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Predominant Bacteria Associated with Black Rockcod (Notothenia Coriiceps, Richardson 1844) from King George Island, Antarctica

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Predominant Bacteria Associated with Black Rockcod (Notothenia Coriiceps, Richardson 1844) from King George Island, Antarctica 246, Bull. Eur. Ass. Fish Pathol., 36(6) 2016 Predominant bacteria associated with black rockcod (Notothenia coriiceps, Richardson 1844) from King George Island, Antarctica R. Avendaño-Herrera1,2,3*, R. Irgang1,2, M. Poblete-Morales1,2, Y. Vásquez-Martínez4, C. Robles4, D. Toro-Ascuy4 and M. Cortez-San Martín4* 1Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile; 2 Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, Chile; 3 Centro de Investigación Marinas de Quintay (CIMARQ), Quintay, Chile; 4 Laboratorio de Virología Molecular, Centro de Biotecnología Acuícola, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile Abstract The present study isolated bacteria from black rockcod Notothenia coriiceps (Richardson, 1844) to determine if the relative proximity of salmon farms to natural habitats has resulted in the spread of known salmon pathogens. For this, bacteria were isolated from 60 N. coriiceps collected from seven sites along Fildes Bay, Antarctica (62°12´S, 58°57´W). Bacterial cultures were obtained from the kidney, liver, and spleen. The predominant bacterium of each culture was 16S rRNA gene se- quenced, with 35 isolates obtained from 14 specimens. The predominant genera were Pseudomonas (9), Psychrobacter (7), and Staphylococcus (7), followed by Sporosarcina (4), and Bacillus (3). Notably, the Aliivibrio sp. (99.85% similarity with A. logei) and Chryseobacterium scophthalmus isolates were found and subsequently used to challenge rainbow trout (Oncorhynchus mykiss) and fine flounder (Paralichthys adspersus) species, which showed no clinical signs of disease nor death. Although kidney and skin lesion isolates were recovered (10 and 8 isolates, respectively), N. coriiceps challenges are needed to assess real infection risks. To our knowledge, this is the first description of these bacterial microorganisms from N. coriiceps. Introduction Over the last decade, the Chilean aquaculture relocating farther south to the Magallanes and industry has rapidly grown due to favorable Chilean Antarctic Regions (Ibieta et al., 2011). geography and climate. Initially, fresh and Antarctica is relatively close to Chile, and the seawater salmonids were farmed in the Los southernmost aquaculture center is only 950 Lagos Region of southern Chile. However, the km from the Chilean Antarctic Professor Julio infectious salmon anemia virus crisis in the Escudero Base on King George Island. mid-2000s resulted in many production centers * Corresponding author’s e-mail: R. Avendaño-Herrera ([email protected] - [email protected]) and M. Cortez-San Martín ([email protected]) Bull. Eur. Ass. Fish Pathol., 36(6) 2016, 247 Most farmed fish are transported, often long- laboratory facilities at the Profesor Julio Escudero distance, during production (Avendaño-Herrera Base (Chilean Antarctic research station) for et al., 2014). This increases the risk of introduc- bacteriological examination. ing bacterial or viral pathogens to wild species susceptible to disease or to becoming natural Bacteriological analysis reservoirs. For example, Streptococcus phocae, the The specimens were aseptically dissected in a etiological agent of warm water streptococcosis, biosafety cabinet. Bacterial samples were taken was related to Atlantic salmon (Salmo salar) from any external lesions observed, liver, spleen, mortalities at a farm near Puerto Natales in the and kidney and streaked onto Marine Agar 2216 Magallanes Region, where water temperatures (Difco™), Tryptic Soy Agar supplemented with are < 10°C (Yáñez et al., 2013). 1% NaCl (TSA-1), and Columbia Sheep Blood Agar Plates (CSBA; AES Laboratories) and aero- Most southern Chile/Antarctic fauna are per- bically incubated at 4°C and 18°C for 10 days. ciform Notothenioidei fish (Eastman, 1993). Plate-grown bacteria were transported from While Notothenioidei fish have been studied Antarctica to the Laboratorio de Patología de in relation to global climate change in polar Organismos Acuáticos y Biotecnología Marina ecosystems (Huth and Place, 2013), there is little of the Universidad Andrés Bello (Viña del Mar, information regarding the potential of Antarctic Chile). Afterwards, a representative colony of fish species acting as bacterial pathogen carriers. the predominant morphotype from each plate Therefore, the present study sampled different was selected, streaked onto a new TSA-1 plate organs from the abundant and widespread to obtain pure cultures, and then stored at –80°C Antarctic black rockcod (Notothenia coriiceps), in Criobille tubes (AES Laboratories). which was captured with species-specialised, environmentally friendly equipment during Phenotypic characterisation the 2014 ECA-50 expedition to Chile’s Antarctic All stored isolates were characterised using base (62°12´S, 58°57´W). Predominant bacteria phenotypic tests as described by MacFaddin were identified from N. coriiceps tissues and (2003). Biochemical reaction tests included compared against known salmonid pathogens colony morphology and pigmentation; Gram- to determine incidence in this wild fish. staining; cytochrome oxidase; a catalase reaction (3% H2O2); and cell morphology and motility. Materials and methods Growth was also tested on TSA-1 under aerobic Fish specimens conditions at different temperatures (4, 15, 18, Sixty adult and juvenile black rockcod speci- 20, 30, and 37°C) and in TSB supplemented mens (26.7 ± 4.9 cm) were caught through with different NaCl concentrations (0, 1, 3, 5, 7, longline fishing at depths of 1-25 m from seven and 10%). All phenotypic tests were incubated sites (numbered 1 to 7) near Fildes Bay on King aerobically at 18°C and observed at 1 to 10 days George Island, Antarctica. Fish were killed fol- before being recorded as negative. lowing American Veterinary Medical Asso- ciation standards (Leary et al., 2013), cooled If an isolate was suspected of belonging to the with ice, and transported immediately to the Flavobacteriaceae family, additional biochemical 248, Bull. Eur. Ass. Fish Pathol., 36(6) 2016 tests were performed according to Bernardet fine flounder (Paralichthys adspersus; weighing et al. (2002), including gliding motility, the 5–6 g) maintained in fresh and seawater, respec- presence of cell-wall-associated flexirubin- tively. To ensure that fish were uninfected with type pigments, and Congo red adsorption. For any pathogen described in Chile (see details in potentially pathogenic isolates, biochemical Avendaño-Herrera, 2011), samples were sub- analyses were carried out using API® 20E and jected to standard microscopic and bacteriologi- API® ZYM (bioMérieux) strips according to cal examinations, as well as PCR analysis. Fish the manufacturer’s instructions, with excep- were randomly allocated in groups of 15 per tion of the incubation temperature, which was 10-L plastic tank and acclimatised for seven days fixed at 18°C. prior to bacterial challenge. A group of rainbow trout and fine flounder were inoculated by intra- 16S rDNA sequencing and phylogenetic peritoneal injection with 0.1 mL of 5 x 107 cells analysis per fish of each isolate. Another two tanks with DNA from pure cultures was extracted using fish injected with TSB-1 alone were included as the InstaGene Matrix (Bio-Rad) according to negative controls. All trials were maintained in the manufacturer’s instructions. The 16S rRNA a closed system with 50 µm filtered seawater gene was amplified with universal pA and pH (salinity of 35‰) or dechlorinated water for up primers (Edwards et al., 1989), and PCR prod- to 28 days at 17 ± 1°C. Fish were fed daily at 1.5% ucts were purified using the Wizard® SV Gel body weight, and tank water was changed once and PCR Clean-Up System Kits (Promega). The every two days. Cultures of each internal organ amplicon was sequenced by Macrogen (Seoul, (i.e. kidney, spleen, liver) for all fish (dead or Korea) using the same amplifying primers and alive) were prepared by direct streaking onto the sequences were then analysed using the TSA-1 plates. online tool EzTaxon (http://www.ezbiocloud. net/eztaxon). The 16S rRNA gene sequences Results and discussion were also aligned using the MEGA v5 software Despite growing Antarctic-based research, (Tamura et al., 2011). Genetic distances were this is the first study to report on marine mi- obtained using Kimura’s two-parameter model croorganisms in Antarctic fish. Some studies and clustered with the neighbour-joining algo- have described bacterial species in the water rithm. The accuracy of the resulting tree was (Brinkmeyer et al., 2003) and in the intestinal measured by bootstrap resampling of 1,000 flora of Nothothenia neglecta (now N. coriiceps) replicates. The nucleotide sequences generated (MacCormack and Fraile, 1990; Ward et al., for 16S rRNA were deposited in the GenBank 2009), but none made reference to the predom- database under accession numbers KP745564 inant bacteria in N. coriiceps organs, such as the to KP745598 (Figure 1). kidney, spleen, and liver, all with filtration and immune functions. Pathogenicity in fish To determine the pathogenicity of 43Ls.9 and This study sampled seven geographic sites, col- 27K.1, infection trials were conducted in rainbow lecting 59 N. coriiceps specimens with no outward trout (Oncorhynchus mykiss; weighing 6–8 g) and signs of disease. One specimen (fish 43), present- Bull. Eur. Ass. Fish Pathol., 36(6) 2016, 249 27S.1 (KP745584) 89 27S.3 (KP745582) 4Lv.1 (KP745568) 37 3K2 (KP745569)
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