Institute for Microbiology1, Clinic for small Animals2, University of Veterinary Medicine Hannover, Foundation
(in preparation)
Abstract
Bartonella species are being recognized as zoonotic pathogens with increasing importance in veterinary and human medicine. These bacteria can be transmitted by arthropods or alternatively by animal scratches or bites. In general, cats are associated with the transmission of Bartonella henselae to humans, typically resulting in cat scratch disease; however, there have also been sporadic reports of Bartonella transmission by dogs. In this study we analysed randomly collected EDTA-blood samples from dogs for Bartonella bacteraemia by detecting Bartonella DNA using real time PCR and PCR-REA. Out of 159 blood samples 50 show amplification in real time PCR. After species differentiation B. henselae were detected in 45 samples and B. elizabethae in 5 samples. Furthermore, we were able to detect 3 cases of clinical B. henselae infections in dogs with symptoms comparable to those occurring in Bartonella infection of humans. These are described in detail as case reports. In this study we were able to demonstrate a higher detection rate of Bartonella in dogs in comparison to the cat population. The role of Bartonella spp. in humans is not finally clarified.
Introduction:
Bacteria of the genus Bartonella are fastidious, gram-negative rod shaped bacteria encompassing more than 20 described species (Abbott et al., 1997; Chomel et al., 2003;
Chomel et al., 2004; Boulouis et al., 2005; Chomel et al., 2006). Bartonella are highly adapted to mammals as reservoir hosts, in which they commonly cause long-lasting intraerythrocytic bacteremia (Kordick and Breitschwerdt, 1995; Breitschwerdt and Kordick, 2000; Dehio, 2001; Jacomo et al., 2002). Bartonella spp. were first isolated from a dog in 1993 suffering from endocarditis (Breitschwerdt et al., 1995). Since the, several Bartonella species, including B. clarridgeiae, B. elizabethae, B. henselae, B. vinsonii subspecies
berkhoffii and B. washoensis have been shown to infect dogs. In contrast to clinical manifestation in cats, Bartonella may contribute to the pathogenesis of a wide spectrum of diseases in dogs, including polyarthritis, cutaneous vasculitis, endocarditis, myocarditis, epistaxis, pelisosis hepatis and granulomatous inflammatory disease (Breitschwerdt et al., 1995; Kordick et al., 1996; Breitschwerdt et al., 1999; Kitchell et al., 2000; Pappalardo et al., 2000; Chomel et al., 2001; Mexas et al., 2002; Gillespie et al., 2003). The transmission of B. henselae from dogs to humans has occasionally been suggested. Thus, B. henselae was found to be aetiological agent in a case of human osteomyelitis following a dog scratch (Keret et al., 1998). Furthermore, seroreactivity for B. henselae, and Bartonella spp has been described in a dog owner suffering from lymphadenopathy. Thereupon, DNA was amplified from gingival swabs of his dogs (Tsukahara et al., 1998). In Germany no study addresses the prevalence of Bartonella infection in the dog population. Therefore, we investigated the prevalence of Bartonella spp. in dogs in northern and western part of Germany. We were able to show that the prevalence of Bartonella infected dogs is high and that B. henselae is the most prevalent species.
Material and Methods
One hundred fifty nine EDTA blood samples were randomly collected from dogs with different clinical backgrounds in the small animal clinic of the University of Veterinary Medicine, Hannover. All EDTA blood samples were analysed by quantitative real time PCR.
Additionally, EDTA blood or lymph node aspirate was taken from four dogs with specific clinical symptoms, such as lymphadenopathy and granulomatous inflammatory diseases, potentially associated with Bartonella infection.
Genomic DNA was extracted from 125 µl of the deep frozen canine EDTA-anticoagulated blood after thawing using a DNA extraction kit (innuprep dna mini kit, Analytic Jena, Germany). DNA was eluted from the columns with 60 µl elution buffer.
Molecular detection of Bartonella spp. was performed by quantitative real time PCR analysis targeting the citrate synthase gene and species differentiation by restriction analysis of PCR (PCR-REA) (Mietze et al., 2010)
Immunohistochemistry was carried out with anti-B. henselae rabbit serum and the avidin-biotin-peroxidase complex method (Alldinger et al., 1996).
Results
We were able to detect Bartonella spp. in 50 of 159 blood samples, resulting in a prevalence of 31.4%. The species differentiation by PCR-REA showed the restriction pattern of B.
henselae (28.3%) in 45 samples and the pattern of B. elizabethae in 5 samples (3.1%).
Analyses of the clinical data showed no correlation between specific clinical signs and the detection of Bartonella DNA in the blood of the dogs.
Case1:
A four year old Welsh terrier showed recurrent febrile episodes of ambiguous nature as well as hyperesthesia. During the ultrasonographic examination, alterations of the abdominal organs were recognized. Therefore, a diagnostical laparotomy was performed and samples of the organs were taken for histpathological analysis. Histology revealed a purulent-necrotizing splenitis, a pyogranulomatous perihepatitis, a purulent hepatitis and purulent necrotizing pancreatitis. It was suggested, that the cause of these alteration could be a primary or secondary bacterial infection yet microbiological examination showed no aerobic or anaerobic agent content. Using a quantitative real time PCR from EDTA blood from this dog as well as PCR-REA we were able to detect B. henselae. In contrast, detection by culture from the EDTA blood was negative. In immunohistochemistry we were able to detect Bartonella spp.
in the liver tissue of the dog. The dog was treated with marbofloxacin, metamizol and prednisolon. After discharge the dog died suddenly.
Case2:
A seven year old Doberman was delivered to the clinic for small animals of our University with clinical symptoms of inappetence and apathy, and a body temperature of 40.5°C. The tentative diagnosis was an ileus. A diagnostic laparotomy was performed and identified an enlargement of the mesenteric lymph nodes caused by a purulent necrotizing lymphadenitis.
The fine needle biopsy confirmed the purulent necrotizing lymphadenitis and revealed bacterial colonisation. EDTA blood as well as an aspirate of the lymphatic tissue were used for the quantitative real time PCR for Bartonella spp.. After PCR-REA, both revealed the pattern of B. henselae. Unfortunately, the detection by culture was negative. The pathohistological examination identified a malignant lymphoma. The dog was treated with doxycyclin and metronidazol. After one week the dog was euthanized.
Case3:
A nine year old toy poodle was delivered to the clinic for small animals with hyperesthesia.
The abdominal ultrasound revealed a cyst within the pancreas. Bacteriological culture of an aspirate of the cyst showed moderate Clostridium perfringens and B. henselae colonisation.
Western blot analysis of the dog serum confirmed reactivity with surface proteins of B. henselae. The culture and PCR of the EDTA blood were negative. After antimicrobial treatment with amoxicillin the hyperesthesia declined. In a repeated puncture and culture of the cyst, two weeks later, no bacteria were detected and the diameter of the cyst was reduced.
Four weeks afterwards, hyperesthesia had reappeared in the animal. The cyst was then sugically removed in the clinic. Intraoperatively, strong adherences to the pancreas were observed. Until now the dog is under observation.
Discussion
The major reservoir host of B. henselae is the cat. Infections in cats often remain undetected, because infected animals do not display characteristic clinical symptoms. We previously found a detection rate of Bartonella DNA in cats of 16.6 % (Mietze et al., 2010). As only a few studies exist about the prevalence of the dog population (Bai et al., 2010), we analysed 159 canine blood samples. We were able to detect an infection with B. henselae or B.
elizabethae in 50 of 159 dogs. While commonly cats are regarded as the main zoonotic reservoir for B. henselae infection, this study demonstrates that the prevalence of Bartonella spp. in dogs in Germany is at the same level or even higher than in cats. We were able to detect B. henselae in 28.3 % of the analysed dogs and B. elizabethae in 3.1 % of the analysed dogs. In a comparable study conducted in Thailand, a prevalence of 31.3 % of Bartonella spp
was found in a stray dog population, yet none of these dogs was infected with B. henselae (Bai et al., 2010). The fact, that asymptomatic dogs were tested positive for Bartonella spp.
implies dogs may also act as zoonotic reservoir for Bartonella spp.. On the other hand, we were able to detect B. henselae in three out of four dogs suffering from symptoms which may be associated with Bartonella infection. However, only in one dog the symptoms improved after antimicrobial treatment. Interestingly, we exclusively detected B. henselae. Therefore it remains unclear, if dogs similar to cats are only reservoirs for Bartonella or if they are a non-reservoir host and develop the same manifestations of the diseases as humans. It may possibly depend upon the animals’ immune status, as immunocompetent dogs stay asymptomatic and immunocompromised dogs develop the different clinical symptoms. The clinical relevance derived from the microbiologic evaluation of the three cases is difficult to infer due to the small number of cases and it may be necessary to apply molecular Koch’s postulates to establish the pathogenicity of these highly adapted organisms. In comparison to the cat population where we were able to detect only B. henselae, except of one cat, described previously (Mietze et al., 2010), in the dog population we found also B. elizabethae.
B. elizabethae, of which rats are the reservoir and the transmission occurs though rat flea.
Thus, further investigation into the transmission should be carried out to clarify if vectors other than the cat flea, which are also often found on dogs (Just et al., 2008), are involved.
Specifically, ticks must be considered as vector for B. henselae (Mietze et al., 2010; Dietrich et al., 2010), while rat flea may execute the transmission of B. elizabethae. B. elizabethae is another recognized pathogen, causing endocarditis and other pathologies in both humans and dogs (Daly et al., 1993; Mexas et al., 2002). Although the extent to which dogs mediate the transmission of Bartonella spp. to humans is unclear, the obtained results suggest that dogs should be considered as potential source for Bartonella infections in humans.
Reference List
Abbott, R.C., Chomel, B.B., Kasten, R.W., Floydhawkins, K.A., Kikuchi, Y., Koehler, J.E., Pedersen, N.C., 1997. Experimental and natural infection with Bartonella henselae in domestic cats. Comparative Immunology Microbiology and Infectious Diseases 20, 41-51.
Alldinger, S., Wunschmann, A., Baumgartner, W., Voss, C., Kremmer, E., 1996. Up-regulation of major histocompatibility complex class II antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis.
Acta Neuropathologica 92, 273-280.
Bai, Y., Kosoy, M.Y., Boonmar, S., Sawatwong, P., Sangmaneedet, S., Peruski, L.F.,
Enrichment culture and molecular identification of diverse Bartonella species in stray dogs. Veterinary Microbiology In Press, Corrected Proof.
Boulouis, H.J., Chang, C.C., Henn, J.B., Kasten, R.W., Chomel, B.B., 2005. Factors
associated with the rapid emergence of zoonotic Bartonella infections. Vet. Res. 36, 383-410.
Breitschwerdt, E.B., Atkins, C.E., Brown, T.T., Kordick, D.L., Snyder, P.S., 1999. Bartonella vinsonii subsp. berkhoffii and related members of the alpha subdivision of the
Proteobacteria in dogs with cardiac arrhythmias, endocarditis, or myocarditis. J. Clin.
Microbiol. 37, 3618-3626.
Breitschwerdt, E.B., Kordick, D.L., 2000. Bartonella infection in animals: Carriership, reservoir potential, pathogenicity, and zoonotic potential for human infection. Clin.
Microbiol. Rev. 13, 428-438.
Breitschwerdt, E.B., Kordick, D.L., Malarkey, D.E., Keene, B., Hadfield, T.L., Wilson, K., 1995. Endocarditis in a dog due to infection with a novel Bartonella subspecies. J.
Clin. Microbiol. 33, 154-160.
Chomel, B.B., Boulouis, H.J., Breitschwerdt, E.B., 2004. Cat scratch disease and other zoonotic Bartonella infections. Javma-Journal of the American Veterinary Medical Association 224, 1270-1279.
Chomel, B.B., Boulouis, H.J., Maruyama, S., Breitschwerdt, E.B., 2006. Bartonella spp. in pets and effect on human health. Emerg. Infect. Dis. 12, 389-394.
Chomel, B.B., Kasten, R.W., Sykes, J.E., Boulouis, H.J., Breitschwerdt, E.B., 2003. Clinical impact of persistent Bartonella bacteremia in humans and animals. Rickettsiology:
Present and Future Directions 990, 267-278.
Chomel, B.B., Mac Donald, K.A., Kasten, R.W., Chang, C.C., Wey, A.C., Foley, J.E., Thomas, W.P., Kittleson, M.D., 2001. Aortic valve endocarditis in a dog due to Bartonella clarridgeiae. J. Clin. Microbiol. 39, 3548-3554.
Daly, J.S., Worthington, M.G., Brenner, D.J., Moss, C.W., Hollis, D.G., Weyant, R.S., Steigerwalt, A.G., Weaver, R.E., Daneshvar, M.I., Oconnor, S.P., 1993. Rochalimaea elizabethae sp. nov Isolated from A Patient with Endocarditis. Journal of Clinical Microbiology 31, 872-881.
Dehio, C., 2001. Bartonella interactions with endothelial cells and erythrocytes. Trends Microbiol. 9, 279-285.
Dietrich, F., Schmidgen, T., Maggi, R.G., Richter, D., Matuschka, F.R., Vonthein, R., Breitschwerdt, E.B., Kempf, V.A.J., 2010. Prevalence of Bartonella henselae and Borrelia burgdorferi Sensu Lato DNA in Ixodes ricinus Ticks in Europe. Appl.
Environ. Microbiol. 76, 1395-1398.
Gillespie, T.N., Washabau, R.J., Goldschmidt, M.H., Cullen, J.M., Rogala, A.R., Breitschwerdt, E.B., 2003. Detection of Bartonella henselae and Bartonella
clarridgeiae DNA in hepatic specimens from two dogs with hepatic disease. Journal of the American Veterinary Medical Association 222, 47-51.
Jacomo, V., Kelly, P.J., Raoult, D., 2002. Natural history of Bartonella infections (an exception to Koch's postulate). Clinical and Diagnostic Laboratory Immunology 9, 8-18.
Just, F.T., Gilles, J., Pradel, I., Pfalzer, S., Lengauer, H., Hellmann, K., Pfister, K., 2008.
Molecular evidence for Bartonella spp. in cat and dog fleas from Germany and France. Zoonoses and Public Health 55, 514-520.
Keret, D., Giladi, M., Kletter, Y., Wientroub, S., 1998. Cat-scratch disease osteomyelitis from a dog scratch. Journal of Bone and Joint Surgery-British Volume 80B, 766-767.
Kitchell, B.E., Fan, T.M., Kordick, D., Breitschwerdt, E.B., Wollenberg, G., Lichtensteiger, C.A., 2000. Peliosis hepatis in a dog infected with Bartonella henselae. J. Am. Vet.
Med. Assoc. 216, 519-23, 517.
Kordick, D.L., Breitschwerdt, E.B., 1995. Intraerythrocytic Presence of Bartonella henselae.
Journal of Clinical Microbiology 33, 1655-1656.
Kordick, D.L., Swaminathan, B., Greene, C.E., Wilson, K.H., Whitney, A.M., O'Connor, S., Hollis, D.G., Matar, G.M., Steigerwalt, A.G., Malcolm, G.B., Hayes, P.S., Hadfield, T.L., Breitschwerdt, E.B., Brenner, D.J., 1996. Bartonella vinsonii subsp. berkhoffii subsp. nov., isolated from dogs; Bartonella vinsonii subsp. vinsonii; and emended description of Bartonella vinsonii. Int. J. Syst. Bacteriol. 46, 704-709.
Mexas, A.M., Hancock, S.I., Breitschwerdt, E.B., 2002. Bartonella henselae and Bartonella elizabethae as potential canine pathogens. J. Clin. Microbiol. 40, 4670-4674.
Mietze, A., Morick, D., Köhler, H., Harrus, S., Dehio, C., Nolte, I., Goethe, R., 2010.
Combined MLST and AFLP typing of Bartonella henselae isolated from cats reveals new sequence types and suggests clonal evolution. Vet. Microbiol. accepted.
Pappalardo, B.L., Brown, T., Gookin, J.L., Morrill, C.L., Breitschwerdt, E.B., 2000.
Granulomatous disease associated with Bartonella infection in 2 dogs. J. Vet. Intern.
Med. 14, 37-42.
Tsukahara, M., Tsuneoka, H., Iino, H., Ohno, K., Murano, I., 1998. Bartonella henselae infection from a dog. Lancet 352, 1682.
Abb. 1.: IHC of the liver, case1 (A) and negative control (B) A)
B)
4 Occurrence of Bartonella henselae and Borrelia burgdorferi sensu lato