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. 1 Short communication – Revision 1 (JFD-2017-92) 2 3 4 5 6 7 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* 8 9 10 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 11 12 1 13 University of the Negev, Midreshet Ben Gurion, Israel 14 2 The French Associates Institute for Agriculture and Biotechnology of Drylands, Ben-Gurion Institute of Aquaculture, University of Stirling, FK9 4LA, Scotland 15 16 17 18 19 20 21 22 *Corresponding author D. Zilberg Email: dzilberg@bgu.ac.il Tel: +972-8-6596818 Fax: +972-8-6596742 23 24 Keywords: aquaculture, fish health, histopathology, Streptococcus parauberis, streptococcosis 1 25 Since the first report of an outbreak of a streptococcal infection in rainbow trout 26 (Oncorhynchus mykiss) in Japan in 1958 (Hoshina et al. 1958), streptococcosis has been 27 responsible for significant mortalities resulting in considerable losses to the aquaculture 28 industry (Salati 2006; Noga 2010). Numerous species from the family Streptococcaceae have 29 been identified as etiological agents of streptococcosis in fish (Toranzo et al. 2005; Salati 30 2006; Noga 2010), susceptibility to which was documented in both food (Inglis et al. 1993) 31 and ornamental fish species (Russo et al. 2006). Streptococcus parauberis is a coccoid, non- 32 motile, alpha-hemolytic Gram-positive bacterium belonging to the Streptococcacea family 33 (Nho et al. 2011) and has been reported as the etiological agent of streptococcosis in a few 34 fish species, including turbot (Scophthalmus maximus), olive flounder (Paralichthys 35 olivaceus), sea bass (Sebastes ventricosus) and striped bass (Morone saxatilis) (Domeénech et 36 al. 1996; Mata et al. 2004; Baeck et al. 2006; Park et al. 2009; Haines et al. 2013; Oguro et al. 37 2014). S. parauberis has been previously identified as the etiologic agent of bovine mastitis 38 (Bradley 2002). It was formerly known as Streptococcus uberis Type II until comparative 39 analysis of the sequence data of Streptococcus uberis Types I and II showed that both were 40 phylogenetically distinct, and the new species Streptococcus parauberis was proposed 41 (Williams and Collins 1990). 42 This report describes the first occurrence of septicemic disease associated with S. 43 parauberis in a cultured freshwater ornamental fish, the ram cichlid (Mikrogeophagus 44 ramirezi). This small, colorful omnivorous fish is popular among aquarists. The 45 histopathological changes associated with the infection are presented, as well as the 2 46 preliminary bacteriological characteristics of this first isolate of S. parauberis from a 47 freshwater ornamental fish. 48 Mortalities had been reported following a routine sorting procedure at a commercial 49 fish farm culturing the ram cichlid in Southern Israel in January 2014. Fish were seen to be 50 exhibiting apparent signs of sickness that included weakness, loss of equilibrium, skin redness 51 and ecchymotic hemorrhaging as well as lepidorthosis and exophthalmia (Supplementary 52 material 1). Fish were brought for examination to the Fish Health Laboratory at The Jacob 53 Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev (Midreshet Ben- 54 Gurion, Israel) monthly between January and June of 2014 and a total of around 30 fish were 55 examined. From amongst these, around 20 fish underwent a direct microscopic examination 56 of wet mounts and aseptic bacterial isolation and around 10 fish were processed for 57 histopathological analysis. For bacteriological examination, sterile swabs from the liver and 58 kidney were streaked onto tryptone soy agar (Oxoid, Hampshire, UK) and the plates were 59 incubated at 25˚C for 24 h. Biochemical analyses were performed with API 20 STREP and API 60 50 CH test (API system, La Balme les Grottes, France). The isolate was sent to Hy Laboratories 61 Ltd. (Rehovot, Israel) for 16s rRNA gene sequencing and the resulting sequence was 62 subjected to comparative phylogenetic analysis. Whole fish were fixed in formalin for 48 h 63 and stored in 70% ethanol until processing by routine histological techniques. 64 Histopathological analysis revealed infiltration of macrophages, which was mostly 65 evident in liver, kidney, and muscle (Fig. 1a-e). Gram staining demonstrated the presence of 66 densely packed, Gram-positive bacteria in the infiltrating macrophages (Fig 1b, d). Focal 3 67 necrosis occurred in muscle fibers (Fig. 1f) and vacuolization was seen in the liver (Fig. 1c). 68 There was no evident damage to kidney tubules or stroma (Fig. 1a). 69 Gram positive cocci were isolated from symptomatic fish. On TSA plates, 70 morphological characteristics of the colony of around 1 mm in diameter included whitish-to- 71 yellowish coloration, a circular shape with a raised cross sectional elevation and a smooth 72 surface. The isolate was molecularly identified as S. parauberis and, from here onwards will 73 be referred to as S. parauberis RC. The partial 16s rRNA sequence was deposited in GenBank 74 under accession no. MF102143. Partial sequences of several S. parauberis isolates from 75 aquatic and terrestrial environments were retrieved from the National Center for 76 Biotechnology Information (NCBI) database to perform phylogenetic and molecular 77 evolutionary analyses in Phylogeny.fr (Dereeper et al. 2008). The S. parauberis RC was closely 78 related to other S. parauberis strains of aquatic origin (Fig. 2a) although it formed a separate 79 clade from the rest of the group. In the API 20 STREP test, S. parauberis RC was Voges- 80 Proskauer, hippuric acid and esculin positive. All other tests were negative. Furthermore, the 81 isolate was able to metabolize a number of carbohydrates including galactose, glucose, 82 fructose, mannosen-acetylglucosamine, amygdalin, arbutin, esculin ferric citrate, salicin, 83 cellobiose, maltose, lactose, saccharose, trehalose, amidon, glycogen, and gentiobiose. The 84 isolate was able to grow in a wide range of temperatures (17-33˚C), though growth (OD620) 85 was affected in temperatures lower than 21˚C (Fig. 2b). It was found to thrive at various NaCl 86 concentrations (0-40 ppt) in the culture media, however, growth was negatively affected at 87 the highest salinity tested (i.e. 40 ppt) (Fig. 2c). 4 88 We evaluated the susceptibility of S. parauberis RC to several antibiotics including 89 SXT: trimethoprim/sulphamethoxazole; T30: oxytetracycline; N30: neomycin; NOR1: 90 norfloxacin; FFC30: florfenicol by the disc diffusion method. An overnight bacterial inoculum 91 (approx. 108 CFU ml-1) was applied onto the surface of Mueller-Hinton agar plate before 92 placement of the antibiotic discs (BBL™ Sensi-Disc™, BD, NJ). Streptococcus parauberis RC 93 was resistant to T30 but susceptible to SXT, N30, NOR1 and FFC30. A strain of S. parauberis 94 from olive flounder (Paralichthys olivaceus) had similarly been previously identified to be 95 resistant to tetracycline (Park et al. 2009). Based on the results of the biogram, on-farm 96 treatment with florfenicol was applied through medicated feed. The treatment reduced the 97 mortalities, but the infection reoccurred when treatment was withdrawn. After four cycles of 98 repeated antibiotic treatments and reoccurrence of the disease, the farm started feeding the 99 fish with a diet supplemented with rosemary (Rosmarinus officinalis). Rosemary has been 100 previously reported to be effective against Streptococcus iniae and Streptococcus agalactiae 101 (Abutbul et al. 2004; Zilberg et al. 2010). Bacteria could not be isolated from fish during and 102 soon after the application of rosemary, but infection reoccurred once rosemary 103 supplementation was withdrawn. 104 Basic factors contributing to bacterial virulence were comparatively analyzed in our S. 105 parauberis RC isolate and the most common causative agents of streptococcosis in fish, 106 including S. iniae and S. agalactiae. Intra-community (i.e. biofilm, autoaggregation) and inter- 107 community interactions (i.e. co-aggregation) are common mechanisms of bacterial survival in 108 nature and have been identified to play a part in the virulence of pathogens, including in the 109 streptococci (Cvitkovitch et al. 2003; Khemaleelakul et al. 2006). Many aquatic bacteria are 5 110 capable of forming a biofilm, a dense aggregate of surface-adherent microorganisms 111 embedded in an exopolysaccharide matrix (Cvitkovitch et al. 2003; Branda et al. 2005). 112 Biofilm-forming ability was determined by a modified crystal violet assay protocol (Lazado et 113 al. 2010). S. parauberis RC was shown to be capable of forming biofilms under static (Fig. 2d) 114 or mobile (Fig. 2e) conditions. The biofilm forming potential of S. parauberis RC was similar to 115 that of S. iniae at both static and mobile conditions, and to S. agalactiae under mobile 116 conditions (Fig. 2d). Auto-aggregation allows cell-cell interactions to occur and has properties 117 similar to those of biofilms, providing protection from the host defense factors and from 118 external treatments, such as antibiotics (Aparna and Yadav 2008; Lazado et al. 2010). A 119 spectrophotometric-based assay was adopted to evaluate this feature (Lazado et al. 2011). 120 Streptococcus parauberis RC auto-aggregating index was calculated to be 23.4±5.68% (Lazado 121 et al. 2011), indicating that around 23% of the individual bacteria clumped together. 122 Comparing to the other pathogenic streptococci, the capability is 19% higher than S. iniae but 123 43% lower than S. agalactiae. The ability of S. parauberis RC to aggregate provides insight to 124 the documented re-occurrence of infection following treatment withdrawal, i.e. this ability 125 may have provided protection and allowed the bacteria to survive the treatment. 126 Interestingly, S. parauberis RC was also capable to co-aggregating with the 2 pathogenic 127 streptococci (Rickard et al. 2003), with S. iniae 41.8±13.8% and with S. agalactiae 128 41.7±5.68%. This interaction suggests the potential for co-infection to occur. 129 We are speculating two probable causes of the presence of S. parauberis on the farm 130 where the bacterium was isolated. One likely scenario was that the bacteria originated from 131 incoming fish. Phylogenetic relationship of S. parauberis RC with other aquatic-derived 6 132 strains lends support to such a speculation. The farm personnel reported that there was no 133 delivery of fish to the farm for a long time period prior the outbreak, but its correlation with 134 fish handling could suggest that the bacterial infection was latent, and the resulting stress 135 may have caused an outbreak. Another likely scenario is the possibility of transmission of the 136 infection from an adjacent dairy facility. 137 The only reported fish-derived S. parauberis isolate (GenBank accession no. 138 JQ780604) in Israel was from a diseased broomtail wrasse (Cheilinus lunulatus). However, 139 this is the first report to discuss the histopathological changes associated with the infection 140 caused by an S. parauberis isolate from Israel in a freshwater ornamental fish. In addition, 141 some of the fundamental microbiological features characterized in S. parauberis RC may 142 offer insights in the subsequent study of the virulence and pathogenesis associated with this 143 pathogen. 144 145 Acknowledgements 146 The study was supported by research grants from the Ramat Negev Research and 147 Development, Israel, and the Central and Northern Arava Research and Development, Israel. 148 C. Lazado would like to thank the Jacob Blaustein Center for Scientific Cooperation for his 149 postdoctoral fellowship. 150 151 152 7 153 References 154 155 156 Abutbul S., Golan-Goldhirsh A., Barazani O. & Zilberg D. (2004) Use of Rosmarinus officinalis as a treatment against Streptococcus iniae in tilapia (Oreochromis sp.). Aquaculture 238, 97105. 157 158 Aparna M.S. & Yadav S. 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Journal of Fish Diseases 33, 361-369. 217 9 218 List of figure legends 219 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. 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 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. 239 10 240 241 Figure 1. 242 11 243 244 Figure 2. 245 12 246 Supplementary information 247 248 249 250 Supplementary material 1. Gross pathology of ram cichlid (Mikrogeophagus ramirezi) infected with Streptococcus parauberis. 251 252 13