Case Report Rapport de cas Bilateral mandibular pyogranulomatous lymphadenitis and pulmonary nodules in a dog with Bartonella henselae bacteremia Melissa D. Tucker, Rance K. Sellon, Russell L. Tucker, Tamara B. Wills, Andrea Simonsen, Ricardo G. Maggi, Edward B. Breitschwerdt Abstract — This report describes a 2-year-old collie dog with pulmonary nodules, visualized by computed tomographic (CT) scan, with evidence of Bartonella henselae bacteremia and pyogranulomatous lymphadenitis. Clinical signs resolved with antimicrobial therapy. Résumé — Lymphadénite pyogranulomateuse mandibulaire latérale et nodules pulmonaires chez un chien atteint de bactériémie à Bartonella henselae. Ce rapport décrit un chien Collie âgé de 2 ans atteint de nodules pulmonaires, visualisés par tomodensitométrie, avec des signes de bactériémie à Bartonella henselae et de lymphadénite pyogranulomateuse. Les signes cliniques se sont résorbés avec un traitement antimicrobien. (Traduit par Isabelle Vallières) Can Vet J 2014;55:970–974
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artonella infections appear to contribute to a wide variety of clinical signs and pathological abnormalities in cats, dogs, and humans (1–15). The dog described in this report had an acute onset of fever, lethargy, mandibular pyogranulomatous lymphadenitis, multifocal, discrete pulmonary nodules visible only with computed tomography (CT). The dog began to improve 2 weeks after beginning new antimicrobial therapy with enrofloxacin. Despite a thorough diagnostic evaluation, a specific etiologic agent is often not identified in dogs with pyogranulomatous inflammation, regardless of the tissues involved. While a causal association between Bartonella and this dog’s illness cannot be confirmed, this report highlights the importance of considering Bartonella infection in dogs from areas in which this organism has been infrequently diagnosed.
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-7060, USA (MD Tucker, Sellon, RL Tucker); IDEXX Reference Laboratories Inc., Pullman, Washington, USA (Wills); Dillon Small Animal Hospital, Dillon, Montana, USA (Simonsen); Intracellular Pathogens Research Laboratory, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA (Maggi, Breitschwerdt) Address all correspondence to Dr. Rance K. Sellon; e-mail: [email protected] Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. 970
Case description A 2-year-old spayed female collie dog from Montana was referred to Washington State University for evaluation of a 9-day history of lethargy and inappetence and a 3-day history of fever. The referring veterinarian had identified oral ulcerations, ptyalism, fever (40.8°C) and a mild enlargement of mandibular lymph nodes. The dog was born in British Columbia and had moved to southwestern Montana at 6 mo of age, after which the dog had no history of out-of-state travel. All complete blood cell counts (CBCs), serum biochemistry profile, and urinalysis results provided by the referring veterinarian were within reference intervals and thoracic radiographs were unremarkable. Serological tests for Ehrlichia canis, Rickettsia rickettsia, and Borrelia burgdorferi were negative; a SNAP® cPL test (IDEXX Laboratories, Westbrook, Massachusetts, USA) for pancreatitis was normal. Drugs administered prior to referral included doxycycline, 8.2 mg/kg body weight (BW), PO, q12h, maropitant 2.4 mg/kg BW, PO, q24h, amoxicillin 30.6 mg/kg BW, PO, q12h, ciprofloxacin 20.4 mg/kg BW, PO, q12h, tramadol 2 mg/kg BW PO, q12h, sucralfate 40.8 mg/kg BW, PO, q8h, famotidine 0.8 mg/kg BW, PO, q12h, and aspirin 13.3 mg/kg BW, PO, q12h. The day prior to referral, the dog was given injectable maropitant 1 mg/kg BW, SQ, q24h, ampicillin 24.5 mg/kg BW, IV, q8h and carprofen, 2 mg/kg BW, SQ, once. Due to a lack of response to therapy, progressive lethargy, inappetence, and persistent fever, the dog was referred for additional evaluation. On referral physical examination, the dog was quiet but alert and had a rectal temperature of 39.9°C. There was mild ptyalism, but oral ulcerations were no longer seen. There was moderate to severe diffuse enlargement of both mandibular CVJ / VOL 55 / OCTOBER 2014
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Figure 1. Computed tomography (CT) post-contrast para-sagittal 2-mm reconstruction image at the level of the left tympanic bulla demonstrating the enhancing enlarged mandibular and medial retropharyngeal lymph nodes (arrows). LTB — left tympanic bulla.
Figure 2. Transverse pre-contrast computed tomography (CT) 3-mm slice at the level of the 7th thoracic vertebra. There is a 7-mm soft tissue nodule in the right cranial lung (arrow).
lymph nodes, which were firm and appeared to be moderately painful. A CBC was normal with the exception of mild lymphopenia [651/mL, reference interval (RI): 900 to 4000/mL), attributed to a glucocorticoid response secondary to illness, and thrombocytopenia (98 000/mL, RI: 160 000 to 500 000/mL) accompanied by a high mean platelet volume (15.3/fl, RI: 7.4 to CVJ / VOL 55 / OCTOBER 2014
13/fl). Platelet clumps, infectious agents, or cellular inclusions were not observed during blood smear examination. Venous blood gas values were normal. On the first day of hospitalization, needle aspiration cytology of each mandibular lymph node was consistent with lymphoid hyperplasia and neutrophilic lymphadenitis characterized by marked infiltration with primarily moderately degenerate neutrophils, and moderate to marked cellular degeneration indicative of necrosis. There were low numbers of macrophages, lymphocytes, and plasma cells. There was no cytological evidence of neoplasia, and microorganisms were not seen. On the second day of hospitalization, an abdominal ultrasound and thoracic radiographs were unremarkable. A CT scan of the head, neck, and thorax identified marked mandibular and medial retropharyngeal lymphadenopathy with surrounding tissue pathology attributed to inflammation or hemorrhage (Figure 1). Five pulmonary nodules measuring up to 7 mm in diameter were also visualized. There was 1 nodule in each of the right cranial, right middle, and left cranial lung lobes, and 2 nodules in the right caudal lung lobe (Figure 2). Due to the small size of the pulmonary nodules, ultrasoundguided aspiration was not considered diagnostically sensitive and a surgical lung biopsy was delayed pending infectious disease test results. Based on the cytology of the initial lymph node aspirate, while under general anesthesia following the CT scan, blood and needle aspirate samples were collected aseptically from each mandibular lymph node. Gram-stained lymph node aspirates, and aerobic and anaerobic bacterial cultures, were negative. These samples were also tested for Bartonella spp. DNA by PCR of the original samples and from each BAPGM (Bartonella alpha Proteobacteria Growth Medium) enrichment culture (Galaxy Diagnostics, Research 971
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Triangle Park, North Carolina, USA) of each sample. Using DNA extracted from the blood and the lymph node aspirates, PCR failed to amplify Bartonella spp. DNA. However, following incubation in liquid BAPGM growth medium for 8 d, Bartonella henselae (SA2 strain) DNA was amplified and sequenced from the enrichment blood culture. Subculture isolation was not attempted and Bartonella serological testing was not requested. As an optimal treatment regimen has not been defined for dogs, and while pending completion of the Bartonella spp. test results, the dog was treated with azithromycin (Zithromax; Pfizer Animal Health, New York, New York, USA), 10 mg/kg BW, PO, once daily for 9 d then every other day for 5 wk starting on the third day of hospitalization. On the 5th day of hospitalization the dog showed no clinical improvement and had then been sick for a total of 13 d. As prior diagnostic efforts had not identified an etiology and the Bartonella PCR was pending, the left mandibular lymph node was surgically removed and submitted for histopathology. A thorough oropharyngeal examination during anesthesia was unremarkable. Because of sustained anorexia, an esophageal feeding tube was placed to provide nutritional support. Esophagoscopy was used to confirm proper placement of the feeding tube and to examine the esophagus, which appeared normal. Histologic evaluation of the lymph node identified rare remnants of lymphoid follicles with nodular infiltrates consisting of large numbers of neutrophils and epithelioid macrophages surrounded by numerous plasma cells and small lymphocytes. There were low numbers of similar inflammatory nodules in the adjacent subcutis and muscle. The histological diagnoses were severe, pyogranulomatous mandibular lymphadenitis and cellulitis. Special stains, including Gomori methenamine silver stain, Periodic Acid-Schiff reaction, and a Gram stain, were negative. Tests for antigens of Histoplasma and Blastomyces in urine and for Cryptococcus in serum were negative. Over the following 8 d, the remaining mandibular lymph node decreased in size from approximately 5 cm in diameter to 3 cm in diameter. However, the dog remained anorexic and febrile at 39.5°C. Enrofloxacin (Bayer Animal Health, Shawnee Mission, Kansas, USA), 10 mg/kg BW, PO, once daily for 6 wk was started on the 12th day of hospitalization and administered concurrently with azithromycin. The dog required esophageal tube feeding for a total of 2 wk, after which the tube was removed because the dog was eating normally and had also resumed normal daily activities. Dramatic improvement in the dog’s appetite began 7 d after starting on enrofloxacin (16 d after starting on azithromycin). Although an optimal post-treatment time to retest infected dogs has not been established, 2 wk after completing the 6-week course of antibiotic therapy, blood was retested for Bartonella spp. with PCR and BAPGM enrichment culture, both of which were negative. Nine months following treatment for B. henselae bacteremia, repeat thoracic radiographs were normal and the dog remained clinically healthy.
Discussion To the authors’ knowledge, this is the first reported case of B. henselae bacteremia in a dog with acute-onset fever, bilateral mandibular pyogranulomatous lymphadenitis, and multifocal 972
pulmonary nodules that were suspected to be small granulomas. Based upon the CT scan, an inflammatory etiology for the pulmonary nodules was suspected and as the nodules did not become visible on radiographs over time, other causes for inflammatory pulmonary nodules, such as fungal disease or metastatic cancer, seemed less likely. Documenting granulomatous inflammation in biopsied pulmonary nodules in conjunction with identifying B. henselae organisms in various inflammatory cells within the granulomas by immunohistochemistry or fluorescence in-situ hybridization would have been necessary to make a clear association between this dog’s pulmonary nodules and B. henselae infection. However, as the clinical signs (fever, lethargy, and inappetence) and mandibular lymphadenopathy resolved after initiation of antibiotics that achieve intracellular concentration, and because there was no radiographic increase in size of the pulmonary nodules 9 mo after antibiotic treatment, our results may support a potential role for B. henselae infection as a cause or cofactor of the fever, granulomatous lymphadenitis, and pulmonary nodules in this dog. Although fever is an inconsistent disease manifestation in dogs with chronic Bartonella spp. bacteremia (1), B. henselae is a documented cause of cat scratch disease (a triad of fever, lymphadenopathy, and a history of a cat scratch or bite) in humans and is a frequent cause of fever of unknown origin, particularly in children (2). The authors offered to repeat the thoracic CT scan at no cost to the client, but due to resolution of the disease manifestations and the long distance that the owners had to travel to obtain a repeat study, re-evaluation was declined. Granulomatous lymphadenitis has been previously described in a dog from North Carolina infected with Bartonella vinsonii subsp. berkhoffii and in a dog from Minnesota infected with B. henselae (3,4). The North Carolina case had unilateral mandibular lymph node involvement, whereas the dog herein had bilateral mandibular and medial retropharyngeal lymphadenopathy. In contrast, the dog from Minnesota had generalized lymphadenopathy, which has also been reported in humans in association with infection with B. henselae and B. alsatica (5,6). Taken together, these cases suggest that infection with Bartonella in dogs could potentially cause focal, regional, or generalized lymphadenomegaly. Bartonella spp. infection has also been previously linked to granulomatous inflammation and nodular lesions in tissues other than lymph nodes. Granulomatous hepatitis has been reported in association with B. henselae infections in some dogs and humans (7,8). Discrete nodular granulomatous lesions in the myocardium and diaphragm have been reported in association with B. henselae infection in 2 cats (9). In the human literature, 1 case of B. henselae associated with nodular granulomatous pneumonitis was described in an immunocompromised renal transplant patient with a prolonged history of fever, vomiting, and diarrhea, followed by eventual CT identification of multiple discrete pulmonary nodules that were subsequently confirmed as granulomas following excisional biopsy (10). In that patient, other diagnostic test results were negative and B. henselae DNA was amplified and sequenced from a pulmonary homogenate obtained from a surgical lung biopsy (10). As granulomatous inflammation has been documented in cats, dogs, and humans CVJ / VOL 55 / OCTOBER 2014
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ent but at low prevalence rates (22,23). The owners had applied fipronil (Merial, Duluth, Georgia, USA) in the summer months and had never seen fleas or ticks on any of their dogs. All known Bartonella spp. are believed to be transmitted by an insect vector or by bites, scratches or needle stick; however, the mode of transmission of B. henselae for this patient remains unclear (24). With this dog having originated in British Columbia and resided only in Montana, B. henselae transmission had to have occurred in British Columbia or Montana. Recently, B. henselae DNA was amplified and sequenced from stored paraffin blocks containing heart valves from dogs that died due to endocarditis or myocarditis in Colorado where flea and tick exposure is also infrequent (25). Although perhaps uncommon, dogs in the western United States, where flea and tick infestations are reportedly infrequent, can be infected with B. henselae, through a currently unknown mode of transmission. We conclude that, although other etiologies or co-infection with other bacteria cannot be ruled out in this dog, this case suggests a possible role for Bartonella infection in dogs with pyogranulomatous lymphadenitis and pulmonary nodules of undetermined cause. Additional research is necessary to define modes of transmission, and the geographic prevalence of these bacteria throughout the US and Canada, particularly in regions with low flea and tick prevalence. CVJ
References 1. Perez C, Diniz PP, Pultorak EL, Maggi RG, Breitschwerdt EB. An unmatched case controlled study of clinicopathologic abnormalities in dogs with Bartonella infection. Comp Immunol Microbiol Infect Dis 2013;36:481–487. 2. Florin TA, Zaoutis TE, Zaoutis LB. Beyond cat scratch disease: Widening spectrum of Bartonella henselae infection. Pediatrics 2008;121:413–425. 3. Pappalardo BL, Brown T, Gookin JL, Morrill CL, Breitschwerdt EB. Granulomatous disease associated with Bartonella infection in 2 dogs. J Vet Intern Med 2000;14:37–42. 4. Morales SC, Breitschwerdt EB, Washabau RJ, Matise I, Maggi RG, Duncan AW. Detection of Bartonella henselae DNA in two dogs with pyogranulomatous lymphadenitis. J Am Vet Med Assoc 2007;230:681–685. 5. Rolain JM, Lepidi H, Zanaret M, et al. Lymph node biopsy specimens and diagnosis of cat-scratch disease. Emerg Infect Dis 2006;12:1338–1344. 6. Angelakis E, Lepidi H, Canel A, et al. Human case of Bartonella alsatica lymphadenitis. Emerg Infect Dis 2008;14:1951–1953. 7. Gillespie TN, Washabau RD, Goldschmidt MH, Cullen JM, Rogala AR, Breitschwerdt EB. Detection of Bartonella clarridgeiae DNA in hepatic specimens from two dogs with hepatic disease. J Am Vet Med Assoc 2003;222:47–51. 8. VanderHeyden TR, Yong SL, Breitschwerdt EB, et al. Granulomatous hepatitis due to Bartonella henselae infection in an immunocompetent patient. BMC Infectious Diseases 2012;12:17. Available from: http:// www.biomedcentral.com/1471–2334/12/17 Last accessed July 22, 2014. 9. Varanat M, Broadhurst J, Linder KE, Maggi RG, Breitschwerdt EB. Identification of Bartonella henselae in 2 cats with pyogranulomatous myocarditis and diaphragmatic myositis. Vet Pathol 2012;49:608–611. 10. Caniza MA, Granger DL, Wilson KH, et al. Bartonella henselae: Etiology of pulmonary nodules in a patient with depressed cell-mediated immunity. Clin Infect Dis 1995;20:1505–1511. 11. Chomel BB, Kasten RW. Bartonellosis, an increasingly recognized zoonosis. J Appl Microbiol 2010;109:743–750. 12. Kaiser PO, Riess T, O’Rourke F, Linke D, Kempf VA. Bartonella spp.: Throwing light on uncommon human infections. Int J Med Microbiol 2011;301:7–15. 13. Lin JW, Chen CM, Chang CC. Unknown fever and back pain caused by Bartonella henselae in a veterinarian after a needle puncture:
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naturally infected with Bartonella spp., these bacteria satisfy the recently proposed Postulate of Comparative Infectious Disease Causation in the context of causation and granulomatous inflammatory lesions (16). Diagnosis of bartonellosis can be challenging. In the 2 previous canine cases of Bartonella-associated lymphadenitis, the diagnosis was confirmed by amplification of Bartonella spp. DNA from surgically excised granulomatous lymph nodes, whereas in our case PCR amplification was not successful using a sample acquired by lymph node aspiration. In humans, it has been reported that bacteria are most readily observed within the first 2 to 3 wk following the onset of lymphadenopathy, with numbers decreasing rapidly as the suppurative inflammation progresses (17). The dog in this case report was presented with a 9-day history of illness, but the true time course of lymphadenopathy is unknown. It is possible that bacterial numbers in the nodes had decreased significantly by the time of sampling, thus making them difficult to detect by PCR. It is also plausible that Bartonella was present at the time of lymph node sampling, but was missed due to the large size of the nodes and inherent potential for sampling error when performing tissue aspirates with a 22-gauge needle. A portion of the lymph node biopsy material should have been cultured for Bartonella (18); however, this was not done in this case. Acute and convalescent B. henselae serology may have also been supportive of a diagnosis of acute bartonellosis, but was not requested in this case. In the dog of this report, Bartonella bacteremia was confirmed by PCR and DNA sequencing after culturing blood in an optimized insect cell culture-based growth medium (BAPGM) (19). Enrichment culture increases bacterial numbers to a high enough level to allow for successful PCR amplification. In an effort to rule out other bacterial pathogens as a potential cause for disease in this patient, cultures using standard aerobic and anaerobic culture techniques were performed but were negative. However, a recent study evaluating dogs by BAPGM enrichment culture and 16S rDNA PCR amplification of subcultured isolates identified dogs that were concurrently infected with B. henselae and other fastidious bacteria. In that study, the BAPGM method appeared to preferentially grow organisms within the Class alpha-Proteobacteria, which were rarely isolated using conventional microbiological methods. The authors acknowledged that the prolonged incubation times required with enrichment cultures may increase the incidence of isolating non-pathogenic commensal bacteria, normal flora, contaminants, or environmental bacteria. The pathogenicity of the isolates identified in that study was unclear and a causal relationship between an organism and clinical disease could not be made (20). The commercial company performing the BAPGM enrichment blood culture used to test this dog specifically targets Bartonella spp. by PCR and does not perform subcultures from the enrichment sample; therefore, it is possible that this dog could have been co-infected with another fastidious bacterium, as reported previously for human patients (21). Of additional clinical and microbiological relevance, this dog was from Montana, where cat and dog fleas (Ctenocephalides felis and Ctenocephalides canis, respectively) and tick vectors (Dermacentor andersoni and Rhipicephalus sanguineus) are pres-
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A case report and literature review. Vector Borne Zoonotic Dis 2011;11:589–591. 14. Breitschwerdt EB, Maggi RG, Chomel BB, Lappin MR. Bartonellosis: An emerging infectious disease of zoonotic importance to animals and human beings. J Vet Emerg Crit Care 2010;20:8–30. 15. Cross JR, Rossmeisl JH, Maggi RG, Breitschwerdt EB, Duncan RB. Bartonella-associated meningoradiculoneuritis and dermatitis or panniculitis in 3 dogs. J Vet Intern Med 2008;22:674–678. 16. Breitschwerdt EB, Linder KL, Day MJ, Maggi RG, Chomel BB, Kempf VA. Koch’s postulates and the pathogenesis of comparative infectious disease causation associated with Bartonella species. J Comp Path 2013;148:115–125. 17. Karem KL, Paddock CD, Regnery RL. Bartonella henselae, B. quintana, and B. bacilliformis: Historical pathogens of emerging significance. Microbes Infect 2000;2:1193–1205. 18. Perez C, Maggi RG, Diniz PP, Breitschwerdt EB. Molecular and serological diagnosis of Bartonella infection in 61 dogs from the United States. J Vet Intern Med 2011;25:805–810. 19. Duncan AW, Maggi RG, Breitschwerdt EB. A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: Pre-enrichment liquid culture followed by PCR and subculture onto agar plates. J Microbiol Methods 2007;69:273–281.
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20. Davenport AC, Mascarelli PE, Maggi RG, Breitschwerdt EB. Phylogenetic diversity of bacteria isolated from sick dogs using the BAPGM enrichment culture platform. J Vet Intern Med 2013;4:854–861. 21. Cadenas MB, Maggi RG, Diniz PP, Breitschwerdt KT, Sontakkes S, Breitschwerdt EB. Identification of bacteria from clinical samples using Bartonella alpha-Proteobacteria growth medium. J Microbiol Methods 2007;71:147–155. 22. Jameson PH, Greene CE, Regnery RL, et al. Prevalence of Bartonella henselae antibodies in pet cats throughout regions of North America. J Infect Dis 1995;172:1145–1149. 23. Greene CE. Infectious Diseases of the Dog and Cat. 2nd ed. Philadelphia, Pennsylvania: WB Saunders, 1998:155–157. 24. Oliveira AM, Maggi RG, Woods CW, Breitschwerdt EB. Suspected needle stick transmission of Bartonella vinsonii subspecies berkhoffii to a veterinarian. J Vet Intern Med 2010;24:1229–1232. 25. Fenimore A, Varanat M, Maggi R, Schultheiss P, Breitschwerdt E, Lappin MR. Bartonella spp. DNA in cardiac tissues from dogs in Colorado and Wyoming. J Vet Intern Med 2011;25:613–616.
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artonella infections appear to contribute to a wide variety of clinical signs and pathological abnormalities in cats, dogs, and humans (1–15). The dog described in this report had an acute onset of fever, lethargy, mandibular pyogranulomatous lymphadenitis, multifocal, discrete pulmonary nodules visible only with computed tomography (CT). The dog began to improve 2 weeks after beginning new antimicrobial therapy with enrofloxacin. Despite a thorough diagnostic evaluation, a specific etiologic agent is often not identified in dogs with pyogranulomatous inflammation, regardless of the tissues involved. While a causal association between Bartonella and this dog’s illness cannot be confirmed, this report highlights the importance of considering Bartonella infection in dogs from areas in which this organism has been infrequently diagnosed.
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-7060, USA (MD Tucker, Sellon, RL Tucker); IDEXX Reference Laboratories Inc., Pullman, Washington, USA (Wills); Dillon Small Animal Hospital, Dillon, Montana, USA (Simonsen); Intracellular Pathogens Research Laboratory, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA (Maggi, Breitschwerdt) Address all correspondence to Dr. Rance K. Sellon; e-mail: [email protected] Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. 970
Case description A 2-year-old spayed female collie dog from Montana was referred to Washington State University for evaluation of a 9-day history of lethargy and inappetence and a 3-day history of fever. The referring veterinarian had identified oral ulcerations, ptyalism, fever (40.8°C) and a mild enlargement of mandibular lymph nodes. The dog was born in British Columbia and had moved to southwestern Montana at 6 mo of age, after which the dog had no history of out-of-state travel. All complete blood cell counts (CBCs), serum biochemistry profile, and urinalysis results provided by the referring veterinarian were within reference intervals and thoracic radiographs were unremarkable. Serological tests for Ehrlichia canis, Rickettsia rickettsia, and Borrelia burgdorferi were negative; a SNAP® cPL test (IDEXX Laboratories, Westbrook, Massachusetts, USA) for pancreatitis was normal. Drugs administered prior to referral included doxycycline, 8.2 mg/kg body weight (BW), PO, q12h, maropitant 2.4 mg/kg BW, PO, q24h, amoxicillin 30.6 mg/kg BW, PO, q12h, ciprofloxacin 20.4 mg/kg BW, PO, q12h, tramadol 2 mg/kg BW PO, q12h, sucralfate 40.8 mg/kg BW, PO, q8h, famotidine 0.8 mg/kg BW, PO, q12h, and aspirin 13.3 mg/kg BW, PO, q12h. The day prior to referral, the dog was given injectable maropitant 1 mg/kg BW, SQ, q24h, ampicillin 24.5 mg/kg BW, IV, q8h and carprofen, 2 mg/kg BW, SQ, once. Due to a lack of response to therapy, progressive lethargy, inappetence, and persistent fever, the dog was referred for additional evaluation. On referral physical examination, the dog was quiet but alert and had a rectal temperature of 39.9°C. There was mild ptyalism, but oral ulcerations were no longer seen. There was moderate to severe diffuse enlargement of both mandibular CVJ / VOL 55 / OCTOBER 2014
CA S E R E P O R T
Figure 1. Computed tomography (CT) post-contrast para-sagittal 2-mm reconstruction image at the level of the left tympanic bulla demonstrating the enhancing enlarged mandibular and medial retropharyngeal lymph nodes (arrows). LTB — left tympanic bulla.
Figure 2. Transverse pre-contrast computed tomography (CT) 3-mm slice at the level of the 7th thoracic vertebra. There is a 7-mm soft tissue nodule in the right cranial lung (arrow).
lymph nodes, which were firm and appeared to be moderately painful. A CBC was normal with the exception of mild lymphopenia [651/mL, reference interval (RI): 900 to 4000/mL), attributed to a glucocorticoid response secondary to illness, and thrombocytopenia (98 000/mL, RI: 160 000 to 500 000/mL) accompanied by a high mean platelet volume (15.3/fl, RI: 7.4 to CVJ / VOL 55 / OCTOBER 2014
13/fl). Platelet clumps, infectious agents, or cellular inclusions were not observed during blood smear examination. Venous blood gas values were normal. On the first day of hospitalization, needle aspiration cytology of each mandibular lymph node was consistent with lymphoid hyperplasia and neutrophilic lymphadenitis characterized by marked infiltration with primarily moderately degenerate neutrophils, and moderate to marked cellular degeneration indicative of necrosis. There were low numbers of macrophages, lymphocytes, and plasma cells. There was no cytological evidence of neoplasia, and microorganisms were not seen. On the second day of hospitalization, an abdominal ultrasound and thoracic radiographs were unremarkable. A CT scan of the head, neck, and thorax identified marked mandibular and medial retropharyngeal lymphadenopathy with surrounding tissue pathology attributed to inflammation or hemorrhage (Figure 1). Five pulmonary nodules measuring up to 7 mm in diameter were also visualized. There was 1 nodule in each of the right cranial, right middle, and left cranial lung lobes, and 2 nodules in the right caudal lung lobe (Figure 2). Due to the small size of the pulmonary nodules, ultrasoundguided aspiration was not considered diagnostically sensitive and a surgical lung biopsy was delayed pending infectious disease test results. Based on the cytology of the initial lymph node aspirate, while under general anesthesia following the CT scan, blood and needle aspirate samples were collected aseptically from each mandibular lymph node. Gram-stained lymph node aspirates, and aerobic and anaerobic bacterial cultures, were negative. These samples were also tested for Bartonella spp. DNA by PCR of the original samples and from each BAPGM (Bartonella alpha Proteobacteria Growth Medium) enrichment culture (Galaxy Diagnostics, Research 971
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Triangle Park, North Carolina, USA) of each sample. Using DNA extracted from the blood and the lymph node aspirates, PCR failed to amplify Bartonella spp. DNA. However, following incubation in liquid BAPGM growth medium for 8 d, Bartonella henselae (SA2 strain) DNA was amplified and sequenced from the enrichment blood culture. Subculture isolation was not attempted and Bartonella serological testing was not requested. As an optimal treatment regimen has not been defined for dogs, and while pending completion of the Bartonella spp. test results, the dog was treated with azithromycin (Zithromax; Pfizer Animal Health, New York, New York, USA), 10 mg/kg BW, PO, once daily for 9 d then every other day for 5 wk starting on the third day of hospitalization. On the 5th day of hospitalization the dog showed no clinical improvement and had then been sick for a total of 13 d. As prior diagnostic efforts had not identified an etiology and the Bartonella PCR was pending, the left mandibular lymph node was surgically removed and submitted for histopathology. A thorough oropharyngeal examination during anesthesia was unremarkable. Because of sustained anorexia, an esophageal feeding tube was placed to provide nutritional support. Esophagoscopy was used to confirm proper placement of the feeding tube and to examine the esophagus, which appeared normal. Histologic evaluation of the lymph node identified rare remnants of lymphoid follicles with nodular infiltrates consisting of large numbers of neutrophils and epithelioid macrophages surrounded by numerous plasma cells and small lymphocytes. There were low numbers of similar inflammatory nodules in the adjacent subcutis and muscle. The histological diagnoses were severe, pyogranulomatous mandibular lymphadenitis and cellulitis. Special stains, including Gomori methenamine silver stain, Periodic Acid-Schiff reaction, and a Gram stain, were negative. Tests for antigens of Histoplasma and Blastomyces in urine and for Cryptococcus in serum were negative. Over the following 8 d, the remaining mandibular lymph node decreased in size from approximately 5 cm in diameter to 3 cm in diameter. However, the dog remained anorexic and febrile at 39.5°C. Enrofloxacin (Bayer Animal Health, Shawnee Mission, Kansas, USA), 10 mg/kg BW, PO, once daily for 6 wk was started on the 12th day of hospitalization and administered concurrently with azithromycin. The dog required esophageal tube feeding for a total of 2 wk, after which the tube was removed because the dog was eating normally and had also resumed normal daily activities. Dramatic improvement in the dog’s appetite began 7 d after starting on enrofloxacin (16 d after starting on azithromycin). Although an optimal post-treatment time to retest infected dogs has not been established, 2 wk after completing the 6-week course of antibiotic therapy, blood was retested for Bartonella spp. with PCR and BAPGM enrichment culture, both of which were negative. Nine months following treatment for B. henselae bacteremia, repeat thoracic radiographs were normal and the dog remained clinically healthy.
Discussion To the authors’ knowledge, this is the first reported case of B. henselae bacteremia in a dog with acute-onset fever, bilateral mandibular pyogranulomatous lymphadenitis, and multifocal 972
pulmonary nodules that were suspected to be small granulomas. Based upon the CT scan, an inflammatory etiology for the pulmonary nodules was suspected and as the nodules did not become visible on radiographs over time, other causes for inflammatory pulmonary nodules, such as fungal disease or metastatic cancer, seemed less likely. Documenting granulomatous inflammation in biopsied pulmonary nodules in conjunction with identifying B. henselae organisms in various inflammatory cells within the granulomas by immunohistochemistry or fluorescence in-situ hybridization would have been necessary to make a clear association between this dog’s pulmonary nodules and B. henselae infection. However, as the clinical signs (fever, lethargy, and inappetence) and mandibular lymphadenopathy resolved after initiation of antibiotics that achieve intracellular concentration, and because there was no radiographic increase in size of the pulmonary nodules 9 mo after antibiotic treatment, our results may support a potential role for B. henselae infection as a cause or cofactor of the fever, granulomatous lymphadenitis, and pulmonary nodules in this dog. Although fever is an inconsistent disease manifestation in dogs with chronic Bartonella spp. bacteremia (1), B. henselae is a documented cause of cat scratch disease (a triad of fever, lymphadenopathy, and a history of a cat scratch or bite) in humans and is a frequent cause of fever of unknown origin, particularly in children (2). The authors offered to repeat the thoracic CT scan at no cost to the client, but due to resolution of the disease manifestations and the long distance that the owners had to travel to obtain a repeat study, re-evaluation was declined. Granulomatous lymphadenitis has been previously described in a dog from North Carolina infected with Bartonella vinsonii subsp. berkhoffii and in a dog from Minnesota infected with B. henselae (3,4). The North Carolina case had unilateral mandibular lymph node involvement, whereas the dog herein had bilateral mandibular and medial retropharyngeal lymphadenopathy. In contrast, the dog from Minnesota had generalized lymphadenopathy, which has also been reported in humans in association with infection with B. henselae and B. alsatica (5,6). Taken together, these cases suggest that infection with Bartonella in dogs could potentially cause focal, regional, or generalized lymphadenomegaly. Bartonella spp. infection has also been previously linked to granulomatous inflammation and nodular lesions in tissues other than lymph nodes. Granulomatous hepatitis has been reported in association with B. henselae infections in some dogs and humans (7,8). Discrete nodular granulomatous lesions in the myocardium and diaphragm have been reported in association with B. henselae infection in 2 cats (9). In the human literature, 1 case of B. henselae associated with nodular granulomatous pneumonitis was described in an immunocompromised renal transplant patient with a prolonged history of fever, vomiting, and diarrhea, followed by eventual CT identification of multiple discrete pulmonary nodules that were subsequently confirmed as granulomas following excisional biopsy (10). In that patient, other diagnostic test results were negative and B. henselae DNA was amplified and sequenced from a pulmonary homogenate obtained from a surgical lung biopsy (10). As granulomatous inflammation has been documented in cats, dogs, and humans CVJ / VOL 55 / OCTOBER 2014
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ent but at low prevalence rates (22,23). The owners had applied fipronil (Merial, Duluth, Georgia, USA) in the summer months and had never seen fleas or ticks on any of their dogs. All known Bartonella spp. are believed to be transmitted by an insect vector or by bites, scratches or needle stick; however, the mode of transmission of B. henselae for this patient remains unclear (24). With this dog having originated in British Columbia and resided only in Montana, B. henselae transmission had to have occurred in British Columbia or Montana. Recently, B. henselae DNA was amplified and sequenced from stored paraffin blocks containing heart valves from dogs that died due to endocarditis or myocarditis in Colorado where flea and tick exposure is also infrequent (25). Although perhaps uncommon, dogs in the western United States, where flea and tick infestations are reportedly infrequent, can be infected with B. henselae, through a currently unknown mode of transmission. We conclude that, although other etiologies or co-infection with other bacteria cannot be ruled out in this dog, this case suggests a possible role for Bartonella infection in dogs with pyogranulomatous lymphadenitis and pulmonary nodules of undetermined cause. Additional research is necessary to define modes of transmission, and the geographic prevalence of these bacteria throughout the US and Canada, particularly in regions with low flea and tick prevalence. CVJ
References 1. Perez C, Diniz PP, Pultorak EL, Maggi RG, Breitschwerdt EB. An unmatched case controlled study of clinicopathologic abnormalities in dogs with Bartonella infection. Comp Immunol Microbiol Infect Dis 2013;36:481–487. 2. Florin TA, Zaoutis TE, Zaoutis LB. Beyond cat scratch disease: Widening spectrum of Bartonella henselae infection. Pediatrics 2008;121:413–425. 3. Pappalardo BL, Brown T, Gookin JL, Morrill CL, Breitschwerdt EB. Granulomatous disease associated with Bartonella infection in 2 dogs. J Vet Intern Med 2000;14:37–42. 4. Morales SC, Breitschwerdt EB, Washabau RJ, Matise I, Maggi RG, Duncan AW. Detection of Bartonella henselae DNA in two dogs with pyogranulomatous lymphadenitis. J Am Vet Med Assoc 2007;230:681–685. 5. Rolain JM, Lepidi H, Zanaret M, et al. Lymph node biopsy specimens and diagnosis of cat-scratch disease. Emerg Infect Dis 2006;12:1338–1344. 6. Angelakis E, Lepidi H, Canel A, et al. Human case of Bartonella alsatica lymphadenitis. Emerg Infect Dis 2008;14:1951–1953. 7. Gillespie TN, Washabau RD, Goldschmidt MH, Cullen JM, Rogala AR, Breitschwerdt EB. Detection of Bartonella clarridgeiae DNA in hepatic specimens from two dogs with hepatic disease. J Am Vet Med Assoc 2003;222:47–51. 8. VanderHeyden TR, Yong SL, Breitschwerdt EB, et al. Granulomatous hepatitis due to Bartonella henselae infection in an immunocompetent patient. BMC Infectious Diseases 2012;12:17. Available from: http:// www.biomedcentral.com/1471–2334/12/17 Last accessed July 22, 2014. 9. Varanat M, Broadhurst J, Linder KE, Maggi RG, Breitschwerdt EB. Identification of Bartonella henselae in 2 cats with pyogranulomatous myocarditis and diaphragmatic myositis. Vet Pathol 2012;49:608–611. 10. Caniza MA, Granger DL, Wilson KH, et al. Bartonella henselae: Etiology of pulmonary nodules in a patient with depressed cell-mediated immunity. Clin Infect Dis 1995;20:1505–1511. 11. Chomel BB, Kasten RW. Bartonellosis, an increasingly recognized zoonosis. J Appl Microbiol 2010;109:743–750. 12. Kaiser PO, Riess T, O’Rourke F, Linke D, Kempf VA. Bartonella spp.: Throwing light on uncommon human infections. Int J Med Microbiol 2011;301:7–15. 13. Lin JW, Chen CM, Chang CC. Unknown fever and back pain caused by Bartonella henselae in a veterinarian after a needle puncture:
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naturally infected with Bartonella spp., these bacteria satisfy the recently proposed Postulate of Comparative Infectious Disease Causation in the context of causation and granulomatous inflammatory lesions (16). Diagnosis of bartonellosis can be challenging. In the 2 previous canine cases of Bartonella-associated lymphadenitis, the diagnosis was confirmed by amplification of Bartonella spp. DNA from surgically excised granulomatous lymph nodes, whereas in our case PCR amplification was not successful using a sample acquired by lymph node aspiration. In humans, it has been reported that bacteria are most readily observed within the first 2 to 3 wk following the onset of lymphadenopathy, with numbers decreasing rapidly as the suppurative inflammation progresses (17). The dog in this case report was presented with a 9-day history of illness, but the true time course of lymphadenopathy is unknown. It is possible that bacterial numbers in the nodes had decreased significantly by the time of sampling, thus making them difficult to detect by PCR. It is also plausible that Bartonella was present at the time of lymph node sampling, but was missed due to the large size of the nodes and inherent potential for sampling error when performing tissue aspirates with a 22-gauge needle. A portion of the lymph node biopsy material should have been cultured for Bartonella (18); however, this was not done in this case. Acute and convalescent B. henselae serology may have also been supportive of a diagnosis of acute bartonellosis, but was not requested in this case. In the dog of this report, Bartonella bacteremia was confirmed by PCR and DNA sequencing after culturing blood in an optimized insect cell culture-based growth medium (BAPGM) (19). Enrichment culture increases bacterial numbers to a high enough level to allow for successful PCR amplification. In an effort to rule out other bacterial pathogens as a potential cause for disease in this patient, cultures using standard aerobic and anaerobic culture techniques were performed but were negative. However, a recent study evaluating dogs by BAPGM enrichment culture and 16S rDNA PCR amplification of subcultured isolates identified dogs that were concurrently infected with B. henselae and other fastidious bacteria. In that study, the BAPGM method appeared to preferentially grow organisms within the Class alpha-Proteobacteria, which were rarely isolated using conventional microbiological methods. The authors acknowledged that the prolonged incubation times required with enrichment cultures may increase the incidence of isolating non-pathogenic commensal bacteria, normal flora, contaminants, or environmental bacteria. The pathogenicity of the isolates identified in that study was unclear and a causal relationship between an organism and clinical disease could not be made (20). The commercial company performing the BAPGM enrichment blood culture used to test this dog specifically targets Bartonella spp. by PCR and does not perform subcultures from the enrichment sample; therefore, it is possible that this dog could have been co-infected with another fastidious bacterium, as reported previously for human patients (21). Of additional clinical and microbiological relevance, this dog was from Montana, where cat and dog fleas (Ctenocephalides felis and Ctenocephalides canis, respectively) and tick vectors (Dermacentor andersoni and Rhipicephalus sanguineus) are pres-
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A case report and literature review. Vector Borne Zoonotic Dis 2011;11:589–591. 14. Breitschwerdt EB, Maggi RG, Chomel BB, Lappin MR. Bartonellosis: An emerging infectious disease of zoonotic importance to animals and human beings. J Vet Emerg Crit Care 2010;20:8–30. 15. Cross JR, Rossmeisl JH, Maggi RG, Breitschwerdt EB, Duncan RB. Bartonella-associated meningoradiculoneuritis and dermatitis or panniculitis in 3 dogs. J Vet Intern Med 2008;22:674–678. 16. Breitschwerdt EB, Linder KL, Day MJ, Maggi RG, Chomel BB, Kempf VA. Koch’s postulates and the pathogenesis of comparative infectious disease causation associated with Bartonella species. J Comp Path 2013;148:115–125. 17. Karem KL, Paddock CD, Regnery RL. Bartonella henselae, B. quintana, and B. bacilliformis: Historical pathogens of emerging significance. Microbes Infect 2000;2:1193–1205. 18. Perez C, Maggi RG, Diniz PP, Breitschwerdt EB. Molecular and serological diagnosis of Bartonella infection in 61 dogs from the United States. J Vet Intern Med 2011;25:805–810. 19. Duncan AW, Maggi RG, Breitschwerdt EB. A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: Pre-enrichment liquid culture followed by PCR and subculture onto agar plates. J Microbiol Methods 2007;69:273–281.
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20. Davenport AC, Mascarelli PE, Maggi RG, Breitschwerdt EB. Phylogenetic diversity of bacteria isolated from sick dogs using the BAPGM enrichment culture platform. J Vet Intern Med 2013;4:854–861. 21. Cadenas MB, Maggi RG, Diniz PP, Breitschwerdt KT, Sontakkes S, Breitschwerdt EB. Identification of bacteria from clinical samples using Bartonella alpha-Proteobacteria growth medium. J Microbiol Methods 2007;71:147–155. 22. Jameson PH, Greene CE, Regnery RL, et al. Prevalence of Bartonella henselae antibodies in pet cats throughout regions of North America. J Infect Dis 1995;172:1145–1149. 23. Greene CE. Infectious Diseases of the Dog and Cat. 2nd ed. Philadelphia, Pennsylvania: WB Saunders, 1998:155–157. 24. Oliveira AM, Maggi RG, Woods CW, Breitschwerdt EB. Suspected needle stick transmission of Bartonella vinsonii subspecies berkhoffii to a veterinarian. J Vet Intern Med 2010;24:1229–1232. 25. Fenimore A, Varanat M, Maggi R, Schultheiss P, Breitschwerdt E, Lappin MR. Bartonella spp. DNA in cardiac tissues from dogs in Colorado and Wyoming. J Vet Intern Med 2011;25:613–616.
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