Differentiation of Ehrlichia platys and E. equi infections in dogs by using 16S ribosomal DNA-based PCR.

We have encountered a previously unrecognized specificity problem when using the small-subunit ribosomal DNA (16S rDNA)-based PCR primers recommended for use in the identification of Ehrlichia equi in clinical samples. These PCR primers annealed to E. platys 16S rDNA in blood samples containing high levels of E. platys organisms. Therefore, we designed and tested new PCR primers for the identification of E. equi.

Serologic and molecular evidence of coinfection with multiple vector-borne pathogens in dogs from Thailand.

Forty-nine dogs from Thailand were evaluated for serologic evidence of exposure or polymerase chain reaction (PCR) evidence of infection with vectorborne pathogens, including Ehrlichia sp. (Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, and Ehrlichia risticii), Bartonella vinsonii subsp. berkhoffi (Bvb), spotted fever group (SFG) rickettsiae (Rickettsia rickettsii), Typhus group (TG) rickettsiae (Rickettsia canada, Rickettsia prowazekii, and Rickettsia typhi), and Babesia sp. (Babesia canis and Babesia gibsonii). All study dogs had at least 1 of 3 entry criteria: fever, anemia, or thrombocytopenia. By immunofluorescence antibody (IFA) testing, seroreactivity was most prevalent to E chaffeensis (74%) and E canis (71%) antigens, followed by E equi (58%), Bvb (38%), E risticii (38%), R prowazekii (24%), B canis (20%), R rickettsii (12%), R canada (4%), and B gibsonii (4%) antigens. There was 100% concordance between E canis IFA and Western blot immunoassay (WI) for 35 of 35 samples; 2 samples were IFA and WI reactive only to E equi antigens. By PCR amplification, 10 dogs were found to be infected with E canis, 5 with Ehrlichia platys, and 3 with B canis. Sequencing of PCR products was undertaken to compare Ehrlichia strains from Thailand to strains originating from the United States. Partial DNA sequence analysis confirmed infection with E canis and E platys, with identical 16S rRNA sequence alignment to E canis (U26740) and to E platys (M83801), as reported in GenBank. Partial E canis P28.1 and P28.2 amino acid sequences from Thai dogs were divergent from analogous sequences derived from North American E canis (AF082744) strains, suggesting that the Thai dogs were infected with a geographically distinct strain of E canis compared to North American strains. The results of this study indicate that dogs in Thailand have substantial exposure to vectorborne diseases and that coinfection with these pathogens may be common.

Infection with Bartonella weissii and detection of Nanobacterium antigens in a North Carolina beef herd.

Very recently, Bartonella organisms have been isolated from large ruminants (deer, elk, and dairy and beef cattle) located in the United States and in France. In this study, we report the serologic, microbiologic, and molecular findings related to the isolation of a Bartonella species in North Carolina beef cattle and the detection of nanobacterial antigen using a commercially available enzyme-linked immunosorbent assay. Between August 1998 and September 1999, blood was collected from 38 cattle ranging in age from 1 month to 6.5 years. After a 1-month incubation period, a Bartonella sp. was isolated on a 5% rabbit blood agar plate from three of six EDTA blood samples. PCR amplification of the 16S rRNA gene from all three isolates resulted in a DNA sequence that was 100% identical to that of B. weissii 16S rRNA (GenBank no. AF199502). By IFA testing, 36 of 38 cattle had antibodies (> or =1:64) to Bartonella weissii (bovine origin) antigens. Nanobacterial antigen was detected in 22 of 22 serum samples. We conclude that infection with an organism similar or closely related to B. weissii can occur in North Carolina cattle and that although their actual existence is still controversial Nanobacterium antigens were detected with a commercially available test kit. The epidemiology, vector biology, and potential pathogenicity of these organisms in cattle deserve future consideration.

Coinfection with three Ehrlichia species in dogs from Thailand and Venezuela with emphasis on consideration of 16S ribosomal DNA secondary structure.

As part of a larger study to investigate tick-borne infections in dogs from Thailand and Venezuela, documentation of coinfection with three Ehrlichia species in two dogs, one from each country, became the focus of the present study. Although neither dog had clinical signs attributable to ehrlichiosis, both dogs were anemic and neutropenic and the Thai dog was thrombocytopenic. Genus- and species-specific PCR targeting the 16S rRNA genes indicated that both dogs were coinfected with Ehrlichia canis, E. platys, and E. equi. To our knowledge, these results provide the first molecular documentation for the presence of E. equi in dogs from these countries. Using universal bacterial PCR primers, one nearly full-length 16S rRNA gene could be amplified from each dog. The sequences were identical to each other and almost identical to that of E. platys (AF156784), providing the first E. platys 16S ribosomal DNA (rDNA) sequences reported from these two geographically divergent countries. To determine whether these sequence differences allow differentiation between these two strains and other published 16S rDNA E. platys sequences, we performed a phylogenetic analysis of the rRNA, incorporating the consideration of secondary structure.

Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina.

Both dogs and humans can be coinfected with various Ehrlichia, Bartonella, Rickettsia, and Babesia species. We investigated a kennel of sick Walker Hounds and their owners in southeastern North Carolina for evidence of tick-borne infections and associated risk factors. A high degree of coinfection was documented in the dog population. Of the 27 dogs, 26 were seroreactive to an Ehrlichia sp., 16 to Babesia canis, and 25 to Bartonella vinsonii, and 22 seroconverted to Rickettsia rickettsii antigens. According to PCR results, 15 dogs were infected with Ehrlichia canis, 9 with Ehrlichia chaffeensis, 8 with Ehrlichia ewingii, 3 with Ehrlichia equi, 9 with Ehrlichia platys, 20 with a Rickettsia species, 16 with a Bartonella species, and 7 with B. canis. The detection of DNA from any Ehrlichia species was associated with clinical illness and with concurrent B. canis infection (by PCR). Both E. canis and an uncharacterized Rickettsia species appeared to result in chronic or recurrent infection. Death in the dog population was associated with living in a dirt lot rather than the concrete kennel. Of 23 people on whom serologic testing was conducted, eight were seroreactive to Bartonella henselae, one to E. chaffeensis, and one to R. rickettsii antigen; however, none had clinical or hematologic abnormalities consistent with illness caused by these organisms. We conclude that kennel dogs with heavy tick exposure can be infected at a high rate with multiple, potentially zoonotic, tick-borne pathogens. In addition, our findings further illustrate the utility of PCR for documenting coinfection with tick-transmitted pathogens.

Efficacy of doxycycline, azithromycin, or trovafloxacin for treatment of experimental Rocky Mountain spotted fever in dogs.

Dogs were experimentally inoculated with Rickettsia rickettsii (canine origin) in order to compare the efficacies of azithromycin and trovafloxacin to that of the current antibiotic standard, doxycycline, for the treatment of Rocky Mountain spotted fever. Clinicopathologic parameters, isolation of rickettsiae in tissue culture, and PCR amplification of rickettsial DNA were used to evaluate the response to therapy or duration of illness (untreated infection control group) in the four groups. Concentrations of the three antibiotics in plasma and blood cells were measured by high-performance liquid chromatography. Doxycycline and trovafloxacin treatments resulted in more-rapid defervescence, whereas all three antibiotics caused rapid improvement in attitudinal scores, blood platelet numbers, and the albumin/total-protein ratio. Based upon detection of retinal vascular lesions by fluorescein angiography, trovafloxacin and doxycycline substantially decreased rickettsia-induced vascular injury to the eye, whereas the number of ocular lesions in the azithromycin group did not differ from that in the infection control group. As assessed by tissue culture isolation, doxycycline resulted in the earliest apparent clearance of viable circulating rickettsiae; however, rickettsial DNA could still be detected in the blood of some dogs from all four groups on day 21 postinfection, despite our inability to isolate viable rickettsiae at that point. As administered in this study, trovafloxacin was as efficacious as doxycycline but azithromycin proved less efficacious, possibly due to the short duration of administration.

Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii.

Historically, disease manifestations in dogs seroreactive to Ehrlichia canis antigens by indirect immunofluorescent antibody testing have been attributed to infection with either E. canis or Ehrlichia ewingii. A 1996 study by Dawson and colleagues provided PCR evidence that healthy dogs from southeastern Virginia could be naturally infected with Ehrlichia chaffeensis. This observation stimulated us to determine which Ehrlichia spp. infected sick dogs that were referred to our hospital from the same region. Based upon PCR amplification with species-specific primers, sick dogs seroreactive to E. canis antigens were determined to be infected with four Ehrlichia species: E. canis, E. chaffeensis, E. equi, and E. ewingii. Coinfection with three Ehrlichia species (E. canis, E. ewingii, and E. equi) was documented for one dog. An additional canine pathogen presumed to be tick transmitted, Bartonella vinsonii subsp. berkhoffii, was identified in 7 of 12 dogs. Importantly, our results indicate that in naturally infected dogs, E. chaffeensis can cause severe disease manifestations that are clinically and serologically indistinguishable from disease manifestations of E. canis or E. ewingii. In addition, our findings support the efficacy of doxycycline for treatment of E. canis, E. equi, and E. ewingii infections but indicate that, based upon the persistence of E. chaffeensis DNA for 1 year following treatment, E. chaffeensis infection in dogs may be more refractory to doxycycline treatment. Undetected coinfection with Bartonella may also complicate the evaluation of treatment efficacy while resulting in disease manifestations that mimic ehrlichiosis.

Doxycycline hyclate treatment of experimental canine ehrlichiosis followed by challenge inoculation with two Ehrlichia canis strains.

Dogs were experimentally inoculated with Ehrlichia canis Florida to assess the efficacy of doxycycline hyclate for the treatment of acute ehrlichiosis. Treatment with doxycycline eliminated infection in eight of eight dogs. Untreated infected control dogs appeared to eliminate the infection or, alternatively, suppress the degree of ehrlichiemia to a level not detectable by tissue culture isolation or PCR or by transfusion of blood into recipient dogs. Prior infection did not infer protection against homologous (strain Florida) or heterologous (strain NCSU Jake) strains of E. canis. We conclude that doxycycline hyclate is an effective treatment for acute E. canis infection; however, these results may not be applicable to chronic infections in nature. Spontaneous resolution of infection, induced by the dog's innate immune response, provides evidence that an E. canis vaccine, once developed, might potentially confer protective immunity against the organism.

Effect of a live attenuated intranasal vaccine on latency and shedding of feline herpesvirus 1 in domestic cats.

A prospective study was conducted that evaluated duration of virus shedding through acute and experimentally-induced recurrent disease episodes in 12 cats, and tissue distribution of latent infections, following intranasal vaccination with a temperature sensitive (ts) mutant strain of feline herpesvirus 1 (FHV1). Six of these cats were challenged with a virulent field strain of the agent to assess the extent to which vaccination affected subsequent shedding of virus and the establishment of latent infections. Virus isolation (VI) tests were done in parallel with a polymerase chain reaction (PCR) assay to compare the performance of each diagnostic method. The PCR confirmed that all 12 cats shed virus throughout the periods of vaccination, challenge or mock-challenge, and a cyclophosphamide-dexamethasone stress protocol to reactivate latent infections. Shedding to the tsFHV1 was documented by VI for up to 25 days following vaccination and for up to 15 days following challenge, but not after experimental stress. Overall, FHV1 was present in 144 of 300 (48%) cat-days of testing by PCR compared to 32 of 300 (11%) by VI. The frequency and distribution of latent FHV1 detected in neurologic, ophthalmic, and other tissues by PCR were identical for vaccine-only and vaccine-challenge groups, thereby disproving previous hypotheses that tsFHV1 mutants administered by this route protect against latency.