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1 Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana USA 46556
3 Corresponding author (email: Carina_Blackmore{at}doh.state.fl.us)
ABSTRACT:
To evaluate the importance of eastern cottontails (Sylvilagus floridanus) as amplifying hosts for Cache Valley virus (CVV), we tested hunter-provided blood samples from northern Indiana for specific neutralizing (N) antibodies against this mosquito-borne bunya-virus. Samples were collected during the winter of 1994–95. Two seronegative eastern cottontails, captured in July 1995, were also infected with CVV by subcutaneous inoculation, and two others were infected by allowing CVV-infected mosquitoes to feed on them. The results indicate that eastern cottontails probably are not important amplifying hosts for CVV. The prevalence of N antibodies against CVV was low (6.0%, n=82) among the hunter-killed animals. Low viremia (<1.8 log10 plaque-forming units/ml) of short duration (1–3 days) were seen in three of four experimentally infected eastern cottontails. The viremias were insufficient for infecting Coquillettidia perturbans, a mosquito species commonly found naturally infected with CVV.
Key words: Bunyaviridae, Cache Valley virus, Orthobunyavirus, serology, Sylvilagus floridanus.
Cache Valley virus (CVV), a North American orthobunyavirus (family Bunyaviridae, genus Orthobunyavirus) with a wide geographic distribution, cycles between mosquitoes and mammals in nature (Calisher et al., 1986). The amplifying vertebrate host(s) is/are not known; however, high prevalence of antibodies against CVV have been reported in domestic and wild ungulates (Kokernot et al., 1969b; Hoff et al., 1973; McLean et al., 1987; Crandell et al., 1989). This virus also has been isolated from horses, cattle, and sheep (McConnell et al., 1987; McLean et al., 1987). Because most large mammals are long-lived and populations of various species can maintain high levels of herd immunity, they generally are not considered adequate amplifying hosts for enzootic arboviruses. Experimental CVV infections of cattle, swine, and goats have shown that these animals develop a very low viremia, probably of insufficient magnitude to infect potential vector mosquitoes (Kokernot et al., 1969a).
Little is known about the prevalence of CVV infections in small mammals. Antibodies to the virus have been detected in woodchucks (Marmota monax), raccoons (Procyon lotor), gray foxes (Urocyon cinereoargenteus), and various rodent species (Kokernot et al., 1969b; Buescher et al., 1970). A number of small animal species have been experimentally infected including raccoons and opossums (Didelphis virginiana) (Kokernot et al., 1969b), as well as desert cottontails (Sylvilagus audubonii), jackrabbits (Lepus californicus), Fresno kangaroo rats (Dipodomys nitratoides exilis), California ground squirrels (Spermophilus beecheyi), Nelson’s antelope ground squirrels (Ammospermophilus nelsoni), deer mice (Peromyscus maniculatus), and laboratory white mice (Mus musculus) (Francy, 1972). The opossums’ virus titers were low; however, desert cottontails, Fresno kangaroo rats, and Nelson’s antelope ground squirrels were more susceptible to CVV, with two-to-three-day mean viremia titers ranging from 3–5 log10 plaque-forming units (pfu)/ml.
The last three species, those that developed elevated viremia levels, occur in the western United States (Hall, 1981) and could serve as amplifying hosts for CVV there. However, species of Sylvilagus occur in most parts of the North American continent where CVV has been isolated. In the Midwest the eastern cottontail (Sylvilagus floridanus) is common (Hall, 1981). Two Anopheles spp. (An. quadrimaculatus and An. punctipennis) and Coquillettidia perturbans mosquitoes have been implicated as potentially important Midwestern CVV vectors (Blackmore et al., 1998). Coquillettidia perturbans feed on a variety of birds and mammals, including rabbits (Edman, 1971). While these two Anopheles spp. feed mostly on large mammals, blood meal analyses have revealed that rabbits also are common blood meal hosts (Kokernot et al., 1969b; Robertson et al., 1993). The objective of this study was to determine, through serosurveillance and experimental infections, whether eastern cottontails could serve as vertebrate amplifying hosts of CVV.
Blood samples for the serosurvey were collected from eastern cottontails in five counties of northern Indiana during December 1994 and January 1995. Local hunters were supplied with Nobuto® (Microfiltration Systems, Dublin, CA, USA) filter paper strips. After these were soaked with blood from harvested animals, strips were air-dried, placed in envelopes containing information about the collector and the site of the kill, and mailed to the University of Notre Dame, Notre Dame, Indiana, USA. In the laboratory, they were stored at 4 C until eluted and tested.
Female Cq. perturbans were collected in CO2-baited CDC miniature light traps (Sudia and Chamberlain, 1962) 2.5 km northwest of Edwardsburg, MI (41.5°N, 86.0°W) in July 1995 and 1996. Old female mosquitoes with worn wings or missing scales were excluded from the experiment. The mosquitoes were held at 21 C and 80%R.H. until use.
Eastern cottontails used in the infection experiment were captured in July 1995 (n=2) and June 1996 (n=2) by a licensed, professional trapper in New York State and shipped to the University of Notre Dame. The animals were held individually in 91 cm3x91 cm3x91 cm3 rabbit cages with free access to food and water. A 30 cm3 nesting box was also supplied to each cage. All work was performed following an experimental protocol approved by the University of Notre Dame IACUC.
The CVV strain used in these experiments (strain MI80-1-450 smb-1, Vero-1), obtained from the Centers for Disease Control and Prevention, Fort Collins, Colorado, USA, was isolated from a horse in St. Joseph County, Michigan, USA, in 1980 (McLean et al., 1987).
Mosquitoes were infected orally with 6.1 log10 pfu/ml virus in a 1:10 CVV/defibrinated rabbit blood mixture as described by Grimstad et al. (1989). To increase the likelihood of feeding, the mosquitoes were sugar-starved 24 hr prior to the experiment. Samples of unfed field-collected females were also frozen (–70 C) and assayed as controls. Fully engorged females were held in an insectary at 27 C and 80% R.H. for 21 days. A 16 hr light:8 hr dark photoperiod was used in all experiments.
The two rabbits trapped in 1995 were infected with CVV by mosquito bite (six infected mosquitoes were removed at random from the holding cage; two fed on one rabbit, four were allowed to feed on the other). Engorged mosquitoes were frozen at –70 C until assayed for virus. In 1996, the rabbits were infected subcutaneously with 5.1 log10 median tissue culture infectious dose (TCID50) CVV.
Blood samples were obtained from each eastern cottontail by ear vein puncture for five consecutive days post-infection (PI). The blood was immediately mixed with an equal volume of Medium 199 supplemented with 10% Seru-Max-2, 2.2 g NaHCO3, and 2.5 mg amphotericin B (Sigma Chemical Company, St. Louis, Missouri, USA) and 75 mg gentamycin (J.R.H. Biosciences, Inc., Lenexa, Kansas, USA)/liter (M199) and frozen at –70 C. Blood was collected prior to the mosquito feedings and on day 9 PI to determine whether seroconversion had occurred. These samples were diluted twofold with M199 centrifuged at 3,000 x G for 10 minutes and stored at 4 C until tested.
To establish whether the viremias of the eastern cottontails were sufficiently high to infect vector mosquitoes, field-collected female Cq. perturbans were allowed to feed on the rabbits for 30–60 min on days 1 through 5 PI. Fully engorged mosquitoes were held for 48 hr and then stored at –70 C until assayed to allow time for virus penetration into midgut cells and enzyme digestion of unabsorbed virions (Edman and Spielman, 1989). For blood sampling and subsequent mosquito feeding, the animals were sedated intramuscularly with 0.1–0.3 ml of either a combination of Fentanyl (0.4 mg/ml) and Droperidol (20 mg/ml) (Inovar®, Pitman Moore, Mundelin, Illinois, USA) in 1995 or Acepromazine maleate (10 mg/ml) (Fermenta Animal Health Company, Kansas City, Missouri, USA) in 1996.
Rabbit sera from inoculated and hunter-killed rabbits were assayed for CVV antibodies as described by Blackmore and Grimstad (1998). Mosquitoes, the artificial blood meal and rabbit blood samples were assayed for virus by plaque assay on confluent Vero cell cultures (Blackmore et al. 1998; Blackmore and Grimstad, 1998).
Blood samples were obtained from 82 Sylvilagus floridanus hunted in five northern Indiana counties. Neutralizing antibodies against CVV were detected in 14% (4/28) of the animals from LaGrange County and 14% (1/7) of the eastern cottontails harvested in Jasper County; no seropositive eastern cottontails were found among those collected in Newton (n=44), Porter (n=1), or Noble counties (n=2).
The six mosquitoes utilized for transmitting the virus to eastern cottontails all had disseminated CVV infections. However, despite being fed upon by multiple mosquitoes capable of transmitting CVV, only low levels of viremia were induced in the two rabbits (Table 1
). The viremia was too low to subsequently infect Cq. perturbans mosquitoes fed on the viremic animals (n=50 mosquitoes/animal/day) (Table 1). The subcutaneously inoculated rabbits also developed a low viremia, and the mosquitoes fed on these animals did not become infected either (n=45, 42 mosquitoes/animal/day; Table 1). All eastern cottontails had seroconverted by day 9 PI.
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Financial support for this study was provided by the University of Notre Dame and by an NIH grant (AI 02753) to the late George B. Craig, Jr., and to Paul R. Grimstad.
2 Current address: Florida Department of Health, 4052 Bald Cypress Way Bin A-08 Tallahassee, Florida 32399 
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Received for publication 22 June 2005.
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