Seasonal Activity of Ixodes dammini in Relation to Lyme Disease-Infected White-Footed Mice, Peromyscus leucopus

This study evaluates the seasonal infestation rates of whitefooted mice, Peromyscus leucopus, harboring immature stages of the deer tick, Ixodes dammini, at three sites on Prudence Island, Rhode Island, USA, a Lyme disease endemic area. Larval infestation rates varied from 46.0 larvae per mouse in August to none at all sites during November, while nymphal infestations ranged from 11.0 per mouse in June to none at all three sites in September through November. At all three study sites, the whitefooted mouse appeared to be the most common host species in the wooded areas where larvae and nymphs of I. dammini are also abundant. The seasonal infectivity of white-footed mice with the Lyme disease spirochete, Borrelia burgdorferi, was also monitored using the larval stage of the deer tick. Mice were trapped at four sites: three on Prudence Island, a Lyme disease end~mic area with a large population of white-tailed deer; and one on Conanicut Island (Jamestown) with no white-tailed deer. All mice taken from Prudence Island were infective to laboratory-reared larvae with the highest infectivity from May to August. Larvae that fed on juvenile mice collected in September, October, and November were not infective. Mice originating from Conanicut Island were also not infective. This suggests, at least at these study sites, that the presence of host-seeking I. dammini is a prerequisite to perpetuating spirochetes in white-footed mice. Furthermore, this study demonstrates that mice are infective to larval ticks during the peak larval season which extends from July to late October. It is presumed that these infected larvae would become infected nymphal ticks which emerge the following season. Risk of Lyme disease transmission can be expressed as an Entomological Risk Index (ERi), a term that describes the relative abundance of infected deer tick nymphs per unit time. Variations in ERi were observed among the three sites and it was also found to change seasonally. The peak in ERi coincided with peak density of nymphal I. dammini and corresponded with the reported occurrence of Lyme disease in humans. ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Dr. Kerwin Hyland for his continuous support and guidance throughout this study. My appreciation is also extended to Dr. Robert Bullock for his encouragement and best of all, his friendship. My thanks are extended to the committee members: Dr. Thomas Mather for providing me with larval ticks, anti-Borrelia antibody, and his valuable suggestions, Dr. Clifford Katz for his comments on the statistical treatment of the data, Dr. David Laux for his valuable comments during the course of this study, Dr. Frank Heppner, Dr. Roger LeBrun and the Defense Committee Chairman, Dr. Richard Casagrande, for their editorial comments and critical review of the


LIST OF TABLES
Lyme disease, also known as Lyme borreliosis, is a human affliction caused by the spirochete, Borrelia burgdorferi, and is transmitted by several species of ticks of the genus Ixodes (Burgdorfer et al., 1982;Spielman et al., 1985). This disease has a wide spectrum of pathological effects and may involve multiple organ systems (Duray and Steere, 1986). Early manifestations of Lyme disease may include the appearance of a characteristic circular rash, erythema chronicum mi gr ans (ECM), an erythematus papule or macule that forms within four to 20 days after the initial bite of an infected tick . Such a rash is reported in 50 to 70% of the Lyme disease cases . Histologically, ECM is an infiltration of lymphoplasmacytes around dermal vessels (Duray, 1987).
About half of the Lyme disease patients who developed this rash may show smaller multiple annular secondary lesions at different sites on the skin. If the patient remains untreated, the rash will spontaneously disappear within a month (it may last for one day to 14 months). Malaise, fatigue, fever, chills, sore throat, headache and generalized achiness are some of the other associated early symptoms (Steere et al., 1977a;. A few months after the initial rash, 10 to 15% of patients may experience neurologic manifestations (Stiernstedt et al., 1986). Unilateral blindness caused by Lyme disease has been identified  where spirochetes were recovered from vitreous debris of one patient.
Unilateral or bilateral facial paralysis may occur in up to 11 % of patients , and bilateral keratitis and optic neuropathy are also seen 1 among Lyme disease patients . Patients without the characteristic ECM may develop aseptic meningitis, encephalitis, cranial neuritis, motor and sensory radiculitis and myelitis of various combinations . One acute case of meningoencephalitis accompanied by slurred speech, seizure, hemiparesis and cerpospinal fluid pleocytosis due to Lyme disease was described by .
Moreover, B. burgdorferi organisms were seen in a brain autopsy of an Alzheimer's disease patient .  presented an extensive review on the neurologic manifestations of Lyme disease.
About 60% of Lyme disease patients develop arthritis. The initial signs usually appear within a few weeks to two years after onset of the disease (Steere et al., 1977a;. Disabling arthritis and erosive joint disease were reported by . Cardiac involvement may occur in about 10% of patients, usually two to three months after the appearance of the skin rash. Reversible and fatal myocarditis have been reported . Additionally, mild congestive heart failure and multi-focal damage proximal to the bundle of His and ventricular condition system were described among Lyme disease victims (Steere et al., 1980b). Other clinical observations associated Lyme disease with atrioventricular block, myopericarditis and left ventricular dysfunction . Acute necrotizing splenitis, with extensive necrosis and inflammation of the spleen and a massive number of Borrelia organisms, was observed in a Lyme disease patient by .
Erythema migrans, a migratory annular skin rash following a tick bite by the sheep tick, Ixodes ricinus, was originally described in Sweden by . Subsequently, this syndrome was refereed to as "Erythema Chronicum Migrans". Later, Lipschutz (1913) suggested that this skin illness is caused by bacteria transmitted by ticks. Gelbjerg-  suggested that ECM was an allergic reaction in certain individuals.  reported ECM cases following tick bites and made reference to a positive response of these patients to penicillin treatment. The first evidence that ECM is caused by an infectious agent that responded to penicillin treatment was given by Binder et al. (1955). He transplanted pieces of skin from patients with the rash to healthy individuals, who later developed the characteristic rash.  referred to spirochetal elements in skin rashes obtained from ECM patients, but it was over 30 years later that Burgdorfer et al. (1982) finally confirmed that a spirochete was the etiologic agent for this disease.
The first human case of ECM in the United States was reported from Wisconsin . In 1975, Allen_ Steere and his associates observed "juvenile rheumatoid arthritis" following an outbreak in southwestern Connecticut and it was referred to as "Lyme disease" or "Lyme arthritis" . Although the etiologic agent was not known,    (Burgdorfer et al., 1982). Further studies demonstrated the presence of similar spirochetes in both nymphal and adult stages of the deer tick . Meanwhile in Europe, spirochetes were seen in the sheep tick, I. ricinus .  demonstrated the irrefutable evidence that these spirochetes reacted immunologically with sera obtained from Lyme disease patients and that this malady is caused by tick-borne spirochetes.
Subsequently, the etiologic agent was described and named Borrelia burgdorferi in recognition of its discoverer, Dr. Willy Burgdorfer of the Rocky Mountain Laboratory in Hamilton, Montana . In subsequent years much efforts has been undertaken at several institutions to understand the epidemiology of Lyme disease, including the enzootic interaction of wild animals and other arthropods.
B. Global Distribution of Lyme Disease.
Lyme borreliosis has been reported from many northeastern states including Massachusetts , Connecticut, Rhode Island , New York Williams et al., 1986) and New Jersey Bowen et al., 1984). It has also been found in the West (California, Oregon, Nevada and Utah) and in the Midwest including Minnesota  and Wisconsin  .. Currently, the disease is known from 43 states . It seems that 4 this illness exhibits a geographic progression and new foci are formed in areas where the disease was not previously known  Indigenous cases of Lyme disease have been diagnosed in Canada . In Europe, this illness was reported from Belgium , Ireland , the Soviet Union Deknenko et al., 1988), Czechoslovakia ), Germany (Caflisch et al., 1984Herzer et al., 1986;Munchhoff et al., 1986;, Austria Schumutzhard et al., 1988), Finland , France , Italy , Sweden (Hovmark et al., 1988;Stiemstedt, 1985), Switzerland (Aeschlimann et al., 1986), Hungary  and Denmark (Hansen et al., 1986). In the Far. East, Lyme disease was described from Japan (Kawabata et al., 1987) and China (Ai et al., 1988). Moreover, local human cases were reported from Australia .
C. Some Epidemiological Aspects of Lyme Disease.
The risk of acquiring Lyme disease is_ correlated with the distribution of I. dammini   .
Various approaches have been suggested to quantify the risk of Lyme disease transmission.  described "Encounter distance" to determine the relative risk of Lyme disease transmission. It is defined as 6 the mean number of meters traveled before encountering a nymphal or adult deer tick.  also recommended using carbon dioxide baited tick traps to determine the human ·risk for Lyme disease.  proposed the use of the brush rabbit, Sylvilagus bachmani (Waterhouse), and the black-tailed rabbit, Lepus californicus Gray, as sentinels for Lyme disease surveillance. They showed that 90% of both sentinels revealed a significant titer against B. burgdorferi.
D. Etiologic Agent of Lyme Disease.
Spirochetes are helically-shaped bacterial organisms with a characteristic anatomy and pattern of locomotion. They belong to the order Spirochaetales, which consists of two families comprising five genera. The genera, Borrelia and Treponema, contain spirochetes that are pathogenic to man and animals. Morphologically, they have a multilayered outer envelope that surrounds the protoplasmic cylinder, which consists of a peptidoglycan layer, cytoplasmic membrane and the enclosed cytopla~mic contents. Axial filaments and axial fibrils (periplasmic flagella) occur between the outer envelope and the protoplasmic cylinder .
Evolutionarily, some workers believe that Borrelia developed primarily as a parasite of ticks and that mammals are accidental hosts (Felsenfield, 1971) while  claimed that Borrelia developed as a symbiont of ticks but acts as a parasite of mammals.
The Lyme disease spirochete, Borrelia burgdorferi  was described from an isolate originating from an adult I. dammini tick collected on Shelter Island, New York. This spirochete is helical with a 7 diameter of 0.18 to 0.25 and a length of 2 to 30 µm (Fig. 1). An average of seven periplasmic flagella are positioned at each end of the organism and overlap around the central area of the cell. Borrelia burgdorferi is gram negative, with optimal growth temperature of 340 C to 370 C and a generation time of 11 to 12 hours at 350 C (Barbour, 1984).
Transovarial transmission of B. burgdorferi in J. dammini was studied by  and . Both studies indicated that this phenomenon seems to be insignificant in disease transmission and may be of a limited importance in maintaining B. burgdorferi in nature. Of 2297 larval J. dammini examined for the presence of B. burgdorferi, 44 were positive with an overall rate of 1.9% . Similarly,  detected spirochetes in 0.7% of field collected larvae.
However,  reported transovarial infection of 100% and · 60% of the larval progeny of two Ixodes ricinus females that had fed on B.
burgdorferi-infected white rabbits. Likewise,  showed that one of three field-collected I. pacificus females with spirochetes produced 100% infected progeny that also maintained the spirochetes transstadially. Moreover, they discovered that the longer the interval between engorgement and dissection, the greater the number of ticks demonstrating generalized infection.
Borrelia burgdorferi have been isolated from a number of birds and wild mammals. White-footed mice are the primary animals harboring the spirochete , but it has also been found in meadow voles , the woodland jumping mouse , the eastern chipmunk , raccoons and white-tailed deer . Borrelia burgdorferi was isolated and cultured from three species of birds, a veery, a rose-breasted grosbeak and a yellow-throat .
Several domestic animals are affected by B. burgdorferi infection.
Dogs were found to be at higher risk for infection than humans . In Wisconsin, 53% of 380 dogs examined showed a significant titer for B. burgdorferi  and similar results were obtained in France .  reported an infection rate of 34.7% with the spirochete among dogs from seven municipalities in New Jersey.
They recommended the use of dog serosurveys for epidemiological surveillance for Lyme disease. Clinically, dogs develop arthritis similar to that seen in humans .  suggested an association with encephalitis in a horse heavily infected with B. burgdorferi. Lindenmeyer et al. (1989) indicated significant variation in Lyme disease infection rates among horses in two counties in Massachusetts where Lyme disease is prevalent. Horses and cows are reported to show lameness, swollen joints and arthritis  The genus Ixodes includes about 250 species worldwide, of which 35 are known to occur in North America . Ticks belonging to this genus are characterized by the absence of festoons, and the presence of an anal groove .
Jxodes dammini   The distribution of Ixodes dammini follows that of the white-tailed deer Spielman et al., 1985). Ealier in the century when the deer population was low, this tick species likely had a limited distribution.
However, the enormous increase in white-tailed deer populations in recent years has allowed the tick to expand its range along the coastal regions of the east coast .  observed that larval ticks were abundant on islands inhabited by deer and absent or scarce on islands lacking deer. They also noted a decline in immature ticks infesting mice a year after removal of deer, however questing adults were abundant for four winters after being denied the deer hosts . 1 8  investigated the effect of climatic factors on the spatial distribution of I. dammini in New Jersey. They stated that rainfall and temperature have little impact on the distribution of the deer tick, and they concluded that physiogeographic regions are the key element determining the spatial distribution of the tick. This conclusion was based upon habitat and physiographic similarities of Lyme disease endemic regions throughout the Northeast. Moreover, they questioned the distanceelevation theory  that was proposed to be the major limiting factor for I. dammini distribution Ixodes dammini is a three-host tick that requires two or more years to complete its life cycle (Fig. 4). This tick species takes three blood meals throughout its life cycle, one during each stage. The life cycle commences when the engorged overwintering female deposits its eggs in the spring Spielman et al., 1985). These eggs hatch into the six-legged larval stage, which generally find either a small mammal or bird for its first blood meal. Although larvae are found on mice from May to July, they are particularly abundant during August, and virtually non-existed by November. Spielman (personal communication) suggested that larvae found in May and June are the winter survivors of the previous summer season, and that August larvae hatch from eggs produced by the fall or spring-engorged females.
After feeding to repletion, larvae drop from the host on soil and leaf litter or inside the rodent burrows. They molt into the eight-legged nymphal stage in the spring of the ensuing year. Nymphs will feed to repletion upon a variety of hosts, including humans, in three to four days, after which they detach, dropping to the ground, where they will transform into the adult.
Adults are active during the fall and throughout winter until the following spring. Foxes, raccoons, dogs and humans are acceptable hosts, however, the white-tailed deer is the preferred host for the female tick. Males are not known to feed on blood, but are found mating with feeding females .
The deer tick has a broad range of mammalian and avian hosts Main et al., 1982;; Wiesbrod and . Usually, the immature stages feed on small-medium-and large-sized mammals and ground-feeding birds.
Twenty-nine mammals belonging to seven orders and 54 bird species representing 14 families have been found infested with immature I.
dammini (Tables 1 and 2). However, some species are more commonly utilized than others . Several studies confirmed that the white-footed mouse is the preferred host for I. dammini subadults (Main et al., 1982;. Moreover, eastern chipmunks and gray squirrels were found to harbor a noticeable number of the immature stages (Main et al., 1982).
Adult I. dammini females are known to feed on medium and large sized mammals. They have been taken from opossums, woodchucks, gray squirrels, gray foxes, red foxes, stripped skunks, dogs, cats, black bears, horses, white-tailed deer and humans Main et al., 1982; burgdorferi from Amblyomma specimens collected from Texas. The spirochete has also been cultivated from American dog ticks, Dermacentor variabilis (Say), feeding on white-footed mice .  has reported unidentified spirochetes isolated from D. variabilis from Texas. Moreover, Rhipicephalus sanguineus (Latreille) and Dermacentor parumapertus Neumann, were infected with B. burgdorferi in east Texas where R. sanguineus is common .
Recently,  showed that larval A.
americanum from Alabama are capable of acquiring B. burgdorferi, but they
White-throated Sparrow Zonotrichia albicollis x Anderson&:   x x x .

Microtus brewai
x .  do not maintain it transstadially. They also found that lone star ticks originating from Texas fail to acquire the Lyme disease spirochete.
c. Other Arthropod Vectors for Lyme Disease.
A number of hematophagus arthropods harbor B. burgdorferi.
Spirochetes isolated from the cat flea, Ctenocephalidis felis, react positively with monoclonal antibody against B. burgdorferi .
Additionally, the spirochete has been detected in the flea, Orchopeas leucopus  burgdorferi .
Although the presence of Lyme disease spirochetes has been demonstrated in mosquitoes, it was shown experimentally that the spirochete survived for less than a week after infection. Furthermore, infected mosquitoes fed on spirochete-free hamsters failed to transmit the infectious agent .
D. The Role of the White-footed Mouse.
Although I. dammini has a broad range of hosts (Main et al., 1982;, white-footed mice appear to be the principal hosts of the pre-adult forms .   In Rhode Island,   burgdorferi. Twenty-five serum samples obtained from white-footed mice collected from inland areas in Rhode Island plus Block Island, Conanicut and Prudence Islands revealed an infection rate of 20% when examined using enzyme-linked immunosorbent assay (ELISA) but first 12% when using an indirect fluorescent antibody assay (IFA) . Of seven mice trapped from Prudence Island during November, 1984, six were found to harbor the spirochete .

29
The ability of a host animal to transmit infection effectively to a vector is termed "competence." However, even though an animal demonstrates competence, this term is insufficient to describe the potential for a species or population of hosts to serve as a zootic resource for an infection. The term "reservoir host" implies that the animal contributes in some way to the maintenance or perpetuation of the infection. Thus, white-footed mice are not only tick hosts, but are competent reservoirs for Lyme disease spirochetes . Several other hosts parasitized by immature deer ticks including white-tailed deer , catbirds , and raccoons (Mather, personal communication) appear incompetent to serve as reservoirs.
Many other species of small mammals including chipmunks, shrews and meadow voles may be competent in transmitting spirochetes to ticks, but demonstrate a low reservoir potential relative to white-footed mice . It is because of this unique position in the dynamics of zoonotic Lyme disease spirochete transmission that mice are the focus of the present study.

INTRODUCTION
Lyme disease has become an important public health concern in many parts of the United States (Schmid et al., 1985;.
Moreover, it seems that this illness exhibits a geographic progression and new foci are formed in areas where the disease was not previously known . This malady is caused by the spirochete, Borrelia burgdorferi, which in the Northeast is transmitted by the deer tick, Ixodes dammini . The disease is a complex, multisystem affliction with broad clinical manifestations including arthritis, cardiac and neurologic complications Steere et al., 1980b;.
The risk of acquiring Lyme disease is correlated with the distribution of I. dammini . The larvae are most abundant in July and August, and by October their numbers have declined. The nymph, the second stage, is common from May to late July. The adult stage, including males and females, is active during fall and through the ensuing spring . Most human cases of Lyme disease are attributed to nymphal tick bites. This may be attributed to the small size of this tick, permitting them to go unnoticed for· several days, and their abundance during the early summer season when people favor outdoor oriented activities. Seasonal transmission of Lyme disease has been positively correlated with the seasonal activity of the nymphal stage.  showed that Lyme disease transmission is highest during May and June. Moreover,    ).
Different techniques have been suggested to quantify the risk of Lyme disease transmission. Encounter distance, a term coined by , was used to determine the relative risk of Lyme disease transmission. It is defined as the mean number of meters traveled by a person before encountering a nymphal or adult deer tick.  recommended the use of carbon dioxide baited tick traps to determine population levels for calculating the human risk of acquiring Lyme disease.  proposed the use of the brush rabbit, Sylvilagus bachmani, and the black-tailed rabbit, Lepus californicus, as sentinels for 32 Lyme disease surveillance. They showed that 90% of both sentinels revealed a significant titer against B. burgdorferi.
Recently,  compared four sampling methods (walking, flagging, trapping and mouse collecting) to determine tick abundance. They concluded that flagging rather than walking or C02 traps is the method of choice for sampling I. dammini subadults.
Entomological Risk Index (ERI), is a novel term introduced by Mather Although I. dammini has a broad host range (Main et al., 1982, Anderson and, the white-footed mouse, Peromyscus leucopus, appears to be the principal host_  in areas where the deer tick is abundant (Main et al., 1982).  evaluated the role of the white-footed mouse as a reservoir host for the Lyme disease spirochete. Based upon the relative abundance of this host and parasitism by I. dammini, they concluded that the white-footed mouse is the principal reservoir host for B. burgdorferi. Moreover, they reported the seasonality of subadult infestations and abundance of I. dammini on P.
leucopus from Crane's Beach, Massachusetts. Here, the larval stage of the deer tick acquires the Lyme disease spirochete while feeding on an infected competent host. After repletion, the larva molts into the nymph the 33 following season and seeks a host for its second blood meal .
Peromyscus leucopus is widely distributed throughout the eastern United States, from Missouri to North Carolina and northward to southern Canada . Fluctuations in population density varies from 38 mice/hectare to 6 mice/hectare within two years ). Populations of this species are not strictly regulated by food supply (Krohne et al., 1988), rather by behavioral territoriality of both sexes (Metzgar, 1971). Home range for males is 634 m2 and 511 m2 for females .
It is evident that white-footed mice are competent reservoir hosts for Lyme disease spirochetes . Larval I. dammini ticks recovered from deer inhabiting a Lyme disease endemic area showed an infection rate of 1 % . Non-infected larval ticks which were allowed to feed on field-captured catbirds, did not become infected with B. burgdorferi, while 76% of larvae derived from white-footed mice trapped from the same area did .
Although several studies have examined the association of I.  , few have investigated the seasonal infestation of white-footed mice .
Determining an animal's infectivity to feeding vectors, and not merely its susceptibility to infection, is critical to establishing its reservoir potential. In this context, reservoir potential defines the contribution made by a species or population of animals towards infecting vector populations , while infectivity, or reservoir competency, is a biologic property of a particular animal or species. It is generally agreed that whitefooted mice (P. leucopus) serve as the principal reservoir for the Lyme disease spirochete, B. burgdorferi . As soon as three weeks after inoculation by tick-bite, this animal efficiently infects nearly all host-feeding I. dammini, the tick vector. Furthermore, infectivity in the laboratory may extend, albeit with lower efficiency, for several months . Of course, the reservoir potential of P. leucopus for the Lyme disease spirochete will be greatest where this mouse exhibits a uniformly high degree of infectivity that persists for the tick season, particularly through the peak late summer period of larval tick activity Amr et al., unpublished).
Even though mice are highly susceptible to infection with the Lyme disease spirochete (Donahue et al., 1985), and this infection appears to persist , the infectivity of this animal to ticks may not be uniformly high nor complete. In previous studies, spirochetal infectivity of mice has varied considerably. From one location in Massachusetts, 46.3% of nymphal I. dammini derived from mouse-fed larvae were infected with B. burgdorferi , while at a different location, mice infected 75.8% of all ticks feeding upon them . However, in each of these studies the infectivity of just one population of mice was examined and for just one point in time.
There are several factors that could affect an animal's infectivity. Agerelated, geographic, or even individual variation in susceptibility might 35 influence spirochetal infectivity, as could temporal or seasonal factors.
Furthermore, the level, or number of inoculations or the animal's immune status could also affect infectivity. Thus, before assuming that spirochetal infectivity exhibited by mice is constant, it seemed reasonable to evaluate concurrently the infectivity of several populations at various points in time.
Therefore, we set out to assess both the seasonal and geographic variation in the spirochetal infectivity of white-footed mice.
The present study investigates the infection rate of I. dammini hostseeking nymphs with B. burgdorferi to : (1) determine the seasonal risk pattern of Lyme disease in an endemic area, (2) to examine and compare the seasonal infestation of immature stages of I. dammini, and 3) to determine the seasonal infectivity of white-footed mice to larval deer ticks with B.
burgdorferi, in three sites on Prudence Island, Rhode Island, USA.

MATERIALS AND METHODS
The primary field sampling site selected for this study is Prudence Island, located in Narragansett Bay, Rhode Island, is 8 kilometers long, 0.8 kilometer wide and comprises approximately 1,528 hectares. About onethird of the island consists of several wildlife reserves and sanctuaries. The white-tailed deer, Odocoilis virginianus, is abundant on this island, with a density of approximately 27 deer/square kilometer .
Additionally, mice were trapped on Conanicut Island, also situated in the Narragansett Bay, where there is no breeding population of white-tailed deer.
Three areas on Prudence Island ( To study the infestation rates of mice with immature deer ticks and their infectivity to larval ticks, mice from the three sampling areas were livetrapped using large folding Sherman box traps (23.2 X 9.0 X 7.7 cm) baited with oatmeal. Sampling was undertaken in similar habitats where nymphal tick abundance was determined prior to trapping. In the laboratory, captured mice were placed individually in wire mesh cages and held over water.
Immature ticks (Fig. 6) were collected daily for five days as they detached from their hosts. Collected ticks were washed with distilled water, counted, placed in 24-ml glass vials and kept in a humid chamber, and allowed to molt naturally. Molted and dead ticks were sorted by stage, identified and counted.
After the collection of naturally-infesting ticks from field-captured mice was complete, larval I. dammini originating from laboratory reared cohorts previously determined to be spirochete-free, were allowed to feed on these mice. Mice were placed individually in wire mesh cages and held over water (Fig. 7). Replete immature ticks from the mice were collected daily for at least five days as they detached from their hosts. Ticks were washed with distilled water, counted, placed in glass vials in a humid chamber, and allowed to molt naturally.    However, larval infestation rates ranged from as low as 5.77 + 2.7 (SE) in May to a high of 46.0 + 5.00 (SE) in August (Fig. 9). Larval and nymphal tick infestation rates among mice collected from Prudence Park also changed significantly during the sampling period were significant, P < 0.0002 and < 0.0001, respectively (Appendix 5).
c: .c .c A total of 35 mice was examined from South Prudence Park. All mice collected South PrudencePark (n = 35) from May through August were infective to larval ticks. Infectivity was calculated as the number of infected nymphs derived from a mouse divided by the total number of examined nymphs taken from that mouse.These mice infected 90.6, 100, 80 and 79 percent of the xenodiagnostic ticks, respectively (Fig. 11). Three of four, five of nine and one of four mice collected from September through November, respectively, infected ticks (  (Figure 11). All the mice examined from May through August were infective to larval ticks while 66.7%, 60.0% and 66.7% yielded infected larvae (Appendix 9). Larval I.
dammini infectivity after feeding on mice taken from this site (Appendix 11) during the different months was significant (P = 0.0081).
Mice trapped at North Prudence showed infectivity rates of 87.2% in May, 70.5% in June, 68.8% in July, 83.3% in August, 25.1% in September, 27.7% in October and 30.2% in November (Fig. 11). All mice trapped from May throughout August were infective, while 33.3%, 40% and 50% of those trapped in September through November, respectively, were capable of infecting larvae (Table 6, Appendix 10). Infectivity of mice to larval ticks throughout the study period (Appendix 11) was significant (P = 0.0194).
A total of 18 mice collected from May through August on Conanicut Island was also examined. Two mice were infested with I. dammini larvae in June (1 larva) and August (1 larva). Otherwise, these mice were either infested by the American dog tick, Dermacentor variabilis, or were free of any tick infestation. All laboratory fed I. dammini larvae derived from these mice were spirochete-free (Table 6).
Juvenile mice trapped at all three Prudence Island sites from September to November were not infective to larval I. dammini, although juveniles taken during July and August from the same sites were infective but to varying degrees (83.3%·in August at North Prudence, 40% and 58.8% during July at Prudence Park and 63.1 and 78.5% in July at South Prudence).
Despite this, the overall infectivity of mice trapped from all sites on Prudence Island during the entire study period did not differ significantly (P = 0.5235).

DISCUSSION
Human infection is caused primarily by the bite of a B. burgdorferiinfected nymphal deer tick . However, female ticks may also transmit spirochetes during the fall and winter or whenever they are active . Larval I.
dammini play no major role in the epidemiology of this disease since transovarial transmission is very low . Thus, determining nymphal tick abundance is an appropriate and practical measure for describing the risk for B. burgdorferi transmission to human populations during late spring and mid-summer.
The role of other tick species in B. burgdorferi transmission is controversial. Borrelia burgdorferi has been detected in or isolated from the lone star tick, Amblyomma americanum. In New Jersey, Schulze et al.
(1984a) isolated B. burgdorferi from specimens recovered from patients who developed characteristic Lyme disease ECM. Moreover, they found 9.1 % of field collected nymphal and adult A. americanum carrying the spirochete.  detected the spirochete in 3.5% of 173 lone star ticks taken from North Carolina. Also, Rawlings (1986) isolated B. burgdorferi from A. americanum specimens collected in. Texas.
Additionally, the spirochete was identified from the pre-adult American dog tick, Dermacentor variabilis, feeding on white-footed mice .  reported on unidentified spirochetes isolated from D. variabilis in Texas. Moreover, Rhipicephalus sanguineus and Dermacentor parumapertus were found 64 infected with B. burgdorferi , in eastern Texas where R.
sanguineus is common.
Recently,  showed that larval A.
americanum from Alabama were capable of acquiring the spirochete, but that the ticks did not maintain B. burgdorferi and pass it transstadially.
Likewise, ticks originating from Texas failed to acquire the Lyme disease spirochete. In a separate study, a total of 179 nymphal A. americanum collected from Prudence Island were examined for the presence of B.
burgdorferi. All nymphs were spirochete-free (Hyland, personal communication Additionally, deer fecal pellets were more frequent at South Prudence and Prudence Park compared to North Prudence Park.  showed that observation of "pellet groups" provides an index to the relative density of the white-tailed deer which could be correlated with the abundance of larval but not to nymphal ticks. populations has been observed by several workers . This is perhaps due to the availability and abundance of infected competent reservoir hosts.
The Entomological Risk Index described and used herein can provide health officials and other concerned authorities with information to base control or treatment strategies in particular areas. By itself, ERI gives a numerical value for the possibility of being bitten by spirochete-carrying nymphal I. dammini. Other measures of risk, namely encounter distance, as proposed by  fail to include the proportion of Borrelia infected ticks, providing just a numerical value for the tick density per unit area. This latter measure would also need to define the type of habitat being sampled. An "encounter distance" obtained in grassy habitat might not accurately reflect risk in other types of habitat at the same site.
From January to September 1989, a total of 291 Lyme disease cases were reported to the Rhode Island Department of Health (Dr. E. Jost, personal communication). The higher frequencies (Fig. 12) of diagnosed cases were seen during May (49), June (92) and July (67). The onset of the clinical manifestation of Lyme disease corresponds with the ERI. Winter cases may be attributed to bites by adult females . Transmission of Lyme disease is highest during May to July, coinciding with the peak season for nymphal 1. dammini, and the number of reported human cases. Outdoor activities should be undertaken with care during these months.   reduce the number of spirochete-infected nymphs in the following season . Efforts in educating the public to avoid high risk areas should be undertaken by posting warning signs.
Previous studies have shown that Prudence Island is a focus for Lyme disease . However, these investigations were shorttermed and were not conducted on a regular basis.  reported an average of six (SD +i.2) larvae of I. dammini burgdorferi antibodies. Of seven mice trapped on the island during November, 1984, six were found to harbor the spirochetes .   when utilizing IFA .
Differences in infestation rates among mice within the same site are perhaps due to the clustering behavior exhibited by I. dammini larvae. After feeding on deer or other medium or large-sized animals, the replete female deer tick drops to the forest floor and oviposits. Hatched larvae tend to 70 cluster near the site of oviposition. While foraging at night, white-footed mice may encounter such clusters, resulting in a higher infestation rate than those mice which did not encounter such clusters. Mice trapped from South Prudence Park were heavily infested with larval deer ticks (32.33, 37.80 and 26.00 during July, August and September), while mice taken from Prudence Park were less infested for the months of July (8.62) and September (30.50).
Mice collected from North Prudence had the least number of larvae in July and September.
Tick abundance on P. leucopus is perhaps related to mouse densities and home range. Indeed, home range may be an important factor in determining mouse infestation rates with immature I. dammini, especially with the larvae. White-footed mice have a home range of 634 m2 and 511 m2 for males and females, respectively ). Such home range may explain variations in the number of immature ticks depending on their spatial distribution in a particular location. Moreover, fluctuations in P. leucopus population density vary from one season and one year to another.
According to Metzgar (1971) these fluctuations can be attributed to behavioral territoriality of both sexes of this mouse species.
Additionally, mouse infestation rates are related to the density of white-tailed deer.  stated that "pellet group" provides an index to the relative density of the white-tailed deer in relation to larval but not for the nymphal tick abundance. More deer fecal pellets were observed at the South Prudence site compared to North Prudence Park. No attempts were made to quantify these observations. 7 1  suggested that differences in the number of ticks per mouse may be related to trapping procedures and that tick abundance varies yearly. Larval densities on white-footed mice trapped from the three study sites on Prudence Island were higher than those reported from mice examined in Connecticut (Main et al., 1982;, and Wisconsin . Averages higher than 12 larvae per mouse were recorded from Nantucket Island, Massachusetts  and Shelter Island, New York .
Owing to the high number of larvae attached and feeding on mice, it seems that P. leucopus is tolerant to the bites of I. dammini larvae.
Immature deer ticks can feed repeatedly on this rodent species . The low number of American dog ticks parasitizing mice on Prudence Island does not imply that this rodent species may have become immune or refractory to D. variabilis.
Although ,  and  proposed that larval J. darnmini develop in a one season cohort, the sharp increase· in larvae parasitizing P. leucopus during August and September (Fig. 5) suggests that those larvae hatched during the summer months. May and June larvae, however, are from the previous summer and represent those which survived the winter. Larval infestation rates increased several fold during July through September among mice trapped from the three sites. This is in agreement with the bimodal pattern suggested earlier by Main et al. (1982) and Spielman (personal communication).

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Although gray squirrels and white-tailed deer were found to harbor a significantly high number of immature deer ticks, they are less abundant than white-footed mice in wooded areas. Main et al. (1982) reported an average of 13.8 larval and 27.3 nymphal I. dammini per squirrel in Connecticut. White-tailed deer were infested with an average of 342 larvae.
The contribution of white-footed mice to the seasonal activity of I. dammini seems to be significant. In the study sites, this rodent species appears to be the most common species in wooded areas where these stages are abundant.
This study demonstrates the contribution of P. leucopus in maintaining immature tick populations, where one mouse may host as many as 14.7 larvae per day from May to late October and 3.6 nymphs from May to August. Replete immature deer ticks detach from their hosts during the time of the day when the mice are in their burrows ). This finding is important in directing tick and Lyme disease control strategies as proposed by .
Infectivity of white-footed mice captured from an endemic area to larval l. dammini can reveal the importance of the enzoonotic interaction between these two species in the epidemiology of Lyme disease in the Northeast. In the wooded habitat of all three Prudence Island study sites, P.
leucopus was the only rodent species encountered. One specimen of the meadow vole, Microtus pennsylvanicus, was trapped from North Prudence.
White-footed mice are efficient reservoir hosts capable of infecting the immature stages of the deer tick.   xenodiagnosis. An interesting further study would measure the level of host-seeking nymphal ticks necessary to result in infectious reservoir mice.
White-footed mice remained infective for the entire period of larval activity. Mice trapped during May and early summer were more infective to larval ticks possibly due to a higher spirochetemia caused by repeated exposure to host-seeking nymphal deer ticks that are in abundance during these months. This finding has epizootiological importance because larval ticks that feed on mice during the summer will emerge as nymphal ticks the following season, yielding a high proportion of spirochete infected nymphs that are capable of transmitting B. burgdorferi. Whether all infected ticks can retain the infection over the winter period should be investigated.
Moreover, these findings are important in implementing plans for Lyme disease control strategies._ Using cotton balls impregnated with insecticides  that are utilized in nest building by the white-footed mouse could certainly result in mortality of engorged larval ticks that have perhaps detached and dropped in the mouse nest . Despite the variation in the number of host-seeking nymphs among the three study sites, differences in infectivity of mice taken from these locations to ticks was not significant.  exposed whitefooted mice to various number of infected infected nymphs and found that a bite from one infected nymphal tick is sufficient to cause an infection in these mice and consequently these mice were infective to larval tick.

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Ticks derived from white-footed mice were heavily infected with B.
burgdorferi. This is perhaps attributed to the fresh infection of these ticks, which were maintained under laboratory conditions that allowed the spirochetes to multiply more readily rather than under field conditions. This observation is based on spirochetal infection sought among hostseeking nymphs flagged from the study sites, where the density of spirochetes per nymph was not as high as those seen in larval ticks that were collected from mice.
Juvenile mice trapped from September through November were not infective to larval ticks due to the absence of host-seeking nymphs during this period. The breeding cycle of P. leucopus extends from April to mid-November, with peaks in April, June, September and October .
Additionally, juveniles may acquire immunity from the female during gestation. Further studies are needed to explore this aspect of transplacental immunity. On the other hand, finding infected females during late summer and early fall suggests that spirochetes are not transmitted from mice females to their offspring. Spielman et al. (1985) suggested that the Lyme disease spirochetes overwinter in nymphal I. dammini. However, it is possible, that B. burgdorferi can survive the winter in P. leucopus.  isolated the spirochete year-round from white-footed mice. Other mammals, such as chipmunks, were found to infect larval J. dammini, but not as efficiently as white-footed mice . Thus, these animals would likely serve as less efficient overwintering hosts. the principal vector for B. burgdorferi transmission among mice and these mice, in turn, perpetuate the infection by infecting feeding vectors. None of the mice trapped from Conanicut Island were infested with nymphal deer ticks at the time of capture and none infected larvae feeding in a xenodiagnosis. This site was flagged during the summer on several occasions with no success in recovering host-seeking nymphs, In addition,  obtained negative titers against B. burgdorferi for mice collected from this island. On the other hand, mice originating from Prudence Island were infective to ticks from May to November. The presence of host-seeking and host-attached nymphal I. dammini appears to be prerequisite for spirochetal infectivity among white-footed mice.
Susceptibility of vertebrate hosts to B. burgdorferi infection varies.  showed that rabbits became infected with the spirochete, however, this host lost its infectivity to ticks within two weeks postinfection. Hamsters, on the other hand, retained their infection and were able to infect I. dammini ticks for six months. Furthermore, infected whitefooted mice remained infective to larval ticks for at least nine weeks after the initial infection . The hispid cotton rat, Sigmodon hispidus, maintained the spirochetal infection for a maximum of four weeks . On the other hand, other species of hosts although infected, appear incapable of infecting ticks. White-tailed deer were heavily infested with all stages of I. dammini, less than 1 % of detached larvae were infected with B. burgdorferi , suggesting that this host is incompetent to serve as reservoir. Likewise,  76 demonstrated that catbirds were not incompetent reservoir hosts for Lyme disease spirochete. Further studies will be necessary to document the reservoir potential of other tick hosts.
ERI is useful to estimate the infected tick level in parks, recreational and high use areas as well as private properties in endemic regions. Leisure activities, including hiking, camping, hunting, and field-related occupations (military trainees, forestry rangers, naturalists, landscapers and wood cutters) result in high exposure to infected nymphs in woodlands. Prudence Island is considered a popular resort area for summer vacationers, and is a popular hunting ground for deer and game birds. Some of these activities are carried out during early summer, the time of the year when host-seeking nymphs are abundant. Hunting occurs in the Fall when the adults are active. Subsequent, control measures may be suggested and implemented to reduce the risk of transmission. Further studies should address the pattern of exposure and the incidence of Lyme disease infection among these groups.