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Since the 1960s (Belarus) and the 1980s (Poland), male European bison (Bison bonasus) living in the Białowieża forest area suffer from balanoposthitis, a chronically necrotising inflammatory disease of the prepuce and penis, which affects approximately 6.4% of the bulls.
The etiology of the disease is unknown, although two gram-positive bacteria from the genus Trueperella, T. bonasi and T. bialowiezensis, have been suspected to play a crucial role in the pathology of balanoposthitis, since they are already present at the early stages of the disease and are absent from healthy animals.
The study reported in this thesis was therefore designed to identify possible virulence factors of T. bonasi DSM 17163 and T. bialowiezensis DSM 17162: neuraminidase, hemolysin, phospholipase D and desoxyribonuclease (DNAse).
Two closely related bacteria responsible for soft tissue infections and infections of the mucous membranes in humans and animals, T. pyogenes (DSM 20630) and A. haemolyticum (DSM 20595), were used for comparisons. The MUAN-filter paper test detected neuraminidase activity in both bacteria strains. In this study two genomic cosmid libraries were constructed for the first time but only one of them – the genomic library of T. bonasi DSM 17163 – contained neuraminidase-positive cosmid clones.
This suggests that the T. bialowiezensis DSM 17162 cosmid library, even though it contained 8x10³ clones with an average insert length of 32,500 bp, did not cover the entire genome of T. bialowiezensis DSM 17162. After extracting the neuraminidase-positive T. bonasi cosmid clones, fragments of the inserts were subcloned into a plasmid vector and again screened for neuraminidase activity. “primer walking” technique was then used to sequence the neuraminidase-positive clone bon/cos6A/pJET4A which contained an insert with two ORFs. One encoded a gene of 3,312 bp length encoding for the neuraminidase activity; the other one was a gene of 1,161 bp length (“bon-UPF”) which encoded for a protein (T. bonasi-„UPF-like“ protein) with the molecular mass of 98,480 and was assigned to the „UPF0027/RtcB“-protein family (RNA ligases) and the “Hint-superfamily” (Hint = Hedgehog intein domain), respectively.
Further subcloning to separate the two genes showed that the presence of “bon-UPF” is not essential for neuraminidase activity.
Like all bacterial neuraminidases, the amino acid sequence of the T. bonasi neuraminidase contains the conserved RIP/RLP (Arg-Ile/Leu-Pro) sequence as well as five copies of the ASP-Box motif (Ser-X-Asp-X-Gly-X-Thr-Trp), for which two to five are customary. The molecular mass of the protein is 273,309. The T. bonasi DSM 17163 neuraminidase has its highest sequence similarity to that of T. pyogenes neuraminidase NanH – they share 44% of their nucleotide sequence and 59% of their amino acid sequence.
I therefore suggest the name “T. bonasi neuraminidase NanH” for the newly identified protein.
Similar to many bacterial neuraminidases, T. pyogenes NanH acts as a virulence factor, playing an important role in the initial damaging of mucosal surfaces and thus facilitating bacterial colonization of host tissues. It is therefore assumed that T. bonasi NanH also constitutes a virulence factor with similar functions as T. pyogenes NanH although this needs to be investigated further. Although T. bonasi DSM 17163 and T. bialowiezensis DSM 17162 only showed a thin zone of hemolysis around their colonies when cultivated on domestic sheep-blood-agar plates, all tests applied failed to detect a hemolysin. These included the hemolysin test, different PCR-approaches, DotBlots, Southern blot hybridisations and the screening of the genomic cosmid libraries on blood agar plates of bison and other species. There was no evidence for the existence of a phospholipase D, nor for the presence of an extracellular DNAse.
Future studies should include a complete genome sequencing of T. bonasi and T. bialowiezensis, as this would facilitate the detection of virulence factors which lack any similarity to any known virulence factor of related bacteria, for instance a novel hemolysin that does not belong to the MACPF/CDC-superfamiliy.
Until then an unambiguous statement on the pathogenicity or otherwise of T. bonasi DSM 17163 and T. bialowiezensis DSM 17162 as the causative agent of the balanoposthitis in European bison is not feasible.