Berliner und Münchener Tierärztliche Wochenschrift 2020 Chemisches und Veterinäruntersuchungsamt Stuttgart (CVUAS), Fellbach1 Open Access Konsiliarlabor für Corynebacterium pseudotuberculosis (DVG), Fellbach2 Wilhelma – Zoologisch-Botanischer Garten, Stuttgart3 Berl Münch Tierärztl Wochenschr DOI 10.2376/1439-0299-2020-6 Actinomycetes associated with abscess for- © 2020 Schlütersche mation in a goat, a llama and two alpacas Verlagsgesellschaft mbH & Co. KG ISSN 1439-0299 Actinomyceten im Zusammenhang mit Abszessen bei Korrespondenzadresse: einer Ziege, einem Lama und zwei Alpakas [email protected] Eingegangen: 19.02.2020 Reinhard Sting1,2, Anna Katharina Schwalm1, Matthias Contzen1, Marco Roller3, Angenommen: 15.05.2020 Jörg Rau1 Veröffentlicht: 16.07.2020 https://www.vetline.de/berliner-und- muenchener-tieraerztliche-wochenschrift- open-access Abstract The genera Actinomyces and Schaalia, both members of the bacterial order Actino- mycetales, include relevant infectious agents that cause abscesses in small rumi- nants and New World camelids. Due to the high diversity of the Actinomycetales, detection of undescribed members of this order is to be expected. Novel actinomy- cetes species were cultivated from a goat, a llama and two alpacas suffering from abscesses with suspected caseous lymphadenitis (CLA). Analyses carried out on these isolates using MALDI-TOF MS and 16S rRNA gene sequencing revealed actino- mycetes, presumably belonging to the bacterial genera Actinomyces and Schaalia. The data suggest that the caprine isolate is a undescribed Actinomyces species, while the isolates originating from a llama and two alpacas show a close relation- ship to each other within a unique Schaalia cluster, suggesting a host-adapted novel Schaalia species. Both methods proved equally suitable for reliable identification of known and of undescribed Actinomyces and Schaalia species. This study contributes to extending our knowledge about novel species belonging to the bacterial family of Actinomycetaceae (actinomycetes) associated with abscesses in goats and New World camelids. Precise identification of actinomycetes at species level is of high relevance in veterinary practice with regard to differentiation from caseous lymphad- enitis and assessment of treatment success. Keywords: small ruminant, New World camelids, pseudotuberculosis, Actinomyces, Actinomycetes, Actinomycetaceae, Schaalia Zusammenfassung Bakterien der Gattungen Actinomyces und Schaalia gehören zur Ordnung Actino- mycetales und sind wichtige Erreger infektiöser Abszesse bei Wiederkäuern und Neuweltkameliden. Aufgrund des großen Artenreichtums der Actinomycetales sind neue und bisher nicht beschriebene Arten zu erwarten. So konnten wir neue Acti- nomyceten aus Abszessen einer Ziege, eines Lamas und zweier Alpakas mit Verdacht auf Pseudotuberkulose isolieren. Untersuchungen der Isolate mittels MALDI-TOF- Massenspektrometrie und Sequenzierungen des 16S rRNA-Gens ergaben Actino- myceten, die den Gattungen Actinomyces und Schaalia zugeordnet werden können. Die Ergebnisse weisen darauf hin, dass es sich bei dem Ziegen-Isolat um eine bisher noch nicht beschriebene Actinomyces-Spezies handelt. Die Isolate von dem Lama und den beiden Alpakas hingegen erwiesen sich als eng verwandt innerhalb eines gemeinsamen Schaalia-Clusters, was auf eine neue wirtsadaptierte Schaalia-Spezies hinweist. Beide Methoden erwiesen sich als geeignet, bekannte und bisher nicht beschriebene Actinomyces- und Schaalia-Spezies zuverlässig zu identifizieren. Diese Studie trägt dazu bei, unsere Kenntnisse über neue Spezies der Familie der Actinomycetaceae (Actinomyceten) im Zusammenhang mit Abszessen bei Ziegen und Neuweltkameliden zu erweitern. Eine exakte Identifizierung von Actinomyce- ten ist unter dem Hintergrund der Differenzierung zur Pseudotuberkulose und der Einschätzung von Therapieerfolgen von großer Bedeutung. Schlüsselwörter: Kleine Wiederkäuer, Neuweltkameliden, Pseudotuberkulose, Actinomyces, Actinomyzeten, Actinomycetaceae, Schaalia Berliner und Münchener Tierärztliche Wochenschrift 2020 Introduction In October 2018, an adult goat (Caprae aegagrus hircus) suffered from a neck abscess. Material for bacteriological Abscess formation due to bacterial infections can lead examination was taken from the opened abscess. to the development of severe debilitating or even life- In August 2019, an abscess was noticed on the shoul- threatening diseases in animals and humans. Among der and chest area of an alpaca (Vicugna pacos). After causative pathogens, bacteria of the order Actinomy- opening the abscess, material was taken for bacteriolog- cetales are of special relevance for purulent lesions. ical examination. Actinomycetales belong to the Actinobacteria, which In September 2019, another adult alpaca had three represent a diverse phylum of gram-positive bacteria, abscesses, two in the left cheek and one at the left jaw comprising the major pathogen containing bacterial angle. The abscesses were opened by incision and a families Actinomycetaceae, Corynebacteriaceae, Mycobac- smooth, yellowish mass was recovered and sent to our teriaceae and Nocardiaceae (Nouioui et al. 2018). Cer- laboratory for bacteriological examination. tain members of the genus Corynebacterium are well The goat, the llama and the two alpacas lived all on known pathogens which spread within a herd and separate farms without any direct contact. cause abscesses (Braga et al. 2006, Sprake and Gold All samples were submitted for bacteriological exam- 2012), whereas bacterial species of the family Actinomy- ination to clarify the suspicion of CLA. Bacteriological cetaceae (actinomycetes) with the genera Actinomyces, examination was carried out according to standard pro- Arcanobacterium, Trueperella, and the recently described cedures. Abscess material was streaked on 5% sheep genus Schaalia (recently separated from the genus blood agar (Oxoid, Wesel, Germany) and MacConkey Actinomyces) usually affect individuals (Brown 2006, agar (BD BBL, Heidelberg, Germany) for aerobic incu- Fowler 1996, Nouioui et al. 2018). Therefore, detection bation at 37°C for two days. In addition, Schaedler agar of the causative agent and identification at species level (BD) and Wilkins-Chalgren agar with amikacin and 7% is of particular importance if there is suspicion of case- sheep blood (BD) were inoculated and incubated anaer- ous lymphadenitis (CLA) in goats, sheep, and camelids. obically at 37°C for two days. The isolates were stored in The family Actinomycetaceae forms a large group of our culture collection at -70°C using the Microbank™ anaerobic or microaerophilic bacteria producing short, system (Pro-Lab Diagnostics, Neston, Cheshire, U.K.). curved rods or branching filaments in varying degrees. For comparative studies, Schaalia hyovaginalis field Among these, the genera Actinomyces and Schaalia isolates and the type strain DSM 10695 (DSMZ German comprise many species which are widely distributed in Collection of Microorganisms and Cell Cultures, Braun- the environment and have been isolated from natural schweig, Germany) were included in this study (Table 1). habitats like soil, but also from humans and animals All bacterial isolates were analysed by matrix-assisted (Nouioui et al. 2018, Yassin 2014). Infections caused laser desorption ionization-time of flight mass spec- by actinomycetes have been recognised for some time trometry (MALDI-TOF MS) (Bruker Biotyper; Bruker (Smith 1918) and numerous species have since been Daltonik, Bremen, Germany), using the commercial described as causative agents of purulent and suppura- database 8.468 augmented with additional reference tive infections in various hosts (Nouioui et al. 2018, Yas- entries created in this study. The Bruker Biotyper data- sin 2014). However, due to the phylogenetic diversity of base 8.468 does not yet take into account the newer the genus Actinomyces which subdivides into different taxonomy of the revised genus Actinomyces (Nouioui et clusters, lineages and groups, undescribed species are to al. 2018). be expected (Yassin 2014, Zhao et al. 2014). Accordingly, The creation of new reference entries, so called main the taxonomy of the Actinobacteria has undergone revi- spectra projections (MSP), followed the instructions and sion, species have been transferred to other genera and standards of the manufacturer. These procedures and the novel species have been proposed and defined in the software used have been described elsewhere in more last decade (Nouioui et al. 2018, Yassin 2014, Zhao et detail (Pranada et al. 2016, Rau et al. 2016a). Further al. 2014). In view of the large phylum of Actinobacteria information on user-made additional MSP applied in encompassing numerous pathogenic and apathogenic this study (see Table 1) is shown in the MALDI-UP cat- bacterial species, precise identification at species level is alogue on https://maldi-up.ua-bw.de (Rau et al. 2016b). crucial. This is of special relevance for the identification For decoding of 16S rRNA gene sequences, PCR of bacterial pathogens causing abscesses that resemble assays were carried out as described elsewhere (Contzen CLA when considering the impact on control programs et al. 2011) using the primers 27f (5’-AGA GTT TGA implemented for goats, sheep and camelids or assess- TCC TGG CTC AG-3’) and 1522rN (5’-CAT GCG GCC ment of successful treatment (Schumacher et al. 2009). GCA AGG AGG TGA TCC ARC CGC A-3’) according In this study, we describe the occurrence of abscesses to Johnson (1994). The PCR products