Studies on Trueperella pyogenes isolated from an okapi (Okapia johnstoni) and a royal python (Python regius)

Background The present study was designed to characterize phenotypically and genotypically two Trueperella pyogenes strains isolated from an okapi (Okapia johnstoni) and a royal python (Python regius). Case presentation The species identity could be confirmed by phenotypic properties, by MALDI-TOF MS analysis and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. Furthermore, sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region (ISR), the target genes rpoB encoding the β-subunit of bacterial RNA polymerase, tuf encoding elongation factor tu and plo encoding the putative virulence factor pyolysin allowed the identification of both T. pyogenes isolates at species level. Conclusions Both strains could be clearly identified as T. pyogenes. The T. pyogenes strain isolated in high number from the vaginal discharge of an okapi seems to be of importance for the infectious process; the T. pyogenes strain from the royal python could be isolated from an apparently non-infectious process. However, both strains represent the first isolation of T. pyogenes from these animal species.


Background
Trueperella pyogenes is worldwide considered as part of the commensal biota of skin and mucous membranes of the upper respiratory and urogenital tract of animals [1]. However, T. pyogenes is also an important opportunistic pathogen that causes mastitis, abortion and a variety of diverse pyogenic infections in livestock, including cattle, sheep, goats, horses, and pigs [2][3][4]. In cattle, T. pyogenes appears to be responsible for infections of the reproductive tract [5] and the mammary gland [6], as well as cases of pneumonia and liver abscessation of large and small ruminants [7]. In swine, T. pyogenes is well known as a causative agent of different types of inflammation in various organs including the lung, heart, joints, mammary glands, and in the reproductive tract [8,9]. Furthermore, T. pyogenes could be found in companion animals [4]. One of the first reported cases in companion animals was an otitis externa detected in a cat and cystitis in a dog [10]. More recently, Wareth et al. [11] described a co-infection case of T. pyogenes with Brucella abortus in a cat and dog. Additionally, various wildlife animals could harbour T. pyogenes [3]. In 2010, Ülbegi-Mohyla et al. [12] characterized two T. pyogenes strains isolated from a bearded dragon and a gecko. Additionally, T. pyogenes infections were reported from a bison and from camels [13,14], from goitered gazelles [15] and from a white-tailed deer [16]. Likewise, some other sporadic cases of infectious diseases associated with T. pyogenes were described in a galago [17], in gray slender lorises [18,19] and in a eurasian lynx [20].
The present study was designed to identify and further characterize T. pyogenes isolated from wildlife animals phenotypically and genotypically. To the best of our knowledge, the present study provides a first detailed description of T. pyogenes recovered from an okapi and a royal python.

Case presentation
As part of routine examination and diagnostics performed on zoo animals at Frankfurt Zoo (Frankfurt am Main, Germany) in 2019, T. pyogenes 24398 was isolated

Phenotypic characterization
A phenotypic characterization was performed using conventional cultural and biochemical assays as previously described [12,18,20,27]    (HCCA, in 50% acetonitrile and 2.5% trifluoroacetic acid in pure water) followed by drying and loading into the mass spectrometer.

Genotypic properties
The genomic DNA of both isolates and the type strains T. pyogenes DSM 20630 T (pig), T. abortisuis DSM 19515 T (placenta of sow after abortion), T. bernardiae DSM 9152 T (human blood) and T. bonasi DSM 17163 T (european bison) were extracted using the DNeasy blood and tissue kit (Qiagen GmbH, Hilden, Germany), in accordance with the manufacturer's instructions. The concentration and purity of DNA were measured by utilizing a Nano Drop spectrophotometer (ND1000; Thermo Fisher Scientific GmbH, Dreieich, Germany). The detection of gene cpn60 of T. pyogenes was performed using a previously designed loop-mediated isothermal amplification (LAMP) assay [24] with a portable real-time fluorometer (Genie II®, OptiGene Ltd, Horsham, UK) and the reference strains T. pyogenes DSM 20630 T , T. abortisuis DSM 19515 T , T. bernardiae DSM 9152 T and T. bonasi DSM 17163 T .
The PCR products were purified and sequenced by Eurofins Umwelt Nord GmbH (Göttingen, Germany). The obtained sequences were analyzed via the cluster method of the MegAlign program (DNASTAR Inc., ver. 15, Madison, WI, USA) by comparing with the nucleotide sequences of 16S rRNA, ISR, rpoB, tuf and plo from different Trueperella reference strains. Moreover, the resulting amino acid sequences of pyolysin of both T. pyogenes isolates were compared with the respective sequences of pyolysin of T. pyogenes DSM 20630 T , closely related pore-forming toxins of genus Arcanobacterium and with other bacterial pore-forming toxins. All the nucleotide and amino acid sequences were obtained from the NCBI GenBank.

Discussion and Conclusion
Both T. pyogenes strains investigated in the present study showed a narrow zone of complete hemolysis on 5% sheep blood agar and CAMP-like reactions in the staphylococcal β-hemolysin zone with Rhodococcus hoagii as an indicator strain. The conventional biochemical properties and the results of the commercial identification system revealed almost identical results to previously investigated T. pyogenes of various origins and T. pyogenes DSM 20630 T [12,18,20,27] (Table. 1). The T. pyogenes isolates yielded positive reactions for pyrrolidonyl arylamidase, alkaline phosphatase, β-glucuronidase, β-galactosidase, α-glucosidase and N-acetyl-β-glucosaminidase and negative reactions for nitrate reduction and pyrazinamidase. Additionally, the isolates hydrolyzed gelatine, but not esculin and urea. The isolates also fermented D-glucose, D-ribose, D-xylose, D-maltose, D-  (Table 1). A postitive reaction on Löffler agar is typical for T. pyogenes and widely used for phenotypic identification of this species [2,18]. 29 for the first hit and log score values of 2.28 and 1.9 for the second hit, respectively (data not shown). These log score values confirmed, in accordance with the current decision rules of the manufacturer, the species designation. Comparable to the present results, MALDI-TOF MS had already been shown to be a rapid and reliable technique for identifying bacteria of genera Arcanobacterium and Trueperella, including T. pyogenes [20,21].
The previously described cpn60-specific LAMP assay could successfully be used to identify the species-specific gene cpn60 of T. pyogenes 24,398 and T. pyogenes 171, 003,246 in the present investigation. This was comparable to the LAMP assay for detecting gene cpn60 of the previously described T. pyogenes of various origins [24], a T. pyogenes strain isolated from an adult roebuck (Capreolus capreolus) [23], and a T. pyogenes strain isolated from a eurasian lynx (Lynx lynx) [20]. The results of the cpn60 LAMP assay are shown in Fig. 1; Table 2.
Both strains T. pyogenes 24398 and T. pyogenes 171003246 were further identified by sequencing ISR, the genes tuf and rpoB and the putative virulence factor pyolysin encoding gene plo.  Dendrograms of the ISR, tuf and rpoB genes are presented in Fig. 3.
T. pyogenes 24398 was isolated in high numbers from vaginal discharge of an okapi and seems to be responsible for the infectious process; T. pyogenes 171003246 was isolated from a non-infectious process of a royal python suffering from a throat swelling, possibly caused by trauma. Both T. pyogenes isolates were identified by a biochemical test, LAMP and MALDI-TOF MS. The genomic targets of the two isolates, 16S rRNA gene, ISR, tuf, rpoB and plo were sequenced and compared to the respective targets of reference and other strains. Thus, the report is the first to provide a detailed characterization of T. pyogenes strains of these origin.