Ricardo G. Maggi

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.

Molecular Prevalence of Bartonella,Babesia, and Hemotropic Mycoplasma sp. in Dogs with Splenic Disease

BACKGROUNDnAmong diseases that cause splenomegaly in dogs, lymphoid nodular hyperplasia (LNH), splenic hemangiosarcoma (HSA), and fibrohistiocytic nodules (FHN) are common diagnoses. The spleen plays an important role in the immunologic control or elimination of vector-transmitted, blood-borne pathogens, including Bartonella sp., Babesia sp., and hemotropic Mycoplasma sp.nnnOBJECTIVEnTo compare the prevalence of Bartonella sp., Babesia sp., and hemotropic Mycoplasma sp. DNA in spleens from dogs with LNH, HSA, and FHN.nnnMATERIALS AND METHODSnParaffin-embedded, surgically obtained biopsy tissues from LNH (N = 50), HSA (N = 50), and FHN (N = 37) were collected from the anatomic pathology archives. Spleens from specific pathogen-free (SPF) dogs (N = 8) were used as controls. Bartonella sp., Babesia sp., and Mycoplasma sp. DNA was amplified by PCR, followed by DNA sequencing.nnnRESULTSnBartonella sp. DNA was more prevalent in FHN (29.7%) and HSA (26%) as compared to LNH (10%) (P = .019, .0373, respectively) or control spleens (0.0%). The prevalence of Babesia sp. and hemotropic Mycoplasma sp. DNA was significantly lower than Bartonella sp. DNA in HSA (P = .0005, .006, respectively) and FHN (P = .003, .0004, respectively). There was no statistically significant difference in DNA prevalence among the 3 genera in the LNH group.nnnCONCLUSIONSnThe higher prevalence of Bartonella sp. in FHN and HSA warrants future investigations to determine if this bacterium plays a role in the development of these splenic diseases.

Cross-contamination in the Molecular Detection of Bartonella from Paraffin-embedded Tissues

The genus Bartonella comprises a group of gram-negative, fastidious bacteria. Because of diagnostic limitations of culture and serologic testing, polymerase chain reaction (PCR) has become a powerful tool for the detection of Bartonella spp. in blood and tissue samples. However, because many wild and domestic animals harbor Bartonella spp., transfer of Bartonella DNA during sample collection or histologic processing could result in false-positive PCR test results. In this study, we describe evidence of Bartonella DNA dissemination and transfer in the necropsy room and during the subsequent processing of formalin-fixed paraffin-embedded tissues. Bartonella DNA was amplified from different areas of the necropsy room, from the liquid paraffin in the tissue processor, and from different parts of the microtome. Unless stringent procedures are established and followed to avoid cross-contamination, the molecular detection of Bartonella spp. from tissue samples obtained at necropsy or processed in a multispecies histopathology laboratory will not be reliable.

Identification of Bartonella henselae in 2 Cats With Pyogranulomatous Myocarditis and Diaphragmatic Myositis

Most cats infected with Bartonella henselae remain outwardly healthy carriers for years; however, self-limiting fever, transient anemia, neurologic dysfunction, lymphadenopathy, reproductive disorders, aortic valvular endocarditis, and neutrophilic myocarditis have been described in experimentally or naturally infected cats. Two cats in a North Carolina shelter died with pyogranulomatous myocarditis and diaphragmatic myositis. Bacteria were visualized in the lesions by Warthin-Starry silver impregnation and by B. henselae immunohistochemistry. B. henselae DNA was amplified and sequenced from the heart of 1 cat and from multiple tissue samples, including heart and diaphragm, from the second cat. This study supports a potential association between B. henselae and what has been historically described as “transmissible myocarditis and diaphragmitis” of undetermined cause in cats.

Bartonella DNA in the blood and lymph nodes of Golden Retrievers with lymphoma and in healthy controls.

BACKGROUNDnAlthough lymphoma is the most common neoplastic process reported in dogs, its precise etiology is unknown. Golden Retrievers are more likely to develop lymphoma, suggesting a breed predisposition; however, other factors, including environment, immunity, and infection, are likely contributors to oncogenesis.nnnHYPOTHESISnWe hypothesized that the development of lymphoma in Golden Retrievers may be associated with vector-borne infections, specifically Bartonella, Anaplasma, or Ehrlichia species infections.nnnANIMALSnGolden Retrievers with lymphoma and healthy Golden Retrievers from across the United States were recruited for study participation.nnnMETHODSnA matched, case-control study was performed to determine the association of lymphoma and the presence of Bartonella, Anaplasma, and Ehrlichia species in serum, blood, and lymph node aspirates.nnnRESULTSnUsing PCR analyses and DNA sequencing, single and coinfections with Bartonella henselae, Bartonella elizabethae, Bartonella quintana, and/or Bartonella vinsonii (berkhoffii) were detected in the blood and lymph node aspirates of Golden Retrievers with lymphoma (5/28 dogs, 18%) and in healthy Golden Retrievers (10/56 dogs, 18%); no Anaplasma or Ehrlichia DNA was detected in any dog. When compared with dogs with lymphoma, a higher (P <.001) proportion of healthy Golden Retrievers were receiving monthly acaricide treatments (2.6 times higher).nnnCONCLUSIONS AND CLINICAL IMPORTANCEnBartonella DNA can be detected in blood and lymph nodes; importantly, in this report, Bartonella was detected in the same proportion of clinically healthy dogs and dogs with lymphoma. Longitudinal studies should be conducted to determine the mode of transmission of Bartonella in dogs, whether lymphatic infection is persistent, or whether these bacteria may contribute to the development of lymphoma.

Bacillary angiomatosis in an immunosuppressed dog

A dog being treated with immunosuppressive doses of prednisone and azathioprine for pancytopenia of unknown origin, developed, over a 2-week period, multiple erythematous nodular lesions in the skin including footpads. Skin samples revealed lesions identical to those of human bacillary angiomatosis (BA). The nodules were composed of multifocal proliferations of capillaries, each lined by protuberant endothelial cells. The capillary clusters were separated by an oedematous connective tissue, lightly infiltrated with degenerate inflammatory cells, including neutrophils and macrophages. Tissue sections stained with Warthin-Starry silver stain revealed large numbers of positively stained bacilli in the stromal tissue, most heavily concentrated around the proliferating capillaries. Lesions of vascular degeneration and inflammation were evident. Bartonella vinsonii subsp. berkhoffii genotype 1 was independently amplified and sequenced from the blood and the skin tissue. The pathognomonic nature of the histological lesions, demonstration of compatible silver-stained bacilli in the tissue, and identification of B. vinsonii subsp. berkhoffii in the blood and tissue indicates that this is most likely the aetiologic agent responsible for the lesions. Antibiotic therapy was successful in resolving the nodules. It would appear that B. vinsonii subsp berkhoffii, like Bartonella henselae and Bartonella quintana, has the rare ability to induce angioproliferative lesions, most likely in association with immunosuppression. The demonstration of lesions identical to those of human BA in this dog is further evidence that the full range of clinical manifestations of human Bartonella infection occurs also in canines.

Prevalence of Bartonella spp. by culture, PCR and serology, in veterinary personnel from Spain

BackgroundThe genus Bartonella includes fastidious, facultative intracellular bacteria mainly transmitted by arthropods and distributed among mammalian reservoirs. Bartonella spp. implicated as etiological agents of zoonoses are increasing. Apart from the classical Bartonella henselae, B. bacilliformis or B. quintana, other species (B. elizabethae, B. rochalimae, B. vinsonii arupensis and B. v. berkhoffii, B. tamiae or B. koehlerae, among others) have also been associated with human and/or animal diseases. Laboratory techniques for diagnosis (culture, PCR assays and serology) usually show lack of sensitivity. Since 2005, a method based on a liquid enrichment Bartonella alphaproteobacteria growth medium (BAPGM) followed by PCRs for the amplification of Bartonella spp. has been developed. We aimed to assess culture, molecular and serological prevalence of Bartonella infections in companion animal veterinary personnel from Spain.MethodsEach of 89 participants completed a questionnaire. Immunofluorescence assays (IFA) using B. vinsonii berkhoffii (genotypes I, II and III), B. henselae, B. quintana and B. koehlerae as antigens were performed. A cut-off of 1:64 was selected as a seroreactivity titer. Blood samples were inoculated into BAPGM and subcultured onto blood agar plates. Bartonella spp. was detected using conventional and quantitative real-time PCR assays and DNA sequencing.ResultsAmong antigens corresponding to six Bartonella spp. or genotypes, the lowest seroreactivity was found against B. quintana (11.2%) and the highest, against B. v. berkhoffii genotype III (56%). A total of 27% of 89 individuals were not seroreactive to any test antigen. Bartonella spp. IFA seroreactivity was not associated with any clinical sign or symptom. DNA from Bartonella spp., including B. henselae (nxa0=xa02), B. v. berkhoffii genotypes I (nxa0=xa01) and III (nxa0=xa02), and B. quintana (nxa0=xa02) was detected in 7/89 veterinary personnel. PCR and DNA sequencing findings were not associated with clinical signs or symptoms. No co-infections were observed. One of the two B. henselae PCR-positive individuals was IFA seronegative to all tested antigens whereas the other one was not B. henselae seroreactive. The remaining PCR-positive individuals were seroreactive to multiple Bartonella spp. antigens.ConclusionsHigh serological and molecular prevalences of exposure to, or infection with, Bartonella spp. were found in companion animal veterinary personnel from Spain. More studies using BAPGM enrichment blood culture and PCR are needed to clarify the finding of Bartonella PCR-positive individuals lacking clinical symptoms.

Identification of Hemotropic Mycoplasmas in an Eastern Box Turtle (Terrapene carolina carolina) and a Yellow-bellied Slider (Trachemys scripta scripta) from North Carolina, USA

Abstract:u2003 Mycoplasma spp. are known from several chelonian and other reptilian species. We determined if turtles obtained by the Turtle Rescue Team at North Carolina State University are carriers of hemotropic Mycoplasma or Bartonella spp. Spleen samples were collected at necropsy during May through July, 2014 from 53 turtles of seven species. All turtles were dead or were euthanized upon arrival due to severe traumatic injuries, or they died shortly after beginning treatment. We used PCR amplification for both bacterial genera; Bartonella spp. DNA was not amplified. Based upon sequencing of the 16S rRNA subunit, one eastern box turtle (Terrapene carolina carolina) and one yellow-bellied slider (Trachemys scripta scripta) were infected with Mycoplasma spp. that have genetic similarities to strains that infect other animals.

Protozoal nodular dermatitis and panniculitis in a Rottweiler puppy caused by Caryospora bigenetica.

BACKGROUNDnCaryospora bigenetica is an intracellular protozoan parasite in snakes and raptors (primary hosts) and rodents (secondary host). Experimental infection has been documented in mice, pigs and goats; natural infection in dogs is rare.nnnOBJECTIVESnTo describe the clinical presentation, histological features, treatment and outcome of a case of protozoal nodular dermatitis and panniculitis in a Rottweiler puppy caused by C. bigenetica.nnnRESULTSnThe puppy presented with generalized subcutaneous nodules measuring up to 2 cm in diameter. Histopathology revealed marked suppurative to pyogranulomatous dermatitis and panniculitis with intralesional protozoal organism. PCR and DNA sequencing confirmed infection with C. bigenetica. Treatment with a combination of oral trimethoprim-sulfamethoxazole (TMS), pyrimethamine and high-dose clindamycin (20 mg/kg twice daily) resulted in resolution of lesions in 6 weeks. Discontinuation of the treatment 2 weeks later was followed by a rapid relapse of skin lesions. Clindamycin and TMS were restarted and all lesions resolved within 2 weeks; TMS was discontinued 4 weeks later due to adverse effects. The lesions remained in remission for 2 months while the puppy received clindamycin monotherapy before a second relapse of skin lesions occurred.nnnCONCLUSION AND CLINICAL IMPORTANCEnTo the best of the authors knowledge, this is the first documentation of the treatment and outcome of C. bigenetica cutaneous infection in a dog. Although remission of clinical signs can be achieved with combination therapy of clindamycin and TMS, long-term management is challenging and relapses should be anticipated.