Distribution and prevalence of Cytauxzoon felis in bobcats (Lynx rufus), the natural reservoir, and other wild felids in thirteen states.

Cytauxzoon felis, a protozoan parasite of wild and domestic felids, is the causative agent of cytauxzoonosis in domestic and some exotic felids in the United States. The bobcat (Lynx rufus) is the natural reservoir for this parasite, but other felids such as Florida panthers (Puma concolor coryii) and domestic cats may maintain long-term parasitemias and serve as reservoirs. Experimentally, two tick species, Dermacentor variabilis and Amblyomma americanum, have demonstrated the ability to transmit C. felis. These two tick species have overlapping distributions throughout much of the southeastern United States. The objective of the current study was to determine the distribution and prevalence of C. felis in free-ranging bobcat populations from 13 states including California, Colorado, Florida, Georgia, Kansas, Kentucky, Missouri, North Carolina, North Dakota, Ohio, Oklahoma, South Carolina, and West Virginia. These states were selected because of differential vector presence; D. variabilis is present in each of these states except for the region of Colorado sampled and A. americanum is currently known to be present only in a subset of these states. Blood or spleen samples from 696 bobcats were tested for C. felis infection by a polymerase chain reaction (PCR) assay which targeted the first ribosomal internal transcribed spacer region (ITS-1). Significantly higher prevalences of C. felis were detected from Missouri (79%, n=39), North Carolina (63%, n=8), Oklahoma (60%, n=20), South Carolina (57%, n=7), Kentucky (55%, n=74), Florida (44%, n=45), and Kansas (27%, n=41) compared with Georgia (9%, n=159), North Dakota (2.4%, n=124), Ohio (0%, n=19), West Virginia (0%, n=37), California (0%, n=26), and Colorado (0%, n=67). In addition to bobcats, seven cougars (Puma concolor) from Georgia, Louisiana, and North Dakota and one serval (Leptailurus serval) from Louisiana were tested for C. felis. Only one cougar from Louisiana was PCR positive, which represents the first report of an infected cougar outside of the Florida panther population. These data also indicate that C. felis is present in North Dakota where infection has not been reported in domestic cats. Based on a nonparametric analysis, prevalence rates were significantly higher in states where there are established populations of A. americanum, which supports recent data on the experimental transmission of C. felis by A. americanum and the fact that domestic cat clinical cases are temporally associated with A. americanum activity. Collectively, these data confirm that bobcats are a common reservoir for C. felis and that A. americanum is likely an epidemiologically important vector.

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.

Rocky mountain spotted fever in the United States, 1997-2002.

The increased incidence of Rocky Mountain spotted fever (RMSF) in 1997-2002 compared with previous years may be related to enhanced awareness and reporting of RMSF as well as changes in human-vector interaction. However, reports on RMSF mortality underscore the need for physician vigilance in considering a diagnosis of RMSF for febrile individuals potentially exposed to ticks and stress the importance of treating such persons regardless of the presence of a rash.

Vaccination of small Asian mongoose (Herpestes javanicus) against rabies.

Oral vaccination of free-ranging wildlife is a promising technique in rabies control. The small Asian mongoose (Herpestes javanicus) is an important reservoir of rabies on several Caribbean islands, but no vaccines have been evaluated for this species. Captive mongooses were used to test the safety and efficacy of the commercially licensed vaccinia-rabies glycoprotein (V-RG) recombinant vaccine and a newly developed genetically engineered oral rabies virus vaccine (SPBNGA-S). In one study using V-RG, no vaccinated animals developed detectable rabies virus-neutralizing antibodies, and all but one died after experimental challenge with rabies virus. In contrast, all animals given SPBNGA-S demonstrated seroconversion within 7 to 14 days after vaccination and survived rabies virus challenge. On the basis of these preliminary results indicating the greater efficacy of SPBNGA-S vs. V-RG vaccine, additional investigations will be necessary to determine the optimal dose and duration of vaccination, as well as incorporation of the SPBNGA-S vaccine into edible bait.

Molecular detection and characterization of Cytauxzoon felis and a Babesia species in cougars from Florida.

Piroplasms, morphologically indistinguishable from Cytauxzoon felis, previously were detected in 36% of cougars in Florida. We utilized a nested 18S rRNA assay, which amplifies DNA from all piroplasms, to screen blood samples collected from 41 cougars from Florida (39 native Florida panthers [Puma concolor coryi] and two translocated Texas cougars [P. c. stanleyana]) from 1989-2005. Thirty-nine of the 41 cougars (95%) were positive for piroplasms; however, sequence analysis and restriction enzyme digestion revealed that only five were positive for C. felis. Samples from 32 cougars were positive for a Babesia sp. Two cougars were co-infected with both C. felis and the Babesia sp. Phylogenetic analysis of 18S rRNA gene sequence indicated that the Florida panther Babesia sp. was most closely related to a Babesia sp. reported from Ixodes ovatus from Japan, Babesia divergens, and Babesia odocoilei. This study indicates that Florida panthers harbor two distinct piroplasms, C. felis and a Babesia sp., and that some individuals are infected with both organisms. The infectivity and pathogenicity of this Babesia sp. for domestic cats is unknown. This represents the first report of a feline Babesia sp. in North America.

Rocky Mountain spotted fever in the United States, 1997-2002.

Rocky Mountain spotted fever (RMSF) is the most commonly reported fatal tick-borne disease in the United States. During 1997-2002, 3,649 cases of RMSF were reported to the Centers for Disease Control and Prevention via the National Electronic Telecommunications System for Surveillance; 2,589 case report forms, providing supplemental information, were also submitted. The average annual RMSF incidence during 1997-2002 was 2.2 cases/million persons. The annual incidence increased during 1997-2002 to a rate of 3.8 cases/million persons in 2002. The incidence was lowest among persons aged<5 and 10-29 years, and highest among adults aged 60-69 years. The overall case-fatality rate was 1.4%; the rate peaked in 1998 at 2.9% and declined to 0.7% in 2001 and 2002. Children<5 years of age had a case-fatality rate (5%) that was significantly greater than the rates for age groups<60 years of age, except for that for 40-49 years of age. Continued national surveillance is needed to assess the effectiveness of prevention efforts and early treatment in decreasing severe morbidity and mortality associated with RMSF.

Scrub typhus, Republic of Palau.

Scrub typhus, caused by Orientia tsutsugamushi, is a severe febrile illness transmitted to humans by trombiculid mites, which normally feed on rodents. The first known outbreak of scrub typhus in Palau occurred in 2001 to 2003 among residents of the remote southwest islands. To determine the extent of scrub typhus distribution in Palau, we tested serum samples from humans and rodents for antibodies to O. tsutsugamushi. Of 212 Palau residents surveyed in 2003, 101 (47.6%) had immunoglobulin G (IgG) antibody titers >1:64, and 56 (26.4%) had concurrent IgG and IgM antibody titers >1:512 and 1:64, respectively. Of 635 banked serum samples collected from Palau residents in 1995, 34 (5.4%) had IgG antibody titers >1:64. Sera collected from rodents (Rattus norvegicus and R. rattus) in 2003 and 2005 were tested, and 18 (28.6%) of 63 had IgG antibody titers >1:64. These findings suggest that scrub typhus is endemic in Palau.