Owosso woodlot - subdivision (Table 8), but no attempt was made to 59 obtain serum samples from them. The two seropositive pheasants were collected in 1970 near Wacousta, Clinton County, Michigan. These birds, found in widely separated areas of Michigan, may indicate the need for extensive and intensive surveys of bird pOpulationS in areas suspected of California Group virus activity. The role of birds in natural cycles of the California Group viruses has been adequately investigated only in Europe in connection with the Tahyna subtype. Bardos gt gt. (1960) reported neutralizing antibodies in the sera of 31 species of migratory and nonmigratory birds in Czechoslovakia, five of which are species which occur com- monly throughout North America: Coot (Fulica atra), Barn Swallow (Hirundo rustica), Bank Swallow (Rtparia riparia), Starling (Sturnus vulgaris), and House Sparrow (Passer domesticus). Malkova and Marhoul (1966) failed to experi- mentally infect pheasants with Tahyna virus, but Chernesky (1968) did succeed in transmitting an untyped California virus to newly hatched white Leghorn chickens by the bite of infected Aedes aegypti (L.). Although published North American studies have not reported seropositive wild birds (Henderson and Coleman, 1971), the numbers of birds tested thus far do not seem to be an adequate sample. Studies in this country regarding the susceptibility of birds to Cal- ifornia Group viruses are as yet insufficient to establish their importance as natural hosts. SUMMARY AND CONCLUSIONS In the summer of 1971 a study was conducted to assess the epidemiological conditions existing in a woodlot - subdivision near Owosso, Michigan, where clinical Cal- ifornia encephalitis in children had occurred in 1970. Three clinical cases of California encephalitis in subdivision children are reported. Two cases occurred in late August, 1970, in male neighborhood playmates, ages 10 and 13. The other, involving a 2-year old girl, occurred in early September, 1971, one mile east of the homes of the 1970 cases. In each infection the patient experienced headache, high fever and seizures, and recovery was uneventful. Epidemiological evidence for each patient suggests that virus transmission occurred in the vicinity of their home. A serological survey of 30 residents of the woodlot - subdivision revealed significant hemagglutination inhibition antibody titers to La Crosse subtype antigen (a 1:20) in 4 individuals (13.3%). These seropositive individuals were children between the ages of 8 and 12 years, all of whom had lived in the subdivision Since 1967 or earlier. One of these children, a lO-year old 60 61 girl whose titer was 1:80, had experienced an encephalitis — like illness in 1967 and can be considered a presumptive case of California encephalitis. Encephalitis-like diseases of unknown origin occurred in 2 Owosso children and l Ovid child at times when con- firmed clinical California encephalitis was present in Owosso children. This suggests the need for further epidemiological investigations in these communities. 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