2007 SOUTHEASTERN NATURALIST 6(4):737–742 Survey for West Nile Virus Infection in Free-ranging American Alligators in Louisiana Rachel M. McNew1, Ruth M. Elsey2, Thomas R. Rainwater3, Eric J. Marsland1, and Steven M. Presley1,* Abstract - West Nile virus (WNV) is an endemic arboviral pathogen that occurs throughout most of the United States and is typically maintained through a birdmosquito- bird transmission cycle. The ecological significance of the virus is high due to its ability to infect and cause disease in humans, livestock, and wildlife. West Nile virus infection of many vertebrate species causes signs of viral illness, including encephalitis that may result in mortality. Infection by WNV has recently been detected in captive Alligator mississippiensis (American Alligators) in Georgia and Louisiana, and in both captive and free-ranging alligators in Florida. However, additional surveys for WNV in populations of free-ranging alligators within the southeastern USA have not been conducted. The purpose of this study was to survey free-ranging alligators in south Louisiana for active WNV infection. Blood samples were collected from 93 alligators captured at Rockefeller Wildlife Refuge in Cameron Parish, LA, during May 2006 and were screened for WNV using reverse transcriptase polymerase chain reaction (RT-PCR). All samples (100%) tested negative for WNV, indicating a lack of detectable active infection in these animals. Additional surveys of the occurrence of WNV in alligators throughout the southeastern USA are needed to determine the susceptibility of these reptiles to the virus, effects on the health of infected populations, and the potential role of alligators in the maintenance and transmission of the virus. Introduction West Nile virus (WNV) is an arboviral (arthropod-borne viral) pathogen (flavivirus) that is primarily maintained in a natural transmission cycle involving ornithophillic mosquito species and birds, but other vertebrate hosts, including humans and mammals, may be incidentally infected by mosquitoes (Huchzermeyer 2002, Komar 2003); thus, this zoonotic disease poses a threat to both wildlife and human health (Goddard 2000). The severity of the disease associated with WNV infection ranges from mild fever and malaise to life-threatening illness. Encephalitis and meningitis may occur in humans and other animals, particularly mammals, as a result of WNV infection (Marra et al. 2004). Other signs associated with the virus include fever, internal hemorrhaging, and tissue necrosis, which may lead to eventual brain damage (Marra et al. 2004). Reptiles may play a significant role in arboviral disease transmission cycles (Bowen 1977, Nevarez et al. 2005). Japanese encephalitis virus has 1The Institute of Environmental and Human Health, Box 41163, Lubbock, TX 79409. 2Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, 5476 Grand Chenier Highway, Grand Chenier, LA 70643. 3The Institute of Environmental and Human Health Field Station, Jeffersonian Institute, PO Box 764, Jefferson, TX 75657. *Corresponding author - steve.presley@tiehh.ttu.edu. 738 Southeastern Naturalist Vol. 6, No. 4 been reported in several reptilian species (Doi et al.1983, Mifune et al.1969, Shortridge et al. 1977). Recently, WNV has been recovered from the blood of multiple crocodilian species including Crocodylus niloticus Laurenti (Nile Crocodile) in Africa and Crocodylus moreletii Dumeril and Bibron (Morelet’s Crocodile) in Mexico (Steinman et al. 2003). In addition, WNV has also been detected in both captive and free-ranging Alligator mississippiensis (Daudin) (American Alligators) in the USA (Jacobson et al. 2005a,b, Miller et al. 2003, Nevarez et al. 2005). Alligators are typically affected by WNV in ways that are visually discernable, with the most common signs being neurological changes such as tremors, tilting of the head, and swimming in circles (Jacobson et al. 2005a, Nevarez et al. 2005). Human cases of WNV infection have been documented in Louisiana since 2002 (Zohrabian et al. 2004), and the virus has been detected in captive alligators at several ranches in the state. However, free-ranging alligators in Louisiana have not yet been examined for WNV infection. Thus, the objective of this study was to conduct a preliminary survey of the incidence of active WNV infection in an alligator population in southwestern Louisiana. Methods Blood collection During May 2006, 93 free-ranging American Alligators were captured at night from impoundments on the Louisiana Department of Wildlife and Fisheries’ Rockefeller Wildlife Refuge in Cameron Parish, LA. Alligators were located with high-intensity spotlights, approached by airboat, and captured with a self-locking cable snare (Decker Manufacturing, Keokuk, IA). The alligators ranged in size from 49.5 cm to 208.3 cm total length. A 10-mL blood sample was immediately collected from the spinal vein of the larger alligators into a heparin-coated syringe and placed on ice for transport. After blood was collected, measurements and sex were recorded, and then the larger alligators were released at the capture site. The smallest alligators were retained in holding boxes and later bled with smaller bore needles not available during the field capture. Of the 10-mL blood sample taken from each alligator, 2–3 mL of whole blood was placed into a vial and frozen for analyses. Blood analysis Reverse-transcriptase polymerase chain reaction (RT-PCR) was used to detect viral infection in the blood at the time of capture. RT-PCR is an assay that has been documented to provide accurate and timely results in cell cultures and infected mosquito pools (Hadfield 2001). Other studies have utilized RT-PCR for blood meal analyses in mosquitoes that fed on reptilian blood as a means of detecting other flavivirus species (Cupp et al. 2004). RNA extraction and assay Total RNA was extracted from 0.2 mL of whole blood using TriReagent® BD (Sigma-Aldrich), per manufacturer’s directions. Approximate RNA yield was determined to be 3–4 μg for the majority of samples, and the concentrations were adjusted to 0.2 ng/μL for RT-PCR screening. RT-PCR was performed on 2007 R.M. McNew, R.M. Elsey, T.R. Rainwater, E.J. Marsland, and S.M. Presley 739 the total RNA extracted from the alligator blood to determine presence of WNV. Specimens were assayed using the primer set FLV1/FLV2, developed by Platonov et al. (2001), which targets the West Nile/Kunjin virus’ conserved region of the envelope (E) gene; FLV1 consists of 5’- GGI AGC AGI GCC ATI TGG T(A/T)C ATG TGG - 3’ and FLV2 consists of 5’- C(G/T)I GTG TCC CAI CCI GCI GTG TCA TC - 3’. Forty-five μL of a prepared RT-PCR mixture was combined with 5 μL (1 ng) of the blood sample and dispensed into a 0.2-mL PCR tube. Using a PTC-100 thermal cycler (MJ Research Waltham, MA), cycling procedures for the RT-PCR reactions included a cycle at 49 °C for 45 minutes to synthesize the first-strand cDNA; a second cycle at 95 °C for 3 minutes for initial denaturation; 40 cycles at 94 °C for 45 seconds, 56 °C for 45 seconds, and 72 °C for 1 minute for PCR amplification; and then a final elongation step at 72 °C for 10 minutes. Twenty-five μL of the RT-PCR product from each sample was then loaded along with bromophenol blue onto a 2% agarose gel in Tris-borate- EDTA (TBE) buffer and processed for approximately 1.5 hr at 50 volts. Results All of the 93 alligator blood samples assayed (one per animal) proved negative for the presence of active WNV infection by RT-PCR (Fig. 1). Discussion Results from this study suggest that alligators tested for WNV did not have a detectable viremia at the time of sampling, and that active infection by WNV was not occurring in free-ranging alligators in the Rockefeller Figure 1. Gel electrophoresis of an RT-PCR assay for WNV from whole blood samples of alligators. Shown are the results of 12 samples. Lane 1 is the molecular ladder. Lanes 2–13 are negative alligator samples. Lane 14 is the WNV positive control at 220 bp, and lane 15 is the negative control. 740 Southeastern Naturalist Vol. 6, No. 4 Wildlife Refuge. This may be due to a lack of WNV presence in mosquito species in the specific area sampled during this study. These results support the low prevalence of WNV-infected mosquitoes in early summer months, as peak viral activity has been reported in the months of July through October in the United States (Hayes et al. 2005). Alligators are known to be potential WNV amplifiers, and one source of viral infection may be the consumption of infected prey species (Klenk et al. 2004). Although birds are most often used as indicator species for WNV (Guptill et al. 2003), they may not be likely the most appropriate choice for Louisiana (LA Department of Health and Hospitals 2006). Several species of mosquitoes occurring in the New Orleans area prefer ectothermic blood meals, including Uranotaenia sapphirina (Osten Sacken) and Culex erraticus (Dyar and Knab) (Darsie and Ward 2005). Both of these species have tested positive for WNV infection, and Culex spp. mosquitoes have been shown to be the principal vectors of the virus (CDC 2005, Hayes et al. 2005). Various other arboviral infections have been reported in other reptiles (Bowen 1977, Doi et al. 1983). The ability for a pathogen to overwinter in an area is essential to the persistence of a disease in temperate environments, and is a critical factor in the study and understanding of endemic diseases (Kitron et al. 1998). The potential for reptiles to serve as overwintering reservoirs for specific arboviruses has been previously proposed. Bowen (1977) reported Gopherus berlandieri (Agassiz) (Texas Tortoise) served as an overwintering reservoir for western equine encephalitis (WEE), and Doi et al. (1982) reported Takydromus sp. (lacertid lizards) and Eumeces sp. (skinks) as overwintering reservoirs for Japanese encephalitis virus (JEV). However, the results of the present study do not support such a relationship between WNV and the alligator population. A recent report (Jacobson et al. 2005b) of WNV antibodies detected in blood of free-ranging alligators in Florida (a low seroprevalence of 1.5%) suggests the potential for exposure to and infection of alligators with WNV in other areas within the species’ range. In the present study, antibodies were not examined, as we were focused on active WNV infection at the time of sampling. Recent studies have reported that the serum of American Alligators exhibit antibacterial, amoebacidal, and antiviral properties (Merchant et al. 2003, 2004, 2005). The alligators sampled in this study may have never been infected with WNV, or they may have been exposed to the virus, but both innate and acquired immunity may have prevented progression of infection. It is clear that reptiles are susceptible to infection by WNV, and they may also be involved in the maintenance and transmission dynamics of the virus (Bowen 1977, Nevarez et al. 2005). The lack of WNV detection in alligators tested in this study suggests that active infection among alligators in this geographic area was not occurring at the time of sampling. However, given the high density of alligator populations in many areas of the southeastern US and the documented occurrence of WNV in free-ranging and 2007 R.M. McNew, R.M. Elsey, T.R. Rainwater, E.J. Marsland, and S.M. Presley 741 captive alligators from numerous geographic locations, additional research is needed to more closely examine the epizoological role of these reptiles in WNV transmission dynamics. Future studies should include IgG, IgM, and protein electrophoresis so that the history of infection in sampled animals can be determined. 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