Actinomycetes associated with abscess formation in a goat, a llama and two alpacas

Abscess formation due to bacterial infections can lead to the development of severe debilitating or even life-threatening diseases in animals and humans. Among
causative pathogens, bacteria of the order Actinomycetales are of special relevance for purulent lesions. Actinomycetales belong to the Actinobacteria, which represent a diverse phylum of gram-positive bacteria, comprising the major pathogen containing bacterial families Actinomycetaceae, Corynebacteriaceae, Mycobacteriaceae and Nocardiaceae (Nouioui et al. 2018). Certain members of the genus Corynebacterium are well known pathogens which spread within a herd and cause abscesses (Braga et al. 2006, Sprake and Gold 2012), whereas bacterial species of the family Actinomycetaceae (actinomycetes) with the genera Actinomyces, Arcanobacterium, Trueperella, and the recently described genus Schaalia (recently separated from the genus Actinomyces) usually affect individuals (Brown 2006, Fowler 1996, Nouioui et al. 2018). Therefore, detection of the causative agent and identification at species level is of particular importance if there is suspicion of caseous lymphadenitis (CLA) in goats, sheep, and camelids. The family Actinomycetaceae forms a large group of anaerobic or microaerophilic bacteria producing short, curved rods or branching filaments in varying degrees. Among these, the genera Actinomyces and Schaalia comprise many species which are widely distributed in the environment and have been isolated from natural habitats like soil, but also from humans and animals (Nouioui et al. 2018, Yassin 2014). Infections caused by actinomycetes have been recognised for some time (Smith 1918) and numerous species have since been described as causative agents of purulent and suppurative infections in various hosts (Nouioui et al. 2018, Yassin 2014). However, due to the phylogenetic diversity of the genus Actinomyces which subdivides into different clusters, lineages and groups, undescribed species are to be expected (Yassin 2014, Zhao et al. 2014). Accordingly, the taxonomy of the Actinobacteria has undergone revision, species have been transferred to other genera and novel species have been proposed and defined in the last decade (Nouioui et al. 2018, Yassin 2014, Zhao et al. 2014). In view of the large phylum of Actinobacteria encompassing numerous pathogenic and apathogenic bacterial species, precise identification at species level is crucial. This is of special relevance for the identification of bacterial pathogens causing abscesses that resemble CLA when considering the impact on control programs implemented for goats, sheep and camelids or assessment of successful treatment (Schumacher et al. 2009).

In this study, we describe the occurrence of abscesses suspicious for CLA in a goat, a llama and two alpacas caused by undescribed actinomycetes. These cases show that a broader spectrum of actinomycetes than previously known poses a determining cause of abscess formation and pyogenic lesions in small ruminants and New World camelids.

In November 2011, material from an abscess on the neck of a llama (Lama glama) was submitted for bacteriological examination.

In October 2018, an adult goat (Caprae aegagrus hircus) suffered from a neck abscess. Material for bacteriological examination was taken from the opened abscess.
In August 2019, an abscess was noticed on the shoulder and chest area of an alpaca (Vicugna pacos). After opening the abscess, material was taken for bacteriological examination.

In September 2019, another adult alpaca had three abscesses, two in the left cheek and one at the left jaw angle. The abscesses were opened by incision and a smooth, yellowish mass was recovered and sent to our laboratory for bacteriological examination.

The goat, the llama and the two alpacas lived all on separate farms without any direct contact.

All samples were submitted for bacteriological examination to clarify the suspicion of CLA. Bacteriological examination was carried out according to standard procedures. Abscess material was streaked on 5% sheep blood agar (Oxoid, Wesel, Germany) and MacConkey agar (BD BBL, Heidelberg, Germany) for aerobic incubation at 37°C for two days. In addition, Schaedler agar (BD) and Wilkins-Chalgren agar with amikacin and 7% sheep blood (BD) were inoculated and incubated anaerobically at 37°C for two days. The isolates were stored in our culture collection at -70°C using the Microbank™ system (Pro-Lab Diagnostics, Neston, Cheshire, U.K.).
For comparative studies, Schaalia hyovaginalis field isolates and the type strain DSM 10695 (DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were included in this study (Table 1).

All bacterial isolates were analysed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Biotyper; Bruker Daltonik, Bremen, Germany), using the commercial database 8.468 augmented with additional reference entries created in this study. The Bruker Biotyper database 8.468 does not yet take into account the newer taxonomy of the revised genus Actinomyces (Nouioui et al. 2018).

The creation of new reference entries, so called main spectra projections (MSP), followed the instructions and standards of the manufacturer. These procedures and the software used have been described elsewhere in more detail (Pranada et al. 2016, Rau et al. 2016a). Further information on user-made additional MSP applied in this study (see Table 1) is shown in the MALDI-UP catalogue on https://maldi-up.ua-bw.de (Rau et al. 2016b).

For decoding of 16S rRNA gene sequences, PCR assays were carried out as described elsewhere (Contzen et al. 2011) using the primers 27f (5’-AGA GTT TGA TCC TGG CTC AG-3’) and 1522rN (5’-CAT GCG GCC GCA AGG AGG TGA TCC ARC CGC A-3’) according to Johnson (1994). The PCR products were sequenced on demand (Microsynth, Balgach, Switzerland) and the sequence data obtained was compared with sequence entries in GenBank (http://www.ncbi.nlm.nih.gov) using the Basic Local Alignment Search Tool for nucleotides (BLASTN) on the NCBI website (Pruitt et al. 2002).

MALDI-TOF MS and 16S rDNA dendrograms were created using Actinomyces and Schaalia reference isolates including the isolates which originated from the goat, llama and the two alpacas. In addition, a Corynebacterium pseudotuberculosis and a Trueperalla pyogenes strain were included (Table 1).

The MALDI-TOF MS dendrogram was created with the “BioTyper MSP Dendrogram Creation Standard Method”, as provided by the manufacturer within the Biotyper software (vers. 3.1, Bruker), using the newly created MSP from this study and a collection of MSP from the commercial Bruker MBT Compass Reference Library that was released in April 2019 and contains 2,969 species and 8,468 MSP.

The phylogenetic tree was constructed using the neighbour-joining distance algorithm with standard settings in Geneious Prime 2020.1.1.

The bacteria cultivated from the goat’s abscess (isolate CVUAS 31303) grew in a whitish, chalk-like layer of discernible pinpoint-sized, non-haemolytic colonies under
aerobic conditions. The bacteria appeared as catalase-positive, gram-positive, short, club-shaped rods. This primary culture was accompanied by an equivalent strong growth of the obligate anaerobic bacteria Fusobacterium (F.) necrophorum and Prevotella (P.) heparinolytica detected under anaerobic atmospheric conditions.

The bacteria originating from the abscess of the llama (isolate CVUAS 8688) and the alpacas (isolate CVUAS 31838 and CVUA 31845.2) showed profuse and strong growth of pinpoint-sized, non-haemolytic colonies on blood sheep agar after a two-days incubation period in an aerobic atmosphere. None of the three isolates showed catalase activity. Gram staining revealed gram-positive, short or slightly curved, club-shaped rods. Additionally, strong growth of Trueperella (T.) pyogenes and moderate growth of Bibersteinia (B.) trehalosi was observed with the llama’s abscess. In contrast, the isolates originating from the alpacas grew in pure culture.

The isolate CVUAS 31303 (goat) could be distinguished from Schaalia isolates using MALDI-TOF MS and was located within the Actinomyces cluster (Fig. 1). The isolates originating from the llama (isolate CVUAS 8688) and the alpacas (isolates CVUAS 31838 and CVUAS 31845.2) exhibit a close relationship clustering on a branch belonging to a separate cluster close to several Schaalia (S.) species, and are therefore referred to as Schaalia species. In contrast, caprine, sheep and porcine S. hyovaginalis field isolates and the S. hyovaginalis type strain DSM 10695, which were identified and verified as S. hyovaginalis by MALDI-TOF MS, are located within a common cluster clearly separated from the isolates originating from the New World camelids (Fig. 1). The MALDI TOF mass-spectra of the isolates and several reference strains are available by exchange via the MALDI-TOF user platform (Rau et al. 2016b).

In the 16S rDNA dendrogam, the goat isolate CVUAS 31303 showed a close relationship to Actinomyces species, including Actinomyces bovis. In contrast, the llama and alpaca isolates showed a close relationship in a common sub-cluster within a Schaalia cluster, but were clearly distinct from the cluster comprising sheep, caprine and porcine S. hyovaginalis field isolates including the type strain DSM 10695 (Fig. 2).

A considerable proportion of abscess formation in small ruminants and camelids is caused by bacteria belonging to the phylum Actinobacteria. A prominent member of the order Actinomycetales is Corynebacterium (C.) pseudotuberculosis, the causative agent of CLA, which is highly relevant in small ruminants and camelids worldwide (Al-Harbi 2011, Al-Tuffyli and Shekhan 2012, de la Fuente et al. 2017, de Lima e Silva et al. 2016). Control programmes have been implemented in numerous countries worldwide to effectively combat CLA in small ruminants and camelids. Therefore, abscess formation resembling CLA has to be verified by identification of the pathogenic agent, which must be reliably distinguished from C. pseudotuberculosis. Numereous actinomycetes have been described as causative agents of suppurative lesions in humans and animals (Nouioui et al. 2018, Yassin 2014). Among the genera within the family Actinomycetaceae, the recently described novel genus Schaalia has been separated from the genus Actinomyces and reported in connection with abscess formation in humans and various animal species. Case reports have been published on abscess formation due to Actinomyces and Schaalia infections in different localisations such as the brain, subcutis or lymph nodes of goats (Alssahen et al. 2020, Hirai et al. 2007, Ndegwa et al. 2001, Oyekunle et al. 2010, Schumacher et al. 2009), New World camelids (Brown 2006, Fowler 1996), sheep (Alssahen et al. 2020, Collins et al. 2001, Foster et al. 2012), pigs (Hommez et al. 1991, Reichel and Wragg 2007), horses (Chung et al. 2018, Fielding et al. 2008), dogs (Hoyles et al. 2000, Song et al. 2015) and captive and free-ranging wildlife (Alssahen et al. 2020, Gamble and Clancy 2013, Wickhorst et al. 2017). The microorganisms reported in this study, which had been isolated from abscesses in a goat, a llama and two alpacas, attracted our attention because of their similarity to Actinomyces spp. and Schaalia spp., respectively. Further studies on these isolates using MALDI-TOF MS and 16S rDNA analysis initially yielded inconclusive results at species level, despite comprehensive databases that are available for evaluation procedures. However, the isolates could be assigned to bacteria of the genera Actinomyces (goat isolate) and Schaalia (llama and alpaca isolates). The isolate originating from the abscess in the goat is located within the MALDI-TOF MS and 16S rDNA Actinomyces cluster, but is separate to other concrete Actinomyces spp. In contrast, the isolates obtained from the llama and the alpacas show very close relationships to each other in a separate branch next to other Schaalia spp. in both the MALDI-TOF MS and 16S rDNA dendrogram. The close relationship of these actinomycetes in New World camelids, which were isolated at different times and places, suggest the presence of a novel host-adapted Schaalia species. Fowler (1996) also reported on abscess formation in the throat region, lungs or liver and dental abscesses caused by Actinomyces sp. unique to New World camelids and provisionally called these isolates Actinomyces (lamae). The author pointed out that these bacterial isolates may be under-reported due to their morphological appearance as gram-positive, short rods which may be mistaken for cocci.

It is notable that the actinomycetes growth reported in this study was accompanied by F. necrophorum and P. heparinolytica (goat) or T. pyogenes and B. trehalosi (llama). However, no accompanying bacterial aerobic or anaerobic flora could be detected in the abscesses from the two alpacas. Other researchers also report growth of actinomycetes in pure cultures (Brown 2006, Chung et al. 2018, Foster et al. 2012, Oyekunle et al. 2010, Song et al. 2015) or growth in mixed cultures including T. pyogenes, Fusobacterium spp., Bacteroides spp. and Prevotella spp. or Staphylococcus spp. and Streptococcus spp. (Al-Harbi 2011, Collins et al. 2001, Foster et al. 2012, de la Fuente et al. 2017, Gamble and Clancy 2013, Roeder et al. 1989, Schumacher et al. 2009, Wickhorst et al. 2017).

The abscess wounds in the two alpacas were successfully treated by rinsing with iodine solution without additional antibiotic treatment. Unfortunately, no data is available on
the treatment of the abscesses in the goat and the llama. MALDI-TOF MS and 16S rRNA gene analyses suggest that the actinomycetes described in this study represent undesignated novel Actinomyces and Schaalia species. However, this assumption has to be proven by further comprehensive investigations to elucidate the taxonomic status of these interesting, obviously pathogenic actinomycetes.

Actinomycetes represent important bacteria associated with abscess formation. This report shows that so far undescribed actinomycetes are also responsible for abscess formation in addition to already described pyogenic bacteria. Precise and reliable identification at species level is therefore crucial with special reference to CLA (pseudotuberculosis), which is controlled in large-scale control programmess in many countries. Reliable identification of Actinomyces and Schaalia species and detection of undescribed species can be achieved by MALDI-TOF MS and 16S rDNA analyses.

The present study extends our current knowledge about novel actinomycetes associated with abscess formation in small ruminants and New World camelids.

The authors thank Jana Ade and Mandy Hailer for performance and evaluation of the PCR assays. We would also like to thank Martin Dyk for carrying out the MALDI-TOF MS analyses and the evaluation of the spectra.

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of this paper.

Dr. Reinhard Sting
Chemisches und Veterinäruntersuchungsamt Stuttgart
Schaflandstr. 3/3
70736 Fellbach
Germany
reinhard.sting@cvuas.bwl.de

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