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Pseudo-membranes on internal organs associated with Rhodococcus qingshengii infection in Atlantic salmon (Salmo salar)
Veterinary Microbiology 147 (2011) 200–204
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Pseudo-membranes on internal organs associated with Rhodococcus qingshengii infection in Atlantic salmon (Salmo salar) ˜ o-Herrera a, Sabela Balboa b, Alejandra Doce b, Pedro Ilardi c, Rube´n Avendan Pablo Lovera d, Alicia E. Toranzo b, Jesu´s L. Romalde b,* a
Universidad Andre´s Bello, Facultad de Ciencias Ba´sicas, Departamento de Ciencias Ba´sica, Vin˜a del Mar, Chile Departamento de Microbiologı´a y Parasitologı´a, CIBUS, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain c Laboratorio de Patologı´a de Peces, Veterquı´mica S.A., Santiago, Chile d Laboratorio Biovac, Puerto Montt, Chile b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 October 2009 Received in revised form 28 May 2010 Accepted 2 June 2010
This paper describes a pathological condition in intensive reared Atlantic salmon (Salmo salar), restricted to the appearance of pseudo-membranes covering internal organs (i.e. spleen, liver, heart and others) associated with the presence of large numbers of a Grampositive bacteria. Isolate 79043-3, obtained as pure culture from affected fish, was subjected to a polyphasic taxonomic study in order to determine its exact taxonomic position, as well as to experimental challenges leading to determine its pathogenic potential for cultured fish. Based on this characterization, we report the first isolation of Rhodococcus qingshengii, from a farmed population of Atlantic salmon in Chile. Virulence studies demonstrated that the isolate fulfilled the Koch’s postulates, suggesting that this bacterial species could be considered as an opportunistic pathogen for Atlantic salmon. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Rhodococcus qingshengii Atlantic salmon Pseudo-membranes
1. Introduction Disease caused by Gram-positive bacteria has become a major problem affecting the freshwater and marine fish farming through the world (Austin and Austin, 2007). Amongst the Gram-positive microorganisms that affect salmon farming in Chile, Renibacterium salmoninarum was first recognized two decades ago (Sanders and Barros, 1986), provoking a chronic disease capable of causing significant mortalities in trout (Onchorhynchus mykiss) and salmon (Salmo salar and Oncorhynchus kisutch). More recently, we characterized Streptococcus phocae, an important emerging pathogen for salmonid culture only in Chile (Romalde et al., 2008). To date, economic losses caused by these fish pathogens have decreased; however, S. phocae remains one of the most important risk factor in the salmon industry in Chile during the summer season. * Corresponding author. Tel.: +34 981563100x16908; fax: +34 981528085. E-mail address: [email protected] (J.L. Romalde). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.06.003
The intensive exploitation of salmonids is an activity of high economical importance in Chile, with an estimated production of approximately 655 ton during the year 2007 (www.salmonchile.cl). The production mainly takes place in the south of the Country and is dominated by the marine culture of Atlantic salmon. In the last years, the management of salmonid farming in Chile is changing in order to increase regular vaccination, as well as to reduce its environmental impact. In 2008, fish farmed in estuary cages and previously immunized with a multivalent commercial vaccine were randomly sampled during the spring–summer season. Atlantic salmon (Salmo salar) showed pseudomembranes on different organs (i.e. spleen, liver, heart and others), which were presumptively associated with side effects provoked by the injection with the oil-adjuvanted vaccine. Bacteriological analysis of these pseudo-membranes revealed the presence of a Gram-positive bacterium. A polyphasic taxonomic analysis was performed to identify this bacterium and its pathogenic potential for farmed fish was investigated in experimental challenges. Based on this characterization, we report the first isolation of Rhodococcus
R. Avendan˜o-Herrera et al. / Veterinary Microbiology 147 (2011) 200–204
qingshengii, from a farmed population of Atlantic salmon in Chile. 2. Materials and methods 2.1. Fish As part of the Chilean surveillance programme for infectious salmonid anemia (ISA) virus detection, 20 Atlantic salmon (70–80 g) were randomly sampled during the spring–summer season from a farm with history of Piscirickettsia salmonis problems, and were transported alive to the laboratory facilities for bacteriological examination. Fish had been previously intraperitoneally (i.p.) vaccinated in the fresh water stage with a multivalent commercial vaccine against infectious pancreatic necrosis virus (IPNV), Vibrio ordalii, Aeromonas salmonicida and P. salmonis. The fish, farmed in floating cages in estuary water at salinity 22% and 8 1 8C with natural light regime, did not show any clinical signs or gross pathology. 2.2. Microscopical and bacteriological analysis For microscope observations, smears from the internal organs and from the pseudo-membranes were examined using a phase contrast microscope at 400 magnification. A selected number of slides were also stained using Gram stain. At the same time, all samples from liver, kidney and spleen were streaked onto tryptic soy agar supplemented with 1% NaCl (TSA-1) and Columbia sheep blood agar plates (AES Laboratories) and incubated aerobically at 20 8C for 72 h. Bacterial isolates were subsequently stored at 70 8C in Criobilles tubes (AES Laboratories, France). 2.3. Phenotypical characterization of the isolate The main phenotypic characteristics of the bacterial isolates were determined as described previously (MacFaddin, 1993; Romalde et al., 2008). The following tests were performed: colony morphology, pigmentation, Gram staining, cell morphology, motility (phase contrast microscopy), cytochrome oxidase, catalase reaction (3% H2O2), oxidation/ fermentation reactions, nitrate reduction, methyl red and Voges–Proskauer reactions, hydrolysis of gelatin, agar, starch, Tween 80, casein and DNA, growth at different temperatures (4, 15, 28 and 37 8C), salt tolerance (0, 1, 1.5, 3, 6 and 10% of NaCl) and growth on Columbia blood agar, MacConkey agar, Sabouraud agar, marine agar 2216, thiosulfate citrate bile salts sucrose agar (TCBS), cysteine agar with and without 5% of fetal serum bovine, brain heart infusion agar and tryptone yeast extract salts agar. In addition, the API miniaturized systems (bioMe´rieux, including API 20 STREP and API ZYM) were employed according to the manufacturer’s instructions. Vibrio anguillarum ATCC 43307, Yersinia ruckeri CECT 955, and Streptococcus phocae ATCC 51973T were employed as controls in the characterization tests. The drug sensitivities of the isolate were determined by the disc diffusion method on Mueller–Hinton agar (MHA) as recommended by the Clinical and Laboratory Standards Institute (NCCLS, 2006) to use for fish pathogens. The
201
chemotherapeutic agents used (micrograms per disc) (Oxoid) were: amoxicillin (25), trimethoprim-sulfamethoxazole (1.25/23.75), enrofloxacin (5), florfenicol (30), oxytetracycline (30), oxolinic acid (2) and eritromicin (15). Reference strain Aeromonas salmonicida subsp. salmonicida ATCC 33658T was used as a control. 2.4. 16S rDNA sequencing and phylogenetic analysis Genomic DNA extraction, amplification and sequencing reactions of the 16S rRNA gene were performed as previously described (Beaz-Hidalgo et al., 2009). Sequencing reactions were performed using the kit GenomeLab DTCS-Quick Start Kit (Beckman Coulter, USA). Sequence corrections and analysis were performed with DNAstar Seqman program (Lasergene, USA). The obtained sequence was subjected to a BLAST search against the latest release of the GenBank. Phylogenetic relationships were determined by using MEGA version 4.0 software program (Tamura et al., 2007). Distance matrices were determined (Kimura, 1980) and these matrices were used to elaborate dendrograms by using the neighbour-joining (NJ) method (Saitou and Nei, 1987). A bootstrap analysis to investigate the stability of the trees was performed on 1000 replicates. 2.5. DNA–DNA hybridization Genomic DNA for DNA–DNA hybridizations was prepared using the Easy DNA (Invitrogen) kit. DNA–DNA hybridization experiments were done by the hydroxyapatite/microtitre plate method (Ziemke et al., 1998) with a hybridization temperature (Tm) of 60 8C. Levels of DNA– DNA relatedness were determined between isolate 790433 and the type strains of Rhodococcus baikonurensis (DSM 44587T), Rhodococcus erythropolis (DSM 43066T) and R. qingshengii (DSM 45222T). 2.6. Characterization of fatty acids by MIDI analysis The fatty acids of the isolate 79043-3 was extracted from cells grown on TSA-1 at 20 8C for 48 h, methylated and analysed using the standard protocol of the Microbial Identification System (MIDI, Newark, DE, USA) as described by Sasser (1990). 2.7. Fulfillment of Koch’s postulates In order to investigate the virulence capacities of the Gram-positive isolate, experimental infection trials were conducted using unvaccinated Atlantic salmon (average weight 60 g) obtained from a commercial farm, with no history of disease problems, located at central Chile. Fish were divided into four groups of 5 each and were held in 20 l tanks with aerated seawater (temperature 16 8C) and acclimatized during 72 h prior to bacterial challenge. The inoculum was prepared by suspending cells from a 24 h culture onto TSA-1 in sterile saline solution (SS; 0.85% NaCl) and adjusting it to a concentration of 5 109 cfu per millilitre by comparison with a McFarland scale. Appropriate decimal dilutions were then prepared in SS. Three groups were inoculated by intraperitoneal injection
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with 0.2 ml of dilutions ranging from 5 105 to 5 107 cfu per fish. Another tank with fish injected with 100 ml of SS was included as control. All trials were maintained for 21 days. Fish were fed daily at 1.5% body weight and the water in each tank was changed once every 2 days to remove faecal material. The fish dead during the trials, as well as the surviving fish, sacrificed as before by anesthetic overdose 3 weeks post-challenge, were submitted to bacteriological and histopathological examination. The Ethics Committees for Animal Experiments of the Universities Andre´s Bello and Santiago de Compostela approved the protocols of this study. 2.8. Histolopathogy Liver, pyloric caecae, pancreas, spleen and heart were processed using standard procedures and the sections (5 mm) stained with Haematoxylin & Eosin (H&E), in order to describe the significant morphological changes. The cells were checked at 2.5 until 63 magnifications in a Leica DM2000 microscopy. 3. Results and discussion The intensification of fish farming in Chile has resulted in growing problems of infectious and/or non-infectious diseases, which causes significant economic losses due to mortalities of eggs, fry and post-smolt in seawater. This paper describes a pathological condition in intensive reared Atlantic salmon (30 kg/m3), characterized by a severe peritonitis, visible as a whitish, loose to compact pseudo-membrane, in some cases containing reddish-tan exudates, located surrounding the internal organs such as spleen, heart, liver and pyloric caecae. Microscopic examinations of wet mount smears from the internal lesions revealed the presence of high quantities of short-rod bacteria, while those Gram stained indicated their Gram-positive character. In all cases, a microorganism was obtained in pure culture after 2–3 days from the internal organs of all fish examined, particularly from the pseudo-membranes. All the isolates were identical (data not shown) and considered as clones, being the representative isolate coded as isolate 79043-3. Colonies were smooth, circular with regular edges, dry and creamy in colour. The phenotypic tests showed that the isolate was a non-motile, non-fermentative, Grampositive bacterium that produced catalase, but did not present cytochrome oxidase. Reactions for arginine dihydrolase, lysine decarboxylase, ornithine decarboxilase and Voges Proskauer were negative, but urease activity was present. Reduction of nitrate and methyl red tests were positive. Gelatin, casein, elastin, Tween 80 and DNA were not hydrolysed. Growth occurred at 15–37 8C and with 0– 3% NaCl. No growth was observed on TCBS, MacConkey and Simmon’s citrate agars. On API 20 STREP identification strips, the isolate 79043-3 was consistently positive for pyruvate, esculin, 2-naphthyl-b-D-galactopyranoside, 2naphthyl phosphate and L-leucine-2-naphthylamide, being classified according to the API database as belonging as Streptococcus salivarius. On the other hand, miniaturized API ZYM kit reacted positively for utilization of 2-naphthyl
phosphate, 2-naphthyl caprylate, L-leucyl-2-naphthylamide, L-valyl-2-naphthylamide, 2-naphthyl phosphate, naphthol-AS-BI-phosphate, 2-naphthyl-aD-glucopyranoside and 6-Br-2-naphthyl-bD-glucopyranoside. Results of the agar disk diffusion test indicated multiresistance of the isolate to the majority of the chemotherapeutic agents studied, being only susceptible to enrofloxacin, oxytetracycline and trimethoprim-sulfamethoxazole. Analysis of the 16S rRNA gene sequence allocated the microorganism to the genus Rhodococcus. Data from the sequence similarity analysis indicated that the closest relatives of the strain were R. qingshengii djl-6T, R. jialingiae djl-6-2T, R. baikonurensis GTG 1041T and R. erythropolis DSM 43066T, exhibiting 99.79, 99.79, 99.47 and 99.31% sequence similarities, respectively (Fig. 1). The partial 16SrRNA gene sequence for isolate 79043-3 (1455 bp) is available in the databases under accession number FN555393. Characterization of fatty acids showed that major (>10%) FAME were C16:0 (22.43%), C18:1 w9c (24.48%), 10-methyl C18:0 (13.13%) and summed feature 3 (16.40%; comprising C16:1 w6c/C16:1 w7c), showing moderate similarity (0.75%) with R. erythropolis and R. globerulus. DNA–DNA hybridization analysis revealed that the isolate 79043-3 showed high DNA–DNA relatedness with R. qingshengii (88.7% re-association), while with R. erythropolis and R. baikonurensis were 61.0% and 39.3%, respectively. All these results clearly indicated that the isolate 79043-3 belongs to the species R. qingshengii. In genus Rhodococcus there are species known to be pathogenic for fish (Olsen et al., 2006), as well as other Rhodococcus unidentified at species level, but still causing chronic granulomatous nephritis lesions in juvenile Atlantic salmon (Claveau, 1991; Speare et al., 1992) and panophthalmitis in Chinook salmon (Oncorhynchus tshawitscha) (Backman et al., 1990). Interestingly, when the sequence of Chilean strain was compared with those of Rhodococcus strains isolated from diseased Atlantic salmon in Norway and Scotland (Olsen et al., 2006), it shared nearly 99.8% sequence similarity with strains 00/50/6670 and 4115 (accession numbers AY147846 and AJ505559, respectively). Further studies would be needed in order to establish if the Rhodococcus strains isolated by Olsen et al. (2006) belong also to R. qingshengii, taking into account that also the general phenotypic characteristics of the isolate 79043-3 are coincident with those described by these authors for the strains from diseased Atlantic salmon in Europe. On the other hand, phenotypic and biochemical tests did not result in an acceptable match for identification of the isolate as R. qingshengii, mainly when the miniaturized API 20 STREP was employed, or as R. jianlingiae the most recently described species within the genus. A reason for this misidentification can be that R. qingshengii is not included in the API Database, which is focused in human pathogens. In addition, the morphology and the colour of the colony as well as the activity observed on Tween 80, reduction of nitrate and methyl red are in disagreement with the biochemical description made by Xu et al. (2007). The results of the virulence assays showed that the Grampositive isolate was able to induce disease or to cause mortality (1 and 3 out of 5 fish at concentrations of 5 106 and 5 107 cfu per fish, respectively), 3 days after the
[(Fig._1)TD$IG]
R. Avendan˜o-Herrera et al. / Veterinary Microbiology 147 (2011) 200–204
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Fig. 1. Phylogenetic tree based on partial 16S rRNA gene sequences including related Rhodococcus and Nocardia species. Segniliparus rotundus was used as out-group. Horizontal branch lengths are proportional to evolutionary divergence. Bootstrap values >50% from 1000 replicates appear next to the corresponding branch.
exposure to the bacterium. On external examination, gross lesions were seen in all fish, including petechiae and hemorrhages on the surface around the mouth and opercula. Marked hemorrhages around the eyes were more pronounced (Fig. 2(A)). Internal findings showed pale liver, splenomegaly, melanosis in abdominal cavity, ascities and internal hemorrhaging. The most common lesion was the presence of the pseudo-membranes on spleen, liver, heart and swinbladder (Fig. 2(B–D)). In all cases, inoculated Grampositive bacteria could be recovered from the pseudomembranes of the dead fish. No mortalities and no gross external or internal pathology were recorded for fish receiving 5 105 cfu per fish as well as the non-challenged fish. Histopathology revealed perivascular leucocytic infiltration in spleen (data not shown). Other changes in the
spleen included marked granulomas and increased fibroblastic reticular cells. Inter lamellar hyperplasia with fusion of lamellae was also observed in gill tissues (data not shown). No significant morphologic changes were observed in heart and liver. No histopathological signs were observed in tissues from control fish. To date, R. qingshengii has only been isolated from a carbendazim-contaminated soil samples obtained from vegetable field in Japan (Xu et al., 2007) This compound is one of the most widely used amongst the benzimidazole family of fungicides in the control of agricultural and forestry diseases. However, the origin of the R. qingshengii in marine environment as well as the possible route of infection in Atlantic salmon is not yet clarified. We can speculate that a possible origin can be the repeated application of pesticides in the environment around the salmonid farms. On the other
[(Fig._2)TD$IG]
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Fig. 2. Experimentally infected Atlantic salmon with haemorrhages around the eye (A) and characteristic pseudo-membranes and/or haemorrhage on liver (B); heart (C) and spleen (D). Arrows indicate the pseudo-membranes.
hand, the sporadic appearance of disease attributed to Rhodococcus sp. infection in salmonid aquaculture suggests that it is not a major pathogen of farmed fish (Speare et al., 1995). However, based on the course of the experimental challenge studies, R. qingshengii could be considered as an opportunistic pathogen for Atlantic salmon. Despite the mortality rate associated with Rhodococcus species appears to be very low, the sub-clinical effects on production could be significant. In fact, the prevalence of infections by this bacterium could be underrated due to their possible confusion with side effects from vaccination with oil-adjuvanted vaccines. Additional studies are necessary to evaluate the risk for salmonid cultures, particularly because our isolate exhibit multi-drug resistance that may make effective treatments difficult. Acknowledgements This work was supported in part by Grants AGL200613208-C02-01 from the Ministerio de Ciencia e Innovacio´n (Spain) and FONDECYT 1090054 from the Comisio´n Nacional de Investigacio´n Cientı´fica y Tecnolo´gica (CON˜ ez for her help with ICYT) (Chile). We thank S. Nun biochemical characterizations. S. Balboa and A. Doce acknowledge the Ministerio de Ciencia e Innovacio´n and Xunta de Galicia (Spain) for research fellowships. References Austin, B., Austin, D.A., 2007. Bacterial Fish Pathogens: Diseases of Farmed and Wild Fish. Springer Praxis Publishing, Chichester, UK. Backman, S., Ferguson, H.W., Prescott, J.F., Wilcock, B.P., 1990. Progressive panophthalmitis in Chinook salmon, Oncorhynchus tshawytscha (Walbaum): a case report. J. Fish Dis. 13, 345–353. Beaz-Hidalgo, R., Alperi, A., Figueras, M.J., Romalde, J.L., 2009. Aeromonas piscicola sp. nov., isolated from diseased fish. Syst. Appl. Microbiol. 32, 471–479.
Claveau, R., 1991. Ne´phrite granulomateuse a` Rhodococcus spp. dans um e´levage de saumons de l’ Atlantique (Salmo salar). Me´d. Vet. Que´. 21, 160–161. Kimura, M., 1980. A simple methods for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. MacFaddin, J.F., 1993. Pruebas bioquı´micas para la identificacio´n de bacterias de importancia clı´nica. The William & Wilkins Company, Baltimore (translation by Me´dica Panamericana S.A.). NCCLS, 2006. Methods for Antimicrobial Disk Susceptibility Testing of Bacteria Isolated from Aquatic Animal; Approved Guideline. NCCLS Document M42-A. NCCLS, Wayne, Pennsylvania. Olsen, A.B., Birkbeck, T.H., Nilsen, H.K., MacPherson, H.L., Wangel, C., Myklebust, C., Laidler, L.A., Aarflot, L., Thoen, E., Nyga˚rd, S., Thayumanavan, T., Colquhoun, D., 2006. Vaccine-associated systemic Rhodococcus erythropolis infection in farmed Atlantic salmon. Dis. Aquat. Organ. 72, 9–17. ˜ os, B., de la Fuente, E., San Romalde, J.L., Ravelo, C., Valde´s, I., Magarin ˜ o-Herrera, R., Toranzo, A.E., 2008. Streptococcus Martı´n, C., Avendan phocae, an emerging pathogen for salmonid culture. Vet. Microbiol. 130, 198–207. Saitou, N., Nei, M., 1987. The neighbour-joining methods: a new method for reconstructing phylognetic tree. Mol. Biol. Evol. 4, 406– 425. Sanders, J.E., Barros, J., 1986. Evidence by fluorescent antibody test for the occurrence of Renibacterium salmoninarum among salmonid fish in Chile. J. Wild Dis. 22, 255–257. Sasser, M., 1990. Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. Technical Note No. 101. MIDI Inc., Newark, DE. Speare, D.J., Brocklebank, J., MacNair, N., Claveau, R., Backman, S., Ewan, P.E., Bernard, K.A., 1992. Granulomatous nephritis associated with Rhodococcus sp. infection in Atlantic salmon (Salmo salar). Can. Vet. J. 33, 192. Speare, D.J., Brocklebank, J., MacNair, N., Bernard, K.A., 1995. Experimental transmission of a salmonid Rhodococcus sp. isolate to juvenile Atlantic salmon, Salmo salar. J. Fish Dis. 18, 587–597. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. Mega 4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Xu, J.-L., He, J., Wang, Z.-C., Wang, K., Li, W.-J., Tang, S.-K., Li, S.-P., 2007. Rhodococcus qingshengii sp. nov., a carbendazim-degrading bacterium. Int. J. Syst. Evol. Microbiol. 57, 2754–2757. Ziemke, F., Hofle, M.G., Lalucat, J., Rosello´-Mora, R., 1998. Reclassification of Shewanella putrefaciens Owen’s genomic group II as Shewanella baltica sp. nov. Int. J. Syst. Bacteriol. 48, 179–186.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Pseudo-membranes on internal organs associated with Rhodococcus qingshengii infection in Atlantic salmon (Salmo salar) ˜ o-Herrera a, Sabela Balboa b, Alejandra Doce b, Pedro Ilardi c, Rube´n Avendan Pablo Lovera d, Alicia E. Toranzo b, Jesu´s L. Romalde b,* a
Universidad Andre´s Bello, Facultad de Ciencias Ba´sicas, Departamento de Ciencias Ba´sica, Vin˜a del Mar, Chile Departamento de Microbiologı´a y Parasitologı´a, CIBUS, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain c Laboratorio de Patologı´a de Peces, Veterquı´mica S.A., Santiago, Chile d Laboratorio Biovac, Puerto Montt, Chile b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 October 2009 Received in revised form 28 May 2010 Accepted 2 June 2010
This paper describes a pathological condition in intensive reared Atlantic salmon (Salmo salar), restricted to the appearance of pseudo-membranes covering internal organs (i.e. spleen, liver, heart and others) associated with the presence of large numbers of a Grampositive bacteria. Isolate 79043-3, obtained as pure culture from affected fish, was subjected to a polyphasic taxonomic study in order to determine its exact taxonomic position, as well as to experimental challenges leading to determine its pathogenic potential for cultured fish. Based on this characterization, we report the first isolation of Rhodococcus qingshengii, from a farmed population of Atlantic salmon in Chile. Virulence studies demonstrated that the isolate fulfilled the Koch’s postulates, suggesting that this bacterial species could be considered as an opportunistic pathogen for Atlantic salmon. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Rhodococcus qingshengii Atlantic salmon Pseudo-membranes
1. Introduction Disease caused by Gram-positive bacteria has become a major problem affecting the freshwater and marine fish farming through the world (Austin and Austin, 2007). Amongst the Gram-positive microorganisms that affect salmon farming in Chile, Renibacterium salmoninarum was first recognized two decades ago (Sanders and Barros, 1986), provoking a chronic disease capable of causing significant mortalities in trout (Onchorhynchus mykiss) and salmon (Salmo salar and Oncorhynchus kisutch). More recently, we characterized Streptococcus phocae, an important emerging pathogen for salmonid culture only in Chile (Romalde et al., 2008). To date, economic losses caused by these fish pathogens have decreased; however, S. phocae remains one of the most important risk factor in the salmon industry in Chile during the summer season. * Corresponding author. Tel.: +34 981563100x16908; fax: +34 981528085. E-mail address: [email protected] (J.L. Romalde). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.06.003
The intensive exploitation of salmonids is an activity of high economical importance in Chile, with an estimated production of approximately 655 ton during the year 2007 (www.salmonchile.cl). The production mainly takes place in the south of the Country and is dominated by the marine culture of Atlantic salmon. In the last years, the management of salmonid farming in Chile is changing in order to increase regular vaccination, as well as to reduce its environmental impact. In 2008, fish farmed in estuary cages and previously immunized with a multivalent commercial vaccine were randomly sampled during the spring–summer season. Atlantic salmon (Salmo salar) showed pseudomembranes on different organs (i.e. spleen, liver, heart and others), which were presumptively associated with side effects provoked by the injection with the oil-adjuvanted vaccine. Bacteriological analysis of these pseudo-membranes revealed the presence of a Gram-positive bacterium. A polyphasic taxonomic analysis was performed to identify this bacterium and its pathogenic potential for farmed fish was investigated in experimental challenges. Based on this characterization, we report the first isolation of Rhodococcus
R. Avendan˜o-Herrera et al. / Veterinary Microbiology 147 (2011) 200–204
qingshengii, from a farmed population of Atlantic salmon in Chile. 2. Materials and methods 2.1. Fish As part of the Chilean surveillance programme for infectious salmonid anemia (ISA) virus detection, 20 Atlantic salmon (70–80 g) were randomly sampled during the spring–summer season from a farm with history of Piscirickettsia salmonis problems, and were transported alive to the laboratory facilities for bacteriological examination. Fish had been previously intraperitoneally (i.p.) vaccinated in the fresh water stage with a multivalent commercial vaccine against infectious pancreatic necrosis virus (IPNV), Vibrio ordalii, Aeromonas salmonicida and P. salmonis. The fish, farmed in floating cages in estuary water at salinity 22% and 8 1 8C with natural light regime, did not show any clinical signs or gross pathology. 2.2. Microscopical and bacteriological analysis For microscope observations, smears from the internal organs and from the pseudo-membranes were examined using a phase contrast microscope at 400 magnification. A selected number of slides were also stained using Gram stain. At the same time, all samples from liver, kidney and spleen were streaked onto tryptic soy agar supplemented with 1% NaCl (TSA-1) and Columbia sheep blood agar plates (AES Laboratories) and incubated aerobically at 20 8C for 72 h. Bacterial isolates were subsequently stored at 70 8C in Criobilles tubes (AES Laboratories, France). 2.3. Phenotypical characterization of the isolate The main phenotypic characteristics of the bacterial isolates were determined as described previously (MacFaddin, 1993; Romalde et al., 2008). The following tests were performed: colony morphology, pigmentation, Gram staining, cell morphology, motility (phase contrast microscopy), cytochrome oxidase, catalase reaction (3% H2O2), oxidation/ fermentation reactions, nitrate reduction, methyl red and Voges–Proskauer reactions, hydrolysis of gelatin, agar, starch, Tween 80, casein and DNA, growth at different temperatures (4, 15, 28 and 37 8C), salt tolerance (0, 1, 1.5, 3, 6 and 10% of NaCl) and growth on Columbia blood agar, MacConkey agar, Sabouraud agar, marine agar 2216, thiosulfate citrate bile salts sucrose agar (TCBS), cysteine agar with and without 5% of fetal serum bovine, brain heart infusion agar and tryptone yeast extract salts agar. In addition, the API miniaturized systems (bioMe´rieux, including API 20 STREP and API ZYM) were employed according to the manufacturer’s instructions. Vibrio anguillarum ATCC 43307, Yersinia ruckeri CECT 955, and Streptococcus phocae ATCC 51973T were employed as controls in the characterization tests. The drug sensitivities of the isolate were determined by the disc diffusion method on Mueller–Hinton agar (MHA) as recommended by the Clinical and Laboratory Standards Institute (NCCLS, 2006) to use for fish pathogens. The
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chemotherapeutic agents used (micrograms per disc) (Oxoid) were: amoxicillin (25), trimethoprim-sulfamethoxazole (1.25/23.75), enrofloxacin (5), florfenicol (30), oxytetracycline (30), oxolinic acid (2) and eritromicin (15). Reference strain Aeromonas salmonicida subsp. salmonicida ATCC 33658T was used as a control. 2.4. 16S rDNA sequencing and phylogenetic analysis Genomic DNA extraction, amplification and sequencing reactions of the 16S rRNA gene were performed as previously described (Beaz-Hidalgo et al., 2009). Sequencing reactions were performed using the kit GenomeLab DTCS-Quick Start Kit (Beckman Coulter, USA). Sequence corrections and analysis were performed with DNAstar Seqman program (Lasergene, USA). The obtained sequence was subjected to a BLAST search against the latest release of the GenBank. Phylogenetic relationships were determined by using MEGA version 4.0 software program (Tamura et al., 2007). Distance matrices were determined (Kimura, 1980) and these matrices were used to elaborate dendrograms by using the neighbour-joining (NJ) method (Saitou and Nei, 1987). A bootstrap analysis to investigate the stability of the trees was performed on 1000 replicates. 2.5. DNA–DNA hybridization Genomic DNA for DNA–DNA hybridizations was prepared using the Easy DNA (Invitrogen) kit. DNA–DNA hybridization experiments were done by the hydroxyapatite/microtitre plate method (Ziemke et al., 1998) with a hybridization temperature (Tm) of 60 8C. Levels of DNA– DNA relatedness were determined between isolate 790433 and the type strains of Rhodococcus baikonurensis (DSM 44587T), Rhodococcus erythropolis (DSM 43066T) and R. qingshengii (DSM 45222T). 2.6. Characterization of fatty acids by MIDI analysis The fatty acids of the isolate 79043-3 was extracted from cells grown on TSA-1 at 20 8C for 48 h, methylated and analysed using the standard protocol of the Microbial Identification System (MIDI, Newark, DE, USA) as described by Sasser (1990). 2.7. Fulfillment of Koch’s postulates In order to investigate the virulence capacities of the Gram-positive isolate, experimental infection trials were conducted using unvaccinated Atlantic salmon (average weight 60 g) obtained from a commercial farm, with no history of disease problems, located at central Chile. Fish were divided into four groups of 5 each and were held in 20 l tanks with aerated seawater (temperature 16 8C) and acclimatized during 72 h prior to bacterial challenge. The inoculum was prepared by suspending cells from a 24 h culture onto TSA-1 in sterile saline solution (SS; 0.85% NaCl) and adjusting it to a concentration of 5 109 cfu per millilitre by comparison with a McFarland scale. Appropriate decimal dilutions were then prepared in SS. Three groups were inoculated by intraperitoneal injection
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with 0.2 ml of dilutions ranging from 5 105 to 5 107 cfu per fish. Another tank with fish injected with 100 ml of SS was included as control. All trials were maintained for 21 days. Fish were fed daily at 1.5% body weight and the water in each tank was changed once every 2 days to remove faecal material. The fish dead during the trials, as well as the surviving fish, sacrificed as before by anesthetic overdose 3 weeks post-challenge, were submitted to bacteriological and histopathological examination. The Ethics Committees for Animal Experiments of the Universities Andre´s Bello and Santiago de Compostela approved the protocols of this study. 2.8. Histolopathogy Liver, pyloric caecae, pancreas, spleen and heart were processed using standard procedures and the sections (5 mm) stained with Haematoxylin & Eosin (H&E), in order to describe the significant morphological changes. The cells were checked at 2.5 until 63 magnifications in a Leica DM2000 microscopy. 3. Results and discussion The intensification of fish farming in Chile has resulted in growing problems of infectious and/or non-infectious diseases, which causes significant economic losses due to mortalities of eggs, fry and post-smolt in seawater. This paper describes a pathological condition in intensive reared Atlantic salmon (30 kg/m3), characterized by a severe peritonitis, visible as a whitish, loose to compact pseudo-membrane, in some cases containing reddish-tan exudates, located surrounding the internal organs such as spleen, heart, liver and pyloric caecae. Microscopic examinations of wet mount smears from the internal lesions revealed the presence of high quantities of short-rod bacteria, while those Gram stained indicated their Gram-positive character. In all cases, a microorganism was obtained in pure culture after 2–3 days from the internal organs of all fish examined, particularly from the pseudo-membranes. All the isolates were identical (data not shown) and considered as clones, being the representative isolate coded as isolate 79043-3. Colonies were smooth, circular with regular edges, dry and creamy in colour. The phenotypic tests showed that the isolate was a non-motile, non-fermentative, Grampositive bacterium that produced catalase, but did not present cytochrome oxidase. Reactions for arginine dihydrolase, lysine decarboxylase, ornithine decarboxilase and Voges Proskauer were negative, but urease activity was present. Reduction of nitrate and methyl red tests were positive. Gelatin, casein, elastin, Tween 80 and DNA were not hydrolysed. Growth occurred at 15–37 8C and with 0– 3% NaCl. No growth was observed on TCBS, MacConkey and Simmon’s citrate agars. On API 20 STREP identification strips, the isolate 79043-3 was consistently positive for pyruvate, esculin, 2-naphthyl-b-D-galactopyranoside, 2naphthyl phosphate and L-leucine-2-naphthylamide, being classified according to the API database as belonging as Streptococcus salivarius. On the other hand, miniaturized API ZYM kit reacted positively for utilization of 2-naphthyl
phosphate, 2-naphthyl caprylate, L-leucyl-2-naphthylamide, L-valyl-2-naphthylamide, 2-naphthyl phosphate, naphthol-AS-BI-phosphate, 2-naphthyl-aD-glucopyranoside and 6-Br-2-naphthyl-bD-glucopyranoside. Results of the agar disk diffusion test indicated multiresistance of the isolate to the majority of the chemotherapeutic agents studied, being only susceptible to enrofloxacin, oxytetracycline and trimethoprim-sulfamethoxazole. Analysis of the 16S rRNA gene sequence allocated the microorganism to the genus Rhodococcus. Data from the sequence similarity analysis indicated that the closest relatives of the strain were R. qingshengii djl-6T, R. jialingiae djl-6-2T, R. baikonurensis GTG 1041T and R. erythropolis DSM 43066T, exhibiting 99.79, 99.79, 99.47 and 99.31% sequence similarities, respectively (Fig. 1). The partial 16SrRNA gene sequence for isolate 79043-3 (1455 bp) is available in the databases under accession number FN555393. Characterization of fatty acids showed that major (>10%) FAME were C16:0 (22.43%), C18:1 w9c (24.48%), 10-methyl C18:0 (13.13%) and summed feature 3 (16.40%; comprising C16:1 w6c/C16:1 w7c), showing moderate similarity (0.75%) with R. erythropolis and R. globerulus. DNA–DNA hybridization analysis revealed that the isolate 79043-3 showed high DNA–DNA relatedness with R. qingshengii (88.7% re-association), while with R. erythropolis and R. baikonurensis were 61.0% and 39.3%, respectively. All these results clearly indicated that the isolate 79043-3 belongs to the species R. qingshengii. In genus Rhodococcus there are species known to be pathogenic for fish (Olsen et al., 2006), as well as other Rhodococcus unidentified at species level, but still causing chronic granulomatous nephritis lesions in juvenile Atlantic salmon (Claveau, 1991; Speare et al., 1992) and panophthalmitis in Chinook salmon (Oncorhynchus tshawitscha) (Backman et al., 1990). Interestingly, when the sequence of Chilean strain was compared with those of Rhodococcus strains isolated from diseased Atlantic salmon in Norway and Scotland (Olsen et al., 2006), it shared nearly 99.8% sequence similarity with strains 00/50/6670 and 4115 (accession numbers AY147846 and AJ505559, respectively). Further studies would be needed in order to establish if the Rhodococcus strains isolated by Olsen et al. (2006) belong also to R. qingshengii, taking into account that also the general phenotypic characteristics of the isolate 79043-3 are coincident with those described by these authors for the strains from diseased Atlantic salmon in Europe. On the other hand, phenotypic and biochemical tests did not result in an acceptable match for identification of the isolate as R. qingshengii, mainly when the miniaturized API 20 STREP was employed, or as R. jianlingiae the most recently described species within the genus. A reason for this misidentification can be that R. qingshengii is not included in the API Database, which is focused in human pathogens. In addition, the morphology and the colour of the colony as well as the activity observed on Tween 80, reduction of nitrate and methyl red are in disagreement with the biochemical description made by Xu et al. (2007). The results of the virulence assays showed that the Grampositive isolate was able to induce disease or to cause mortality (1 and 3 out of 5 fish at concentrations of 5 106 and 5 107 cfu per fish, respectively), 3 days after the
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Fig. 1. Phylogenetic tree based on partial 16S rRNA gene sequences including related Rhodococcus and Nocardia species. Segniliparus rotundus was used as out-group. Horizontal branch lengths are proportional to evolutionary divergence. Bootstrap values >50% from 1000 replicates appear next to the corresponding branch.
exposure to the bacterium. On external examination, gross lesions were seen in all fish, including petechiae and hemorrhages on the surface around the mouth and opercula. Marked hemorrhages around the eyes were more pronounced (Fig. 2(A)). Internal findings showed pale liver, splenomegaly, melanosis in abdominal cavity, ascities and internal hemorrhaging. The most common lesion was the presence of the pseudo-membranes on spleen, liver, heart and swinbladder (Fig. 2(B–D)). In all cases, inoculated Grampositive bacteria could be recovered from the pseudomembranes of the dead fish. No mortalities and no gross external or internal pathology were recorded for fish receiving 5 105 cfu per fish as well as the non-challenged fish. Histopathology revealed perivascular leucocytic infiltration in spleen (data not shown). Other changes in the
spleen included marked granulomas and increased fibroblastic reticular cells. Inter lamellar hyperplasia with fusion of lamellae was also observed in gill tissues (data not shown). No significant morphologic changes were observed in heart and liver. No histopathological signs were observed in tissues from control fish. To date, R. qingshengii has only been isolated from a carbendazim-contaminated soil samples obtained from vegetable field in Japan (Xu et al., 2007) This compound is one of the most widely used amongst the benzimidazole family of fungicides in the control of agricultural and forestry diseases. However, the origin of the R. qingshengii in marine environment as well as the possible route of infection in Atlantic salmon is not yet clarified. We can speculate that a possible origin can be the repeated application of pesticides in the environment around the salmonid farms. On the other
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Fig. 2. Experimentally infected Atlantic salmon with haemorrhages around the eye (A) and characteristic pseudo-membranes and/or haemorrhage on liver (B); heart (C) and spleen (D). Arrows indicate the pseudo-membranes.
hand, the sporadic appearance of disease attributed to Rhodococcus sp. infection in salmonid aquaculture suggests that it is not a major pathogen of farmed fish (Speare et al., 1995). However, based on the course of the experimental challenge studies, R. qingshengii could be considered as an opportunistic pathogen for Atlantic salmon. Despite the mortality rate associated with Rhodococcus species appears to be very low, the sub-clinical effects on production could be significant. In fact, the prevalence of infections by this bacterium could be underrated due to their possible confusion with side effects from vaccination with oil-adjuvanted vaccines. Additional studies are necessary to evaluate the risk for salmonid cultures, particularly because our isolate exhibit multi-drug resistance that may make effective treatments difficult. Acknowledgements This work was supported in part by Grants AGL200613208-C02-01 from the Ministerio de Ciencia e Innovacio´n (Spain) and FONDECYT 1090054 from the Comisio´n Nacional de Investigacio´n Cientı´fica y Tecnolo´gica (CON˜ ez for her help with ICYT) (Chile). We thank S. Nun biochemical characterizations. S. Balboa and A. Doce acknowledge the Ministerio de Ciencia e Innovacio´n and Xunta de Galicia (Spain) for research fellowships. References Austin, B., Austin, D.A., 2007. Bacterial Fish Pathogens: Diseases of Farmed and Wild Fish. Springer Praxis Publishing, Chichester, UK. Backman, S., Ferguson, H.W., Prescott, J.F., Wilcock, B.P., 1990. Progressive panophthalmitis in Chinook salmon, Oncorhynchus tshawytscha (Walbaum): a case report. J. Fish Dis. 13, 345–353. Beaz-Hidalgo, R., Alperi, A., Figueras, M.J., Romalde, J.L., 2009. Aeromonas piscicola sp. nov., isolated from diseased fish. Syst. Appl. Microbiol. 32, 471–479.
Claveau, R., 1991. Ne´phrite granulomateuse a` Rhodococcus spp. dans um e´levage de saumons de l’ Atlantique (Salmo salar). Me´d. Vet. Que´. 21, 160–161. Kimura, M., 1980. A simple methods for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. MacFaddin, J.F., 1993. Pruebas bioquı´micas para la identificacio´n de bacterias de importancia clı´nica. The William & Wilkins Company, Baltimore (translation by Me´dica Panamericana S.A.). NCCLS, 2006. Methods for Antimicrobial Disk Susceptibility Testing of Bacteria Isolated from Aquatic Animal; Approved Guideline. NCCLS Document M42-A. NCCLS, Wayne, Pennsylvania. Olsen, A.B., Birkbeck, T.H., Nilsen, H.K., MacPherson, H.L., Wangel, C., Myklebust, C., Laidler, L.A., Aarflot, L., Thoen, E., Nyga˚rd, S., Thayumanavan, T., Colquhoun, D., 2006. Vaccine-associated systemic Rhodococcus erythropolis infection in farmed Atlantic salmon. Dis. Aquat. Organ. 72, 9–17. ˜ os, B., de la Fuente, E., San Romalde, J.L., Ravelo, C., Valde´s, I., Magarin ˜ o-Herrera, R., Toranzo, A.E., 2008. Streptococcus Martı´n, C., Avendan phocae, an emerging pathogen for salmonid culture. Vet. Microbiol. 130, 198–207. Saitou, N., Nei, M., 1987. The neighbour-joining methods: a new method for reconstructing phylognetic tree. Mol. Biol. Evol. 4, 406– 425. Sanders, J.E., Barros, J., 1986. Evidence by fluorescent antibody test for the occurrence of Renibacterium salmoninarum among salmonid fish in Chile. J. Wild Dis. 22, 255–257. Sasser, M., 1990. Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. Technical Note No. 101. MIDI Inc., Newark, DE. Speare, D.J., Brocklebank, J., MacNair, N., Claveau, R., Backman, S., Ewan, P.E., Bernard, K.A., 1992. Granulomatous nephritis associated with Rhodococcus sp. infection in Atlantic salmon (Salmo salar). Can. Vet. J. 33, 192. Speare, D.J., Brocklebank, J., MacNair, N., Bernard, K.A., 1995. Experimental transmission of a salmonid Rhodococcus sp. isolate to juvenile Atlantic salmon, Salmo salar. J. Fish Dis. 18, 587–597. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. Mega 4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Xu, J.-L., He, J., Wang, Z.-C., Wang, K., Li, W.-J., Tang, S.-K., Li, S.-P., 2007. Rhodococcus qingshengii sp. nov., a carbendazim-degrading bacterium. Int. J. Syst. Evol. Microbiol. 57, 2754–2757. Ziemke, F., Hofle, M.G., Lalucat, J., Rosello´-Mora, R., 1998. Reclassification of Shewanella putrefaciens Owen’s genomic group II as Shewanella baltica sp. nov. Int. J. Syst. Bacteriol. 48, 179–186.