Barbara C. Hegarty

Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene.

Ehrlichia species are the etiological agents of emerging and life-threatening tick-borne human zoonoses, in addition to causing serious and fatal infections in companion animals and livestock. We developed the first tricolor TaqMan real-time polymerase chain reaction assay capable of simultaneously detecting and discriminating medically important ehrlichiae in a single reaction. Analytical sensitivity of 50 copies per reaction was attained with templates from Ehrlichia chaffeensis, Ehrlichia ewingii, and Ehrlichia canis by amplifying the genus-specific disulfide bond formation protein gene (dsb). Ehrlichia genus-specific dsb primers amplified DNA from all known Ehrlichia species but not from other rickettsial organisms including Anaplasma platys, Anaplasma phagocytophilum, Rickettsia conorii, or Rickettsia typhi. High species specificity was attained as each species-specific TaqMan probe (E. chaffeensis, E. ewingii, and E. canis) identified homologous templates but did not cross-hybridize with heterologous Ehrlichia templates at concentrations as high as 10(8) copies. Identification of E. chaffeensis, E. ewingii, and E. canis from natural and experimental infections, previously confirmed by polymerase chain reaction and serological or microscopic evidence, demonstrated the comparable specificity and sensitivity of the dsb real-time assay. This assay provides a powerful tool for prospective medical diagnosis for human and canine ehrlichioses and for ecologic and epidemiological studies involving arthropod and mammalian hosts.

Serological and Molecular Prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia Species in Dogs from Minnesota

A population of 731 naturally exposed pet dogs examined at a private practice in Baxter, Minnesota, an area endemic for Lyme disease and anaplasmosis, was tested by serological and molecular methods for evidence of exposure to or infection with selected vector-borne pathogens. Serum samples were tested by enzyme-linked immunosorbent assay (ELISA) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and for Dirofilaria immitis antigen. Blood samples from 273 dogs were also analyzed by polymerase chain reaction (PCR) for Anaplasma and Ehrlichia species DNA. Based on the owner history and the attending veterinarians physical examination findings, dogs exhibiting illness compatible with anaplasmosis or borreliosis were considered clinical cases, and their results were compared to the healthy dog population. Antibodies to only A. phagocytophilum were detected in 217 (29%) dogs; to only B. burgdorferi, in 80 (11%) dogs; and seroreactivity to both organisms, in 188 (25%) dogs. Of 89 suspected cases of canine anaplasmosis or borreliosis, A. phagocytophilum or B. burgdorferi antibodies were detected in 22 dogs (25%) and 8 dogs (9%) respectively, whereas antibodies to both organisms were found in 38 dogs (43%). Ehrlichia canis antibodies and D. immitis antigen were each detected in 11 (1.5%) dogs. Anaplasma phagocytophilum DNA was amplified from 7 of 222 (3%) healthy dogs and 19 of 51 (37%) clinical cases. Seroreactivity to both A. phagocytophilum and B. burgdorferi was detected more frequently in suspected cases of anaplasmosis and/or borreliosis than seroreactivity to either organism alone. Based on PCR testing, A. phagocytophilum DNA was more prevalent in suspected cases of anaplasmosis or borreliosis than in healthy dogs from the same region.

Chronic Canine Ehrlichiosis (Ehrlichia canis): A Retrospective Study of 19 Natural Cases

Nineteen dogs from Greece with chronic ehrlichiosis were studied. The dogs exhibited bicytopenia or pancytopenia, bone marrow hypoplasia, seroreactivity to Ehrlichia canis (E. canis) antigens, and had no history of drug or radiation exposure. Anorexia, depression, severe bleeding tendencies, hypoalbuminemia, and increased serum alanine aminotransferase activity were also hallmarks of the disease. All these animals eventually died, irrespective of the treatment applied. Some dogs were also serologically positive for Rickettsia conorii, Leishmania infantum (L. infantum), and Bartonella vinsonii subspp. berkhoffii. Polymerase chain reaction testing of bone marrow samples revealed E. canis, Anaplasma phagocytophilia, Anaplasma platys, and L. infantum in some dogs. Concurrent infections did not appear to substantially influence the clinical course and final outcome of the chronic canine ehrlichiosis.

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.

A survey of tick-borne bacteria and protozoa in naturally exposed dogs from Israel

Antibody reactivity against seven bacterial or protozoal pathogens was measured in sera derived from 40 dogs suspected of a tick-borne disease. Sera from 73% (29/40) of the dogs reacted with three or more test antigens. Seroreactivity was most prevalent to Babesia canis antigen (90%) followed by Babesia gibsoni (75%), Ehrlichia canis (63%), Rickettsia conorii--Moroccan strain (58%), Rickettsia conorii--Israeli strain no. 2 (28%), Borrelia burgdorferi (10%) or Bartonella vinsonii (berkhoffii) (10%). Seroconversion documented in seven dogs, supported an acute phase diagnosis of ehrlichiosis in four dogs, R. conorii infection in three dogs and babesiosis in one dog. In the remaining dogs, correlation of clinical abnormalities with increased seroreactivity was not established through the design of this study. Although Lyme borreliosis has not been reported in people in Israel, Western blot analysis for antibodies reactive to B. burgdorferi identified genus-specific antiflagellin antibodies indicating that dogs in Israel are exposed to a Borrelia species. Identification of species-specific seroreactivity was not possible and infection with a Borrelia species other than B. burgdorferi is likely. Seroreactivity to B. vinsonii (berkhoffii) in dogs outside the USA is reported here for the first time.

Evaluation of conventional and real-time PCR assays for detection and differentiation of Spotted Fever Group Rickettsia in dog blood

Spotted Fever Group Rickettsia is important cause of emerging and re-emerging infectious disease in people and dogs. Importantly, dogs can serve as sentinels for disease in people. Sensitive and specific diagnostic tests that differentiate among species of infecting Rickettsia are needed. The objective of this study was to develop a sensitive and specific PCR that differentiates SFG Rickettsia infecting dog blood. Conventional and real-time PCR assays were developed using primers that targeted a small region of the ompA gene. Their sensitivity, determined by testing a cloned target sequence in the presence of host DNA, was 15-30 and 5 copies of DNA, respectively. Testing of Rickettsia cultures and analysis of Rickettsia gene sequences deposited in GenBank verified DNA could be amplified and used to differentiate species. DNA from the blood of infected dogs was also tested. Importantly, Rickettsia DNA was detected before seroconversion in some dogs. The species of infecting Rickettsia was also identified. We conclude these assays may assist in the timely diagnosis of infection with SFG Rickettsia. They may also facilitate the discovery of novel SFG Rickettsia infecting dogs, and in the investigation of dogs as sentinels for emerging rickettsioses.

The Incidence of Ehrlichial and Rickettsial Infection in Patients with Unexplained Fever and Recent History of Tick Bite in Central North Carolina

We examined the clinical and laboratory findings of a consecutive series of patients from central North Carolina presenting with fever and a history of tick bite within the preceding 14 days. Evidence of a tick-transmitted pathogen was detected in 16 of 35 patients enrolled over a 2-year period. Nine patients were infected with Ehrlichia chaffeensis, and 6 were infected with a spotted fever group rickettsia; 1 patient had evidence of coinfection with E. chaffeensis and a spotted fever group rickettsia. Four patients had detectable antibodies against the human granulocytic ehrlichiosis agent; however, only 2 had a 4-fold antibody titer rise without detectable antibodies against E. chaffeensis. The other 2 were thought to have cross-reacting antibodies to E. chaffeensis. We conclude that ehrlichial infections may be as common as spotted fever group rickettsial infections in febrile patients from central North Carolina with a recent history of tick bite.

Bartonella vinsonii subsp. berkhoffii and Bartonella henselae bacteremia in a father and daughter with neurological disease

BackgroundBartonella vinsonii subsp. berkhoffii is an important, emerging, intravascular bacterial pathogen that has been recently isolated from immunocompetent patients with endocarditis, arthritis, neurological disease and vasoproliferative neoplasia. Vector transmission is suspected among dogs and wild canines, which are the primary reservoir hosts. This investigation was initiated to determine if pets and family members were infected with one or more Bartonella species.MethodsPCR and enrichment blood culture in Bartonella alpha Proteobacteria growth medium (BAPGM) was used to determine infection status. Antibody titers to B. vinsonii subsp. berkhoffii genotypes I-III and B. henselae were determined using a previously described indirect fluorescent antibody test. Two patients were tested sequentially for over a year to assess the response to antibiotic treatment.ResultsIntravascular infection with B. vinsonii subsp. berkhoffii genotype II and Bartonella henselae (Houston 1 strain) were confirmed in a veterinarian and his daughter by enrichment blood culture, followed by PCR and DNA sequencing. Symptoms included progressive weight loss, muscle weakness, lack of coordination (the father) and headaches, muscle pain and insomnia (the daughter). B. vinsonii subsp. berkhoffii genotype II was also sequenced from a cerebrospinal fluid BAPGM enrichment culture and from a periodontal swab sample. After repeated courses of antibiotics, post-treatment blood cultures were negative, there was a decremental decrease in antibody titers to non-detectable levels and symptoms resolved in both patients.ConclusionsB. vinsonii subsp. berkhoffii and B. henselae are zoonotic pathogens that can be isolated from the blood of immunocompetent family members with arthralgias, fatigue and neurological symptoms. Therapeutic elimination of Bartonella spp. infections can be challenging, and follow-up testing is recommended. An increasing number of arthropod vectors, including biting flies, fleas, keds, lice, sandflies and ticks have been confirmed or are suspected as the primary mode of transmission of Bartonella species among animal populations and may also pose a risk to human beings.

Bartonella spp. bacteremia in high-risk immunocompetent patients.

Serum and blood samples from 192 patients, who reported animal exposure (100.0%) and recent animal bites or scratches (88.0%), were screened for antibodies by indirect immunofluorescence assays and for bacteremia using the BAPGM (Bartonella alpha Proteobacteria growth medium) platform. Predominant symptoms included fatigue (79.2%), sleeplessness (64.1%), joint pain (64.1%), and muscle pain (63.0%). Bartonella spp. seroreactivity or bacteremia was documented in 49.5% (n = 95) and 23.9% (n = 46) of the patients, respectively; however, indirect immunofluorescence antibodies were not detected in 30.4% (n = 14) of bacteremic patients. Regarding components of the BAPGM platform, Bartonella DNA was amplified from 7.5% of blood (n = 21), 8.7% of serum (n = 25), and 10.3% of enrichment culture samples (n = 29). Polymerase chain reaction (PCR) on only extracted blood would not have detected Bartonella infection in 34.7% (16/46) of bacteremic patients. Serology, in conjunction with blood, serum, and BAPGM enrichment culture PCR, facilitates the diagnosis of Bartonella spp. bacteremia in immunocompetent patients.