First description of Bartonella koehlerae infection in a Spanish dog with infective endocarditis

Bartonella koehlerae has been recently described as a new cat- and cat fleas-associated agent of culture-negative human endocarditis. It has been also encountered in one dog from Israel and six dogs from the USA, but other clinically relevant reports involving this bacterium are lacking. A 7-year-old intact male mixed dog presented with clinico-pathological signs consistent with mitral endocarditis and cutaneous hemangiosarcoma. Molecular studies revealed the presence of Bartonella koehlerae DNA in samples from blood and mitral valve tissue. This is the first description of B. koehlerae in Spain, corroborating that it can also be detected in dogs. Bartonella koehlerae infection should also be considered in Spain in humans and dogs presenting with clinical disease suggestive of it, such as culture-negative endocarditis.


Background
Bartonella spp. are the etiological agents in humans and animals of several emergent and re-emergent vectorborne diseases that have a broad spectrum of clinical presentations including endocarditis, granulomatous diseases, meningoencephalitis, polyarthritis, uveitis or hemolytic anemia [1].
Bartonella koehlerae has been documented as a human pathogen and it has been increasingly detected in the last years [2,3]. There are sporadic isolated descriptions of this bacterium in dogs. Bartonella koehlerae has been described in seven dogs from Israel and the USA with endocarditis, splenic disease, with suspicion of having a vector-transmitted infection or with hyperinsulinemic hypoglycemia syndrome [4][5][6][7]. PCR screening of Bartonella ITS region was performed by real-time PCR using oligonucleotides BsppITS325s: 5'-CTT CAG ATG ATG ATC CCA AGC CTT YTG GCG-3' and BsppITS543as: 5'-TAA AYT GGT GGG CCT GGG AGG ACT TG-3' as forward and reverse primers, respectively, to increase the sensitivity of the detection of a broader range of Bartonella species [8]. Amplification was performed in a 25-μl final volume reaction containing 12.5 μl of 2× Sso Advanced Universal Sybr Green Supermix (BioRad), 0.2 μl of 100 μM of each forward and reverse primer (IDT® DNA Technology) and 5 μl of DNA from each sample tested. PCR negative controls were prepared using 5 μl of DNA from blood of a healthy dog. Positive controls for PCR were prepared by serial dilution (using dog blood DNA) of genomic DNA from B. henselae down to 0.001 pg/μl (equivalent to 0.5 bacteria per μl). qPCR was performed in an BioRad CFX96 Real-Time System, under the following conditions: a single hot-start cycle at 95°C for 2 min followed by 55 cycles of denaturing at 94°C for 10 s, annealing at 66°C for 10 s, and extension at 72°C for 15 s. Amplicons products were sequenced to establish species strain identification. The sensitivity of the qPCR assay is 98% at 0.001 pg/μl (0.5 bacteria genomes per μl).

Results
A 7-year-old intact male mixed dog was presented for a three-week history of anorexia, weight loss, polyuria and polydipsia that had worsened to severe generalized weakness at presentation time. There was also a cutaneous mass located in the right flank that had ruptured some days before resulting in serosanguinolent discharge. The dog lived outdoors, was regularly vaccinated and dewormed. Physical examination abnormalities included poor body condition (BCS, 2/9), fever (40°C), tachycardia, grade 3-heart apex systolic murmur, pain in several joints, abdominal discomfort and an ulcerated cutaneous mass in the abdomen.
Abdominal ultrasound evidenced generalized splenomegaly and diffuse nodular hypoechoic peripheral lesions in both kidneys. Thorax radiographs showed an alveolar pattern on right lung lobes and left cranial lung lobes. Thickened mitral valve with regurgitation was detected on echocardiography. Fine needle aspiration of the cutaneous mass and spleen were consistent with a mesenchymal neoplasm and reactive splenomegaly, respectively.
Intensive treatment for presumptive infective endocarditis and septic shock was performed with fluid therapy, antibiotics (ampicillin and enrofloxacin), ranitidine and low molecular weight heparin. However health status deteriorated rapidly and the dog died twelve hours after admission. Necropsy confirmed mitral endocarditis (Figs. 1, 2, 3) and secondary inflammatory, necrotic, hemorrhagic and thrombotic lesions in kidneys (renal infarction, glomerulonephritis), lungs (bronchopneumonia) and spleen. Histopathology of the cutaneous mass revealed a neoplastic proliferation of endothelial cells consistent with hemangiosarcoma.
A polymerase chain reaction (PCR) targeting a fragment of the 16S-23S ribosomal RNA intergenic spacer (ITS) was performed on extracted whole blood and mitral valve tissue (frozen tissue stored with saline) at the SVGM using conserved primers for species of the genus Bartonella as previously described [9], which yielded positive results. In order to ascertain the species involved whole blood samples and mitral valve paraffinembedded tissue were shipped to the Intracellular Pathogens Research Laboratory (Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA). PCR from DNA obtained of Bartonella/ alpha-Proteobacteria growth medium (BAPGM) preenrichment blood culture [8] and from the mitral valve tissue were positive for Bartonella spp. The presence of Bartonella koehlerae DNA was confirmed in blood (homology of 214/219 bp, 100% query cover, identity score 98%), and in tissue (homology of 217/219 bp, 100% query cover, identity score 99% with GenBank AF 312490 sequence) (Fig. 4).

Discussion
Here, we report the first description of Bartonella koehlerae in Spain, as the causative agent of Bartonella endocarditis involving the mitral valve in a dog.
Infective endocarditis is an uncommon and often deadly disease in dogs that represents a diagnostic challenge in most of the cases because echocardiogram can be normal, and blood cultures can yield false-negative results due to the low-grade and intermittent bacteremia associated to Bartonella infection at the time of blood collection. Previous research has emphasized that negative blood cultures are especially frequent in Bartonella-induced endocarditis and other techniques are needed for diagnosis [10]. The combination of a pre-enrichment liquid culture with PCR amplification enhances the detection of Bartonella species [8]. Previous studies have shown that B. vinsonii berkhoffii is one of the most common agents found in canine Bartonella endocarditis, where infection often involves the aortic valve [10]. However other species, such as B. koehlerae and B. henselae, have been recently implicated in canine endocarditis in the USA and Israel [4,11]. In Spain, several cases of endocarditis due to Bartonella spp. has been reported in people in the past ten years and the number of cases is likely to be underestimated [12].
Interestingly, recent reports suggest that Bartonella spp. should be investigated as a cause of vasoproliferative tumors in both humans and dogs, due to their ability of enhancing endothelial cell proliferation in conjunction with inhibition of apoptosis [13,14]. Species of Bartonella involved are B. bacilliformis, B. quintana, B. henselae, B. vinsonii berkhoffi and B. koehlerae. Unfortunately, definitive association of Bartonella and the skin hemangiosarcoma could not be established in this study since Bartonella DNA could not be amplified from the skin tumor. False-negative PCR results could be caused by prolonged period of formalin fixation [15,16]. Further research is needed to test the potential role of the genus Bartonella as cofactor in the development of cancer [13,14].
In Spanish dogs, presence of canine bartonellosis has been demonstrated with serological studies [17]. Moreover, there is molecular evidence of this infection in a recently described dog with generalised pyogranulomatous disease and hyperviscosity syndrome [18]. Several reports have confirmed that Bartonella infection is present in people in the Valencian Community and Catalonia (Spain) [19,20].
Definitive documentation of the causative Bartonella spp. in dogs may prove to be a legitimate medical concern, as dogs have been also implicated in the transmission of bartonellosis following a bite or a scratch [21,22]. Bartonella koehlerae has been described as a cat-associated agent of culture-negative human endocarditis [3], and this case report corroborates that it can also be detected in dogs.
The findings presented in this manuscript reinforce the concept of "One Health" approach, which requires close communication in all aspects of health care for humans and animals, with the aim of supporting public health in general. It is worth highlighting that there are many vectors that could harbor Bartonella organisms as sand flies, human body lice, cat fleas, ear mites, and ticks have been described as potential vectors [1]. Therefore the

Conclusions
To our knowledge, this is the first description of Bartonella koehlerae in Spain, corroborating that the parasite can also be detected in dogs. Bartonella spp. infection should also be considered in Spain in humans and dogs presenting with features suggestive of it, such as blood culture-negative endocarditis.

Additional file
Additional file 1: Figure S1. Alignment of the ITS region sequences comparing the dog sequences with Bartonella henselae and B. koehlerae.

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