VECTOR-BORNE AND ZOONOTIC DISEASES Volume 14, Number 1, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2012.1172
Detection of Eastern Equine Encephalitis Virus Antibodies in Moose (Alces americana), Maine, 2010 Charles Lubelczyk,1 Susan P. Elias,1 Lee Kantar,2 Jennifer Albert,3 Stephen Hansen,3 Kali Saxton-Shaw,4 Katharine MacMillan,4 Leticia B. Smith,1 Rebecca Eisen,4 Bethany Swope,4 Robert Pease Smith,1 and John-Paul Mutebi 4
Abstract
Moose sera were collected from harvested animals during the 2010 hunting season in Maine. Of the 145 serum samples screened by plaque reduction neutralization test (PRNT), 16 (11%) had antibodies to eastern equine encephalitis virus (EEEV). Positive samples were collected from Aroostook County (n = 13), Somerset County (n = 2), and Piscataquis County (n = 1) in northern and central Maine. Preliminary mosquito surveillance revealed the presence of enzootic and bridge vectors mosquitoes, including Culiseta (Climacura) melanura (Coquillett), Aedes (Aedimorphus) vexans (Meigen), and Coquillettidia (Coquillettidia) perturbans (Walker). Select mosquito species were tested by RT-PCR for the presence of EEEV. None were positive. This is the first report of EEEV in moose from Maine. Key Words: Eastern equine encephalitis virus—Moose—Alces Americana—Maine—Mosquitos.
Following an outbreak of the virus in Quebec, Canada, involving horses and emus in 2008 (Chenier et al. 2010), a similar outbreak occurred in Maine the following year. EEEVpositive horses, pheasants, and mosquitoes were reported after surveillance was increased to monitor for virus activity. No human cases were reported during this period despite an unprecedented number of equine fatalities across a wide region of the state (Gibney et al. 2011). Although EEEV infection in humans is rare, it is associated with severe neurological disease and has a mortality rate of up to 33% (Morris 1988). Seasonal mosquito surveillance programs exist in many locations in southern Maine (Holman et al. 2006), but these efforts are very focal with a distribution ranging from York County in southwestern Maine to southern Penobscot County in the interior of the state. Deer have also been used to increase the geographic range of EEEV surveillance in Maine. About 7.1% of deer harvested during the 2009 fall hunting season had antibodies showing exposure to EEEV (Mutebi et al. 2011). Using deer as sentinels for EEEV activity works well in the southern part of the state where densities are in excess of 15 deer/km2 (Rand et al. 2003). In northern and eastern Maine, however, deer densities are well below 5 deer/km2 (Lavigne 1997).
Introduction
E
astern equine encephalitis virus (EEEV) is an arbovirus in the family Togavaridae, genus Alphavirus, whose typical enzootic cycle involves the bite of ornithophilic mosquitoes on birds (Howard and Wallis 1974). Typical enzootic vectors include members of the genera Culiseta and Culex (Howard and Wallis 1974) with bridge vectors including several mammal-biting mosquitoes of the genera Aedes and Coquillettidia (Davis 1940, Chamberlain et al. 1951). Most mammals affected by EEEV are ruminants or cervids (McLean et al. 1985). Horses and llamas, if unvaccinated, may quickly succumb to the disease, whereas other hooved mammals, such as white-tailed deer, may have less severe neurologic symptoms or can be completely asymptomatic after exposure to the virus (Bigler et al. 1975, Tate et al. 2005). Geographically, EEEV is considered a New World arbovirus, with a range extending from southern Canada through the Caribbean to southern Argentina, with foci appearing regularly in coastal regions in the southeastern United States and New England (Morris 1988, Armstrong et al. 2008). Infrequent outbreaks have also been reported from interior portions of North America as well (McLean et al. 1985, Nasci et al. 1993). 1
Maine Medical Center Research Institute, Vector-borne Disease Laboratory, South Portland, Maine. Maine Department of Inland Fisheries and Wildlife, Bangor, Maine. 3 Biology Department, University of Maine, Fort Kent, Maine. 4 Centers for Disease Control and Prevention, Fort Collins, Colorado. 2
77
78 In some of these regions, particularly northern portions of Aroostook, Piscataquis, and Somerset counties, moose (Alces americana L.) have established stable and healthy populations (Wattles and DeStefano 2011). Because EEEV reportedly affects cervids (McLean et al. 1985), it is possible that moose might also be exposed to EEEV. Accordingly, we undertook an expansion of sampling for EEEV at moose hunter registration stations throughout Maine. This effort immediately followed the first season of a pilot program incorporating sites in Aroostook County as part of the state’s seasonal mosquito surveillance following an absence of sampling in the area (Lathrop 1939, Bean 1946). This report details the first detection of EEEV antibodies in A. americana in the northern Maine. It also describes the presence of both potential enzootic and bridge vector mosquito species records collected as part of routine arbovirus surveillance in northern Maine. Methods and Materials Hunter registration station Blood samples were collected from A. americana carcasses at seven hunter registration stations across northern Maine (Ashland, Fort Kent, Houlton, Medway, and Presque Isle in Aroostook County; Greenville in Piscataquis County; and Jackman in Somerset County). These stations were chosen because large numbers of moose had consistently been registered at each in previous years. Sampling began on September 21, 2010, and continued through mid-November, 2010. Whole blood was collected either directly from the heart or from blood pooled in body cavities of the disemboweled carcasses by either sterile syringes and/or sterile pipets and dispensed into vacutainer tubes. Vacutainer tubes were kept on ice in Styrofoam chests and transported to the lab at the end of each day. In the lab, the vacutainer tubes were centrifuged at 3400 rpm for 15 min to separate serum from the blood clot and stored frozen at - 80C. Moose tag numbers were used as ID numbers, and moose approximate ages were estimated using teeth wear and number of antlers for males and body size in females. Serologic tests Moose serum samples diluted 1:10 were screened for EEEVneutralizing antibodies by plaque-reduction neutralization assay (PRNT) (Beaty et al. 1995). Positive specimens were retested and titrated in duplicate for confirmation. Serum samples were considered positive for EEEV antibodies if they neutralized 80% of a challenge dose of & 100 plaque-forming units of EEEV–Sindbis chimeric virus ( Johnson et al. 2011). Mosquito surveillance Beginning in 2010, seasonal mosquito surveys were started from July through September in three communities in Aroostook County (Fort Kent [4715¢31.22†N/6835¢22.08†W], Frenchville [4719¢30.6†N/6829¢49.38†W], and Madawaska [4721¢19.13†N/6819¢18.13†W]). Investigators placed CDC Miniature Light traps (Model #512, J.W. Hock Co. Gainesville, FL) baited with CO2 at wetland sites once per week. Traps were hung at approximately 15:00 hours and retrieved the following morning by 09:00 hours. Female mosquitoes were anesthetized using cold temperatures before identification and then identified on a cold surface with a binocular dis-
LUBELCZYK ET AL. secting microscope. Standard keys (Andreadis et al. 2005, Darsie and Ward 2005) were used to identify specimens. Mosquitoes were then pooled by site and species. From July 1 to July 31, all Culiseta and Culex mosquitoes were submitted to the Maine Health and Environmental Testing Lab (HETL) as part of Maine’s arbovirus surveillance. Aedes spp. and Cq. pertubans were included for testing from August onward. Mosquitoes were screened for EEEV using forward primer 1858 and reverse primer 1926 (Lambert et al. 2003). Statistical analysis Chi-squared tests were used to determine differences in proportions of EEEV antibody-positive moose by county (Aroostook versus other counties), sex, age, and month, accepting differences as significant at p £ 0.05. Collection sites identified by the hunters and marked on a DeLorme Maine Atlas were geocoded using ArcGIS v.10 (ArcGIS, ESRI Redlands, CA). Serological testing results were included as attributes of the moose data and analyzed using the Moran I statistic to determine whether the distribution of EEEV antibody-positive samples was random, dispersed, or clustered. The samples were considered randomly distributed at P > 0.05. Results Hunter registration stations We examined 195 moose from three Maine counties, including northernmost Aroostook (174) and two adjacent counties to the south, Piscataquis (2) and Somerset (19). Of the animals examined, sera were collected from 145. From these, 16 (11%) tested positive for EEEV antibodies by PRNT (Table 1). The proportions of EEEV antibody-positive serum samples in Aroostook (10.4% [13/125]) did not differ from the two counties to the south (15% [3/20]), v2 = 0.37, p = 0.54. Nor did the proportions of positive samples differ significantly between females (4.8% [7/75]) and males (6.2% [9/70], v2 = 0.46, p = 0.50). No positive samples were reported among juveniles ( < 2 years), and the proportions of positive samples did not differ by month (September 7.1% [1/14], October 16.1% [5/ 31], and November 10.0% [10/100], v2 = 1.14, p = 0.56). At the minor civil division (MCD) level, positive moose were found across a wide area of northern Maine (Fig. 1), indicating EEEV activity occurring in the state north to the Canadian border. There were large areas where collections were made that resulted in no positive samples, especially in Aroostook County (Fig.1), suggesting focal distribution of enzootic EEEV activity in northern Maine. Table 1. Number of Moose Serum Samples Collected, Number and Percentage of EEEV-Antibody-Positive Sera from Different Counties in Maine, 2010
County Aroostook Somerset Piscataquis Total
Moose serum samples screened
No. EEEVantibodypositive sera
% EEEV positive (95% CI)
125 18 2 145
13 2 1 16
10.4 (5.0, 15.7) 11.1 (7.4, 25.6 50.0 (50.0, 100.0)
EEEV, eastern equine encephalitis virus; CI, confidence interval.
EEEV IN MOOSE
FIG. 1.
79
Location of geocoded moose samples collected during 2010 in three northern Maine counties.
Of the 195 moose samples collected, only 136 locations could be accurately identified and geocoded; the remainder lacked specific map coordinates. Samples without a specific location of kill were excluded from the spatial analysis. Analysis of the spatial distribution of PRNT results collected from moose revealed a random distribution (Moran I = - 0.0099, p value = 0.9109). Mosquito surveillance CO2/light traps were hung for 26 trap nights, beginning on June 30 and ending on September 22. A total of 722 female
mosquitoes were collected and identified, with the majority of collections composed of Aedes (Aedimorphus) vexans (Meigen) (n = 343), Aedes (Ochlerotatus) canadensis (Theobald) (n = 54), Anopheles (Anopheles) punctipennis (Say) (n = 111), and Coquillettidia (Coquillettidia) perturbans (Walker) (n = 104). Known enzootic vectors of EEEV collected included Culiseta melanura (n = 1), Culiseta (Climacura) morsitans (Theobald) (n = 8), Culiseta (Culicella) minnesotae Barr (n = 1), and members of the Culex pipiens/restuans complex (n = 10). For analysis, we pooled the lone sample date in June with samples in July, and the one September collection with collections from August. No seasonal differences were noted except a decline in Cq.
80 perturbans captures of 7.8 to 1.1 per trap night (trap nights = 12 for June/July, 14 for August/September). Across species, no other seasonal differences were noted. No mosquitoes tested positive by RT-PCR for either West Nile virus or EEEV. Discussion Whereas previous studies have examined exposure of other cervids, primarily white-tailed deer, to EEEV (Mutebi et al. 2011), this study demonstrates that moose are also exposed to EEEV in Maine and establishes that potential enzootic and bridge vector mosquitoes are present in northern Maine to facilitate a zoonotic cycle of the disease. No mosquitoes collected during this study tested positive for EEEV, but the collection of Cs. melanura in northern Maine expands the known distribution of this species in the state (Darsie and Ward 2005). Culiseta mosquitoes are regarded as essential for the emergence and establishment of this virus, Cs. melanura in particular, because of its capacity to maintain high level of virus titers (Armstrong and Andreadis 2010). In addition, Cs. melanura are also regarded as a possible bridge vector, because they will occasionally take a blood meal from humans (Armstrong and Andreadis 2010). We recognize that a small number of specimens were collected during this survey so, given that there is a low prevalence of virus in endemic regions, it is not surprising that none of the mosquitoes were infected with EEEV. Two ubiquitous mammal-biting mosquitoes were found (Ae. vexans and Cq. perturbans) throughout the summer in northern Maine, inhabiting opportunistic wetlands and cattail marshes (Moore 1993). Both species are bridge vectors for the virus (Armstrong and Andreadis 2010) and frequently feed upon white-tailed deer (Molaei and Andreadis 2006, Molaei et al. 2008), but little data exist on these species’ preference for moose. The small sample numbers of mosquitoes collected in this study were insufficient to examine seasonality for species, but future efforts will attempt to expand geographic sampling and to study seasonal patterns. It is interesting though to note that the presence of EEEV antibodies in moose in the three counties examined occurred in a year with limited arboviral activity reported in northern New England (Centers for Disease Control and Prevention 2011). This stands in contrast to 2009, when 16 livestock animals and three pheasant flocks succumbed to the virus in Maine (Gibney et al. 2011). Given that EEEV was documented in horses approximately 160 km (100 miles) northwest of some collection sites (Chenier et al. 2010), it was not surprising that seropositive animals are present. Although persistence of antibodies to EEEV has been documented in reptiles and birds (Hayes et al. 1964, Calisher et al. 1986), there is little information of its persistence in cervids. Boadella et al. (2012) reported possible persistence of flavivirus antibodies well over 1 year in European ungulates, but persistence of EEEV (an alphavirus) antibodies in moose and white-tailed deer remains uncertain. One possibility is that the adult cohort of moose, with higher antibody prevalence, might have been exposed to EEEV prior to 2010. While there is conflicting evidence that EEEV might impact white-tailed deer (Tate 2005, Schmitt 2007), there has been no indication, at present, that the virus debilitates moose the way it does equines and South American camelids (llamas and alpacas) (Moore 1993). The apparent widespread distribution
LUBELCZYK ET AL. of the virus, however, is a cause for concern and emphasizes the need for expanded surveillance in the state. Furthermore the nonclustered distribution of positive moose indicates that the virus is well established in Maine. The appearance of this virus can be highly localized (Nasci et al. 1993). It remains to be determined if, through continued collections of geocoded samples, we will see sites of reliable foci appear. Because moose are the dominant ungulate in northern forest systems in eastern North America, an understanding of the role that moose play in the ecology of EEEV is important in understanding how useful this species can be as part of a comprehensive wildlife surveillance program. To date, this is the first inclusion of this species as part of a comprehensive program for arbovirus surveillance in the northeastern Unites States. In addition, serum and tissues from hunter-harvested animals can provide public health professionals a source of samples that would be difficult to obtain through other survey methods (live-capture or trapping methods). Author Disclosure Statement No competing financial interests exist. References Andreadis, TG, Thomas, MC, Shepard, JJ. Identification guide to the mosquitoes of Connecticut. CAES Bulletin 966. New Haven, CT: 2005;173. Armstrong, PM, Andreadis, TG. Eastern equine encephalitis virus in mosquitoes and their role as bridge vectors. Emerg Inf Dis 2010; 16:1869–1874. Armstrong, PM, Andreadis, TG, Anderson, JF, Stull, J, et al. Tracking Eastern equine encephalitis virus perpetuation in the Northeastern United States by phylogenetic analysis. Am J Trop Med Hyg 2008; 79:291–296. Bean, JL. A preliminary list of the mosquitoe of Maine (Culicidae Diptera. Can. Entomol. 1946; 78:25–28. Beaty, BJ, Calisher, CH, Shope, RE. (1995) Arboviruses. In: Lennette, EH, Lennette, DA, Lennette, ET, eds. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infection, 7th ed. Washington DC: American Public Health Association, 1995:204–205. Bigler, WJ, Lassing, EB, Lewis, AL, Hoff, GL. Arbovirus surveillance in Florida: Wild vertebrate studies 1965–1974. J Wildlife Dis 1975; 11:348–356. Boadella M, Dı´ez-Delgado I, Gutie´rrez-Guzma´n AV, Ho¨fle U, et al. Do wild ungulates allow improved monitoring of flavivirus circulation in Spain? Vector Borne Zoonotic Dis 2012; 12:490–495. Calisher, CH, Berardi, VP, Muth, DJ, Buff, EE. Specificity of immunoglobulin M and G antibody response in humans infected with eastern and western equine encephalitis viruses: application to rapid serodiagnosis. J Clin Microbiol 1986; 23: 369–372. Centers for Disease Control and Prevention [CDC]. Summary of Notifiable Diseases—United States, 2010. MMWR. 2012; 59: 1–111. Chamberlain, RW, Rubin, H, Kissling, RE. Recovery of virus of Eastern equine encephalomyelitis from a mosquito, Culiseta melanura (Coquillett). Proc Soc Exp Biol Med 1951; 77:396–397. Chenier, SG, Cote, J, Vanderstock, S, Maciera, A, et al. An eastern equine encephalomyelitis (EEE) outbreak in Quebec in the fall of 2008. Can Vet J 2010; 51:1011–1015. Darsie, RF, Jr., Ward, RA. Identification and Geographical Distribution of Mosquitoes of North America, North of Mexico. Gainesville, FL: University Press of Florida, 2005:380.
EEEV IN MOOSE Davis, WA. A study of birds and mosquitoes as hosts for the virus of eastern equine encephalomyelitis. Am J Hyg 1940; 32:45–59. Gibney, KB, Robinson, S, Mutebi, J-P, Hoenig, DE, et al. Eastern equine encephalitis: An emerging arboviral disease threat, Maine, 2009. Vector Borne Zoonotic Dis 2011; 11:637–639. Holman, MS, Darsi, RF, Foss, KA. A checklist of the mosquitoes of Maine with new state records. J Am Mosquito Control Assoc 2006; 22:327–329. Howard, JJ, Wallis, RC. Infection and transmission of eastern equine encephalomyelitis virus with colonized Culiseta melanura (Coqillett). Am J Trop Med Hyg 1974; 23:522–525. Johnson, BW, Kosoy, O, Wang, E, Delorey, M, et al. Use of Sindbis/Eastern Equine Encephalitis chimeric viruses in plaque reduction neutralization tests for arboviral disease diagnostics. Clin Vaccine Immunol 2011; 18:1486–1491. Lathrop, FH. Mosquitoes collected in Maine. ME Agr Expt Sta Bull 1939; 397:828–829. Lambert AJ, Martin DA, Lanciotti RS. Detection of North American Eastern and Western equine encephalitis viruses by nucleic acid amplification assays. J Clin Microbiol 2003; 41:379–385. Lavigne, GL. 1997. White-tailed deer assessment and strategic plan. Augusta, ME: Maine Department of Inland Fisheries and Wildlife, 111 pp. McLean, RG, Frier, G, Parham, GL, Francy, DB, et al. Investigations of the vertebrate hosts of eastern equine encephalitis during an epizootic in Michigan, 1980. Am J Trop Med Hyg 1985; 34:1190–1202. Moore, CG, Marfin, AA, Mitchell, CJ, McLean, RG, et al. Guidelines for arbovirus surveillance programs in the United States. National Center for Infectious Diseases (U.S.), Division of Vector-Borne Infectious Diseases. 1993; 83p. Morris, CD. Eastern equine encephalomyelitis. In: Monath, TP, ed. The Arboviruses: Epidemiology and Ecology. Boca Raton, FL: CRC Press, 1988:1–20. Molaei, G, Andreadis, TG. Identification of avian- and mammalian-derived bloodmeals in Aedes vexans and Culiseta mela-
81 nura (Diptera:Culicidae) and its implications for West Nile virus transmission in Connecticut, U.S.A. J Med Entomol 2006; 43:1088–1093. Molaei, G, Andreadis, TG, Armstrong, PM, Diuk-Wasser, M. Host-feeding patterns of potential mosquito vectors in Connecticut, U.S.A.: Molecular analysis of bloodmeals from 25 species of Aedes, Anopheles, Culex, Coquillettidia, Psorophora, and Uranotaenia. J Med Entomol 2008; 45:1143–1151. Mutebi, JP, Lubelczyk, C, Eisen, R, Panella, N, et al. Using wild white-tailed deer to detect eastern equine encephalitis virus activity in Maine. Vector Borne Zoonotic Dis 2011; 11:1403– 1409. Nasci, RS, Berry, RL, Restifo, RA, Parsons, MA, et al. Eastern equine encephalitis virus in Ohio during 1991. J Med Ent 1993; 30:217–222. Rand, PW, Lubelczyk, C, Lavigne, GR, Elias, SP, et al. Deer density and the abundance of Ixodes scapularis (Acari: Ixodidae). J Med Entomol 2003; 40:179–184. Schmitt, SM, Cooley TM, Fitzgerald SD, Bolin SR, et al. An outbreak of Eastern Equine Encephalitis Virus in free-ranging White-Tailed Deer in Michigan. J Wildlife Dis 2007; 43:635– 644. Tate, CM, Howerth EW, Stallknecht DE, Allison AB, et al. Eastern Equine Encephalitis in a free-ranging White-Tailed Deer (Odocoileus virginianus). J Wildlife Dis 2005; 41:241–245. Wattles, DW, DeStefano, S. Status and management of moose in the northeastern United States. Alces 2011; 47:53–68.
Address correspondence to: Charles Lubelczyk Field Biologist Maine Medical Center Research Institute Vector-Borne Disease Laboratory 81 Research Drive Scarborough, ME 04074 E-mail: [email protected]
Detection of Eastern Equine Encephalitis Virus Antibodies in Moose (Alces americana), Maine, 2010 Charles Lubelczyk,1 Susan P. Elias,1 Lee Kantar,2 Jennifer Albert,3 Stephen Hansen,3 Kali Saxton-Shaw,4 Katharine MacMillan,4 Leticia B. Smith,1 Rebecca Eisen,4 Bethany Swope,4 Robert Pease Smith,1 and John-Paul Mutebi 4
Abstract
Moose sera were collected from harvested animals during the 2010 hunting season in Maine. Of the 145 serum samples screened by plaque reduction neutralization test (PRNT), 16 (11%) had antibodies to eastern equine encephalitis virus (EEEV). Positive samples were collected from Aroostook County (n = 13), Somerset County (n = 2), and Piscataquis County (n = 1) in northern and central Maine. Preliminary mosquito surveillance revealed the presence of enzootic and bridge vectors mosquitoes, including Culiseta (Climacura) melanura (Coquillett), Aedes (Aedimorphus) vexans (Meigen), and Coquillettidia (Coquillettidia) perturbans (Walker). Select mosquito species were tested by RT-PCR for the presence of EEEV. None were positive. This is the first report of EEEV in moose from Maine. Key Words: Eastern equine encephalitis virus—Moose—Alces Americana—Maine—Mosquitos.
Following an outbreak of the virus in Quebec, Canada, involving horses and emus in 2008 (Chenier et al. 2010), a similar outbreak occurred in Maine the following year. EEEVpositive horses, pheasants, and mosquitoes were reported after surveillance was increased to monitor for virus activity. No human cases were reported during this period despite an unprecedented number of equine fatalities across a wide region of the state (Gibney et al. 2011). Although EEEV infection in humans is rare, it is associated with severe neurological disease and has a mortality rate of up to 33% (Morris 1988). Seasonal mosquito surveillance programs exist in many locations in southern Maine (Holman et al. 2006), but these efforts are very focal with a distribution ranging from York County in southwestern Maine to southern Penobscot County in the interior of the state. Deer have also been used to increase the geographic range of EEEV surveillance in Maine. About 7.1% of deer harvested during the 2009 fall hunting season had antibodies showing exposure to EEEV (Mutebi et al. 2011). Using deer as sentinels for EEEV activity works well in the southern part of the state where densities are in excess of 15 deer/km2 (Rand et al. 2003). In northern and eastern Maine, however, deer densities are well below 5 deer/km2 (Lavigne 1997).
Introduction
E
astern equine encephalitis virus (EEEV) is an arbovirus in the family Togavaridae, genus Alphavirus, whose typical enzootic cycle involves the bite of ornithophilic mosquitoes on birds (Howard and Wallis 1974). Typical enzootic vectors include members of the genera Culiseta and Culex (Howard and Wallis 1974) with bridge vectors including several mammal-biting mosquitoes of the genera Aedes and Coquillettidia (Davis 1940, Chamberlain et al. 1951). Most mammals affected by EEEV are ruminants or cervids (McLean et al. 1985). Horses and llamas, if unvaccinated, may quickly succumb to the disease, whereas other hooved mammals, such as white-tailed deer, may have less severe neurologic symptoms or can be completely asymptomatic after exposure to the virus (Bigler et al. 1975, Tate et al. 2005). Geographically, EEEV is considered a New World arbovirus, with a range extending from southern Canada through the Caribbean to southern Argentina, with foci appearing regularly in coastal regions in the southeastern United States and New England (Morris 1988, Armstrong et al. 2008). Infrequent outbreaks have also been reported from interior portions of North America as well (McLean et al. 1985, Nasci et al. 1993). 1
Maine Medical Center Research Institute, Vector-borne Disease Laboratory, South Portland, Maine. Maine Department of Inland Fisheries and Wildlife, Bangor, Maine. 3 Biology Department, University of Maine, Fort Kent, Maine. 4 Centers for Disease Control and Prevention, Fort Collins, Colorado. 2
77
78 In some of these regions, particularly northern portions of Aroostook, Piscataquis, and Somerset counties, moose (Alces americana L.) have established stable and healthy populations (Wattles and DeStefano 2011). Because EEEV reportedly affects cervids (McLean et al. 1985), it is possible that moose might also be exposed to EEEV. Accordingly, we undertook an expansion of sampling for EEEV at moose hunter registration stations throughout Maine. This effort immediately followed the first season of a pilot program incorporating sites in Aroostook County as part of the state’s seasonal mosquito surveillance following an absence of sampling in the area (Lathrop 1939, Bean 1946). This report details the first detection of EEEV antibodies in A. americana in the northern Maine. It also describes the presence of both potential enzootic and bridge vector mosquito species records collected as part of routine arbovirus surveillance in northern Maine. Methods and Materials Hunter registration station Blood samples were collected from A. americana carcasses at seven hunter registration stations across northern Maine (Ashland, Fort Kent, Houlton, Medway, and Presque Isle in Aroostook County; Greenville in Piscataquis County; and Jackman in Somerset County). These stations were chosen because large numbers of moose had consistently been registered at each in previous years. Sampling began on September 21, 2010, and continued through mid-November, 2010. Whole blood was collected either directly from the heart or from blood pooled in body cavities of the disemboweled carcasses by either sterile syringes and/or sterile pipets and dispensed into vacutainer tubes. Vacutainer tubes were kept on ice in Styrofoam chests and transported to the lab at the end of each day. In the lab, the vacutainer tubes were centrifuged at 3400 rpm for 15 min to separate serum from the blood clot and stored frozen at - 80C. Moose tag numbers were used as ID numbers, and moose approximate ages were estimated using teeth wear and number of antlers for males and body size in females. Serologic tests Moose serum samples diluted 1:10 were screened for EEEVneutralizing antibodies by plaque-reduction neutralization assay (PRNT) (Beaty et al. 1995). Positive specimens were retested and titrated in duplicate for confirmation. Serum samples were considered positive for EEEV antibodies if they neutralized 80% of a challenge dose of & 100 plaque-forming units of EEEV–Sindbis chimeric virus ( Johnson et al. 2011). Mosquito surveillance Beginning in 2010, seasonal mosquito surveys were started from July through September in three communities in Aroostook County (Fort Kent [4715¢31.22†N/6835¢22.08†W], Frenchville [4719¢30.6†N/6829¢49.38†W], and Madawaska [4721¢19.13†N/6819¢18.13†W]). Investigators placed CDC Miniature Light traps (Model #512, J.W. Hock Co. Gainesville, FL) baited with CO2 at wetland sites once per week. Traps were hung at approximately 15:00 hours and retrieved the following morning by 09:00 hours. Female mosquitoes were anesthetized using cold temperatures before identification and then identified on a cold surface with a binocular dis-
LUBELCZYK ET AL. secting microscope. Standard keys (Andreadis et al. 2005, Darsie and Ward 2005) were used to identify specimens. Mosquitoes were then pooled by site and species. From July 1 to July 31, all Culiseta and Culex mosquitoes were submitted to the Maine Health and Environmental Testing Lab (HETL) as part of Maine’s arbovirus surveillance. Aedes spp. and Cq. pertubans were included for testing from August onward. Mosquitoes were screened for EEEV using forward primer 1858 and reverse primer 1926 (Lambert et al. 2003). Statistical analysis Chi-squared tests were used to determine differences in proportions of EEEV antibody-positive moose by county (Aroostook versus other counties), sex, age, and month, accepting differences as significant at p £ 0.05. Collection sites identified by the hunters and marked on a DeLorme Maine Atlas were geocoded using ArcGIS v.10 (ArcGIS, ESRI Redlands, CA). Serological testing results were included as attributes of the moose data and analyzed using the Moran I statistic to determine whether the distribution of EEEV antibody-positive samples was random, dispersed, or clustered. The samples were considered randomly distributed at P > 0.05. Results Hunter registration stations We examined 195 moose from three Maine counties, including northernmost Aroostook (174) and two adjacent counties to the south, Piscataquis (2) and Somerset (19). Of the animals examined, sera were collected from 145. From these, 16 (11%) tested positive for EEEV antibodies by PRNT (Table 1). The proportions of EEEV antibody-positive serum samples in Aroostook (10.4% [13/125]) did not differ from the two counties to the south (15% [3/20]), v2 = 0.37, p = 0.54. Nor did the proportions of positive samples differ significantly between females (4.8% [7/75]) and males (6.2% [9/70], v2 = 0.46, p = 0.50). No positive samples were reported among juveniles ( < 2 years), and the proportions of positive samples did not differ by month (September 7.1% [1/14], October 16.1% [5/ 31], and November 10.0% [10/100], v2 = 1.14, p = 0.56). At the minor civil division (MCD) level, positive moose were found across a wide area of northern Maine (Fig. 1), indicating EEEV activity occurring in the state north to the Canadian border. There were large areas where collections were made that resulted in no positive samples, especially in Aroostook County (Fig.1), suggesting focal distribution of enzootic EEEV activity in northern Maine. Table 1. Number of Moose Serum Samples Collected, Number and Percentage of EEEV-Antibody-Positive Sera from Different Counties in Maine, 2010
County Aroostook Somerset Piscataquis Total
Moose serum samples screened
No. EEEVantibodypositive sera
% EEEV positive (95% CI)
125 18 2 145
13 2 1 16
10.4 (5.0, 15.7) 11.1 (7.4, 25.6 50.0 (50.0, 100.0)
EEEV, eastern equine encephalitis virus; CI, confidence interval.
EEEV IN MOOSE
FIG. 1.
79
Location of geocoded moose samples collected during 2010 in three northern Maine counties.
Of the 195 moose samples collected, only 136 locations could be accurately identified and geocoded; the remainder lacked specific map coordinates. Samples without a specific location of kill were excluded from the spatial analysis. Analysis of the spatial distribution of PRNT results collected from moose revealed a random distribution (Moran I = - 0.0099, p value = 0.9109). Mosquito surveillance CO2/light traps were hung for 26 trap nights, beginning on June 30 and ending on September 22. A total of 722 female
mosquitoes were collected and identified, with the majority of collections composed of Aedes (Aedimorphus) vexans (Meigen) (n = 343), Aedes (Ochlerotatus) canadensis (Theobald) (n = 54), Anopheles (Anopheles) punctipennis (Say) (n = 111), and Coquillettidia (Coquillettidia) perturbans (Walker) (n = 104). Known enzootic vectors of EEEV collected included Culiseta melanura (n = 1), Culiseta (Climacura) morsitans (Theobald) (n = 8), Culiseta (Culicella) minnesotae Barr (n = 1), and members of the Culex pipiens/restuans complex (n = 10). For analysis, we pooled the lone sample date in June with samples in July, and the one September collection with collections from August. No seasonal differences were noted except a decline in Cq.
80 perturbans captures of 7.8 to 1.1 per trap night (trap nights = 12 for June/July, 14 for August/September). Across species, no other seasonal differences were noted. No mosquitoes tested positive by RT-PCR for either West Nile virus or EEEV. Discussion Whereas previous studies have examined exposure of other cervids, primarily white-tailed deer, to EEEV (Mutebi et al. 2011), this study demonstrates that moose are also exposed to EEEV in Maine and establishes that potential enzootic and bridge vector mosquitoes are present in northern Maine to facilitate a zoonotic cycle of the disease. No mosquitoes collected during this study tested positive for EEEV, but the collection of Cs. melanura in northern Maine expands the known distribution of this species in the state (Darsie and Ward 2005). Culiseta mosquitoes are regarded as essential for the emergence and establishment of this virus, Cs. melanura in particular, because of its capacity to maintain high level of virus titers (Armstrong and Andreadis 2010). In addition, Cs. melanura are also regarded as a possible bridge vector, because they will occasionally take a blood meal from humans (Armstrong and Andreadis 2010). We recognize that a small number of specimens were collected during this survey so, given that there is a low prevalence of virus in endemic regions, it is not surprising that none of the mosquitoes were infected with EEEV. Two ubiquitous mammal-biting mosquitoes were found (Ae. vexans and Cq. perturbans) throughout the summer in northern Maine, inhabiting opportunistic wetlands and cattail marshes (Moore 1993). Both species are bridge vectors for the virus (Armstrong and Andreadis 2010) and frequently feed upon white-tailed deer (Molaei and Andreadis 2006, Molaei et al. 2008), but little data exist on these species’ preference for moose. The small sample numbers of mosquitoes collected in this study were insufficient to examine seasonality for species, but future efforts will attempt to expand geographic sampling and to study seasonal patterns. It is interesting though to note that the presence of EEEV antibodies in moose in the three counties examined occurred in a year with limited arboviral activity reported in northern New England (Centers for Disease Control and Prevention 2011). This stands in contrast to 2009, when 16 livestock animals and three pheasant flocks succumbed to the virus in Maine (Gibney et al. 2011). Given that EEEV was documented in horses approximately 160 km (100 miles) northwest of some collection sites (Chenier et al. 2010), it was not surprising that seropositive animals are present. Although persistence of antibodies to EEEV has been documented in reptiles and birds (Hayes et al. 1964, Calisher et al. 1986), there is little information of its persistence in cervids. Boadella et al. (2012) reported possible persistence of flavivirus antibodies well over 1 year in European ungulates, but persistence of EEEV (an alphavirus) antibodies in moose and white-tailed deer remains uncertain. One possibility is that the adult cohort of moose, with higher antibody prevalence, might have been exposed to EEEV prior to 2010. While there is conflicting evidence that EEEV might impact white-tailed deer (Tate 2005, Schmitt 2007), there has been no indication, at present, that the virus debilitates moose the way it does equines and South American camelids (llamas and alpacas) (Moore 1993). The apparent widespread distribution
LUBELCZYK ET AL. of the virus, however, is a cause for concern and emphasizes the need for expanded surveillance in the state. Furthermore the nonclustered distribution of positive moose indicates that the virus is well established in Maine. The appearance of this virus can be highly localized (Nasci et al. 1993). It remains to be determined if, through continued collections of geocoded samples, we will see sites of reliable foci appear. Because moose are the dominant ungulate in northern forest systems in eastern North America, an understanding of the role that moose play in the ecology of EEEV is important in understanding how useful this species can be as part of a comprehensive wildlife surveillance program. To date, this is the first inclusion of this species as part of a comprehensive program for arbovirus surveillance in the northeastern Unites States. In addition, serum and tissues from hunter-harvested animals can provide public health professionals a source of samples that would be difficult to obtain through other survey methods (live-capture or trapping methods). Author Disclosure Statement No competing financial interests exist. References Andreadis, TG, Thomas, MC, Shepard, JJ. Identification guide to the mosquitoes of Connecticut. CAES Bulletin 966. New Haven, CT: 2005;173. Armstrong, PM, Andreadis, TG. Eastern equine encephalitis virus in mosquitoes and their role as bridge vectors. Emerg Inf Dis 2010; 16:1869–1874. Armstrong, PM, Andreadis, TG, Anderson, JF, Stull, J, et al. Tracking Eastern equine encephalitis virus perpetuation in the Northeastern United States by phylogenetic analysis. Am J Trop Med Hyg 2008; 79:291–296. Bean, JL. A preliminary list of the mosquitoe of Maine (Culicidae Diptera. Can. Entomol. 1946; 78:25–28. Beaty, BJ, Calisher, CH, Shope, RE. (1995) Arboviruses. In: Lennette, EH, Lennette, DA, Lennette, ET, eds. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infection, 7th ed. Washington DC: American Public Health Association, 1995:204–205. Bigler, WJ, Lassing, EB, Lewis, AL, Hoff, GL. Arbovirus surveillance in Florida: Wild vertebrate studies 1965–1974. J Wildlife Dis 1975; 11:348–356. Boadella M, Dı´ez-Delgado I, Gutie´rrez-Guzma´n AV, Ho¨fle U, et al. Do wild ungulates allow improved monitoring of flavivirus circulation in Spain? Vector Borne Zoonotic Dis 2012; 12:490–495. Calisher, CH, Berardi, VP, Muth, DJ, Buff, EE. Specificity of immunoglobulin M and G antibody response in humans infected with eastern and western equine encephalitis viruses: application to rapid serodiagnosis. J Clin Microbiol 1986; 23: 369–372. Centers for Disease Control and Prevention [CDC]. Summary of Notifiable Diseases—United States, 2010. MMWR. 2012; 59: 1–111. Chamberlain, RW, Rubin, H, Kissling, RE. Recovery of virus of Eastern equine encephalomyelitis from a mosquito, Culiseta melanura (Coquillett). Proc Soc Exp Biol Med 1951; 77:396–397. Chenier, SG, Cote, J, Vanderstock, S, Maciera, A, et al. An eastern equine encephalomyelitis (EEE) outbreak in Quebec in the fall of 2008. Can Vet J 2010; 51:1011–1015. Darsie, RF, Jr., Ward, RA. Identification and Geographical Distribution of Mosquitoes of North America, North of Mexico. Gainesville, FL: University Press of Florida, 2005:380.
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Address correspondence to: Charles Lubelczyk Field Biologist Maine Medical Center Research Institute Vector-Borne Disease Laboratory 81 Research Drive Scarborough, ME 04074 E-mail: [email protected]
