Research Group 3: Wildlife diseases

Research field: Bacteriology

Head: Mirjam Grobbel

Samples from diseased animals from Tierpark Berlin Friedrichsfelde and material from necropsies are examined for bacteriological and mycological pathogens using routine diagnostics. The isolated pathogens are differentiated and identified by recording their physiological and biochemical characteristics. Serological and molecular biological methods are also used.

Additionally, the bacteriology group offers services to external institutions. For example we are consultants for and are performing laboratory work for a project of the Institute for livestock reproduction Schönow e.V. which is dealing with hygiene control in artificial insemination stations.

Projects:

1. Identification and classification of virulence associated genes in Acranobacterium (A.) bonsai and A. bialowiezense

Since 1980, a chronic disease of the external genital organs of male European bison (Bison bonasus) has been observed on the Polish side of the Bialowieza Forest (Kita et al., 1994). Bulls of different age groups are affected; the youngest bull affected only 6 months old. Five to 10% of the bulls are culled annually due to this disease. The disease (balanoposthitis) results in the inability of affected bulls to reproduce. In severe cases, balanoposthitis leads to necrosis and autoamputation of the tip of the penis.

Different bacteria have been cultured from tissues of diseased bulls. In early stages of the disease, Gram-positive cocci and coryneform bacteria predominate whereas in advanced stages Fusoabcterium necrophorum and other anaerobic bacteria prevail (Lehnen et al., 2003).

Two yet undescribed Arcanobacterium (A.) strains were cultured exclusively from the prepuce of bison bulls suffering from balanoposthitis or detected by specific PCR. Comparison of 16S rRNA-gene sequences of these new strains with data accessioned in GenBank exhibited highest sequence similarities to A. pyogenes DSM 20630T (96.1% and 96.4%, respectively) and Arcanobacterium bernardiae DSM 9152T (95.5% and 95.8%, respectively). Ongoing investigation indicate that the new strains are affiliated with the genus Arcanobacterium but are not members of an known species. Based on our findings, we described two novel species of the genus Arcanobacterium for which we proposed the names Arcanobacterium bialowiezense and Arcanobacterium bonasi   (Lehnen et al.; 2006).

Fig 3 Grampositive, pleomorphe rods of Arcanobacterium sp. in Gram’s stain

It is of interest that the new Arcanobacterium species have been cultured only from diseased but not from healthy bulls or females investigated in parallel. Based on our investigation it seems possible that a synergistic interaction between aerobic and anerobic bacteria plays a key role in balanoposthitis in European bison. Synergisms between aerobes and anaerobes have been described in association with different clinical manifestations and lead to advanced growth and increase of virulence of the microorganism involved (Smith et al., 1989). Such an synergistic effect has been described in foot-rot, a disease in sheep caused by Arcanobacterium pyogenes and anaerobic bacteria.

To estimate the virulence and the influence of A. bonasi and A. bialowieziense in these infections a currently running doctoral thesis project deals with the identification and classification of virulence associated genes in these bacteria.

So far the main focus is on haemolysins from the classes of phospholipids and cholesterol depending cytolysins (CDC), as these genes are already known from other Arcanobacterium spp.. Currently the similarity of these factors is analysed by DotBlot and PCR.

Neuraminidase is another important virulence factor in Arcanobacteria, which can be phenotypicaly detected by MUAN (2-(4-methyl-umbelliferyl)-α-d-N-acetylneuraminic acid). Molecular biological analyses of the corresponding genes are in progress.

Publications:

Lehnen A, Busse HJ, Frölich K, Krasinska M, Kämpfer P, Speck S.

Arcanobacterium bialowiezense sp. nov. and Arcanobacterium bonasi sp. nov., isolated from the prepuce of European bison bulls (Bison bonasus) suffering from balanoposthitis, and emended description of the genus Arcanobacterium Collins et al. 1983

2.  Steptococcus equi ssp. ruminatorum in Tieren des Serengeti Nationalparks

In 2002 several spotted hyenas (Crocuta crocuta) in the Tansanian Ngorongoro Crater were found suffering from a strangles like infection.

The causative agent isolated from tissue samples of dead animals was identified as Streptococcus (S.) equi ssp. ruminatorum, a close relative to S. equi ssp. equi which is responsible for equine strangles.

S. equi ssp. ruminatorum was also isolated from different samples of non infected hyenas, other local predators and different prey species. Sequence analyses of the M-like protein genes of the isolates revealed clear differences between healthy and diseased animals.

The next step in our investigation will be the phylogenetic analysis and the identification of virulence associated genes. In order to calculate the prevalence of S. equi ssp. ruminatorum in the Serengeti National Park we are going to quantitatively and geographically expand the sample collection.

Publications:

Speck S, Höner OP, Wachter B, Fickel

Characterization of Streptococcus equi subsp. ruminatorum isolated from spotted hyenas (Crocuta crocuta) and plains zebras (Equus burchelli), and identification of a M-like protein (SrM) encoding gene.

Vet Microbiol. 2008 Apr 1;128(1-2):148-59

Höner OP, Wachter B, Speck S, Wibbelt G, Ludwig A, Fyumagwa RD, Wohlsein P, Lieckfeldt D, Hofer H, East ML.

Severe Streptococcus infection in spotted hyenas in the Ngorongoro Crater, Tanzania

Vet Microbiol. 2006 Jun 15;115(1-3):223-8

3.  Wild animals and antimicrobial resistant bacteria

A future focus of the bacteriology group at the IZW will be resistant bacteria isolated from wild animals.

Occurrence and spread of antimicrobial resistances in bacteria are of major concern for experts as well as for the public. Monitoring programs for antimicrobial resistant bacteria in human medicine and food production have already been established to show the current bacterial resistance situation and to predict their development in the future.

Abb. 6 Susceptible and resistant becteria in the disc agar diffusion test	Abb.7 Extended Spectrum ß-Lactamase producing bacteria in the Epsilon-test

The direct transmission of bacteria from human to human via the food chain is not the only possible route. Transmission from animal to human and between different animals has also been demonstrated. Isolates from companion animals are becoming part of the monitoring programs but there is still only scarce information about antimicrobial drug resistance in bacteria from wild animals. It is very important to include them as they are a possible reservoir and a possible spreading vector for resistant bacteria.

To close this gap in knowledge we will take part in a project planed by the institute for microbiology and epizootics of the Freie Universität Berlin in cooperation with several European partners, which first will focus on samples of migratory bird species.

To supplement knowledge of known routes of antibiotic resistance spread, we hope to shed light on less researched sources for the spread of antimicrobial resistance.

Publications:

Guenther, S., Grobbel, M., Lübke-Becker, A., Goedecke, A., Friedrich N.D., Wieler L.H. and Ewers C.

Antimicrobial resistance profiles of E. coli from common European wild bird species.

International Journal of Antimicrobial Agents, under Review

Schwarz, S., Alešík, E., Grobbel, M., Lübke-Becker, A., Wallmann, J., Werckenthin, C., Wieler, L. H. (2007):

The BfT-GermVet monitoring programm - aims and basics.

Berliner und Münchener Tierärztliche Wochenschrift, 120 (9/10), 2007, 357-362

Grobbel, M., Lübke-Becker, A., Alešík, E., Schwarz, S., Wallmann, J., Werckenthin, C., Wieler, L. H. (2007):

Antimicrobial susceptibility of Escherichia coli from swine, horses, dogs, and cats as determined in the BfT-GermVet monitoring program 2004-2006.

Berliner und Münchener Tierärztliche Wochenschrift, 120 (9/10), 2007, 391-401