This is the peer reviewed version of the following article: Lazado CC, Fridman
S, Sinai T, Zilberg D. First report of Streptococcus parauberis in a cultured
freshwater ornamental fish, the ram cichlid Mikrogeophagus ramirezi (Myers &
Harry, 1948). Journal of Fish Diseases 2018;41:161–164, which has been
published in final form at https://doi.org/10.1111/jfd.12676. This article may be
used for non-commercial purposes in accordance With Wiley Terms and
Conditions for self-archiving.
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Short communication – Revision 1 (JFD-2017-92)
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First report of Streptococcus parauberis in a cultured freshwater
ornamental fish, the ram cichlid Mikrogeophagus ramirezi (Myers &
Harry, 1948)
Carlo C. Lazado1, Sophie Fridman1,2, Tamar Sinai1 and Dina Zilberg1*
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Running title: Streptococcus parauberis infection in the ram cichlid
Accepted for publication in Journal of Fish Diseases published by Wiley. The published version is available at:
https://doi.org/10.1111/jfd.12676
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University of the Negev, Midreshet Ben Gurion, Israel
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The French Associates Institute for Agriculture and Biotechnology of Drylands, Ben-Gurion
Institute of Aquaculture, University of Stirling, FK9 4LA, Scotland
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*Corresponding author
D. Zilberg
Email: dzilberg@bgu.ac.il
Tel: +972-8-6596818
Fax: +972-8-6596742
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Keywords: aquaculture, fish health, histopathology, Streptococcus parauberis, streptococcosis
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Since the first report of an outbreak of a streptococcal infection in rainbow trout
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(Oncorhynchus mykiss) in Japan in 1958 (Hoshina et al. 1958), streptococcosis has been
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responsible for significant mortalities resulting in considerable losses to the aquaculture
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industry (Salati 2006; Noga 2010). Numerous species from the family Streptococcaceae have
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been identified as etiological agents of streptococcosis in fish (Toranzo et al. 2005; Salati
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2006; Noga 2010), susceptibility to which was documented in both food (Inglis et al. 1993)
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and ornamental fish species (Russo et al. 2006). Streptococcus parauberis is a coccoid, non-
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motile, alpha-hemolytic Gram-positive bacterium belonging to the Streptococcacea family
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(Nho et al. 2011) and has been reported as the etiological agent of streptococcosis in a few
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fish species, including turbot (Scophthalmus maximus), olive flounder (Paralichthys
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olivaceus), sea bass (Sebastes ventricosus) and striped bass (Morone saxatilis) (Domeénech et
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al. 1996; Mata et al. 2004; Baeck et al. 2006; Park et al. 2009; Haines et al. 2013; Oguro et al.
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2014). S. parauberis has been previously identified as the etiologic agent of bovine mastitis
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(Bradley 2002). It was formerly known as Streptococcus uberis Type II until comparative
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analysis of the sequence data of Streptococcus uberis Types I and II showed that both were
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phylogenetically distinct, and the new species Streptococcus parauberis was proposed
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(Williams and Collins 1990).
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This report describes the first occurrence of septicemic disease associated with S.
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parauberis in a cultured freshwater ornamental fish, the ram cichlid (Mikrogeophagus
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ramirezi). This small, colorful omnivorous fish is popular among aquarists. The
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histopathological changes associated with the infection are presented, as well as the
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preliminary bacteriological characteristics of this first isolate of S. parauberis from a
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freshwater ornamental fish.
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Mortalities had been reported following a routine sorting procedure at a commercial
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fish farm culturing the ram cichlid in Southern Israel in January 2014. Fish were seen to be
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exhibiting apparent signs of sickness that included weakness, loss of equilibrium, skin redness
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and ecchymotic hemorrhaging as well as lepidorthosis and exophthalmia (Supplementary
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material 1). Fish were brought for examination to the Fish Health Laboratory at The Jacob
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Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev (Midreshet Ben-
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Gurion, Israel) monthly between January and June of 2014 and a total of around 30 fish were
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examined. From amongst these, around 20 fish underwent a direct microscopic examination
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of wet mounts and aseptic bacterial isolation and around 10 fish were processed for
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histopathological analysis. For bacteriological examination, sterile swabs from the liver and
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kidney were streaked onto tryptone soy agar (Oxoid, Hampshire, UK) and the plates were
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incubated at 25˚C for 24 h. Biochemical analyses were performed with API 20 STREP and API
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50 CH test (API system, La Balme les Grottes, France). The isolate was sent to Hy Laboratories
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Ltd. (Rehovot, Israel) for 16s rRNA gene sequencing and the resulting sequence was
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subjected to comparative phylogenetic analysis. Whole fish were fixed in formalin for 48 h
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and stored in 70% ethanol until processing by routine histological techniques.
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Histopathological analysis revealed infiltration of macrophages, which was mostly
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evident in liver, kidney, and muscle (Fig. 1a-e). Gram staining demonstrated the presence of
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densely packed, Gram-positive bacteria in the infiltrating macrophages (Fig 1b, d). Focal
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necrosis occurred in muscle fibers (Fig. 1f) and vacuolization was seen in the liver (Fig. 1c).
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There was no evident damage to kidney tubules or stroma (Fig. 1a).
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Gram positive cocci were isolated from symptomatic fish. On TSA plates,
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morphological characteristics of the colony of around 1 mm in diameter included whitish-to-
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yellowish coloration, a circular shape with a raised cross sectional elevation and a smooth
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surface. The isolate was molecularly identified as S. parauberis and, from here onwards will
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be referred to as S. parauberis RC. The partial 16s rRNA sequence was deposited in GenBank
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under accession no. MF102143. Partial sequences of several S. parauberis isolates from
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aquatic and terrestrial environments were retrieved from the National Center for
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Biotechnology Information (NCBI) database to perform phylogenetic and molecular
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evolutionary analyses in Phylogeny.fr (Dereeper et al. 2008). The S. parauberis RC was closely
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related to other S. parauberis strains of aquatic origin (Fig. 2a) although it formed a separate
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clade from the rest of the group. In the API 20 STREP test, S. parauberis RC was Voges-
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Proskauer, hippuric acid and esculin positive. All other tests were negative. Furthermore, the
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isolate was able to metabolize a number of carbohydrates including galactose, glucose,
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fructose, mannosen-acetylglucosamine, amygdalin, arbutin, esculin ferric citrate, salicin,
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cellobiose, maltose, lactose, saccharose, trehalose, amidon, glycogen, and gentiobiose. The
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isolate was able to grow in a wide range of temperatures (17-33˚C), though growth (OD620)
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was affected in temperatures lower than 21˚C (Fig. 2b). It was found to thrive at various NaCl
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concentrations (0-40 ppt) in the culture media, however, growth was negatively affected at
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the highest salinity tested (i.e. 40 ppt) (Fig. 2c).
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We evaluated the susceptibility of S. parauberis RC to several antibiotics including
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SXT: trimethoprim/sulphamethoxazole; T30: oxytetracycline; N30: neomycin; NOR1:
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norfloxacin; FFC30: florfenicol by the disc diffusion method. An overnight bacterial inoculum
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(approx. 108 CFU ml-1) was applied onto the surface of Mueller-Hinton agar plate before
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placement of the antibiotic discs (BBL™ Sensi-Disc™, BD, NJ). Streptococcus parauberis RC
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was resistant to T30 but susceptible to SXT, N30, NOR1 and FFC30. A strain of S. parauberis
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from olive flounder (Paralichthys olivaceus) had similarly been previously identified to be
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resistant to tetracycline (Park et al. 2009). Based on the results of the biogram, on-farm
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treatment with florfenicol was applied through medicated feed. The treatment reduced the
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mortalities, but the infection reoccurred when treatment was withdrawn. After four cycles of
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repeated antibiotic treatments and reoccurrence of the disease, the farm started feeding the
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fish with a diet supplemented with rosemary (Rosmarinus officinalis). Rosemary has been
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previously reported to be effective against Streptococcus iniae and Streptococcus agalactiae
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(Abutbul et al. 2004; Zilberg et al. 2010). Bacteria could not be isolated from fish during and
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soon after the application of rosemary, but infection reoccurred once rosemary
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supplementation was withdrawn.
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Basic factors contributing to bacterial virulence were comparatively analyzed in our S.
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parauberis RC isolate and the most common causative agents of streptococcosis in fish,
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including S. iniae and S. agalactiae. Intra-community (i.e. biofilm, autoaggregation) and inter-
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community interactions (i.e. co-aggregation) are common mechanisms of bacterial survival in
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nature and have been identified to play a part in the virulence of pathogens, including in the
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streptococci (Cvitkovitch et al. 2003; Khemaleelakul et al. 2006). Many aquatic bacteria are
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capable of forming a biofilm, a dense aggregate of surface-adherent microorganisms
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embedded in an exopolysaccharide matrix (Cvitkovitch et al. 2003; Branda et al. 2005).
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Biofilm-forming ability was determined by a modified crystal violet assay protocol (Lazado et
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al. 2010). S. parauberis RC was shown to be capable of forming biofilms under static (Fig. 2d)
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or mobile (Fig. 2e) conditions. The biofilm forming potential of S. parauberis RC was similar to
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that of S. iniae at both static and mobile conditions, and to S. agalactiae under mobile
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conditions (Fig. 2d). Auto-aggregation allows cell-cell interactions to occur and has properties
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similar to those of biofilms, providing protection from the host defense factors and from
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external treatments, such as antibiotics (Aparna and Yadav 2008; Lazado et al. 2010). A
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spectrophotometric-based assay was adopted to evaluate this feature (Lazado et al. 2011).
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Streptococcus parauberis RC auto-aggregating index was calculated to be 23.4±5.68% (Lazado
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et al. 2011), indicating that around 23% of the individual bacteria clumped together.
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Comparing to the other pathogenic streptococci, the capability is 19% higher than S. iniae but
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43% lower than S. agalactiae. The ability of S. parauberis RC to aggregate provides insight to
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the documented re-occurrence of infection following treatment withdrawal, i.e. this ability
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may have provided protection and allowed the bacteria to survive the treatment.
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Interestingly, S. parauberis RC was also capable to co-aggregating with the 2 pathogenic
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streptococci (Rickard et al. 2003), with S. iniae 41.8±13.8% and with S. agalactiae
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41.7±5.68%. This interaction suggests the potential for co-infection to occur.
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We are speculating two probable causes of the presence of S. parauberis on the farm
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where the bacterium was isolated. One likely scenario was that the bacteria originated from
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incoming fish. Phylogenetic relationship of S. parauberis RC with other aquatic-derived
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strains lends support to such a speculation. The farm personnel reported that there was no
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delivery of fish to the farm for a long time period prior the outbreak, but its correlation with
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fish handling could suggest that the bacterial infection was latent, and the resulting stress
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may have caused an outbreak. Another likely scenario is the possibility of transmission of the
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infection from an adjacent dairy facility.
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The only reported fish-derived S. parauberis isolate (GenBank accession no.
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JQ780604) in Israel was from a diseased broomtail wrasse (Cheilinus lunulatus). However,
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this is the first report to discuss the histopathological changes associated with the infection
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caused by an S. parauberis isolate from Israel in a freshwater ornamental fish. In addition,
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some of the fundamental microbiological features characterized in S. parauberis RC may
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offer insights in the subsequent study of the virulence and pathogenesis associated with this
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pathogen.
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Acknowledgements
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The study was supported by research grants from the Ramat Negev Research and
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Development, Israel, and the Central and Northern Arava Research and Development, Israel.
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C. Lazado would like to thank the Jacob Blaustein Center for Scientific Cooperation for his
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postdoctoral fellowship.
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List of figure legends
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Figure 1. Histopathology from S. parauberis-infected ram cichlid. Infiltrating macrophages in
kidney tissue (a) containing Gram positive bacteria (b). Liver appears vacuolated (c) with
focally occurring infiltrating macrophages, containing Gram positive bacteria (d). Infiltrating
macrophages in the muscle (e) and focally occurring necrosis in muscle fibers (f). Sections are
stained with H&E (a, c, e, f) and Gram stain (b, c); m, macrophages.
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Figure 2. S. parauberis RC: Phylogeny, growth characteristics and biofilm formation. (a)
Phylogram of S. parauberis from ram cichlid and other isolates of terrestrial and aquatic (with
red arrowhead) origins. The isolate with an arrowhead shaded in red and outlined in black
was previously isolated in Israel. Branches corresponding to partitions reproduced in less
than 50% bootstrap replicates are collapsed. The analysis involved 11 nucleotide sequences.
All positions containing gaps and missing data were eliminated. The alignment in MUSCLE
was curated in Glocks 0.91b to include a total of 635 positions, representing 40% of the
alignment. The curated alignment was used for phylogenetic anylysis in PhyML and the tree
was rendered by TreeDyn. Culture conditions, including (b) temperature and (c) NaCl
concentration, affecting the growth of S. parauberis RC. Biofilm formation at 25˚C either in
(d) static or (e) mobile conditions were analyzed in a microplate. For mobile conditions, the
plate was incubated with shaking (80 rpm). Values presented in b, c, d and e are mean ± SE of
observations from three independent experiments each with three replicate set-ups.
Column bars with different letters indicate significant difference (P<0.05) as tested by oneway ANOVA followed by Tukey’s multiple comparison tests.
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Figure 1.
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Figure 2.
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Supplementary information
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Supplementary material 1. Gross pathology of ram cichlid (Mikrogeophagus ramirezi)
infected with Streptococcus parauberis.
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