Supplementary MaterialsFig. of inside a diseased member of the forma is a member of their normal microbiota. Due to unawareness, fastidiousness, antibiotic sensitivity and lack of diagnostics the role of as a cat and human pathogen might be under-reported as with other infections. More studies are necessary to elucidate the role of in domestic cats and other in order to better estimate its zoonotic potential. Electronic supplementary material The online version of this article (10.1007/s10482-020-01454-x) contains supplementary material, which is available to authorized users. (((Woo et al. 2014), (Eisenberg et al. 2014), (Eisenberg et al. 2015b), (Eisenberg et al. 2016) and (Eisenberg et al. 2020b) were described as novel species. Whereas and are closely associated with black rats (has exclusively been isolated from humans (Lau et al. 2016; Woo et al. 2014) and was recently found to belong to a novel genus, (Eisenberg et al. 2020a). and were only once isolated from clinical disease in animals, i.e. from a cat with pneumonia and a dog with phlegmon, respectively (Eisenberg et al. 2015a, 2020b). However, with respect to zoonotic potential, has been found to also cause RBF in humans (Fukushima et al. 2017; Ogawa et al. 2018) and a similar case of RBF could recently be attributed to for the first time (Matt et al. 2020). Interestingly, various phylotypes consistent with 16S rRNA gene sequence based operational taxonomic units (OTU) have been described from humans and various animal species (Fig.?1). We here report a second strain of is used as outgroup. T indicating type strain; Bar, 0.02 nucleotide substitutions per site Materials and methods Case description A breeding group of the endangered rusty-spotted cat (tests for feline parvovirus, coronavirus and protozoa revealed negative results. Due DM1-Sme to disease progression, the animal was euthanized. Pathological investigation A gross pathology examination and histology were performed. For histopathological examination, specimens of multiple organs were fixed in buffered 4% formalin, processed by standard methods and inlayed in paraffin. Microtome areas had been stained with hematoxylinCeosin (HE). Immuno-histochemistry (IHC) for antibody given by Davids Biotechnologie GmbH (Regensburg, Germany). A goat-anti-rabbit IgG biotinylated antibody (Vector Laboratories, Burlingame, USA) offered as a second antibody and allowed the recognition from the antigenCantibody complicated using the Vectastain ABC-Elite Package (Linaris). Diaminobenzidine (DAB; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was added, producing a brown-colored precipitate developing where antibody have bound. FFPE samples of the lung of a C57BL/6 mouse that was experimentally infected with (Fornefett et al. 2017) underwent the same protocol and served as positive controls. For a negative control, FFPE samples of the rusty-spotted cat underwent the described protocol with only the primary antibody being replaced with negative control rabbit immunoglobulin fraction (Dako). Evaluation of the immune-histochemical examination was performed using a transmission light microscope. Phenotypic characterization Bacterial isolation and physiological properties Bacterial isolates were obtained and isolates were identified using standard microbiological examinations. Briefly, native DM1-Sme tissue samples were processed for microbial culture by inoculating flame sterilized, freshly cut tissue surfaces onto culture media (Columbia agar with 5% sheep blood [SBA; Oxoid, Wesel, Germany] and Gassner agar [VWR, Darmstadt, Germany]). Agar plates were incubated for up to 48?h at 20?C using aerobic and microaerobic culture conditions. Phenotypic characterization of streptobacilli is known to yield only few weakly positive reactions (Eisenberg et al. 2015c), however, standard microbiological procedures included tests for hemolysis on SBA, catalase activity with 3% H2O2 on RGS2 microscopic slides and for presence of cytochrome oxidase with the BBL DrySlide? oxidase system (BectonCDickinson, Heidelberg, DM1-Sme Germany). Urease, hydrogen sulfide, indole, motility and oxidative and fermentative glucose assimilation were tested on Christensen agar, SIM and OF medium in slant agar tubes, respectively (all Merck, Darmstadt, Germany). Microscopic examinations of fixed smears were performed using Grams stain. For further identification attempts, the Omnilog GEN III plate identification system (Biolog, Hayward, USA) was utilized for the first time using the most sensitive protocols for fastidious bacteria (C1 and C2) with and without addition of 10% bovine serum according to manufacturers recommendations. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) Mass spectrometry procedure has been recently described in detail (Eisenberg et al. 2018, 2020b). The commercial database used (DB 8,468; BrukerDaltonics) comprised 24 spectra each from 10 strains. Reference spectra from well-characterized, quality-controlled strains of.