Isolation of Aeromonas Salmonicida from Salmon Lice Lepeophtheirus Salmonis and Marine Plankton

DISEASES OF AQUATIC ORGANISMS Vol. 16: 79-81.1993 Published June 24 Dis. aquat. Org.

NOTE

Isolation of Aeromonas salmonicida from salmon lice Lepeophtheirus salmonis and marine plankton

Lars ~ese',Oivind ~nger~,'

'University of Bergen. Department of Fisheries and Marine Biology, Bergen High TechnologyCenter, N-5020 Bergen. Norway 'University of Bergen, Department of Microbiology and Plant Physiology, Jahnebakken5. N-5007 Bergen, Norway

ABSTRACT: Aeromonas sahnonicida was isolated from sal- be important in introducing pathogens such as mon lice Lepeophtheirus salmonis and zooplankton in sam- Aeromonas salmonicida into fish farms (Cusack & ples taken at a fish farm with an outbreak of furunculosis. Approximately 104 cells of A. salmonicida per salmon louse Cone 1986, Nylund et al. 1991). and 600 cells of A. salmonicida per m1 of homogenized plank- In this study we report immunofluorescence detec- ton suspension were detected. By use of immunomagnetic tion of Aerornonas salmonicida in high numbers in sed- beads coated with monoclonal antibodies against the lipo- iment~,seawater, and in surface films. In addition, we polysaccharides of A. salmonicida, the bacteria were retneved in almost pure culture from certain of the heavily have been able to isolate the bacterium from salmon 'contaminated' environmental san~ples. lice and from marine plankton. We also discuss the benefits that immunomagnetic beads provide in the Aerornonas salrnonicida, the etiological agent of fu- isolation of A. salmonicida from environmental sam- runculosis, causes severe losses in cultured fish in ples. many parts of the world (Austin & Austin 1987). Since Materials and methods. Sampling: Atlantic salmon 1985, furunculosis has spread epizootically in Norway. Salrno salar suffering from furunculosis were killed by Furunculosis has now been recorded in most fish farms a blow to the head. Salmon lice were picked from the on the western coast of Norway, where outbreaks re- fish with sterile tweezers, care being taken not to col- sult in the highest mortalities experienced on the lect lice from bloody areas on the fish. Plankton sam- farms. ples were collected by dragging a plankton trawl The survival potential of Aeromonas salrnonicida in (mesh size 40 pm) through the upper 50 cm in a net- water has been thoroughly investigated (McCarthy cage. The samples were kept in the dark and on ice 1977, Sakai 1986, Rose et al. 1990). The bacterium has until they were processed in the laboratory on the never been isolated from any environmental sources same day. Water and sediments were sampled and but it has been detected in high numbers by applica- processed as described by Enger & Thorsen (1992). tion of irnmunofluorescence in samples from sedi- Antibodies: Two monoclonal antibodies, AL2E6 and ments, seawater, and surface films from fish farms ALlB2, directed towards the lipopolysaccharide (LPS) stocked with salmon suffering from furunculosis and the outer protein layer (A-layer), respectively, (Enger & Thorsen 1992). of Aeromonas salrnonicida were kindly donated by Although fish have been regarded as the main vector R. Nilsen, Apothekerenes Laboratorium, Tromsa, in the transmission of furunculosis, ectoparasites may Norway. also serve as vectors because they have been shown to Double staining of samples: The abundance of be likely vectors in the transfer of viral diseases (Ahne Aeromonas salmonicida and a total count in the sam- 1985, Mulcahy et al. 1990). Salmon lice may therefore ples were obtained by a combination of a fluorescent antibody technique (FA) and staining of bacterial DNA Addressee for correspondence by 4',6-diamidino-2-phenylindole(DAPI) (Enger et al.

O Inter-Research 1993 80 Dis. aquat. Org. 16: 79-81. 1993

1989). This method allows simultaneous enumeration ferred to agar plates [Brain Heart Infusion Broth of total number of bacteria and number of fluorescent (Oxoid) + 1.5 % agar (Difco) with 0.01 mg ml-' antibody-stained cells in the same sample. Coomassie Brilliant Blue (Serva) and 0.015 mg Fifteen m1 of surface sample, 20 rnl of water samples, Oleandomycin (Oxoid) ml-' (BHIA-CBB 01 15)]. 1 m1 of plankton sample or 1 m1 of a 1:1000 dilution of Colonies were counted after 48 h of incubation at sediment were filtered through Nuclepore filters with 21 "C. a pore size of 0.2 pm. The monoclonal antibody di- Surface sterilization of the salmon lice was carried rected against the LPS of Aeromonas salmonicida out by dipping them twice in 70 % ethanol. Between (AL2E6) was used in the immunofluorescence assay. the dips the lice were washed in sterile physiological Immunomagnetic beads: Monodispersed latex saline. beads, 2.8 pm in diameter, with covalently linked Plankton samples of 3 m1 were centrifuged (90 X g sheep anti-mouse IgG antibodies (Dynabeads, M-280 for 5 rnin) and the supernatants were removed. The sheep anti-mouse IgG, Dynal A/S Oslo, Norway) were centrifuged plankton samples (ca 1 ml) were homoge- used in the experiment by following the manu- nized in 1 m1 of physiological saline and then incu- facturer's instructions. bated with immunomagnetic beads for l h. The sam- To coat the beads with mouse antibodies specific to ples were then washed and inoculated on agar plates Aeromonas salmonicida, about 10"eads were trans- as described earlier. ferred to a tube containing 12 m1 of monoclonal anti- Imrnuno-colony-blot: A eromonas salmonicida was body preparation [anti-LPS diluted 1:500 in phosphate- identified by the technique described by Rodrigue buffered saline (PBS), pH 7.5, with 0.5 % (v/w) bovine et al. (1989). Nitrocellulose membranes (BioRad, serum albumin (BSA) (Sigma Chemical Co., St. Louis, Richmond, CA, USA) were deposited on BHIA-CBB MO, USA)] (PBS-BSA). The beads were incubated with plates containing possible A. salmonicida colonies. the monoclonal antibodies overnight at 5 to 7 'C on an The membranes were removed and treated with an end-over-end rotator (4 rpm) to avoid settling. After the antibody solution (ALlB2, diluted 1:4000) followed by immunomagnetic beads had been drawn towards the an incubation with peroxidase-conjugated goat anti- inner wall of the tube by a magnet, the residual liquid mouse antibodies (BioRad, diluted 1:2000). After incu- was aspirated and the irnmunomagnetic beads were bation with peroxidase substrate (0.06 % 4-chloro- suspended in 5 m1 of sterile PBS-BSA. naphthanol (BioRad) with 20 % ethanol and 0.015 % Sample preparation: Salmon lice were counted and H202in tris-buffered saline (20 mM Tris, 0.5 M NaC1, homogenized (Thornrnas Tissue Grinders, 343 1-E04, pH = 7.5) colonies of A. salmonicida was observed as Swedesboro, NJ, USA) in sterile physiological saline purple spots on the membranes. (0.9 % NaCl). One m1 of the suspension was incubated Results and discussion. Fluorescent antibody detec- with irnrnunomagnetic beads (2 X 108 beads sample-') tion showed that Aeromonas salmonicida comprised for 1 h. To remove unbound bacteria, the immunomag- approximately 0.1 and 0.02 % of the total bacterial netic beads were washed 5 times (1 m1 of physiological numbers in the water samples and in the sediment, re- saline containing 0.1 % Tween 20). Three replicate spectively (Table 1). The high number of A. salmoni- samples of 0.1 m1 of the bead suspension were trans- cida found in the sediment (Table 1) suggests that

Table 1 Aeromonas salmon~cida. Number of bacteria detected using different detection methods. The samples were collected in a fish farm during an outbreak of furunculosis. Total: total bacterial counts after staining with DAPI. FA: counts with the fluorescent antibody technique. IMB: immunomagnetic beads. ND: not determined

Samples Microscope counts Plate count before IMB Plate count after IMB Colony blot (cells ml-'1 (CFU ml-'1 (CFU ml-l) (CFU ml-l) Total FA Other bact. A. salmonicida Other bact. A. salmon~cida A. salrnonicida

Surface 5.3 X 106 6.3 X 103 5.6 X 102 - 20 - ND Water at 10 cm 3.3 X 105 3.0 X 102 33 - - - ND Water at 3 m 3.0 X lo5 3.9 X 102 66 - 13 - ND Sedimentd 1.3 X lo9 2.9 X 105 4.5 X 104 - 20 - N D Plankton 9.0~10~11x104 ND - ND ca 300 620 Liceb ND ND 4.4X103 6.8~10~ 93 1.1 X 104 1.3 X lo5 Surface-sterilized liceb ND ND 4.8 X 10' 4.0 X 104 3 1.6 X 103 1.7 X 103

-: No colonies Results glven as cells per gram bResults given as cells per louse Nese & Enger: Aeromonas salm onlc~da~n salmon lice and plankton 8 1

the pathogen had been accunlulating there for many on non-selective media. The ability to isolate and grow weeks. In the plankton samples, A. salmonicida com- specific bacteria from environmental samples heavily prised 1.2 % of the total bacterial counts, which is one 'contaminated' with other bacteria is an essential pre- order of magnitude higher than the relative abundance requisite for further work with the specific bacteria. observed in the samples from the water column. Our results show that the often disturbing influence of Despite the fact that A. salmonicida was detected by the background bacterial flora can be reduced sub- FA in water and sediment samples in numbers well stantially by use of this method. within the detection limits of the immunomagnetic beads method, attempts to isolate the bacterium from Acknowledgements. Th~swork was supported in part by a grant from the Norwegian Council of Fisheries Research the water and sediment samples were unsuccessful. (NFFR). We thank the staff of the fish farm for their valuable This is in agreement with previous reports, indicating collaboration. A. Nylund is thanked for his critical comments that A. salrnonicida does not survive indefinitely when on the manuscript. it occurs outside of its host (McCarthy 1977, Rose et al. 1990). LITERATURE CITED Aerornonas salrnonicida was isolated in high numbers from marine plankton and salmon lice (Table 1). Ahne, W. (1985).Argulus foliaceus L.and Pisciola geometra L. In direct plate counts from homogenized salmon lice, as mechanical vectors of spring viraemia of carp virus (SVCV).J. Fish Dis. 8 241-242 A, salmonicida comprised 60 % of the colony forming Austin, B., Aust~n,D. A. (1987). Bacterial fish pathogens. Ellis units (cfu).Using immunomagnetic beads, however, A. Horwood Ltd, Chichester, p. 11 1-195 salmonicida accounted for 98 to 99 % of the cfu. A. sal- Bruno, D. W., Stone, J. (1990). The role of saithe, Pollachius vi- monicida has not previously been isolated from ani- rens L., as a host for the sea lice, Lepeoptheirus salmonis Krayer and Caligus elongatus Nordmann. Aquaculture 89: mals other than fish (Austin & Austin 1987). Because 201 -207 the louse can swim between different hosts (Bruno & Cusack. R.. Cone, D. K. (1986). A review of parasites as vec- Stone 1990), the association of A. salmonicida with sal- tors of viral and bacterial diseases of fish. J. Fish Dis. 9: mon lice suggests that the lice may be important vec- 169-171 tors in the spread of furunculosis. Enger. 0.. Husevbg. B., Goksayr, J (1989). Presence of the fish pathogen Hbrio salmonicida in fish farm sediments. Disinfection of the salmon lice in 70 % ethanol re- Appl. environ. Microbiol. 55: 2815-2818 duced the number of bacteria isolated. However, the Enger. 0.. Thorsen, B. (1992). Possible ecological implications successful isolation of Aeromonas salmonicida from of the high cell surface hydrophobicity of the fish patho- lice that had been surface-disinfected suggests that a gen Aeromonas salmonicida. Can. J. Microbiol. 38: significant proportion of the A. salmonicida cells must 1048-1052 McCarthy, D H. (1977). Some ecological aspects of the bacte- have been residing inside the lice, probably in the gas- rial f~sh pathogen Aeromonas salmonicida. Aquat. trointestinal tract. Detection of live cells of A, salmoni- Microbiol. 6. 299-324 cida in the plankton samples also indicates that marine Mulcahy, D., Klaybor, D., Batts, W. N. (1990). Isolation of in- plankton should be considered as a vector of potential fectious hematopoiet~c necrosis virus from a leech (Pisciola salmosjtlca) and a copepod (Salmonicola sp.),ec- importance in the spread of furunculosis. toparasites of sockeye salmon Oncorhynchus nerka. Dis. The isolates were identified as Aeromonas salmonic- aquat. Org. 8: 29-34 ida subsp. salmonicida based on the API 20 E Nylund, A., Bjarknes, B., Wallace, C. (1991). Lepeophtheirus (Analytical Profile Index Systems S.A., La Balme les salmonis - a possible vector in the spread of diseases on Grottes, France) assay. salmonids. Bull. Eur. Fish Pathol 11: 213-216 Rodrigue, L., Marion, D., Trudel, L., Barthe, C., Lavoie, M. C. It is important to underscore the qualitative differ- (1989). Comparison of method for the evaluation of the ences between the 2 detection methods applied in this oral microbiota of mice. J. microbiol. Meth. 10: 71-82 study. While the immunofluorescence technique pro- Rose, A. S., Ellis, E. E.. Munro. A. L. S. (1990). The survival of vided a useful tool for microscopic enumeration of Aerornonas salmonicida subsp. salrnonicida in seawater. J. Aeromonas salmonicida in preserved samples, the iso- Fish Dis. 13: 205-214 Sakai, D. K. (1986). Electrostatic mechanism of survival of vir- lation techniques based on the use of immunomagnetic ulent Aeromonas salmonicida strains in river water. Appl. beads made it possible to grow the detected bacterium environ. Microbiol. 51: 1343-1349

Responsible Subject Editor: 7: Evelyn, Nanaimo, Manuscript first received: June 12, 1992 B.C., Canada Revised version accepted: March 3, 1993