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Journal of Fish Diseases 2002, 25, 733–736 Identification of Edwardsiella ictaluri from diseased freshwater catfish, Pangasius hypophthalmus (Sauvage), M Crumlish1, T T Dung2, J F Turnbull1, N T N Ngoc2 and H W Ferguson1 1 Institute of Aquaculture, University of Stirling, Stirling, UK2 Aquaculture and Fisheries Science Institute, CanTho University, CanTho, Vietnam Keywords: Edwardsiella ictaluri, freshwater catfish, A newly recognized disease was recently reported in farmed Vietnamese Pangasius sp. (Ferguson,Turnbull, Shinn, Thompson, Dung & Crumlish Hawke (1979) reported the first isolation of 2001). Post-mortem appearance of this condition, Edwardsiella ictaluri from farmed channel catfish, Ôbacillary necrosis of Pangasius Ô, was typified by Ictalurus punctatus (Rafinesque). The bacterium multifocal irregular white lesions of varying sizes on produces an acute septicaemic condition known in several organs including liver, spleen and kidney.
the USA as enteric septicaemia of catfish (ESC) and Histopathologically, the lesions were acute to sub- which results in high mortality, notably in channel acute multifocal areas of necrosis and pyogranulo- catfish (Austin & Austin 1999). The blue catfish, matous inflammation. Associated with these lesions Ictalurus furcatus (Valenciennes), and the white were several species of parasites, but common to all catfish, Ameiurus (¼Ictalurus) catus (L.), are also affected fish were variable numbers of large bacilli, susceptible (Hawke, McWhorter, Steigerwalt & usually seen at the margins of lesions. The patho- Brenner 1981; Plumb & Sanchez 1983). Non- logical findings strongly suggested that the disease ictalurid fish species show no susceptibility as had a bacterial aetiology. Several species of bacteria determined by mortality, clinical disease or estab- were isolated from affected fish, including a novel lished infection following experimental challenge species of Bacillus. These preliminary results formed (Plumb & Sanchez 1983). However, Kasornchandra, the basis for a more detailed study into the Rogers & Plumb (1987) recovered and identified bacteriology of Ôbacillary necrosisÕ. In the results E. ictaluri as the causative agent of a bacterial presented here, bacteria biochemically typical of disease in walking catfish, Clarias batrachus (L.), in E. ictaluri were recovered in large numbers from all Thailand. To date, there is no published description of the fish sampled with gross and histopathological of the isolation or identification of E. ictaluri from signs typical of Ôbacillary necrosis of PangasiusÕ.
In total, 17 P. hypophthalmus with (n ¼ 12) and The freshwater catfish, Pangasius hypophthalmus without (n ¼ 5) clinical and gross signs of disease (Sauvage), is indigenous to the Mekong Delta in were sampled from three farm sites located in two Vietnam. It has been cultured traditionally for provinces in the Mekong Delta, Vietnam. Two farm decades in earthen ponds and more recently in sites were in An Giang province and one farm, which was sampled on two occasions, was located inCanTho Province. Where possible, healthy anddiseased fish were sampled from the same cage or Correspondence Dr M Crumlish, Institute of Aquaculture, pond on each farm. Samples were taken from all University of Stirling, Stirling FK9 4LA, UK(e-mail: mc3@stir.ac.uk) three farms in September 2001 and again from one Journal of Fish Diseases 2002, 25, 733–736 M Crumlish et al. Identification of Edwardsiella ictaluri farm in March 2002. Tissues from all the fish were September 2001) were sent for concise alignment of fixed in 10% buffered formalin, subsequently 500 base pairs and 16S rRNA Genbank analysis by processed for routine histopathology and sections MIDI Laboratory (Newark, USA). This allowed stained with haematoxylin and eosin (Drury & identification of the organisms to species level. All of the isolates were tested for antibiotic sensitivity to Bacterial isolates recovered from the liver of fish furazolidone (FR, 50 and 100 lg), ciprofloxacin sampled in September 2001 were grown only on (CFC, 5 lg), nitrofurantoin (NFT, 50 and 100 lg), tryptone soya agar (TSA, Oxoid, Basingstoke, UK) norfloxacin (NFC, 5 and 100 lg), gentamicin whereas isolates from the fish sampled in March (GMC, 30 and 120 lg), oxolinic acid (OA, 2 lg), 2002 were recovered on TSA as well as on agar oxytetracycline (OT, 30 lg), potentiated sulphon- selective for bacteria belonging to Enterobacteria- amide (SXT, 25 lg), enrofloxacin (ENR, 5 lg), ceae (EMB, Merck, UK). The plates were sealed florfenicol (FFC, 30 lg) and amoxycillin (AML, using Nesco film, incubated at 28 °C, and colony 10 lg). The antibiotics selected were a combination growth observed at both 24 and 48 h post-inocu- of those routinely screened in the diagnostic laboratory at the Institute of Aquaculture, Stirling obtained from the National Collection of Industrial University, and those used by Vietnamese fish and Marine Bacteria (NCIMB, Aberdeen, UK) and farmers (T.T. Dung, personal communication).
used as standards during the characterization tests.
The paper multi-discs impregnated with the anti- The two NCIMB isolates were 13272 (E. ictaluri) biotics were all obtained from Oxoid except FFC, and 2034 (E. tarda). Isolates recovered from the which was from Mast Diagnostics (Liverpool, UK).
clinically sick P. hypophthalmus were inoculated A bacterial lawn on TSA was produced from a onto TSA, EMB and E. ictaluri agar (EIA) (Shotts suspension of two to three colonies emulsified in & Waltman 1990). TSA and EMB were used 5 mL of sterile saline using the spread plate method during sampling at the farm sites, whereas EIA was and the multi-discs were placed on the lawn. Results used subsequently in the laboratory.
were interpreted as sensitive (‡16 mm), partially All the tests were performed following the sensitive (12–15 mm) or resistant (<11 mm) protocols described in Frerichs & Millar (1993).
based on the zone diameters of growth inhibition.
Pure cultures of isolates were produced on TSA and The gross and histopathological lesions of the then single colonies were selected for Gram stains.
clinically affected fish were entirely consistent The oxidation/fermentation (O/F) (Hugh & with those already described for Ôbacillary necrosis Leifson 1953) reaction was tested using O/F basal of PangasiusÕ (Ferguson et al. 2001). Large bacilli media purchased from Difco, UK. Cytochrome were still visible at the margins of typical lesions, oxidase was tested using oxidase strips (Oxoid). The and there were virtually no complications because catalase reaction was determined following the of parasites or other concurrent disease. The method of Gagnon, Hunting & Esselen (1959), isolates recovered on TSA from fish with clinical which used 3% (v/v) hydrogen peroxide. A single signs of disease were predominantly pure. Only bacterial colony was selected and incubated in three fish sampled onto TSA in September 2001 tryptone soya broth (TSB, Oxoid) at 28 and 37 °C produced more than one bacterial species, but and the motility was checked after 24 h using the growth was still dominated by colonies later hanging drop method. The biochemical profiles identified as E. ictaluri. All of the isolates were were determined using the API 20E kit (Bio- incubated at 28 °C, and after 48 h on TSA these Me´rie´ux, UK). Tolerance to salt was measured by produced pinpoint to small sized colonies (average placing two to three colonies into 5 mL of TSB 0.14 Æ 0.03 mm, n ¼ 12) that appeared off-white with 0.5, 1, 1.5, 2, 2.5 and 3% NaCl added. These and translucent with an irregular surface and edge.
suspensions were incubated at 28 °C and checked The colony morphology was very similar for the 12 for growth 7 days post-inoculation. Temperature Vietnamese isolates and NCIMB 13272 (E. ictaluri).
sensitivity was determined by inoculating TSA The fish samples taken in March 2002 on EMB plates with each of the isolates, then incubating yielded pure growth, and all of the isolates grew the plates at 15, 22, 28 and 37 °C; colony growth well on both EMB and EIA, producing similar was checked and recorded daily for 7 days.
colony shape and size, observed as pinpoint, round, Two isolates from Farm 1 (CanTho province, raised, translucent and pale-coloured. Forty-eight September 2001) and Farm 2 (An Giang province, hours at 28 °C was required on all solid agars Journal of Fish Diseases 2002, 25, 733–736 M Crumlish et al. Identification of Edwardsiella ictaluri before individual colonies were clearly visible to the variability in length and width, often with very large rods clearly visible. This variation in size was The bacterial isolates recovered from the clinic- consistently observed in all 12 isolates recovered ally sick P. hypophthalmus were typical of E. ictaluri.
from clinically diseased P. hypophthalmus. Mixed All cultures comprised Gram-negative short or sized rods have not been commonly described in the variable length fermentative, non-motile rods that literature but they have been observed in histopa- were catalase positive but not oxidase positive. All of thology sections from experimental infections in the Vietnamese isolates tested positive for lysine channel catfish (J. Newton, personal communica- decarboxylase and glucose. However, one isolate tion). Biochemically, E. ictaluri has been described from Farm 1 also utilized citrate while two isolates as a homogeneous organism with fewer positive from Farm 3 gave a positive response for urease; this reactions than E. tarda (Waltman, Shotts & Hsu was the same for the NCIMB 13272 (E. ictaluri) 1986). The Vietnamese isolates were biochemically isolate but not the NCIMB 2034 (E. tarda) isolate, compatible with E. ictaluri and two of the isolates which was much more biochemically reactive were identical to the NCIMB E. ictaluri. The compared with the other isolates tested. All bacterial remaining 10 cultures were identical to each other cultures produced growth in TSB at all salt and differed by only one reaction from the NCIMB concentrations tested. The optimal growth tem- perature was determined to be 28 °C, although all On-farm control of ESC outbreaks in the USA isolates grew slowly at 22 and 37 °C, while only the has relied heavily on in-feed antibiotics, particularly NCIMB 2034 isolate produced any growth at oxytetracycline and potentiated sulphonamides (Bowser, Munson, Francis-Floyd & Stiles 1986; The two isolates sent to MIDI Laboratories were Waltman & Shotts 1986). Disturbingly, the identified to species level as E. ictaluri with 99.91% majority of Vietnamese isolates (nine out of 12) confidence, using 530 bp match and 16S rRNA were resistant to these antibiotics, although they analysis (MIDI Laboratory, USA). All of the were found to be sensitive to the remaining eight Vietnamese isolates showed either partial or full antibiotics tested. Despite this in vitro sensitivity, resistance to OT, SXT and OA compared with the albeit somewhat limited, Vietnamese farmers have two type strains, which were sensitive to all of the reported little success combating Ôbacillary necrosisÕ antibiotics tested. Two isolates from Farm 2 were using antibiotics, so alternative methods of preven- resistant to OA only, and one isolate from Farm 1 tion and control will need to be investigated.
was resistant to OT and SXT only. All of theisolates were sensitive to the other antibiotics tested.
The disease identified as Ôbacillary necrosisÕ, recently described from farmed P. hypophthalmus This work was carried out as part of a Department (Ferguson et al. 2001), was first observed in 1999 in for International Development (DFID, UK) fun- Vietnam, where farmers reported high mortality in ded project and the authors would like to thank the fish that had white spots on their internal organs fish farmers in the Mekong delta who participated (T.T. Dung, personal communication). In the in this study. Thanks also to Mrs G. Dreczkowski present study, bacteria were recovered mostly in for help with the antibiotic sensitivity tests.
pure culture, from 12 clinically diseased P. hypoph-thalmus with typical Ôbacillary necrosisÕ lesions. All these isolates have been identified as E. ictaluri.
These results provide further evidence to support Austin B. & Austin D.A. (1999) Enterobacteriacae representatives.
the suggestion that Ôbacillary necrosis of Pangasius Ô In: Bacterial Fish Pathogens: Diseases of Farmed and Wild Fish(ed. by B. Austin & D. A. Austin), 3rd edn, pp. 81–84.
has a bacterial aetiology. Infections in freshwater Springer Praxis Publishing, Chichester.
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(1986) Potentiated sulphonamide therapy of Edwardsiella Although the Vietnamese isolates were typical of ictaluri infection in channel catfish. Mississippi Agricultural E. ictaluri, there were some differences. Normally, and Forestry Experiment Station Research Report 11, 3. [Cited E. ictaluri is described as a small straight rod, which in: Bacterial Diseases of Fish (ed. by V. Inglis, R.J. Roberts & measures 1 lm · 2–3 lm (Plumb 1993). How- N.R.Bromage (1993), p. 312. Blackwell Scientific Publica-tions, Oxford.] ever, the Vietnamese isolates showed much greater Journal of Fish Diseases 2002, 25, 733–736 M Crumlish et al. Identification of Edwardsiella ictaluri Drury R.A.B. & Wallington E.A. (1980) General staining Kasornchandra J., Rogers W.A. & Plumb J.A. (1987) Edward- procedures. In: Carleton’s Histological Techniques (ed. by siella ictaluri from walking catfish, Clarias batrachus L., in R.A.B. Drury & E.A. Wallington ), pp. 125–150. Oxford Thailand. Journal of Fish Diseases 10, 137–138.
Plumb J.A. (1993) Edwardsiella septicaemia. In: Bacterial Dis- Ferguson H.W., Turnbull J.F., Shinn A.P., Thompson K., Dung eases of Fish (ed. by V. Inglis, R.J. Roberts & N.R. Bromage), T.T. & Crumlish M. (2001) Bacillary necrosis in farmed pp. 61–79. Blackwell Scientific Publications, Oxford.
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