Bartonella alsatica in Wild and Domestic Rabbits ( Oryctolagus cuniculus ) in The Netherlands

: Members of the genus Bartonella are Gram-negative facultative intracellular bacteria that are transmitted by arthropod vectors. Bartonella alsatica was detected in the spleens and livers of 7 out of 56 wild rabbits ( Oryctolagus cuniculus ) and in the liver of 1 out of 87 domestic rabbits in the Netherlands. The molecular evidence of B. alsatica infection in wild as well as domestic rabbits indicates the possibility of exposure to humans when these come in close contact with rabbits and possibly their ﬂeas with subsequent risk of Bartonella infection and disease.


Introduction
Bartonella genus members are facultative intracellular Gram-negative bacteria. Many of them have been identified as zoonotic agents. Blood-sucking arthropods transmit the bacteria. The bacteria can infect erythrocytes of mammals potentially causing persistent bacteremia. Bartonella henselae, B. bacilliformis, and B. quintana are the most relevant species for human beings [1], but other species such as B. alsatica can also cause disease. B. alsatica has been described as the causative agent of two human cases of endocarditis, one aortobifemoral graft infection and one case of lymphadenitis [2][3][4][5]. All four patients had been in close contact with wild or domestic rabbits. In continental Europe, the pathogen has been detected both in France and in Spain in live caught, apparently healthy, wild European rabbits (Oryctolagus cuniculus) and in rabbit fleas (Spilopsyllus cuniculi and Xenopsylla cunicularis) [6,7]. Pathology on two euthanized infected rabbits provided no evidence of disease [7].
The incidence of human bartonellosis in the Netherlands is not well-known but estimated to be 2 per 100,000 inhabitants per year [8]. Several different zoonotic Bartonella species are known to circulate in the Netherlands, including B. henselae, B. clarridgeiae, B. schoenbuchensis, B. grahamii and B. washoensis [9,10]. Information on B. alsatica is lacking.
This study retrospectively investigated wild and domestic rabbits examined postmortem in the Netherlands for evidence of B. alsatica infection.

Materials and Methods
Full necropsy was performed following a standard protocol on 51 wild and 87 domestic rabbits. From five wild rabbits only spleen and liver were obtained. The wild rabbits were offered for necropsy to the Dutch Wildlife Health Centre as part of disease surveillance in wildlife, and the domestic rabbits presented to the Veterinary Pathology Diagnostic Centre for diagnostic purposes. Necropsy was performed following a standard protocol on 51 wild rabbits and all 87 domestic rabbits. Necropsy included macroscopic examination, cytological analysis of liver, spleen, lungs, and intestinal contents stained with Hemacolor ® (Hemacolor quick stain, Merck, Darmstadt, Germany), and histological examination of samples of various organs fixed in 4% buffered formalin, embedded in paraffin, cut into 4-µm sections, and stained with hematoxylin and eosin. Organ tissues were frozen at −80 • C.
DNA was extracted from 56 wild rabbits (spleen and liver) and 87 domestic rabbits (liver) using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer's protocol (Qiagen, Venlo, the Netherlands). These samples were then tested for the presence of Bartonella spp. using qPCR [9]. From the qPCR-positive samples, a fragment of the gltA gene was amplified by conventional PCR and sequenced for species identification [11]. Distilled water was used as negative control and B. schoenbuchensis from deer keds (Lipoptena cervi) from a previous study as a positive control. Positive controls and negative water controls were used on every plate tested. To prevent contamination, and false-positive samples, the DNA extraction, PCR mix preparation, sample addition, and (q)PCR analyses were performed in separated air locked dedicated labs.

Results
The PCR-test results are summarized per necropsy result in Table 1. Twelve wild rabbits were positive by qPCR for Bartonella spp. From these, seven could be identified as B. alsatica by conventional PCR and sequencing: The 338 base pairs of the PCR product were 100% identical to GenBank accession number AF204273. Two domestic rabbits were positive by qPCR from which one could be identified as B. alsatica (identical to AF204273). Failure of successful sequencing of the other six of the PCR products might be due to low DNA concentration. The B. alsatica PCR-positive wild animals died of rabbit hemorrhagic disease virus (RHDV) infection (3), myxomatosis (2), or no pathologic examination was performed (2). The positive domestic rabbit died of RHDV infection. None of the six cases examined post-mortem and positive for B. alsatica showed evidence of endocarditis, myocarditis or vascular pathology.

Discussion
This study shows presence of B. alsatica in both domestic and wild rabbits in the Netherlands. The positive rabbits died of common viral diseases of rabbits and showed no evidence for typical Bartonella-associated lesions.
Human infections with Bartonella spp. have been described; however, in an unknown number of cases infection was suspected but proof could not be established by serology, culture or PCR. It is known that B. alsatica can cause blood culture-negative disease and, therefore, the incidence of infection may be under-estimated [3]. Marquez et al. [6] concluded, based on presence of B. alsatica in rabbits and their fleas, that more research should be performed to determine if B. alsatica could be responsible for unknown febrile disease in humans. This study supports the role of wild and domestic rabbits as a reservoir for human infections, and the addition of B. alsatica to the list of zoonotic agents [12]. Moreover, domestic rabbits are also kept in children's farms where people with variable immune status are in close contact with rabbits. The molecular evidence of B. alsatica infection in wild as well as domestic rabbits indicates the possibility of exposure to humans when these come in close contact with rabbits and possibly their fleas with subsequent risk of Bartonella infection and disease. Institutional Review Board Statement: The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as the organs used were derived from wild rabbits offered for necropsy to the Dutch Wildlife Health Centre as part of disease surveillance in wildlife, and 87 domestic rabbits presented to the Veterinary Pathology Diagnostic Centre for diagnostic purposes.

Informed Consent Statement: Not applicable.
Data Availability Statement: GenBank accession number AF204273.