Spring Migration Stopover Ecology of Avian Influenza Virus Shorebird Hosts at Delaware Bay.

Although low pathogenicity avian influenza viruses (LPAIV) are detected in shorebirds at Delaware Bay annually, little is known about affected species habitat preferences or the movement patterns that might influence virus transmission and spread. During the 5-wk spring migration stopover period during 2007-2008, we conducted a radiotelemetry study of often-infected ruddy turnstones (Arenaria interpres morinella; n = 60) and rarely infected sanderlings (Calidris alba; n = 20) to identify locations and habitats important to these species (during daytime and nighttime), determine the extent of overlap with other AIV reservoir species or poultry production areas, reveal possible movements of AIV around the Bay, and assess whether long-distance movement of AIV is likely after shorebird departure. Ruddy turnstones and sanderlings both fed on Bay beaches during the daytime. However, sanderlings used remote sandy points and islands during the nighttime while ruddy turnstones primarily used salt marsh harboring waterfowl and gull breeding colonies, suggesting that this environment supports AIV circulation. Shorebird locations were farther from agricultural land and poultry operations than were random locations, suggesting selection away from poultry. Further, there was no areal overlap between shorebird home ranges and poultry production areas. Only 37% (22/60) of ruddy turnstones crossed into Delaware from capture sites in New Jersey, suggesting partial site fidelity and AIV gene pool separation between the states. Ruddy turnstones departed en masse around June 1 when AIV prevalence was low or declining, suggesting that a limited number of birds could disperse AIV onto the breeding grounds. This study provides needed insight into AIV and migratory host ecology, and results can inform both domestic animal AIV prevention and shorebird conservation efforts.

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of antibodies against the H5 subtype of Influenza A virus in waterfowl.

The ID Screen Influenza H5 Antibody Competition enzyme-linked immunosorbent assay was tested for the detection of antibodies to the H5 subtype of influenza A (IA) virus in waterfowl. Assays were conducted with sera obtained from Mallards (Anas platyrhynchos) and Pekin Ducks (Anas platyrhynchos domestica), experimentally infected with eight low pathogenic (LP) and nine highly pathogenic (HP) H5N1 IA viral strains. Three incubation periods (1, 4 and 18 hours) and two dilutions (1:2 and 1:5) were tested. All serum samples from LP H5-infected birds tested positive; however, improved detection rates were observed for viruses belonging to the HP H5N1 clade 2.2.1 as compared with those belonging to clade 2.1.3.

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.

Annual survival of ruddy turnstones is not affected by natural infection with low pathogenicity avian influenza viruses.

The population of ruddy turnstones (Arenaria interpres morinella) that migrates through Delaware Bay has undergone severe declines in recent years, attributable to reduced availability of horseshoe crab (Limulus polyphemus) eggs at this critical spring migration stopover site. Concurrently, this population has experienced annual low pathogenicity avian influenza virus (AIV) epidemics at this same site. Using a prospective cohort study design with birds individually flagged during May-June 2006-2008, we evaluated resighting rates (a proxy for annual survival) between AIV-infected and uninfected birds at 1 yr after capture, testing, and measurement. Overall resighting rate was 46%, which varied by year and increased with relative mass of the bird when captured. Resighting rates were not different between AIV-infected and uninfected birds in any period. In multivariate analyses, infection status was also unrelated to resighting rate after controlling for year, day, state, sex, body size, mass index, or whether the bird was blood-sampled. Thus, apparent annual survival in ruddy turnstones was not reduced by AIV infection at this migratory stopover. However, it is unknown whether intestinal AIV infection might cause subtle reductions in weight gain which could negatively influence reproduction.

Health assessment and seroepidemiologic survey of potential pathogens in wild Antillean manatees (Trichechus manatus manatus).

The Antillean manatee (Trichechus manatus manatus), a subspecies of the West Indian manatee, inhabits fresh, brackish, and warm coastal waters distributed along the eastern border of Central America, the northern coast of South America, and throughout the Wider Caribbean Region. Threatened primarily by human encroachment, poaching, and habitat degradation, Antillean manatees are listed as endangered by the International Union for the Conservation of Nature. The impact of disease on population viability remains unknown in spite of concerns surrounding the species' ability to rebound from a population crash should an epizootic occur. To gain insight on the baseline health of this subspecies, a total of 191 blood samples were collected opportunistically from wild Antillean manatees in Belize between 1997 and 2009. Hematologic and biochemical reference intervals were established, and antibody prevalence to eight pathogens with zoonotic potential was determined. Age was found to be a significant factor of variation in mean blood values, whereas sex, capture site, and season contributed less to overall differences in parameter values. Negative antibody titers were reported for all pathogens surveyed except for Leptospira bratislava, L. canicola, and L. icterohemorrhagiae, Toxoplasma gondii, and morbillivirus. As part of comprehensive health assessment in manatees from Belize, this study will serve as a benchmark aiding in early disease detection and in the discernment of important epidemiologic patterns in the manatees of this region. Additionally, it will provide some of the initial tools to explore the broader application of manatees as sentinel species of nearshore ecosystem health.

Antibodies to influenza A virus in wild birds across Mongolia, 2006-2009.

Wild waterbirds sampled July 2006-September 2009 in Mongolia were tested for antibodies to avian influenza (AI) virus with the use of a commercially available blocking enzyme-linked immunosorbent assay. Antibodies were detected in 25% (572/2,282) of tested birds representing 26 species, and all antibody-positive samples were from 12 species in the orders Anseriformes and Charadriiformes. The highest antibody prevalence was in Ruddy Shelducks (Tadorna ferruginea; 61.7%; n=261; 95% confidence interval [CI] 55.8-67.6%), Whooper Swans (Cygnus cygnus; 38.4%; n=242; 95% CI 32.3-44.5%), Swan Geese (Anser cygnoides; 15%; n=127; 95% CI 8.6-21.4%), Bar-headed Geese (Anser indicus; 13%; n=738; 95% CI 10.3-15.1%), and Mongolian Gulls (Larus mongolicus; 3.9%; n=255; 95% CI 1.3-6.5%). There was no significant temporal or spatial variation in the presence of antibodies in the sampled species. However, Bar-headed Geese and Mongolian Gulls showed spatial variation in antibody prevalence in 2007 and 2008, respectively. Our study provides insights into the hatch year waterbirds' exposure to AI virus at their natal and molting sites in Mongolia.

Avian influenza virus infection dynamics in shorebird hosts.

To gain insight into avian influenza virus (AIV) transmission, exposure, and maintenance patterns in shorebirds at Delaware Bay during spring migration, we examined temporal AIV prevalence trends in four Charadriiformes species with the use of serial cross-sectional data from 2000 through 2008 and generalized linear and additive models. Prevalence of AIV in Ruddy Turnstones (Arenaria interpres morinella) increased after arrival, peaked in mid-late May, and decreased prior to departure. Antibody prevalence also increased over this period; together, these results suggested local infection and recovery prior to departure. Red Knots (Calidris canutus rufa), Sanderlings (Calidris alba), and Laughing Gulls (Leucophaeus atricilla) were rarely infected, but dynamic changes in antibody prevalence differed among species. In Red Knots, declining antibody prevalence over the stopover period suggested AIV exposure prior to arrival at Delaware Bay with limited infection at this site. Antibody prevalence was consistently high in Laughing Gulls and low in Sanderlings. Both viral prevalence and antibody prevalence in Sanderlings varied directly with those in turnstones, suggesting virus spillover to Sanderlings. Results indicate that, although hundreds of thousands of birds concentrate at Delaware Bay during spring, dynamics of AIV infection differ among species, perhaps due to differences in susceptibility, potential for contact with AIV at this site, or prior exposure. Additionally, Ruddy Turnstones possibly act as a local AIV amplifying host rather than a reservoir.

Comparison of two commercial enzyme-linked immunosorbent assays for detection of Influenza A virus antibodies.

Serologic tools for Influenza A virus (FLUAV) antibody testing of wild birds are currently limited. In the present study, 2 commercial enzyme-linked immunosorbent assays (ELISAs) for detection of FLUAV antibodies, the IDEXX AI MultiS-Screen Ab Test and the ID VET ID Screen Influenza A Antibody Competition, were compared. Sera obtained from mallards (Anas platyrhynchos), experimentally infected with 8 FLUAV subtypes (N = 48), and field serum samples, collected from 11 wild bird species (N = 247), were tested. Overall, a substantial agreement was obtained between the 2 assays as applied to both experimental (86.5% agreement, κ = 0.69) and field samples (89.9% agreement, κ = 0.78). Based on the current study, doubtful results obtained with the ID VET assay should be re-tested to confirm their antibody status. Additionally, increasing the incubation period for the ID VET assay increases the test sensitivity but also increases the likelihood of generating false-positive results. Overall, it is concluded that the 2 ELISAs can be used for FLUAV antibody screening in wild birds and that the sensitivity of the ID VET assay can be increased with slight modifications of the manufacturer's instructions.

Avian paramyxoviruses in shorebirds and gulls.

There are nine serotypes of avian paramyxovirus (APMV), including APMV-1, or Newcastle disease virus. Although free-flying ducks and geese have been extensively monitored for APMV, limited information is available for species in the order Charadriiformes. From 2000 to 2005 we tested cloacal swabs from 9,128 shorebirds and gulls (33 species, five families) captured in 10 states within the USA and in three countries in the Caribbean and South America. Avian paramyxoviruses were isolated from 60 (0.7%) samples by inoculation of embryonating chicken eggs; isolates only included APMV-1 and APMV-2. Two isolates (APMV-2) were made from gulls and 58 isolates (APMV-1 [41 isolates] and APMV-2 [17 isolates]) were made from shorebirds. All of the positive shorebirds were sampled at Delaware Bay (Delaware and New Jersey) and 45 (78%) of these isolates came from Ruddy Turnstones (Arenaria interpres). The APMV-1 infection rate was higher among Ruddy Turnstones compared with other shorebird species and varied by year. Avian paramyxovirus-2 was isolated from two of 394 (0.5%) Ruddy Turnstones at Delaware Bay in 2001 and from 13 of 735 (1.8%) Ruddy Turnstones during 2002. For both APMV-1 and APMV-2, infection rates were higher among Ruddy Turnstones sampled on the south shore of Delaware Bay compared to north shore populations. This spatial variation may be related to local movements of Ruddy Turnstones within this ecosystem. The higher prevalence of APMV in Ruddy Turnstones mirrors results observed for avian influenza viruses in shorebirds and may suggest similar modes of transmission.

Prevalence of antibodies to type a influenza virus in wild avian species using two serologic assays.

Serologic testing to detect antibodies to avian influenza (AI) virus has been an underused tool for the study of these viruses in wild bird populations, which traditionally has relied on virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). In a preliminary study, a recently developed commercial blocking enzyme-linked immunosorbent assay (bELISA) had sensitivity and specificity estimates of 82% and 100%, respectively, for detection of antibodies to AI virus in multiple wild bird species after experimental infection. To further evaluate the efficacy of this commercial bELISA and the agar gel immunodiffusion (AGID) test for AI virus antibody detection in wild birds, we tested 2,249 serum samples collected from 62 wild bird species, representing 10 taxonomic orders. Overall, the bELISA detected 25.4% positive samples, whereas the AGID test detected 14.8%. At the species level, the bELISA detected as many or more positive serum samples than the AGID in all 62 avian species. The majority of positive samples, detected by both assays, were from species that use aquatic habitats, with the highest prevalence from species in the orders Anseriformes and Charadriiformes. Conversely, antibodies to AI virus were rarely detected in the terrestrial species. The serologic data yielded by both assays are consistent with the known epidemiology of AI virus in wild birds and published reports of host range based on virus isolation and RT-PCR. The results of this research are also consistent with the aforementioned study, which evaluated the performance of the bELISA and AGID test on experimental samples. Collectively, the data from these two studies indicate that the bELISA is a more sensitive serologic assay than the AGID test for detecting prior exposure to AI virus in wild birds. Based on these results, the bELISA is a reliable species-independent assay with potentially valuable applications for wild bird AI surveillance.

Canada geese and the epidemiology of avian influenza viruses.

Canada geese (Branta canadensis) are numerous, highly visible, and widely distributed in both migratory and resident populations in North America; as a member of the order Anseriformes, they are often suggested as a potential reservoir and source for avian influenza (AI) viruses. To further examine the role of Canada Geese in the ecology of AI, we re-evaluated existing literature related to AI virus in this species and tested breeding populations of Canada Geese from three states (Georgia, West Virginia, and Minnesota, USA) by virus isolation and serology. The ability of AI virus to persist in goose feces under experimental conditions also was evaluated as an additional measure of the potential for this species to serve as an AI virus reservoir. Virus was not isolated from 1,668 cloacal swabs and type-specific antibody prevalence was low (4/335, 1.2%). Finally, under experimental conditions, AI virus persistence in goose feces and in water contaminated with goose feces was limited as compared to published estimates from duck feces and water. Our results are consistent with historic reports of a low prevalence of AI virus infection in this species, and we suggest that Canada Geese play a minor, if any, role as a reservoir for low pathogenic AI viruses that naturally circulate in wild bird populations.

Antibodies to type A influenza virus in wild waterbirds from Argentina.

Limited information exists on avian influenza (AI) virus infection in South American wild birds. As part of a national surveillance program in Argentina, indigenous waterbirds were screened for antibodies to AI virus. From November 2006 to July 2007, serum samples from 540 waterbirds of 12 species were tested for type-specific antibodies to AI virus with the use of a commercially available blocking enzyme-linked immunosorbent assay (bELISA) and the agar-gel immunodiffusion (AGID) test. Thirty-three percent (176/540) of serum samples were positive with the bELISA and 12% (64/540) were positive with the AGID test. The bELISA detected antibodies to AI virus in eight of the 12 species, and the AGID detected positives in only five species. These results provide insight into AI virus circulation in Argentinean waterbirds and preliminary data to guide further surveillance efforts.

Evaluation of a commercial blocking enzyme-linked immunosorbent assay to detect avian influenza virus antibodies in multiple experimentally infected avian species.

Wild birds of the orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Traditionally, AI virus surveillance in wild birds has relied on virus identification strategies, including virus isolation and detection. To evaluate the accuracy of a commercial blocking enzyme-linked immunosorbent assay (bELISA) and the agar gel immunodiffusion (AGID) test for detection of antibodies in wild birds, which is indicative of AI virus infection, we tested 281 serum samples from various wild avian species that were experimentally infected with AI viruses. Included in these samples were 178 samples from birds with confirmed AI virus infections (122 infected with low-pathogenic AI [LPAI] viruses and 56 infected with highly pathogenic AI [HPAI] viruses) and 103 samples from birds that were uninfected, negative controls. The sensitivities of the bELISA and the AGID test were 0.820 (95% confidence interval [95% CI], 0.756 to 0.874) and 0.674 (95% CI, 0.600 to 0.742), respectively. Both tests had an estimated specificity of 1.00 (95% CI, 0.965 to 1.00). The bELISA was significantly more sensitive than the AGID test for both LPAI virus- and HPAI virus-infected birds. Both assays, however, had a higher sensitivity for birds infected with HPAI virus than for birds infected with LPAI virus. These results demonstrate the potential utility of the bELISA for detection of antibodies to both LPAI and HPAI viruses in multiple avian species, representing five avian orders and 17 genera. Additional studies are warranted to further evaluate the utility of the bELISA for use with naturally infected birds.

Characterization of 'Candidatus Neoehrlichia lotoris' (family Anaplasmataceae) from raccoons (Procyon lotor).

Recently, a novel ehrlichial organism was isolated from a raccoon (Procyon lotor) and the isolate (RAC413) was infectious to two naïve raccoons but not laboratory mice, rats or rabbits. In this study, amplification and sequencing of four gene targets (16S rRNA gene, groESL, gltA and rpoB) confirmed that the novel ehrlichial organism was a member of the family Anaplasmataceae and was most closely related to, but distinct from, 'Candidatus Neoehrlichia mikurensis' TK4456(R) and IS58. RAC413 shared the highest sequence similarity with members of the genus Ehrlichia (94.2-95.1, 80.9-83.1, 67.9-71.9 and 39.9-40.7 % similarity for the 16S rRNA gene, groESL, gltA and rpoB, respectively). No sequence variation in three sequences (16S rRNA gene, groESL and gltA) was observed between the RAC413 isolate and five additional sequences amplified from blood of naturally infected raccoons from several geographically isolated populations in the south-eastern USA. Serum samples from four experimentally infected raccoons did not react to Ehrlichia canis, Ehrlichia chaffeensis, Anaplasma marginale or Anaplasma phagocytophilum antigens in an immunofluorescence assay or an Ehrlichia ewingii peptide in an ELISA format. On the basis of the distinctive molecular and serological characteristics and apparent host specificity of this ehrlichial organism, it is proposed that this organism be designated 'Candidatus Neoehrlichia lotoris' (reference strain RAC413).

Raccoons (Procyon lotor), but not rodents, are natural and experimental hosts for an ehrlichial organism related to "Candidatus Neoehrlichia mikurensis".

"Candidatus Neoehrlichia mikurensis" has been reported from a variety of rodent and Ixodes tick species in Europe and Asia. Recently, an ehrlichial organism closely related to "Candidatus Neoehrlichia mikurensis" was cultured from a raccoon (Procyon lotor) from Georgia, USA. To determine prevalence and distribution, we conducted a molecular survey of free-ranging raccoons (n=197) from 10 populations in 3 states and found that infections were common in tick-infested populations (50-94%). In an effort to determine the host range of this organism, 10 species of rodents (n=137) trapped in 3 areas where positive raccoons had been detected were tested; all were negative. In addition, captive bred raccoons and several common laboratory animals (mice, rats, and rabbits) were inoculated with the raccoon ehrlichial isolate (strain RAC413). Raccoons became infected with the culture isolate but all other hosts were refractory to infection. The 16S rRNA gene sequence (1379bp) of the RAC413 isolate was most similar (98.4-98.8%) to members of the "Candidatus Neoehrlichia mikurensis" group and phylogenetic analysis confirmed this organism was related to, but distinct from, "Candidatus Neoehrlichia mikurensis". Based on the molecular and natural history uniqueness of this organism from raccoons, we propose that this represents a novel species in the "Candidatus Neoehrlichia" group of ehrlichial organisms.

Experimental and field studies on the suitability of raccoons (Procyon lotor) as hosts for tick-borne pathogens.

We investigated the experimental susceptibility and natural exposure of raccoons (Procyon lotor) to five tick-borne pathogens of human and veterinary importance, Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma phagocytophilum (ApVariant 1 and Ap-ha HGE-1 strains), and Borrelia lonestari. Infections were assessed by polymerase chain reaction (PCR), indirect fluorescent antibody (IFA) testing, and/or culture isolation methods for at least 30 days postinoculation (DPI). Two E. chaffeensis-inoculated raccoons seroconverted and were transiently PCR positive. One raccoon was culture positive. Laboratory raised Amblyomma americanum nymphs fed on a third infected raccoon failed to become infected. Two A. phagocytophilum (HGE-1)-inoculated raccoons became PCR positive and seroconverted. Both remained positive for at least 74 DPI. In contrast, raccoons inoculated with A. phagocytophilum (Ap-Variant 1) were only transiently PCR positive and only seroconverted with low titers. No evidence of infection was observed for E. ewingii- and B. lonestari-inoculated raccoons. Only one E. canis-inoculated raccoon was PCR positive 3 DPI. Serologic testing of wild raccoons from five populations (3 infested with ticks) in Georgia and Florida showed antibodies reactive with E. chaffeensis in the 3 tick-infested populations (range of 30%-46%), E. canis in the same three populations (8%-23%), A. phagocytophilum in a single raccoon from Florida (12%), and Borrelia spp. in all 5 populations (8%-53%). All raccoons were PCR negative for tick-borne pathogens. These data suggest that raccoons are likely not important reservoirs of E. canis, E. ewingii, or B. lonestari. However, raccoons are experimentally susceptible and naturally exposed to E. chaffeensis, and these data support the previous finding that raccoons may be involved in the natural history of A. phagocytophilum.

Isolation and establishment of the raccoon Ehrlichia-like agent in tick cell culture.

Feral animals are reservoirs of emerging human pathogens, as well as carriers of closely related wildlife diseases. The latter may interfere with epidemiologic studies by inducing cross-reactive antibodies, or by providing false positive signals in PCR based tests. We cultured a novel intracellular bacterium from the blood of two raccoons (Procyon lotor): RAC413 and RAC414. RAC413 had been experimentally inoculated with blood from a wild-caught raccoon, and provided the material for a blood passage into RAC414. The microbes grew in Ixodes scapularis (black-legged tick) cells, line ISE6, inoculated either with the leukocyte or erythrocyte fraction of anticoagulated blood. Giemsa-stained cells sampled two and three months after initial inoculation of the cultures revealed inclusions similar to those of Ehrlichia sp., except that individual bacteria commonly were elongated and clustered within endosomes. Electronmicroscopy confirmed the presence of irregularly shaped bacteria with evenly granular bacterioplasm bounded by a unit membrane. 16S rDNA sequencing identified the microbes as the raccoon Ehrlichia-like agent previously detected in feral raccoons from Georgia, United States. In conclusion, the availability of a culture isolate of this agent will facilitate future studies to determine its biology, epidemiologic significance, vector association, and host range. The Ehrlichia-like agent infecting raccoons joins a growing list of tick-borne agents cultivable in tick cells.

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.

Persistence of pseudorabies virus in feral swine populations.

Serologic surveys for evidence of exposure to pseudorabies virus (PRV) in feral swine were conducted from November 2001 to April 2002 at 10 sites in the southeastern United States, where evidence of previous PRV exposure had been documented during 1979-89. Sera were tested in the field on the day of collection by latex agglutination. Maximum sample size per site was to be 30 animals, but sampling was discontinued before reaching this number when positive results were obtained. Positive results were obtained at all of the study sites, demonstrating long-term persistence of PRV in feral swine populations. Overall, 38 of 100 (38%) animals were positive for antibodies. Consistent results from latex agglutination tests conducted in the field and laboratory demonstrated that this test was useful as a rapid and reliable diagnostic tool when used in the field.

First culture isolation of Borrelia lonestari, putative agent of southern tick-associated rash illness.

Southern tick-associated rash illness (STARI) is a Lyme disease-like infection described in patients in the southeastern and south-central United States, where classic Lyme disease is relatively rare. STARI develops following the bite of a lone star tick (Amblyomma americanum) and is thought to be caused by infection with an "uncultivable" spirochete tentatively named Borrelia lonestari. In this study, wild lone star ticks collected from an area where B. lonestari is endemic were cocultured in an established embryonic tick cell line (ISE6). The cultures were examined by dark-field microscopy for evidence of infection, and spirochete identity and morphology were evaluated by flagellin B and 16S rRNA gene sequence, by reaction to Borrelia-wide and B. burgdorferi-specific monoclonal antibodies, and by electron microscopy. Live spirochetes were first visualized in primary culture of A. americanum ticks by dark-field microscopy 14 days after the cell culture was inoculated. The sequences of the flagellin B and 16S rRNA genes of cultured spirochetes were consistent with previously reported sequences of B. lonestari. The cultured spirochetes reacted with a Borrelia-wide flagellin antibody, but did not react with an OspA antibody specific to B. burgdorferi, by indirect fluorescent antibody testing. Electron microscopy demonstrated organisms that were free and associated with ISE6 cells, with characteristic Borrelia sp. morphology. This study describes the first successful isolation of B. lonestari in culture, providing a much needed source of organisms for the development of diagnostic assays and forming a basis for future studies investigating the role of the organism as a human disease agent.