Susan I. Hancock

Molecular Evidence Supporting Ehrlichia canis‐Like Infection in Cats

Currently, the pathogenic role of Ehrlichia canis in cats has been proposed predominantly on the basis of the serologic evidence of natural infection and the infrequent detection of morulae‐like structures within the cytoplasm of leukocytes in cats. The purpose of this report was to provide molecular evidence supporting E cams‐like infection in 3 cats that had clinical manifestations consistent with canine ehrlichiosis but lacked antibodies to E canis antigens. Serum from all 3 cats contained antinuclear antibodies (ANAs). The predominant disease manifestation was polyarthritis in 1 cat and bone marrow hypoplasia or dysplasia, accompanied by pancytopenia or anemia and thrombocytopenia, in 1 cat each. The alignment of E canis partial 16S ribosomal DNA (rDNA; 382 nucleotide positions), amplified from EDTA blood samples from each cat, was identical to each other and was identical to a canine isolate of E canis (GenBank accession number AF373613). In 1 cat, concurrent treatment with corticosteroids may have interfered with the therapeutic effectiveness of doxycycline for the elimination of E canis‐like infection. To further define the spectrum of ehrlichiosis in cats, polymerase chain reaction (PCR) testing may be necessary until serologic testing is thoroughly validated in experimentally or naturally infected cats. In addition, until E canis has been isolated from cats and several tissue culture isolates are available from disparate geographic regions for detailed comparative genetic study, the molecular evidence presented in this study supporting E canis‐like infection in cats must be interpreted with caution.

Doxycycline Hyclate Treatment of Experimental Canine Ehrlichiosis Followed by Challenge Inoculation with Two Ehrlichia canis Strains

ABSTRACT 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 acuteE. 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.

Coinfection with Three Ehrlichia Species in Dogs from Thailand and Venezuela with Emphasis on Consideration of 16S Ribosomal DNA Secondary Structure

ABSTRACT 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 ofE. 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.

Bartonella henselae and Bartonella elizabethae as Potential Canine Pathogens

ABSTRACT Bartonella henselae or Bartonella elizabethae DNA from EDTA-anticoagulated blood samples obtained from four dogs was amplified and sequenced. The results showed that B. elizabethae should be added to the list of Bartonella species (i.e., B. vinsonii subsp. berkhoffii, B. henselae, and B. clarridgeiae) that are currently recognized as infectious agents in dogs. Furthermore, these results may have potential zoonotic implications, particularly if dogs can serve as a previously unrecognized reservoir for B. henselae. Although the clinical relevance of these observations remains to be determined, it is possible that molecular diagnostic techniques such as PCR may help to implicate a spectrum of Bartonella spp. as a cause of or a cofactor in chronic canine and human diseases of poorly defined causation.

Efficacy of Doxycycline, Azithromycin, or Trovafloxacin for Treatment of Experimental Rocky Mountain Spotted Fever in Dogs

ABSTRACT 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.

Infection with Bartonella weissii and Detection of Nanobacterium Antigens in a North Carolina Beef Herd

ABSTRACT 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 (≥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 toB. weissii can occur in North Carolina cattle and that although their actual existence is still controversialNanobacterium antigens were detected with a commercially available test kit. The epidemiology, vector biology, and potential pathogenicity of these organisms in cattle deserve future consideration.

Naturally Occurring Ehrlichia chaffeensis Infection in Two Prosimian Primate Species: Ring-tailed Lemurs (Lemur catta) and Ruffed Lemurs (Varecia variegata)

A naturally occurring infection of Ehrlichia chaffeensis in lemurs is described. DNA of Ehrlichia chaffeensis was identified by polymerase chain reaction in peripheral blood from six of eight clinically ill lemurs. Organisms were cultured from the blood of one lemur exhibiting clinical and hematologic abnormalities similar to those of humans infected with E. chaffeensis.

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

ABSTRACT 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 toE. 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.