[show abstract][hide abstract] ABSTRACT: Background: Molecular diagnosis of canine bartonellosis can be extremely challenging and often requires the use of an enrichment culture approach followed by PCR amplification of bacterial DNA. Hypotheses: (1) The use of enrichment culture with PCR will increase molecular detection of bacteremia and will expand the diversity of Bartonella species detected. (2) Serological testing for Bartonella henselae and Bartonella vinsonii subsp. berkhoffii does not correlate with documentation of bacteremia. Animals: Between 2003 and 2009, 924 samples from 663 dogs were submitted to the North Carolina State University, College of Veterinary Medicine, Vector Borne Diseases Diagnostic Laboratory for diagnostic testing with the Bartonella a-Proteobacteria growth medium (BAPGM) platform. Test results and medical records of those dogs were retrospectively reviewed. Methods: PCR amplification of Bartonella sp. DNA after extraction from patient samples was compared with PCR after BAPGM enrichment culture. Indirect immunofluorescent antibody assays, used to detect B. henselae and B. vinsonii subsp. berkhoffii antibodies, were compared with PCR. Results: Sixty-one of 663 dogs were culture positive or had Bartonella DNA detected by PCR, including B. henselae (30/61), B. vinsonii subsp. berkhoffii (17/61), Bartonella koehlerae (7/61), Bartonella volans-like (2/61), and Bartonella bovis (2/61). Coinfection with more than 1 Bartonella sp. was documented in 9/61 dogs. BAPGM culture was required for PCR detection in 32/61 cases. Only 7/19 and 4/10 infected dogs tested by IFA were B. henselae and B. vinsonii subsp. berkhoffii seroreactive, respectively. Conclusions and Clinical Importance: Dogs were most often infected with B. henselae or B. vinsonii subsp. berkhoffii based on PCR and enrichment culture, coinfection was documented, and various Bartonella species were identified. Most infected dogs did not have detectable Bartonella antibodies. M embers of the genus Bartonella are fastidious Gram-negative hemotropic bacteria that are transmitted by several arthropod vectors, by blood transfusion, or via animal scratches or bites. 1,2 A unique peculiarity of this genus is the ability to cause long-last-ing intravascular infection in conjunction with a relapsing pattern of bacteremia in humans, cats, and potentially other mammals. 1–3 The genus Bartonella con-tains a rapidly increasing number of recognized species, many of which are considered emerging animal and hu-man pathogens. Because of the fastidious nature and intracellular tropism of these bacteria for erythrocytes, endothelial cells, and potentially mononuclear phago-cytes, diagnostic confirmation of Bartonella infection has proven to be extremely challenging. Conventional diag-nostic tests, such as bacterial isolation on agar plates, ELISA, and immunofluorescent antibody assays (IFA) for detection of Bartonella spp. antibodies, and PCR amplification of Bartonella DNA after direct extraction from patient samples have considerable limitations, mak-ing these tests relatively insensitive. 4 For example, in 2 small case series, approximately 50% of Bartonella vinsonii subsp. berkhoffii and Bartonella henselae infected dogs did not have detectable IFA antibodies to the infecting Bar-tonella sp. 5,6 One objective of this retrospective study was to compare the results of Bartonella IFA seroreactivity to results of testing with the BAPGM enrichment platform. In 2004, we described a unique diagnostic platform that includes Bartonella PCR after direct extraction of DNA from the patient sample, PCR after enrichment culture in an optimized insect cell culture-based growth medium (Bartonella a-Proteobacteria growth medium [BAPGM]), and PCR if visible growth occurs after sub-culture of the BAPGM-enriched sample onto a blood agar plate, which is incubated for 4 weeks. 4 This diag-nostic platform was developed because conventional microbiological approaches had in most instances failed to isolate Bartonella spp. In addition, unless dogs were therapeutically immunocompromised, PCR after direct extraction of DNA from blood samples generally failed to detect Bartonella spp. DNA. As an insect cell culture-based liquid growth medium, BAPGM supports the
[show abstract][hide abstract] ABSTRACT: We compared clinicopathologic findings in dogs with Bartonella infection to Bartonella spp. negative dogs suspected of a vector-borne disease. Cases (n=47) and controls (n=93) were selected on the basis of positive or negative enrichment culture PCR results, respectively. Signalment, clinicopathologic findings and treatments were extracted from medical records. DNA sequencing identified Bartonella henselae (n=28, 59.6%), Bartonella vinsonii subsp. berkhoffii (n=20, 42.6%), Bartonella koehlerae (n=3, 6.4%), Bartonella volans-like (n=3, 6.4%) and Bartonella bovis (n=1, 2.1%). There were no significant differences in age, breed, size, sex or neuter status between cases and controls. Dogs infected with Bartonella sp. often had a history of weight loss [OR=2.82; 95% CI: 1.08-7.56] and were hypoglobulinemic [OR=4.26; 95% CI: 1.31-14.41]. With the exception of weight loss and hypoglobulinemia, clinicopathologic abnormalities in Bartonella-infected dogs in this study were similar to dogs suspected of other vector-borne infections.
[show abstract][hide abstract] ABSTRACT: Objective-To describe the pharmacokinetics of N-acetylcysteine (NAC) in healthy cats after oral and IV administration. Animals-6 healthy cats. Procedures-In a crossover study, cats received NAC (100 mg/kg) via IV and oral routes of administration; there was a 4-week washout period between treatments. Plasma samples were obtained at 0, 5, 15, 30, and 45 minutes and 1, 2, 4, 8, 12, 24, 36, and 48 hours after administration, and NAC concentrations were quantified by use of a validated high-performance liquid chromatography-mass spectrometry protocol. Data were analyzed via compartmental and noncompartmental pharmacokinetic analysis. Results-Pharmacokinetics for both routes of administration were best described by a 2-compartment model. Mean ± SD elimination half-life was 0.78 ± 0.16 hours and 1.34 ± 0.24 hours for the IV and oral routes of administration, respectively. Mean bioavailability of NAC after oral administration was 19.3 ± 4.4%. Conclusions and Clinical Relevance-The pharmacokinetics of NAC for this small population of healthy cats differed from values reported for humans. Assuming there would be similar pharmacokinetics in diseased cats, dose extrapolations from human medicine may result in underdosing of NAC in cats with acute disease. Despite the low bioavailability, plasma concentrations of NAC after oral administration at 100 mg/kg may be effective in the treatment of chronic diseases.
American Journal of Veterinary Research 02/2013; 74(2):290-3. · 1.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bartonella species are emerging infectious organisms transmitted by arthropods capable of causing long-lasting infection in mammalian hosts. Among over 30 species described from four continents to date, 15 are known to infect humans, with eight of these capable of infecting dogs as well. B. bacilliformis is the only species described infecting humans in Peru; however, several other Bartonella species were detected in small mammals, bats, ticks, and fleas in that country. The objective of this study was to determine the serological and/or molecular prevalence of Bartonella species in asymptomatic dogs in Peru in order to indirectly evaluate the potential for human exposure to zoonotic Bartonella species. A convenient sample of 219 healthy dogs was obtained from five cities and three villages in Peru. EDTA-blood samples were collected from 205 dogs, whereas serum samples were available from 108 dogs. The EDTA-blood samples were screened by PCR followed by nucleotide sequencing for species identification. Antibodies against B. vinsonii berkhoffii and B. rochalimae were detected by IFA (cut-off of 1∶64). Bartonella DNA was detected in 21 of the 205 dogs (10%). Fifteen dogs were infected with B. rochalimae, while six dogs were infected with B. v. berkhoffii genotype III. Seropositivity for B. rochalimae was detected in 67 dogs (62%), and for B. v. berkhoffii in 43 (40%) of the 108 dogs. Reciprocal titers ≥1∶256 for B. rochalimae were detected in 19% of dogs, and for B. v. berkhoffii in 6.5% of dogs. This study identifies for the first time a population of dogs exposed to or infected with zoonotic Bartonella species, suggesting that domestic dogs may be the natural reservoir of these zoonotic organisms. Since dogs are epidemiological sentinels, Peruvian humans may be exposed to infections with B. rochalimae or B. v. berkhoffii.
[show abstract][hide abstract] ABSTRACT: Bartonellae are emerging vector-borne pathogens infecting erythrocytes and endothelial cells of various domestic and wild mammals. Blood samples were collected from domestic and wild canids in Iraq under the United States Army zoonotic disease surveillance program. Serology was performed using an indirect immunofluorescent antibody test for B. henselae, B. clarridgeiae, B. vinsonii subsp. berkhoffii and B. bovis. Overall seroprevalence was 47.4% in dogs (n = 97), 40.4% in jackals (n = 57) and 12.8% in red foxes (n = 39). Bartonella species DNA was amplified from whole blood and representative strains were sequenced. DNA of a new Bartonella species similar to but distinct from B. bovis, was amplified from 37.1% of the dogs and 12.3% of the jackals. B. vinsonii subsp. berkhoffii was also amplified from one jackal and no Bartonella DNA was amplified from foxes. Adjusting for age, the odds of dogs being Bartonella PCR positive were 11.94 times higher than for wild canids (95% CI: 4.55-31.35), suggesting their role as reservoir for this new Bartonella species. This study reports on the prevalence of Bartonella species in domestic and wild canids of Iraq and provides the first detection of Bartonella in jackals. We propose Candidatus Bartonella merieuxii for this new Bartonella species. Most of the Bartonella species identified in sick dogs are also pathogenic for humans. Therefore, seroprevalence in Iraqi dog owners and bacteremia in Iraqi people with unexplained fever or culture negative endocarditis requires further investigation as well as in United States military personnel who were stationed in Iraq. Finally, it will also be essential to test any dog brought back from Iraq to the USA for presence of Bartonella bacteremia to prevent any accidental introduction of a new Bartonella species to the New World.
[show abstract][hide abstract] ABSTRACT: The role of various vector-borne pathogens as a cause of disease in cats has not been clearly determined. The current study evaluated risk factors, clinical and laboratory abnormalities associated with Ehrlichia spp., Anaplasma spp., Neorickettsia spp., Leishmania spp., and Bartonella spp. infection or exposure in 680 client-owned and stray cats from Madrid, Spain. Our results indicate that a large portion (35.1%) of the cat population of Madrid, Spain, is exposed to at least one of the five vector-borne pathogens tested. We found seroreactivity to Bartonella henselae in 23.8%, to Ehrlichia canis in 9.9%, to Anaplasma phagocytophilum in 8.4%, to Leishmania infantum in 3.7%, and to Neorickettsia risticii in 1% of the feline study population. About 9.9% of cats had antibody reactivity to more than one agent. L. infantum DNA was amplified from four cats (0.6%), B. henselae DNA from one cat (0.15%), and B. clarridgeiae DNA from another cat (0.15%).
[show abstract][hide abstract] ABSTRACT: Babesia conradae (B. conradae) causes hemolytic anemia in dogs. This organism has not been reported clinically since it was originally described in southern California in 1991. To date, no anti-protozoal therapies have been associated with clearance of B. conradae. This report describes the use of atovaquone and azithromycin for the treatment of dogs naturally infected with B. conradae and report the re-emergence of B. conradae in southern California. Twelve dogs naturally infected with B. conradae were identified by practicing veterinarians and public health officials in southern California. Treatments consisted of a 10 day course of atovaquone (13.3mg/kg PO q 8h) and azithromycin (10-12.5mg/kg PO q 24h). Four dogs were treated in a randomized blinded placebo-controlled fashion, four additional cases were treated in a non-random, non-blinded fashion and one dog received no treatment. All dogs were tested for B. conradae DNA by polymerase chain reaction (PCR) initially and then once or 3 times post treatment (60-210 days). B. conradae infected dogs that received treatment did not have any detectable Babesia DNA by PCR after treatment. In contrast, dogs receiving placebo had detectable Babesia DNA by PCR throughout the study period. Combination therapy with atovaquone and azithromycin appears to be effective for acute and chronic babesiosis caused by B. conradae.
[show abstract][hide abstract] ABSTRACT: Feline infectious peritonitis (FIP), a fatal disease in cats worldwide, is caused by FCoV infection, which commonly occurs in multicat environments. The enteric FCoV, referred to as feline enteric virus (FECV), is considered a mostly benign biotype infecting the gut, whereas the FIP virus biotype is considered the highly pathogenic etiologic agent for FIP. Current laboratory tests are unable to distinguish between virus biotypes of FCoV. FECV is highly contagious and easily spreads in multicat environments; therefore, the challenges to animal shelters are tremendous. This review summarizes interdisciplinary current knowledge in regard to virology, immunology, pathology, diagnostics, and treatment options in the context of multicat environments.
Veterinary Clinics of North America Small Animal Practice 11/2011; 41(6):1133-69. · 1.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: Molecular diagnosis of canine bartonellosis can be extremely challenging and often requires the use of an enrichment culture approach followed by PCR amplification of bacterial DNA.
(1) The use of enrichment culture with PCR will increase molecular detection of bacteremia and will expand the diversity of Bartonella species detected. (2) Serological testing for Bartonella henselae and Bartonella vinsonii subsp. berkhoffii does not correlate with documentation of bacteremia.
Between 2003 and 2009, 924 samples from 663 dogs were submitted to the North Carolina State University, College of Veterinary Medicine, Vector Borne Diseases Diagnostic Laboratory for diagnostic testing with the Bartonella α-Proteobacteria growth medium (BAPGM) platform. Test results and medical records of those dogs were retrospectively reviewed.
PCR amplification of Bartonella sp. DNA after extraction from patient samples was compared with PCR after BAPGM enrichment culture. Indirect immunofluorescent antibody assays, used to detect B. henselae and B. vinsonii subsp. berkhoffii antibodies, were compared with PCR.
Sixty-one of 663 dogs were culture positive or had Bartonella DNA detected by PCR, including B. henselae (30/61), B. vinsonii subsp. berkhoffii (17/61), Bartonella koehlerae (7/61), Bartonella volans-like (2/61), and Bartonella bovis (2/61). Coinfection with more than 1 Bartonella sp. was documented in 9/61 dogs. BAPGM culture was required for PCR detection in 32/61 cases. Only 7/19 and 4/10 infected dogs tested by IFA were B. henselae and B. vinsonii subsp. berkhoffii seroreactive, respectively.
Dogs were most often infected with B. henselae or B. vinsonii subsp. berkhoffii based on PCR and enrichment culture, coinfection was documented, and various Bartonella species were identified. Most infected dogs did not have detectable Bartonella antibodies.
Journal of Veterinary Internal Medicine 05/2011; 25(4):805-10. · 2.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: "Candidatus Neoehrlichia mikurensis" is a new intracellular pathogen associated with human infection and death. "Candidatus Neoehrlichia mikurensis" infection in a chronically neutropenic dog from Germany was confirmed by DNA sequencing. The same organism was previously described from ticks and two sick human beings from Germany.
Journal of clinical microbiology 03/2011; 49(5):2059-62. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: To evaluate microtiter-plate format ELISAs constructed by use of different diagnostic targets derived from the Ehrlichia ewingii p28 outer membrane protein for detection of E ewingii antibodies in experimentally and naturally infected dogs.
Serum samples from 87 kenneled dogs, 9 dogs experimentally infected with anti-E ewingii, and 180 potentially naturally exposed dogs from Missouri.
The capacities of the synthetic peptide and truncated recombinant protein to function as detection reagents in ELISAs were compared by use of PCR assay, western blot analysis, and a full-length recombinant protein ELISA. Diagnostic targets included an E ewingii synthetic peptide (EESP) and 2 recombinant proteins: a full-length E ewingii outer membrane protein (EEp28) and a truncated E ewingii outer membrane protein (EETp28)
A subset of Ehrlichia canis-positive samples cross-reacted in the EEp28 ELISA; none were reactive in the EESP and EETp28 ELISAs. The EESP- and EETp28-based ELISAs detected E ewingii seroconversion at approximately the same time after infection as the EEp28 ELISAs. In afield population, each of the ELISAs identified the same 35 samples as reactive and 27 samples as nonreactive. Anaplasma and E can is peptides used in a commercially available ELISA platform did not detect anti-E ewingii antibodies in experimentally infected dogs.
The EESP and EETp28 ELISAs were suitable for specifically detecting anti-E ewingii antibodies in experimentally and naturally infected dogs.
American Journal of Veterinary Research 10/2010; 71(10):1195-200. · 1.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Canine vector-borne diseases (CVBDs) are highly prevalent and increasing in distribution worldwide. A longitudinal study was conducted in southern Italy to determine the incidence of and protection against CVBD-causing pathogens in dogs treated with a combination of imidacloprid 10% and permethrin 50% (ImPer). One hundred eleven autochthonous young dogs were divided into group A (n=63) and group B (n=48), both groups containing dogs positive and negative for one or more CVBD-causing pathogens. Additionally, 10 naïve male beagles were introduced in each group in May 2008. Group A was treated with ImPer on day 0 and every 21+/-2 days whereas group B was left untreated. Blood and skin samples were collected at baseline (March-April 2008) and at the first, second and third follow-up times (July and October 2008 and April 2009). Bone marrow was sampled at baseline and at the third follow-up. Serological, cytological and molecular tests were performed to detect Anaplasma platys, Babesia spp., Bartonella spp., Dirofilaria immitis, Ehrlichia canis, Hepatozoon canis and Leishmania infantum. Ectoparasites (fleas, ticks, and sand flies) were monitored throughout the study. The baseline prevalence of CVBDs was 39.6% with 44 dogs positive for at least one pathogen. A. platys (27.5%) and Babesia spp. (15.6%) were the most prevalent species and co-infections with up to two pathogens were detected in 16 (14.7%) individuals. At the end of the evaluation period, there was a 90.7% reduction in overall CVBD incidence density rate (IDR) in group A, as following: 100% reduction in L. infantum; 94.6% in E. canis; 94.4% in Babesia spp.; and 81.8% in A. platys. Initially positive treated dogs showed significantly lower pathogen prevalence at the third follow-up than untreated ones. At the end of the evaluation period, 8 of the 10 untreated beagles were infected with at least one pathogen whereas one of the treated beagles was A. platys positive at a single time point (second follow-up). Overall efficacy against ticks was 97.9%. In October 2009, samples were collected from the remaining 83 dogs (44 from group A and 39 from group B) to investigate the annual incidence of CVBDs in the same, at this time untreated, dog population. A high year incidence for tick-borne diseases (78.1%) and for L. infantum (13.6%) was detected in dogs from group A, seven months after the treatment had been withdrawn. The results demonstrate that ImPer preventive treatment against arthropods protects autochthonous and naïve beagle dogs against CVBD-causing pathogens.
[show abstract][hide abstract] ABSTRACT: This report describes the clinical presentation, diagnosis and treatment of a cat with vegetative valvular endocarditis temporally associated with natural infection with Bartonella henselae. Lethargy, abnormal gait and weakness were the main clinical signs that resulted in referral for diagnostic evaluation. Using a novel and sensitive culture approach, B henselae was isolated from the blood. Following antibiotic therapy there was total resolution of clinical signs, the heart murmur, the valvular lesion by echocardiography, and no Bartonella species was isolated or amplified from a post-treatment blood culture. In conjunction with previous case reports, infective endocarditis can be associated with natural B henselae infection in cats; however, early diagnosis and treatment may result in a better prognosis than previously reported.
Journal of feline medicine and surgery. 02/2010; 12(6):483-6.
[show abstract][hide abstract] ABSTRACT: Vector-transmitted microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, Bartonella, and Borrelia are commonly suspected in dogs with meningoencephalomyelitis (MEM), but the prevalence of these pathogens in brain tissue and cerebrospinal fluid (CSF) of dogs with MEM is unknown.
To determine if DNA from these genera is present in brain tissue and CSF of dogs with MEM, including those with meningoencephalitis of unknown etiology (MUE) and histopathologically confirmed cases of granulomatous (GME) and necrotizing meningoencephalomyelitis (NME).
Hundred and nine dogs examined for neurological signs at 3 university referral hospitals.
Brain tissue and CSF were collected prospectively from dogs with neurological disease and evaluated by broadly reactive polymerase chain reaction (PCR) for Ehrlichia, Anaplasma, Spotted Fever Group Rickettsia, Bartonella, and Borrelia species. Medical records were evaluated retrospectively to identify MEM and control cases.
Seventy-five cases of MUE, GME, or NME, including brain tissue from 31 and CSF from 44 cases, were evaluated. Brain tissue from 4 cases and inflammatory CSF from 30 cases with infectious, neoplastic, compressive, vascular, or malformative disease were evaluated as controls. Pathogen nucleic acids were detected in 1 of 109 cases evaluated. Specifically, Bartonella vinsonii subsp. berkhoffii DNA was amplified from 1/6 dogs with histopathologically confirmed GME.
The results of this investigation suggest that microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, and Borrelia are unlikely to be directly associated with canine MEM in the geographic regions evaluated. The role of Bartonella in the pathogenesis of GME warrants further investigation.
Journal of Veterinary Internal Medicine 01/2010; 24(2):372-8. · 2.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: We evaluated the serological and molecular prevalence of selected organisms in 145 dogs during late spring (May/June) of 2005 and in 88 dogs during winter (February) of 2007 from the Hopi Indian reservation. Additionally, in 2005, 442 ticks attached to dogs were collected and identified as Rhipicephalus sanguineus. Infection with or exposure to at least one organism was detected in 69% and 66% of the dogs in May/June 2005 and February 2007, respectively. Exposure to spotted fever group (SFG) rickettsiae was detected in 66.4% (2005) and 53.4% (2007) of dogs, but rickettsial DNA was not detected using polymerase chain reaction. Active Ehrlichia canis infection (by polymerase chain reaction) was identified in 36.6% (2005) and 36.3% (2007) of the dogs. E. canis infection was associated with SFG rickettsiae seroreactivity (p < 0.001). Anaplasma platys DNA was detected in 8.3% (2005) and 4.5% (2007) of the dogs. Babesia canis and Bartonella vinsonii berkhoffii seroprevalences were 6.7% and 1% in 2005, whereas in 2007 prevalences were 0% and 1.1%, respectively. No Bartonella spp., Ehrlichia chaffeensis, or Ehrlichia ewingii DNA was detected. Dogs on this Hopi Indian reservation were most frequently infected with E. canis or A. platys; however, more than half of the dogs were exposed to a SFG-Rickettsia species.
[show abstract][hide abstract] ABSTRACT: This study reports the occurrence of "Bartonella rochalimae" in Europe and the presence of Bartonella vinsonii subsp. berkhoffii genotypes II and III in dogs in southern Italy and provides DNA sequencing evidence of a potentially new Bartonella sp. infecting dogs in Greece and Italy.
Journal of clinical microbiology 04/2009; 47(5):1565-7. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Broad range 16S rDNA PCR can be used to facilitate the diagnosis of infectious diseases of bacterial origin by detecting 16S rDNA sequences in patient samples. Post amplification sequencing facilitates identification of the infecting organism, but may not allow for differentiation at the species or strain level. This review focuses on the historical use and current applications of broad range 16S rDNA PCR in the diagnosis of bacterial infection. Use of an enrichment liquid culture prior to PCR and the use of real time PCR are also considered. A review of the literature indicates that the diagnostic utility of broad range 16S rDNA PCR is enhanced substantially, if the detected organism is a well-documented pathogen. Frequent detection of environmental organisms of undetermined pathogenicity is currently a limitation. This review also examines weighted criteria developed by different researchers and proposes a decision making tree that establishes the relative importance of various criteria for attributing diagnostic relevance when evaluating individual patient samples. Based upon our review of the literature, a more uniform consensus on the accurate interpretation of broad range 16S rDNA PCR results are needed to improve the microbiological utility of this modality for the diagnosis of bacterial infections in animals and in human patients.
Journal of Microbiological Methods 12/2008; 76(3):217-25. · 2.16 Impact Factor