J. Mitchell Lockhart

TEMPORAL ASSOCIATION OF AMBLYOMMA AMERICANUM WITH THE PRESENCE OF EHRLICHIA CHAFFEENSIS REACTIVE ANTIBODIES IN WHITE-TAILED DEER

From 1981 through 1993, tick infestations and serum antibodies reactive to Ehrlichia chaffeensis, the causative agent of human ehrlichiosis, were monitored among white-tailed deer (Odocoileus virginianus) at Whitehall Experimental Forest, Clarke County, Georgia (USA). Neither ticks nor E. chaffeensis antibodies were detected during the first two years of the study. Infestations of the lone star tick (Amblyomma americanum), a suspected vector of E. chaffeensis, first were noted on deer in 1983. Prevalence and intensity of A. americanum sharply increased from 1985 to 1989, and prevalence was 100% from 1990 to 1993. Antibodies reactive to E. chaffeensis were first detected in 7% of deer sampled in 1986. Antibody prevalence increased to 21% in 1987 and was 100% from 1988 to 1993. This temporal association between the establishment of A. americanum and the appearance of E. chaffeensis antibodies provides evidence to support the concept that A. americanum could be a natural vector of E. chaffeensis. The high prevalence of antibodies among all age classes of deer also reaffirms that white-tailed deer may be sensitive natural sentinels for monitoring the distribution of E. chaffeensis.

Persistent Ehrlichia chaffeensis infection in white-tailed deer.

Four white-tailed deer (Odocoileus virginianus) were inoculated intravenously with a deer-origin isolate (15B-WTD-GA) of Ehrlichia chaffeensis. The course of infection was monitored using indirect fluorescent antibody (IFA), polymerase chain reaction (PCR), and culture over a 9 m period. All deer became rickettsemic within 24 days post inoculation (DPI), and all developed antibody titers >1:64 to E. chaffeensis by 17 DPI. Titers in all deer fell below 1:64 during 87 to 143 DPI. One deer exhibited a second period of seropositivity (peak titer of 1:256) from 207 to 271 DPI but was culture and PCR negative during this period. Rickettsemia was confirmed by reisolation of E. chaffeensis as late as 73 to 108 DPI in three deer. Positive PCR results were obtained from femur bone marrow of one deer and from rumenal lymph node of another deer at 278 DPI. None of the deer developed clinical signs, hematologic abnormalities, or gross or microscopic lesions attributable to E. chaffeensis. Two uninoculated control deer were negative on all tests through 90 DPI at which time they were removed from the study. Herein we confirm that white-tailed deer become persistently infected with E. chaffeensis, have initial rickettsemias of several weeks duration and may experience recrudescence of rickettsemia, which reaffirm the importance of deer in the epidemiology of E. chaffeensis.

Development and use of specific polymerase reaction for the detection of an organism resembling Ehrlichia sp. in white-tailed deer.

The role of white-tailed deer (Odocoileus virginianus) in the epidemiology of Ehrlichia chaffeensis and the agent of human granulocytic ehrlichiosis (HGE) is not fully understood, and diagnostic procedures may be complicated by the recent detection of 16S rDNA sequence from an Ehrlichia sp.-like organism in wild deer. A specific forward primer (DGA) and an Ehrlichia spp. reverse primer (GA1UR) were constructed to amplify this new, distinct Ehrlichia sp.-like 16S rDNA. The DGA primer, a forward primer specific for E. chaffeensis (DCH), and a forward primer specific for the E. phagocytophila genogroup (GE9f) were each used with GA1UR in nested polymerase chain reactions to amplify 16S rDNA sequences from control samples containing the deer Ehrlichia sp.-like organism, E. chaffeensis, or the HGE agent. Primer pairs DGA/GA1UR and DCH/GA1UR specifically amplified 16S rDNA sequences from the corresponding target organism, whereas GE9f/GA1UR amplified 16S rDNA sequence from both the HGE agent and the deer Ehrlichia sp.-like organism. With a nested PCR using DGA/GA1UR and DCH/GA1UR on DNA extracted from white blood cells from 62 deer from 10 populations in four U.S. states, we observed a high prevalence (65%) of 16S rDNA sequences of the deer Ehrlichia sp.-like organism, and a low prevalence (5%) of the E. chaffeensis sequence. In this field survey, E. chaffeensis-reactive antibodies detected by indirect fluorescence assays were associated (P < 0.001) with PCR evidence of the deer Ehrlichia sp.-like organism, but not E. chaffeensis. Infestations of Amblyomma americanum also were associated (P < 0.001) with PCR evidence of the deer Ehrlichia sp.-like organism. The potential for serologic cross-reactions and non-specific PCR products arising from the deer Ehrlichia sp.-like organism should be considered when evaluating the role of deer and their ticks in the epidemiology of ehrlichial pathogens of humans.

SUSCEPTIBILITY OF RED AND GRAY FOXES TO INFECTION BY EHRLICHIA CHAFFEENSIS

Red foxes (Vulpes vulpes) and gray foxes (Urocyon cinereoargenteus) were evaluated for their susceptibility to experimental infection with Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis. Two red foxes and three gray foxes were inoculated intravenously with E. chaffeensis (15B-WTD-GA strain) and were monitored at 7, 14, 21, and 28 days post inoculation (DPI) for evidence of infection using an indirect fluorescent antibody (IFA) assay, light microscopy, polymerase chain reaction (PCR), and cell culture methods. One red fox and one gray fox served as negative controls. Red foxes were susceptible to infection based on reisolation of E. chaffeensis from blood at 7 and 14 DPI, seroconversion by 7 DPI, and positive PCR assays on spleen and lymph nodes at 28 DPI. Morulae were not found in circulating leukocytes and clinical signs or lesions of ehrlichiosis were not observed. In contrast, gray foxes were refractory to infection based on negative results on all culture, PCR, serologic, and microscopic examinations. These findings imply that red foxes, but not gray foxes, are potential vertebrate reservoirs for E. chaffeensis. These findings also illustrate the need to verify serologic evidence of E. chaffeensis infection among wild animals.

Lack of seroreactivity to Ehrlichia chaffeensis among rodent populations.

A retrospective serosurvey for antibodies to Ehrlichia chaffeensis was conducted on eight species of wild rodents (Mus musculus, Oryzomys palustris, Peromysctts leucopns, Rattus norvegicus, Reithrodontomys humulis, Sciurus carolinensis, Sciurus niger, and Sigmodon hispidus) from the southeastern United States. Serum samples (n = 281) collected between 1973 and 1993 were evaluated using an indirect fluorescent antibody test. All samples, screened at a dilution of 1:32, were negative for antibodies to E. chaffeensis. Sixty-three percent of the rodents tested were from areas where E. chaffeensis has been confirmed or is strongly suspected to be endemic. These data suggest limited or no involvement of rodents in the epidemiology of E. chaffeensis.

A recombinant antigen from the heartwater agent (Cowdria ruminatium) reactive with antibodies in some southeastern United States white-tailed deer (Odocoileus virginianus), but not cattle, sera.

Recombinant baculovirus techniques were used to express the 260 amino acid carboxyterminal portion of the 32 kilodalton (kDa) major antigenic protein (MAP 1) of Cowdria ruminantium, the heartwater agent, as a fusion protein. The recombinant MAP 1 was fused to an aminoterminal independently antigenic octapeptide sequence (FLAG® peptide). Recombinant MAP 1 was used as an immunoblotting antigen to evaluate numerous reference antisera against organisms of the tribe Ehrlichieae. Monoclonal and polyclonal C. ruminantium antibodies, monoclonal anti-FLAG® ascites, and antisera to Ehrlichia canis and Ehrlichia chaffeensis reacted with this antigen. Twelve of 79 sera collected 1980 to 1992 from southeastern U.S. white-tailed deer (Odocoileus virginianus) were also unexpectedly immunoblot-positive to MAP 1. These 12 deer sera had, as a group, significantly (P <0.01) greater anti-E. chaffeensis titers (previously determined) than the sera from MAP 1 immunoblot-negative deer living in the same areas. None of the 262 sera from cattle living in the same areas were immunoblot-positive to MAP 1. All of an additional 50 cervine sera from Michigan (USA), 72 bovine sera from northern U.S. cattle, and 72 sera from Puerto Rican cattle were also immunoblot-negative to MAP 1. Sera from African sheep which were falsely seropositive to authentic MAP 1 were also immunoblot-positive to the recombinant MAP 1. Unidentified Ehrlichia spp. capable of serologic crossreactivity with the heartwater agent appear to be present in some southeastern U.S. white-tailed deer but not cattle. These or related Ehrlichia spp. may also be found elsewhere in the world in non-cervine species.

Evaluation of C3H/HeJ Mice for Xenodiagnosis of Infection with Ehrlichia Chaffeensis

Because mice are experimentally susceptible to infection with Ehrlichia species, C3H/HeJmice were evaluated as a potential xenodiagnostic model for detection of infection with and isolation of E. chaffeensis. Intraperitoneal inoculation of mice with E. chaffeensis-infected DH82 cell cultures produced seroconversion, with peak serum antibody titers of 1:256, at high dosages (>1.9 × 104 infected cells) but not at low dosages (1.9 or 1.9 × 102 infected cells). Ehrlichia chaffeensis was not reisolated from blood samples collected from inoculated mice on postinoculation day 21. Nested polymerase chain reaction (PCR), using primers specific for E. chaffeensis, was positive for only 2/70 (2.9%) tissue samples. A field evaluation in which C3H/HeJ mice were inoculated with blood and lymph node suspensions from 5 seropositive white-tailed deer, including 3 deer that were PCR positive for E. chaffeensis, failed to produce seroconversion in mice. The lack of seroconversion at low dosages, the failure to reisolate at any dosage, and the inability to confirm infection in PCR-positive field samples suggests C3H/HeJ mice are not a sensitive model for xenodiagnosis or detection of E. chaffeensis.

Salmonella from gopher tortoises (Gopherus polyphemus) in south Georgia.

From 2002 to 2006, gopher tortoises (Gopherus polyphemus) were collected at Moody Air Force Base, Lowndes/Lanier counties, Georgia, USA, and opportunistically surveyed for the presence of Salmonella species. Four of 155 (2.6%) cloacal swabs collected from 80 tortoises were positive for the presence of Salmonella enterica, and the following serovars were identified: Give, Hartford, Javiana, and Luciana. Female tortoises (5%) were infected at a rate similar to male tortoises (5%). All isolates were obtained from adult tortoises (n=73); subadults (n=7) were all negative. Each isolated serovar is a potential human pathogen, suggesting appropriate precautions should be emphasized when handling these animals.

SEROLOGIC SURVEY OF WILD TURKEYS (MELEAGRIS GALLOPAVO) AND EVIDENCE OF EXPOSURE TO AVIAN ENCEPHALOMYELITIS VIRUS IN GEORGIA AND FLORIDA, USA

Abstract Wild Turkeys (Meleagris gallopavo) are susceptible to many of the same diseases as domestic turkeys. Before 2005, most Wild Turkeys in southern Georgia, US, had little or no exposure to commercial poultry operations. As part of a pathogen survey examining the effects of commercial poultry on Wild Turkeys, samples were collected from Wild Turkeys from March 2005 through May 2008. The turkeys were collected from 13 counties in southern Georgia and Madison County, Florida, and tested for antibodies to various pathogens of poultry. Three (13%) of the turkeys were positive for antibodies to Salmonella. Thirteen turkeys (54%) were positive for Newcastle disease virus antibodies, and 15 turkeys (63%) were positive for antibodies to reticuloendotheliosis virus. One turkey (4%) from Madison County was positive for avian encephalomyelitis virus antibodies.

Severe Moniliformiasis (Acanthocephala: Moniliformidae) in a Gray Squirrel, Sciurus carolinensis, from Arkansas, USA

Three hundred and seventy-five acanthocephalans, Moniliformis clarki, were removed from the small and large intestines of a gray squirrel from Arkansas County, Arkansas (USA). This is the first report of M. clarki from Arkansas. Enteric lesions, including distension, perforating ulcers, enteritis, crypt hypertrophy, goblet cell hyperplasia, and occlusions of the intestinal tract were observed, indicating the pathogenic potential of this parasite.