Anaplasma odocoilei sp. nov. (family Anaplasmataceae) from white-tailed deer (Odocoileus virginianus).

Recently, an undescribed Anaplasma sp. (also called Ehrlichia-like sp. or WTD agent) was isolated in ISE6 tick cells from captive white-tailed deer. The goal of the current study was to characterize this organism using a combination of experimental infection, morphologic, serologic, and molecular studies. Each of 6 experimentally inoculated white-tailed deer fawns (Odocoileus virginianus) became chronically infected (100+ days) with the Anaplasma sp. by inoculation of either infected whole blood or culture. None of the deer showed evidence of clinical disease, but 3 of the 6 deer evaluated had multiple episodes of transient thrombocytopenia. Light microscopy of Giemsa-stained, thin blood smears revealed tiny, dark, spherical structures in platelets of acutely infected deer. Anaplasma sp. was detected in platelets of inoculated deer by polymerase chain reaction, transmission electron microscopy, immunohistochemistry, and in situ hybridization. Five of 6 deer developed antibodies reactive to Anaplasma sp. antigen, as detected by indirect fluorescent antibody testing. Phylogenetic analyses of 16S rRNA, groESL, and gltA sequences confirmed the Anaplasma sp. is related to A. platys. Two attempts to transmit the Anaplasma sp. between deer by feeding Amblyomma americanum, a suspected tick vector, were unsuccessful. Based on its biologic, antigenic, and genetic characteristics, this organism is considered a novel species of Anaplasma, and the name Anaplasma odocoilei sp. nov. is proposed with UMUM76(T) (=CSUR-A1) as the type strain.

Necropsy findings and arbovirus surveillance in mourning doves from the southeastern United States.

Mourning doves (Zenaida macroura) are the most abundant and widespread native member of the columbid family, as well as a major migratory game species, in the United States. However, there is little information on mortality factors in mourning doves. Records of necropsy accessions at the Southeastern Cooperative Wildlife Disease Study (SCWDS) from 15 southeastern states, from 1971 through 2005, were reviewed. One hundred thirty-five mourning doves were submitted from nine states during the 35-yr period. Trichomonosis constituted 40% (n = 54) of all diagnoses and was the most frequent diagnosis. Toxicoses and avian pox constituted 18.5% (n = 25) and 14.8% (n = 20) of all diagnoses, respectively. Remaining diagnoses included trauma, suspected toxicosis, Ascaridia columbae infection, suspected tick paralysis, and undetermined. Adults were observed more frequently with trichomonosis (94.1%) and toxicoses (68%) as compared to juveniles, but a gender predisposition was not apparent for either disease. Age and gender predilections were not apparent for cases of avian pox. The majority of the trichomonosis and avian pox cases were observed in the spring-summer, whereas the majority of the toxicosis cases were observed in the winter-spring. Additionally, the Georgia Department of Human Resources-Division of Public Health and West Virginia Department of Health and Human Resources submitted 809 mourning doves to SCWDS from 2001 through 2005 for West Nile virus surveillance efforts. West Nile virus was isolated from 2.1% (n = 17) and eastern equine encephalitis virus (EEEV) was isolated from 0.2% (n = 2) of the submitted birds.

Avian vacuolar myelinopathy outbreaks at a southeastern reservoir.

Avian vacuolar myelinopathy (AVM) is a neurologic disease of unknown etiology that affects bald eagles (Haliaeetus leucocephalus), American coots (Fulica americana), and several species of waterfowl. An unidentified neurotoxin is suspected as the cause of AVM, which has been documented at several reservoirs in the southeastern United States. We conducted diagnostic and epidemiologic studies annually during October-March from 1998-2004 at Clarks Hill/Strom Thurmond Lake on the Georgia/South Carolina border to better understand the disease. Avian vacuolar myelinopathy was confirmed or suspected as the cause of morbidity and mortality of 28 bald eagles, 16 Canada geese (Branta canadensis), six American coots, two great-horned owls (Bubo virginianus), and one killdeer (Charadrius vociferus). Active surveillance during the outbreaks yielded annual average prevalence of vacuolar lesions in 17-94% of coots, but not in 10 beavers (Castor canadensis), four raccoons (Procyon lotor), and one gray fox (Urocyon cinereoargenteus) collected for the study. Brain lesions were not apparent in 30 Canada geese collected and examined in June 2002. The outbreaks at this location from 1998-2004 represent the most significant AVM-related bald eagle mortality since the Arkansas epornitics of 1994-95 and 1996-97, as well as the first confirmation of the disease in members of Strigiformes and Charadriiformes.

Evaluation of white-tailed deer (Odocoileus virginianus) as natural sentinels for Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the causative agent of human granulocytotropic anaplasmosis, can infect white-tailed deer (WTD; Odocoileus virginianus), and this species is a crucial host for adult Ixodes scapularis, the primary vector of A. phagocytophilum. The goal of this study was to determine the geographic distribution of A. phagocytophilum among WTD across a 19 state region and to evaluate the utility of WTD as natural sentinels. Serologic testing using the indirect fluorescent antibody (IFA) assay was conducted on WTD serum samples and molecular and xenodiagnostic tests were performed to confirm serologic results. The surveillance system was assessed through examination of vital attributes including WTD age and gender associations with serologic status, sample size adequacy for accurate infection status classification, and presence of the vector, I. scapularis. Six hundred thirty-three of 2,666 (24%) WTD in 17 states tested positive for antibodies (>or=128) when tested by IFA assay. Testing for p44 and/or 16S rRNA gene targets identified 73 (16%) PCR positive WTD among 458 animals tested, all of which originated from seropositive populations. Attempts to culture A. phagocytophilum from WTD were unsuccessful; however, xenodiagnostic mice inoculated with blood from 3 WTD became infected. Seroprevalence did not differ by deer age or gender; however, WTD

Experimental infection of white-tailed deer with Anaplasma phagocytophilum, etiologic agent of human granulocytic anaplasmosis.

Serologic and molecular evidence of Anaplasma phagocytophilum has been demonstrated in white-tailed deer (WTD; Odocoileus virginianus), and deer are an important host for the tick vector Ixodes scapularis. In this study, we describe experimental infection of WTD with A. phagocytophilum. We inoculated four WTD with a human isolate of A. phagocytophilum propagated in tick cells. Two additional deer served as negative controls. All inoculated deer developed antibodies (titers, > or =64) to A. phagocytophilum, as determined by an indirect fluorescent antibody test, between 14 and 24 days postinfection [p.i.]), and two deer maintained reciprocal titers of > or =64 through the end of the 66-day study. Although morulae were not observed in granulocytes and A. phagocytophilum was not reisolated via tick cell culture of blood, 16S reverse transcriptase nested PCR (RT-nPCR) results indicated that A. phagocytophilum circulated in peripheral blood of three deer through at least 17 days p.i. and was present in two deer at 38 days p.i. Femoral bone marrow from one deer was RT-nPCR positive for A. phagocytophilum at 66 days p.i. There was no indication of clinical disease. These data confirm that WTD are susceptible to infection with a human isolate of A. phagocytophilum and verify that WTD produce detectable antibodies upon exposure to the organism. Because adults are the predominant life stage of I. scapularis found on deer and because adult I. scapularis ticks do not transmit A. phagocytophilum transovarially, it is unlikely that WTD are a significant source of A. phagocytophilum for immature ticks even though deer have a high probability of natural infection. However, the susceptibility and immunologic response of WTD to A. phagocytophilum render them suitable candidates as natural sentinels for this zoonotic tick-borne organism.

Eastern equine encephalitis in a free-ranging white-tailed deer (Odocoileus virginianus).

Eastern equine encephalitis (EEE) was diagnosed in a free-ranging, adult, male white-tailed deer (Odocoileus virginianus) from Houston County, Georgia, USA, in July 2001. The yearling buck had neurologic disease and died during transport to our diagnostic facility. Eastern equine encephalitis virus (EEEV) was isolated in Vero cell culture and identified by reverse-transcriptase polymerase chain reaction; as well, EEEV antigen was detected in brain by immunohistochemistry. This is the first report of fatal EEEV infection in a white-tailed deer. Antibodies to EEEV were demonstrated by microtiter neutralization in 14 of 99 (14%) of the white-tailed deer from Georgia sampled in fall 2001. Most antibody-positive deer originated from the Coastal Plain physiographic region. Eastern equine encephalitis virus should be considered a possible cause of neurologic disease in white-tailed deer where it may occur.

Isolation of an Anaplasma sp. organism from white-tailed deer by tick cell culture.

We used tick cell culture to isolate a bacterium previously referred to as the "white-tailed deer (WTD) agent" from two captive fawns inoculated with blood from wild WTD (Odocoileus virginianus). Buffy coat cells were added to ISE6 tick cell cultures and incubated at 34 degrees C, and 8 days later, Anaplasma-like inclusions were demonstrated in Giemsa-stained culture samples. The microbes became established and could be continuously passaged in tick cells. The identity of a culture isolate designated WTD76 was verified as the WTD agent by using specific PCR primers and by DNA sequencing. Comparison with sequences available in GenBank indicated that the isolate was most closely related first to Anaplasma platys and second to Anaplasma phagocytophilum, supporting its placement in the genus Anaplasma. Transmission electron microscopy of this Anaplasma sp. organism in tick cell cultures revealed large inclusions filled with pleomorphic and rod-shaped bacteria. Tick cells infected with the Anaplasma sp. organism were used to successfully infect a naive deer, thereby proving the infectivity of the isolate for deer.

Experimental vacuolar myelinopathy in red-tailed hawks.

Avian vacuolar myelinopathy (AVM) was recognized in 1994 as a cause of wild bird mortality when 29 bald eagles (Haliaeetus leucocephalus) succumbed to the disease at DeGray Lake, Arkansas (USA). The cause of AVM and its source remain undetermined despite extensive diagnostic and research investigations. Two years later, when AVM killed 26 eagles in the same area in Arkansas, it became apparent that American coots (Fulica americana) had identical neurologic signs and lesions, and it was hypothesized that eagles acquired AVM via ingestion of affected coots. In order to test this hypothesis, we fed coot tissues (brain, liver, kidney, muscle, fat, and intestinal tract) to rehabilitated, non-releasable red-tailed hawks (Buteo jamaicensis). Five hawks received tissues from coots with AVM lesions, and one hawk received tissues from coots without brain lesions that had been collected at a site where AVM never has been documented. All hawks received 12-70 g/day (mean = 38 g) of coot tissues for 28 days. All six hawks remained clinically normal during the study. The birds were euthanatized on day 29 and microscopic lesions of AVM were found in all hawks that received tissues from affected coots, but not in the hawk that received tissues from unaffected coots. This marks the first time that AVM has been produced in birds under laboratory conditions and proves that birds of prey can acquire AVM via ingestion of tissues from affected coots.

Ehrlichia ewingii infection in white-tailed deer (Odocoileus virginianus).

Two closely related zoonotic ehrlichiae, Ehrlichia chaffeensis and E. ewingii, are transmitted by Amblyomma americanum, the lone star tick. Because white-tailed deer (Odocoileus virginianus) are critical hosts for all mobile stages of A. americanum and are important vertebrate reservoirs of E. chaffeensis, we investigated whether deer may be infected with E. ewingii, a cause of granulocytotropic ehrlichiosis in humans and dogs. To test for E. ewingii infection, we used polymerase chain reaction and inoculation of fawns with whole blood from wild deer. Of 110 deer tested from 20 locations in 8 U.S. states, 6 (5.5%) were positive for E. ewingii. In addition, natural E. ewingii infection was confirmed through infection of captive fawns. These findings expand the geographic distribution of E. ewingii, along with risk for human infection, to include areas of Kentucky, Georgia, and South Carolina. These data suggest that white-tailed deer may be an important reservoir for E. ewingii.