CHARACTERISTICS AND VIRULENCE OF AEROMONAS NYDROPHILA ISOLATES FROM FRESHWATER FISH WITH EPIZOOTIC ULCERATIVE SYNDROME ( EUS )

C!haractcristics and virulence oSA~ronr.o,~r~s hvrlrophida isolates i~ecovered Srom ulcerated fi.eshwatei. fish during the outhreaks of Epizootic Ulcera1,ive Syndrome (EUS) t h a t occurred i n 1390-1992 in S r i Lanka were examined l,o find out whet,llcl. the isolates could he categorized to a single vilulent phenotypic group. Fittg three isolates of A. hyri~~ol)l~~iln l-ec(~vel.ed from ulcerated iish hclonping to 12 diSte~.ent species cullecl.ed from S different ti-cshwater hahitats during t.hc disease outbveaks were tested for ti2 characterislics and the ovcrall similarity of' isolates was determined by numerical taxonomic analysis. At the 96'h similali ty value, the isolates were grouped into seven phenons. Total numhcr niisc~lates categorized undcr the phenuns I, 11,111, IV, V, VI, andVI1 were 18,5,9,5, $1, 4 and 3 rcspectively. Viiulence screening tests of tllc isolates showed that 78% isolates classitjed under plicnon I and all the isolates of' phenon I1 were weakly virulent, 78% isolates of phanon 111 and all the isolates of phenon IV we3.e nloderately viiulc?nt, wllereas 75% isolates oCphenon VI and all the isolates of pl~enons V and VII were higllly vi~.nlent. Antibiotic susceptibility tests showed that the isc.)latrs were i.esistant t o ampicillin, iif'ampicin and t~imet,I~oprim hut sensitive t,o cl~lo~~nmpl~enir :ol , gentamicin, kanamycin, nalidixic acid, ni t rofurantoin, crsytct~acyclin, st:~ept;un~ycin niid tetracycline. Suscc~tit,i!;ty t o c~~ylllronlj~cin and sulplionamide was variable. Phenolypic vnriat ior~s and 1,anKe ol 'oi~.ulcnce cxllihited by A . lylrlrophilu isulatcs recuvel.ed from ulcei.atcd fish collected liom dii'fel,ont hah i ta t s in S1.i Lanka during the outhreaks oJ' EUS indicate that n single highly vilulent phenotypic group ofA, h~~tlropl~i ln is o1 piimmily assc.)cialed with the disease.


INTRODUCTION
Epizootic Ulcerative Syndronle (EUS) characterised by severe ulcerations of the slrin and niuscles is a serious disease of freshwater and brackish water fishes in Asia and P a ~i f i c .' .~ EUS was first reported in Sri Larlka in late 1937.The disease caused heavy mortality in more than 20 species offreshwater and estuarine fish in the South Western Province.::Since then EUS h a s recurred in Sri Lanl~a."~'However the disease incidence appears to be lower than that in the initial years.
" Coi.1.espcmdingauthor EUS seems to have a complex infectious aetiology.Improving the understanding of the complex aetiology of EUS would be useful t o evolve preventive and control measures t o minimize the occurrence of the disease.Bacteria, such as Atrolnonas hydrophila and Vibrio anguillarzsrn, the fungus, Apl~a~aonzyces and rhabdoviruses have been isolated from EUS affected fish."'More recent studies indicate that the Oomycete fungus, Aphanonxycees inuadans is the necessary cause of EUS." However the importance cf bacteria in the pathogenesis of the disease can not be ruled out.'Our previous bacterial investigation on EUS affected fish collected from various freshwater bodies in Sri Lanka showed t h a t A. h.ydroph.ila was the predominant bacteria consistently isolated from EUS affected fish i11 Sri Lanlm4 In the present in~~estigation, detailed characteristics of the A. hydroph.ilaisolates recovered from ulcerated fish collected from different freshwater habitats in Sri Lanka during three outbrealcs of EUS were tested to determine the overall similarity of the isolates by numerical taxonomic analysis.Virulence ofthe isolates was also screened to see whether all the isolates could be cat,egorized to a single virulent strain.In addition, susceptibility of A. hydrophila isolates to different antimicrobial drugs was evaluated.

METHODS AND MATERIALS
Bacteria isolation n7l,cl ider1,tificatiorz: Fish showing gross clinical signs of EUS were collected from eigh.t freshwater bodies in the Western, North-Western, and Sabaragamuwa Provinces in Sri Lanka viz.Boralasgamuwa reservoir, Bemmulla canal, I-1amil.toncanal, shallow streams near paddy fields a t Kirjndiwela and Agalawatte, Lunuwila reservojr, Mahawewa reservoir and.Guruk0d.aOya d.uring three consecutive disease outbreaks that occurred in December 1990, December 1.991-January 1992 and in December 1992.Samples were collected for bacterj.aas stated previouslyVrom muscle below the lesion and internal organs of twenty four ulcerated fish belonging to twelve different species: Anahas testusdineus, Esontu,~ danrica, Etroplus nzacula,tl~s, Etroplus suratensis, 0phiceph.alusstriatus, Puntius f'ilanra~~,tos.u,s,Pur1.tiu.sAinzaculatus, P u n t i u s sarana, Rasbora dan,iconizr,s, Tor Izh.z~dree, Trichogaster pectoralis, and Wallago attu.Samples were cultured on nutrient agar, or trypticase soy agar or Rimler-Shotts agar and incubated a t 27-30 "C for 24 -48 h.!' The dominant isolates were purified and maintained on trypticase soy agar and nutrient agar.Of the total of 83 bacterial isolates recovered from EUS affected fish, 53 isolates were identified and later confirmed as A. hydrophila using the standard m e t h ~d .s .~" -l ~ The 53 isolates included 32 A. Jl.yydr~ph.il~tisolates recovered from our previous bacteriol.ogical.investjgation." In the present study, a total of 82 characteristics includ.inggrowth, morphol.ogica1,pliysjologj.caland, biochemical characteristics of each of the 53 isolates were examined for taxonomic analysis, and similarity values(Sm) between the isolates were computed using the formula described.by Sneath.13Sm =Nj(Na + N , ) x 100, where Ns was the number of similarities between isolates, N , was the number of hssimilar characters between the isolates.After similarity values were calculated pairwise, the data were arranged in a similarity matrix.The data were then transposed into a dendrogram and used as a basis for determlnillg taxonomic arrangements in terms of numerical relationships for all the Isolates of A. h.ydrophiln.
Vii-u.lc71,ce screcn.ilzg:All the isolates of A. hydrophila recovered from EUS-affected fish were screened for virulence.The isolates were subcultured and resuspended.in sterilized saline a t 10" colony forming unitslml.Healthy Etroplus suratensis (an EUS susceptible fish species) weighing 19-27g were injected iiltramuscularly with bacterial isolates at the region 1-1.5cmbelow the anterior part of the dorsal fin a t a dose of 0.05 ml, containing approximately 5 x loG CFU per test fish.Control fish were injected with 0.05 ml of sterilized saline.Ten fish were used for each group.The virulence of the isolates was categorized on the basis of development of lesions and percentage mortalities: 100% mortality within 24 h as highly virulent; over 50% mortality with a h a e m o r r h a ~c lesion within 24 -48 h as moderately virulent; over 50% mortality with a haemorrhagic lesion after 48 h but within 120 h as weakly virulent; less than 50'51 mortality a t 120 h without lesion development as avirulent.Freshly dead fish were isolated for A. hydrophila to satisfy Koch's postulates.Possible relationships between different Siochemical characteristics and the virulence levels of isolates were determined by pairwise cross cor~:elation analysis."'Antibiotic susceptibility tests: Thirteen antimicrobial drugs were evaluated for ef'fectiveness against the A. hydrophila isolates using disk diffusion tec1lnjque.l"The antimicrobial drugs used were ampicillin, chloramphenicol., erythromycin, gentamicin, kanamycin, nalidixic acid, nitrofurantoin, oxytetracyclin, rifampjcin, streptomycin, sulphonamide, tetracycline and trimethoprim.

RESULTS
Profile of' 53 isolates of A .hydrophila recovered from twenty four ulcerated fish including twelve different species are presented in Table 1.The isolates were recovered from the muscular lesion, liver, kidney and spleen.Morphological, growth, physiological and biochemical characteristics followed for the identification of A. h,ydrophila are presented in Table 2. Most of the characteristics were consistent for all the isolates but variable results were obtained for 10 characteristics: hydrolysis of' aesculin, utilization of citrate as sole source of carbon (Simmon's citrate), liquefaction of gelatine, production of H,S from 2.5% peptone water, formation of indole from tryphtophan, methyl red reactioil, formation of acetoin from glucose(Voges Proskauer test), production of' reddish brown pigment on tyrosine agar and fermentation of L-arabinose and salicin.In general, t h e isolates recovered from lesions and internal organs of the same fish showed  Total o f 8 2 cha~~icleric;tics were tested for the total of 53 isolates.+ : 100% positive for the cl~aracteristic, -: 100% negativc fi-,r the charactelistic; d : differs among isolates (5% positive):, A-acid, K-alkaline, TSI triple suKar iron similar characteristics whereas variable characteristics were observed among the isolates ofA.h.ydroph.ilarecovered from the fish sampled from different.locations.The diagrammatic representation of relationships of similarity matrices of'tlze 53 isolates of A. h.ydrophila are given jn Fig. 1.Based on the 10 variable characteristics of the 82 characteristics studied, the 5 3 isolates were classified into 7 phexlorls wit11 mean similarities of more t11, m 96% (Fig. 2).Percent similarity of'the total of 53 isolates was between 88'?r1-100'%.3. Of the inconsistent characteristics found, four characteristics shown by all the isolates of phenon I were similar to the typical characteristics ofA.h,ydrophila (utilization of citrate as sole source of carbon, H,S production from peptone water, indole production, and VP test).However, the isolates were unable to ferment salicin.Only some of the isolates were able to hydrolyse aesculin and ferment arabinose.All the isolates classified under phenon I1 were unable to hydrolyse aesculin and ferment arabinose.All the isolates of phenon I11 showed only one characteristic typical to A. hydroph.ila, the positive reaction for Simmon's citrate test.The isolates of phenon IV were completely positive for the characteristics of typical A. h.ydrophila.The isolates grouped into the phenon V, VI and VII were completely positive for most of the characteristics of typical A. h.ydrophila..However, most of the isolates in phenon V, VI and VII produced reddish brown pi-gment in tyrosine agar, which is an atypical characteristic ofA.lzydrophila. .

. '
The summary of the results of virulence screening among the isolates is shown in Table 4. Thirty six percent of the isolates (19153) were highly.virulent;28% (15153) were moderately virulent and 36% (19153) were weakly virulent.Most of the isolates (78%) in phenon I and all the isolates in phenon I1 were weakly virulent.Most of the moderately virulent isolates belonged to the phenon I11 and IV.Majority of the isolates classified as highly virulent came from phenon V, VI and VII.Most virulent isolates were recovered from severely infected fish.Some inconsjstent biochemical characteristics of the isolates were significantly correlated with their virulerlce potential (Table 5).The statistical analysis indicates that the virulence potential is positively correlated with hydrolysis of aesculin, and fermentation of saljcin and ~iegat~ively correlated with the indole production (pcO.05 ).

DISCUSSION
A. h,ydl-ophiln is an opportunistic bacterium, which produces diseases under stress conditj.ons,often 112 association with other microorganjsms."'Tllere is a inarlred heterogeneity within the species description of A. 1i,~~drophZla.~"'"~~~Numerical taxoilolrlic analysis on strains ofAeronsor1.a.s has been carried out to separate them into difl'erent ge~~ospecies.~~~'Prevjouslyi t was rioted that three c11aracteri.sticsof' the total of46 characteristics tested were inconsisteiltfor theA.h.ydrophila.isolates recovered from EUS affected fish in Sri Lan1m4 In the present study, detailed analysis o-f characteristics of A, h.ydroplzila isolates recovered from EUS affected fish collected from difTerent freshwater habitats in Sri Lanlca indicated.that 10 characteristics of the total of 82 characteristics studied were inconsistent.Based on the inconsistent characteristics, the isolates were grouped into seven phenons WH.S. Cha~zdrakanthi at al.