s o u r c e : h t t p s : / / d o i . o r g / 1 0 . 7 8 9 2 / b o r i s . 1 2 1 5 5 8 | d o w n l o a d e d : 6 . 4 . 2 0 2 1 CASE REPORT Open Access Isolation of Streptococcus agalactiae in a female llama (Lama glama) in South Tyrol (Italy) Alexander Tavella1* , Astrid Bettini1, Monia Cocchi1, Ilda Idrizi1, Stefano Colorio1, Laura Viel1, Claudia Zanardello1 and Patrik Zanolari2 Abstract Background: Streptococcus agalactiae is pathogenic for both animals and humans. In dairy cattle it commonly causes mastitis, with great economic losses, and there is scientific evidence of mastitis, caseous lymphadenitis, contagious skin necrosis and purulent infections associated with S. agalactiae in camels (Camelus dromedarius) as well. In humans, it is a common component of the respiratory and gastrointestinal microflora, but it can also act as a pathogen, especially in elderly people and immunocompromised patients, as well as in pregrant women and newborns. Case presentation: A 10-year old non-pregnant female llama (Lama glama) was conferred to the Institute for Animal Health Control, in Bolzano for necropsy after sudden death. The animal had not shown unusual behaviour and had a low to normal nutritional condition (body condition score 2/5). The breeder had reported a chronic suppurative subcutaneous infection in the intermandibular area, resistant to therapy (therapy unknown). After necropsy, several samples were processed for histological, bacteriological and parasitological examinations. Conclusions: This report describes, to the best of our knowledge, the first isolation of S. agalactiae in llamas (Lama glama). The animal came from a herd that counts approximately 200 South American camelids (llamas, alpacas) along with several horses, chicken, rabbits, cats and dogs; this farm offers services, such as trekking and pet therapy activities. Keywords: Streptococcus agalactiae, Lama glama, South American camelids, Lancefield group B Streptococcus Background Llamas and Alpacas have gained increasing interest in the last 10 years and are now frequently held as farm an- imals and for hobby purposes (trekking, pet therapy) in the alpine regions of Northern Italy. In the Autonomous Province of Bolzano – South Tyrol (Italy), the South American camelids (SACs) population has grown lately and currently counts 800 individuals (583 llamas and 217 alpacas – informations from the local Veterinary Service 2017-databank), representing an important niche product in local livestock breeding. In fact, SACs are often held in multispecies farming systems with sheep and horses, and could come into close contact with humans for trekking and pet therapy reasons. Streptococcus agalactiae (S. agalactiae), a Lancefield Group B Streptococcus (GBS), is an important pathogen affecting both animals and humans [1]. In dairy cattle, it is a major cause of mastitis and an important source of economic loss [2]. GBS have also been found in many other animals, such as camels, dogs, cats, crocodiles, seals, fish and dolphins [3–5]. More in detail, S. agalac- tiae has been frequently observed in camels (Camelus dromedarius), as a causative agent of mastitis, caseous lymphadenitis, contagious skin necrosis and purulent in- fections [6–8]. Moreover, authors described that the camel strains are different from the bovine strains, and that they resemble much more the human strains [3]. To our knowledge, no S. agalactiae strains have been isolated from llamas (Lama glama). S. agalactiae has also been observed in humans. It is a common component of the microflora of the respiratory and gastrointestinal tracts, approximately isolated in * Correspondence: atavella@izsvenezie.it 1Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10, 35020 Legnaro, Italy Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tavella et al. BMC Veterinary Research (2018) 14:343 https://doi.org/10.1186/s12917-018-1676-9 http://crossmark.crossref.org/dialog/?doi=10.1186/s12917-018-1676-9&domain=pdf http://orcid.org/0000-0001-8565-9987 mailto:atavella@izsvenezie.it http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/publicdomain/zero/1.0/ 30% of healthy humans. In the elderly and in immuno- compromised patients, this bacterium can be associated with urinary tract and skin and soft-tissue infections, bacteremia, osteomyelitis, meningitis and endocarditis [9, 10]. Moreover, in pregnant women, invasive maternal infection is associated with abortion, preterm delivery [11], sepsis and meningitis in newborns [12]. In humans, invasive infections caused by S. agalactiae are reported increasingly worldwide [13]. In the pathogenetic mech- anism, the adherence to host epithelial cells is the first critical step of the infectious process, leading to the for- mation of microbial biofilms. These consist of microco- lonies encased in extracellular polysaccharide material. Bacteria inside biofilms have increased resistance to anti- microbial agents and disinfectants [14]. Furthermore, the production of biofilms is correlated with both patho- genicity and virulence of the bacteria. In this report, we describe the case of a 10-year-old non-pregnant female llama (Lama glama), conferred for necropsy to the Institute for Animal Health Control (Istituto Zooprofilattico Sperimentale delle Venezie, IZSVe) in Bolzano (Italy) after sudden death. Case presentation Clinical history The animal had been kept all summer with other six llamas on an alpine pasture, but it originated from a lar- ger llama breeding farm where no S. agalactiae-cases were ever recorded. The affected llama had not shown an unusual behavior. The breeder reported a chronic suppurative subcutaneous infection in the intermandibu- lar area, that had persisted for several months. The nu- tritional condition of the individual was low to normal (body condition score 2/5) and was evaluated through adspection and palpation of the thorax, the abdomen and the back [15]. The animal was found dead by the breeder on the alpine pasture. Necropsy The physical exam revealed a 2–3 cm fistulating lump in the intermandibular area. After removing both hair and skin surfaces, a delimited suppurative infection site was observed in the subcutaneous tissue, in form of an ab- scess. Inspection of the anatomic area did not reveal a connection to the animal’s oral cavity. A diffused sub- cutaneous oedema was noted in the intermandibular area, as well as in the neck and the caudal portion of the head. The retropharyngeal lymph nodes were enlarged and oedematous. After opening the lump, a yellowish creamy content was observed. The exam of the abdominal cavity showed a severe sero-fibrinous ascites. The C3 intestinal compartment showed a catarrhal-haemorrhagic inflammation of the mucous membrane, while the proximal part of the bowel portion was hyperaemic; furthermore, a catarrhal duode- nojejunitis was recognized. A severe nematode larvae in- festation was identified in the intestinal content. The liver had a light brown colour and a brittle consistency and showed a diffused necrosis. In the thoracic cavity, a high-grade of sero-fibrinogenous pleural effusion was identified. The lung showed a pro- nounced oedema, as well as emphysematous areas; further- more, an interstitial pneumonia was observed. The pericardium and heart base were oedematous. No other pathological lesions were observed in any other organ. Selected samples from liver and lung tissues were rou- tinely processed for histopathological examination. Moreover, bacteriological examinations from the inter- mandibular abscess, the liver and the lungs were per- formed by routine laboratory tests, using blood agar (in-house protocol) at 37 ± 1 °C in aerobiosis for 24 h. The flotation procedure was performed, using a solution with specific gravity of 1.3. Histology Samples of lung and liver parenchyma were first col- lected and fixed in 10% neutral buffered formalin, then processed, paraffin embedded, stained with Haematoxy- lin and Eosin (HE) and observed by standard light mi- croscopy for histological examination. A diffuse mild to moderate neutrophilic and lymphocytic interstitial infil- tration with alveolar emphysema was observed in the lung parenchyma (Fig. 1). While examining the liver parenchyma, a multifocal necrosis of hepatocytes with foci of neutrophilic and lymphocytic infiltration and diffuse hydropic degener- ation of hepatocytes were detected (Fig. 2). Bacterial examination After incubation, a pure culture of translucid grey colonies, with a complete ß hemolysis was submitted for identifica- tion. Gram staining revealed Gram positive cocci in linear chains, catalase negative, and esculin negative as well. To confirm the presence of ß hemolytic Streptococci, the CAMP test was performed, indicating a positive re- action, the serological assay (StreptococcalEN Grouping Kit, Oixoid, Wade road, Basingstoke, Hampshire, UK) was performed as well, showing group B antigens. Bio- chemical identification of isolates (api® 32 Strep, bio- Méerieux, Marcy-L’Etoile, France), revealed S. agalactiae (profile: 16122051110; %ID = 81.2; T = 0.77), with an ex- cellent level of genus identification. S. agalactiae was isolated from the purulent material of the intermandibular abscess and from the pulmonary parenchyma. Biofilm formation was evaluated using micro titer plates as described by Stepanovic and coworkers [14]. Tavella et al. BMC Veterinary Research (2018) 14:343 Page 2 of 7 Fig. 1 Lung - Mild to moderate neutrophilic and lymphocytic interstitial infiltration with alveolar emphysema. Presence of a thrombus in the blood vessel. HE, 20X Fig. 2 Liver – Foci of necrosis of hepatocytes with neutrophilic infiltration. HE, 20X Tavella et al. BMC Veterinary Research (2018) 14:343 Page 3 of 7 In order to confirm the identification of the isolated strains obtained from the abscess and from the lung, Matrix-Assisted Laser Desorption/Ionization Time-Of- Flight Mass-Spectrometry (MALDI TOF MS) (Maldi Biotyper, Bruker Daltonics) was performed. This tech- nique allows the identification of a bacterium by deter- mining the molecular mass of fingerprint peptides (mainly ribosomal proteins), and comparing the mass fingerprint of the unknown strains to a database containing reference mass fingerprints. As specified by the manufacturer, a score value of < 1.7 indicates that identification is not reli- able, scores between 1.7 and 2.0 demonstrate that identifi- cation is reliable at the genus level, scores between 2.0 and 2.3 evidence that identification is reliable at the genus level and probable at the species level. Scores higher than 2.3 indicate highly probable species identification. S. agalactiae strains were recognized with a score of 2.454 (abscess isolate), and a score of 2.465 (lung iso- late), indicating a highly probable species identification. The strains formed biofilms under in vitro conditions, and were identified as moderate biofilm producers. Determination of the minimum inhibitory concentration (MIC) Minimum Inhibitory Concentration (MIC) of the lung and abscess isolates was evaluated in accordance with the guidelines of the Clinical and Laboratory Standards Institute [16] for microwell dilution testing, using a commercial plate (Micronaut-S, Merlin, Kleinstrasse 14, Bornheim, Hersel, Germany). The antimicrobials tested were ampicillin, ceftiofur, enrofloxacin, florphenycol, spec- tinomycin, tetracycline, tilmicosin, trimethoprim/sulpha- methoxazole. The MIC results are shown in Table 1. Parasitology Parasitology of the feces reported an elevated number of oval eggs with a thin single wall, measuring approximately 45x30μm, attributable to Strongyloides spp.. Several eggs had transformed to the larval stage. Furthermore, yellow- brownish eggs (approximately 70 × 30 μm) showing a “lemon” shape with evident stoppers at the poles referred to Trichuris spp. were observed. The elevated number of eggs observed at the microscope and the evidence of the se- vere enteral parasitaemia allowed the indirect evaluation of the infestation intensity. Molecular characterization Both isolates were submitted for 16S sequencing. The alignment using the NCBI GenBank BLAST function (Additional files 1 and 2) and the Clustal Omega appli- cation (Additional file 3) confirmed the identification of S. agalactiae. Discussion and conclusions S. agalactiae has already been isolated from skin lesions, periarticular abscesses and mastitic udder in camelids (Camelus dromedarius) [3, 6, 17]. Furthermore, it has been described as an opportunistic pathogen of the upper respiratory tract [18]. S. agalactiae has been iso- lated from caseus lymphadenitis, contagious skin necro- sis and purulent infections in camels [3]; it has also been observed in wounds caused by Hyalomma spp. in drom- edaries in Kenya [19]. So far, no S. agalactiae have been described in llamas, instead S. zooepidemicus, S. pyo- genes, S. faecalis and S. uberis have been isolated from alpacas in Peru [20]. Therefore, to the best of our know- ledge, this represents the first isolation of S. agalactiae from llamas (Lama glama). In this report, the identification of S. agalactiae was performed in an abscess and in the lung parenchima in an animal presenting a parasitic infestation. Parasitism in SACs has become a major health con- cern, as several parasites, in particular gastrointestinal nematodes, are believed to cause mild-to-severe clinical diseases and economic losses. Although death from parasitic gastroenteritis associated with gastrointestinal nematodes may occur, infections tend to be more insidi- ous, often presenting nonspecific clinical signs (i.e. diar- rhoea, anorexia and poor growth) or asymptomatic conditions [21]. Domesticated SACs, comprising llamas, often share pasture with other livestock species and are often farmed under more intensive grazing conditions as in their native countries, factors which may significantly increase the risk of nematode infections [22]. In the present case, an elevated number of eggs attributable to Strongyloides spp. and Trichuris spp. were observed: this has been identified in the large intestine of SACs in pre- vious studies as well [21–23]. Infections with Trichuris spp. can lead to severe anemia [15], even though many clinical signs are often absent at the early stages of infec- tion [24]. The abdominal cavity lesions observed can be ascribed to the intense infestation by gastrointesintal Table 1 Inhibitory concentrations of the isolated S. agalactiae strain Strain Ampicillin μg/ml Ceftiofur μg/ml Enrofloxacin μg/ml Florfenicol μg/ml Spectinomycin μg/ml Tetracycline μg/ml Tilmicosin μg/ml Trimethprim/Sulfamethoxazole μg/ml Abscess 0.125 0.125 0.5 1 128 0.25 4 0.125/2.375 Lung 0.125 0.125 0.5 1 128 0.25 4 0.125/2.375 Tavella et al. BMC Veterinary Research (2018) 14:343 Page 4 of 7 nematodes [23, 25]. Moreover, this could act as a predis- posing factor for septicemic streptococcosis. S. agalactiae is a component of the microflora of the respiratory, genital and gastrointestinal tracts in both humans and ruminants. In humans, GBS is also an im- portant cause of morbidity and mortality in newborns, in the elderly and in immunocompromised adults [26]. Primary manifestations include bacteremia, skin and soft tissue infections, while other signs are referred to pneu- monia, osteomyelitis and urinary tract infections [27, 28]. Furthermore, invasive infections are increasingly reported worldwide: Takahashi and coworkers [13] have highlighted significant differences in clinical aspects, including prog- nosis, between disease-entities caused by S. agalactiae and other Streptococci in human diseases. In veterinary medi- cine, S. agalactiae is the main cause of subclinical mastitis in cattle. To our knowledge, no report of septicemic con- ditions have been observed in SACs. In other species, the bacterial colonization and infection requires the capacity of the bacterium to adhere and to persist. The formation of biofilm-like communities could facilitate microbial sur- vival and proliferation by enhancing resistance to host de- fenses and nutrient deprivation [29]. In our report, the isolates were indeed biofilm producers. The production of biofilm was correlated with pathogenicity and virulence of some bacteria [30]. In fact, microorganisms inside biofilms have increased resistance to antimicrobial agents and dis- infectants, indicating the pathogenicity potential in the isolated strains. The analysis of virulence factors produc- tion was not performed in this report, though previous re- ports have highlighted the potential of virulence factors production by S. agalactiae in bovine mastitis [31]. Fur- thermore, biofilm formation appears to be a prerequisite for colonization of the bovine mammary gland. S. agalac- tiae isolates recovered from bovine subclinical mastitis produced different pH-dependent biofilm levels, sug- gesting that biofilm production is modulated by envir- onmental factors [28, 32]. No data are available for isolates from llamas. Identification of the bacteria was performed by a com- merical kit and by MALDI TOF MS. This last procedure is routinely used in many laboratories, and has shown to be a rapid and reliable technique for the identification of veterinary bacteria [33]. Furthermore, Lartigue and co- workers [34] have identified over 100 S. agalactiae iso- lates, further charachterized by serotyping and multilocus sequence typing, with the use of MALDI TOF MS. Based on the previously described evaluation scores, a score higher than 2.3 allows the highly probable species identifi- cation. In this case report, S. agalactiae strains were iden- tified with a score of 2.454 (abscess isolate), and a score of 2.465 (lung isolate). The determination of MIC was performed in order to evaluate potential antimicrobial resistances. Since no CLSI official breakpoints are available for llamas, and in order to provide a laboratory result, the MIC results (Table 1) were evaluated using the bovine clinical break- points, even though several authors have observed that llamas, but also other SACs, and bovines present differ- ent pharmacokinetics and pharmacodynamics [35, 36]. The evaluation, with bovine breakpoints, highlights sen- sitivity to all antimicrobials tested. While no official breakpoints are available for SACs, non official break- points have been proposed for ampicillin, ceftiofur and enrofloxacin [15]. Based on these data, the MIC results would suggest ampicillin and ceftiofur sensitivity by IV and IM administration, and an intermediate resistance to enrofloxacin by IV administration. The breeding of SACs in the Autonomous Province of Bolzano – South Tyrol (Italy) has grown considerably in the last 10 years, conveying our Province a leading pos- ition in this field. These animals have gained great fame especially as hobby animals in zoos, private hotel gar- dens, as trekking animals and for pet therapy purposes, leading to a close contact between animals and humans, pointing out the possible risk of the transmission to humans. In fact, transmission of S. agalactiae from animals to humans has been briefly described in cattle [37, 38]; the authors describe a probable linkage between cattle expos- ure and detection of GBS in humans, stating that S. agalac- tiae can be transmitted between bovines and humans in a farm environment and that increased cattle exposure is as- sociated with higher risk of infection. To this regard, no data are available for SACs, and molecular characterization of the isolates should be performed in order to establish the zoonotic potential of S. agalactiae. Additional files Additional file 1: BLAST allignment of the abscess isolate. (PDF 387 kb) Additional file 2: BLAST allignment of the lung isolate. (PDF 397 kb) Additional file 3: Clustal Omega allignment. (PDF 62 kb) Abbreviations CAMP test: Christie Atkins Munch Petersen test; CLSI: Clinical and Laboratory Standards Institute; GBS: Lancefield Group B Streptococcus; HE: Haematoxylin and Eosin; IM: Intramuscolar inoculation; IV: Intravenous inoculation; IZSVe: Istituto Zooprofilattico delle Venezie; m/z: Mass to charge ratio; MALDI TOF MS: Matrix- Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry; MIC: Minimum Inhibitory Concentration; S. agalactiae: Streptococcus agalactiae; SACs: South American camelids Acknowledgements N/A Funding N/A Availability of data and materials All data generated or analysed during this study are included and shared in this article. Tavella et al. BMC Veterinary Research (2018) 14:343 Page 5 of 7 https://doi.org/10.1186/s12917-018-1676-9 https://doi.org/10.1186/s12917-018-1676-9 https://doi.org/10.1186/s12917-018-1676-9 Authors’ contributions AT performed necropsy and developed the manuscript, AB co-authored the manuscript, MC performed bacteriological examinations, II performed necropsy and parasitology, SC co-authored the manuscript, LV executed Maldi-Tof and MIC examinations, CZ performed histopathological examinations, PZ provided scientific and technical support. All authors read and approved the final manuscript. Ethics approval and consent to participate No ethical approval was required. Consent for publication No personal data was included in the manuscript; no consent for publication was applicable. Competing interests The authors declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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BMC Veterinary Research (2018) 14:343 Page 7 of 7 https://doi.org/10.1155/2016/4621039 https://doi.org/10.1016/j.bmj.2017.02.004 1 Case presentation Clinical history Necropsy Histology Bacterial examination Determination of the minimum inhibitory concentration (MIC) Parasitology Molecular characterization Discussion and conclusions Additional files Abbreviations Acknowledgements Funding Availability of data and materials Authors’ contributions Ethics approval and consent to participate Consent for publication Competing interests Publisher’s Note Author details References