SCREENING AND CHARACTERIZATION OF POTENTIAL ANT AGONISTIC BACTERIUM FROM FRESHWATER FISH SKIN MUCUS AND ITS APPLICATION IN DISEASE MANAGEMENT
A Thesis Su6mitted to tlie
West Bengal University of Animal and Fishery Sciences, in partia[fu(fil[ment of tlie requirements for tlie CDegree of
MASTER OF FISHERY SCIENCE In
Jlquatic Jlnima[ Hea[tb
(j3y
FARHANA HOQUE, B.F.Sc.
Department of Aquatic Animal Health Faculty of Fishery Sciences West Bengal University of Animal and Fishery Sciences 5 - Budherhat Road, Chakgaria, P.O:- Panchasayar, Kolkata-700094
2013
West Bengal University of Animal and Fishery Sciences Faculty of Fishery Sciences Department of Aquatic Animal Health 5-Budherhat Road, Chakgaria, P.O: Panchasayar, Kolkata-700 094 (Main campus: 68 - Kshudiram Bose Sarani, Belgachia, Kolkata-700 037)
{]Jr.
rr. J. )l6raliam, :M. P.sc. pli/D.
Professor (Pisli :Micro6iofogy)
Rsf. ~"o: FFs,/A"~ -,.j.1.Olg-tq {]Jate:
6- S - 1'3
CERTIFICATE
This is to certify that the work recorded in the thesis entitled "Screening and characterization of potential antagonistic bacterium from freshwater fish skin mucus and its application in disease management" submitted by Ms. Farhana Hoque in partial fulfillment of requirement for the Degree of Master of Fishery Science (Aquatic Animal Health) in the Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, is the faithful and bonafide research work carried out under my supervision and guidance. The results of the investigation reported in this thesis have not so far been submitted for any other Degree or Diploma. The assistance and help received during the course of investigation have been duly acknowledged.
(Prof. T. J. Abraham) Date: b' b . , .> Kolkata- 700 094
Chairman Advisory Committee
}I~Oo/}I£ S1{!F/Er:( Approval of the examiners for the award of the degree of Master of Fishery Science (Aquatic Animal Health)
We, the undersigned have been satisfied with the perfonnance of Ms.
Farhana Hoque in the viva-voce examination, conducted today, the .... ~.9.~ Q.~:". . .... , 2013, recommended that the thesis be accepted for the award of the degree.
Name 1. Prof. T. J. Abraham Chairman of Advisory Committee
External Examiner 3. Dr. O. Dash Member of the Advisory Committee 4. Prof. S. S. Dana Member of the Advisory Committee 5. Dr. T. S. Nagesh Member of the Advisory Committee
Signature
()b~ll)
·::::::::::~~:::£'1~:, ~I~.
jlctnowfedoement It is my proud privifege to ackJlowfecfge inde6tedness to my reverend advisor and respected teaclier (]Jr,
rr: J,
A6raliam, Professor (:Micr06iofogy) and Head,
(]Jepartment of Aquatic Anima{ Hea{tli, CFacu{ty of CFisliery Sciences, West (8enga{ Vniversity of Anima{ and CFisliery Sciences as were as Cliairman of tlie Advisory Committee for liis 6enevofent guidance and ~~en interest to carry out tliis wor~ I wisli to express my foya{ and deepest sense of gratitude to liim for liis affectionate encouragement, untiring supervision, constructive criticism, intimate association and va{ua6fe suggestions tliat liave made tliis tliesis wor/tpossi6fe, I fed deCiglited to express my deep sense ofgratitude to (])r.
q, (]Jasli, Associate
Professor, (]Jepartment of Aquatic Anima{ Hea{tli and mem6er of tlie Advisory committee, for liis precious assistance, effective suggestions and constant inspiration during tlie entire period of researcli wor( I express my lieart fu{{ 'Fislieries
~ension
and (]Jr.
tlian~~
rr: s.
to Prof S. S. (]Jana, Head, (]Jepartment of
:Nagesli, Associate Professor, (]Jepartment of
'Fislien'es 1?.fsource :Management for tlieir precious suggestions and unliesitating lieCp during tlie tliesis work:, I wouUf Cik,§ to express my revering gratitude and proud gratifu{ness to Prof. ((]Jr.) Q~azi :MaliJuzur 1?(lliaman, 1?~gistrar, West (8enga{ Vniversity of HeaCtIi Sciences, for liis va{ua6fe suggestions, constructive Ufeas and constant encouragement rendered tlirougliout tlie course of tliis work~ I desire to express my sincere tliank~ to (]Jr.
(P,
Jaisankgr, Senior Scientist,
Indian Institute of Cliemica{ (]3iofogy (I1C(8), '1(o{k,9-ta Center and (]Jr. :Manas Sark.g.r, Professor and Jfead, (]Jept, of :Micr06iofogy, Calcutta :Nationa{ :Meaica{ Co{fege ana Jfospita{ for tlieir ~ind co-operation dun'ng tlie course of my study, I owe my inde6tness to (]Jr.
q, Jayaseftaran,
Professor, CFislieries Corkge ana 1?/searcli Institute,
Tamifnatfu t£islieries Vniversity, 71iootliuk udi, 'Iamirnadu, {j)r. (:Mrs.) A, Vma, J
jIssociate Professor, Sfirimp (J)isease (J)iagnostic La60ratory, rJami{ :Nacfu Vniversity ofVeterinary and)Inima{Sciences, :Maafiavaram, Cfiennai and tfie Xeaa, (J)epartment of Pisfi Processing rJecfino£ogy for fiefping me witfi 6acteria{ isofa tes. rJfie services rendered 6y tfie q?,JJya{ fife Sciences Pvt. Ltd., Secundera6aafor gene sequencing and PjI:MCE analysis is afso dUly ackrwwfecfged. I am afso grateful to tfie (J)ean, Pacu{ty of Pisfiery Sciences for providing necessary fiefp auring tfie dissertation wor~ I e:(press my sincere (IICCB), 1(o{kflta and
tfiank~
cpnncipa~
to (J)irector, Indian Institute of Cfiemica{ CBiofogy
Cafcutta :Nationa{ :Medica{ Co{fege and Xospita{
(C:N:MC), 1(o{kg.ta for a{fowing me to use tlie fa60ratory faciCities during tfie course of my study. I
e~end my
immense tfiank! to (J)r. CJ0.05) of different fish species and the locations. The proportion of antagonistic bacteria in the total viable counts (TVC) of Catla catla. Labeo rohita. Cirrhinus mrigala. Cyprinus carpio and Clarias batrachus were
observed to be in the range of 1.04% - 6.15%, 1.48% - 7.35%, 2.33% - 16.67%, 1.81 % and 1.16% - 1.18% respectively (Table 4.2).
4.3. Identification of antagonistic bacterial strains A total of 41 antagonistic bacterial strains were isolated out of which 82.93% (n=34) were Gram positive and 17.07% (n=7) were Gram negative (Fig. 3.1). Among the Gram positive bacteria isolated from fish skin mucus, Gram positive rods were the dominant group (Table 4.3; Fig.3.2). The antagonistic bacteria isolated in Catla catla include Bacillus spp. (n=3), Corynebacterium spp. (n=2), Pseudomonas spp. (n=2), Lactobacillus sp. (n= 1) and Enterobacter sp. (n= 1). In Labeo rohita. Gram positive
cocci such as Staphylococcus spp. (n=5), Micrococcus spp. (n=3) were the dominant. The
other
antagonistic
strains
identified
were
Pseudomonas
spp.
(n=2),
Corynebacterium sp. (n=I), Bacillus sp. (n=l) and Enterobacter intermedius (n=I).
All the antagonistic bacteria of Cirrhinus mrigala were Gram positive belonging to the genus Bacillus (n=4), Arthrobacter (n=2) and Lactobacillus (n=I). Likewise, the antagonistic bacterial strains isolated from Cyprinus carpio include Arthrobacter spp. (n=2), Bacillus sp. (n=l) and Lactobacillus sp. (n=1). Eight antagonistic bacterial strains comprising Bacillus spp. (n=2), Lactobacillus spp. (n=2), Staphylococcus spp. (n=2), COIynebacterium sp. (n=l) and Pseudomonas sp. (n=l) were isolated from Clarias batrachus.
In Catla catla, among the isolated antagonistic strains 6 were Gram positive rods and 3 were Gram negative rods. In Labeo rohita, out of the 13 isolated antagonistic strains 2, 8 and 3 strains were Gram positive rods, Gram positive cocci and Gram negative rods, respectively. All the antagonistic bacterial strains isolated from Cirrhinus mrigala (n=7) and Cyprinus carpio (n=4) were Gram positive rods. Out of the 8 antagonistic strains isolated from Clarias batrachus. 5 were Gram positive rods, 2 were Gram positive cocci and the rest was a Gram negative rod.
60
Results
4.4. Screening and selection of potential antagonistic bacterium
4.4.1 In-vitro inhibitory activity of antagonistic bacteria against test bacterial pathogens The results of in-vitro inhibitory activity of the antagonistic (producer) organisms against the test organisms by cross streaking method are presented in Table 4.4. Producer organisms, that exhibited antagonistic activity against the indicator organism, were tested for their ability to inhibit the growth of various Gram positive and Gram negative test organisms. Eighteen opportunistic human and fish pathogenic bacteria belonging to the genera Aeromonas, Bacillus, Edwardsiella, Escherichia, Klebsiella, Pseudomonas, Salmonella, Staphylococcus, Streptococcus and Vibrio were inhibited in-vitro at varying levels by the potential Gram positive antagonistic strains such as Lactobacillus spp. (P32 and P8I), Bacillus spp. (P33, P35, P39, P55, P9I, P92, PIOI, PI34, P136, P201 and P25I), Micrococcus spp. (P52 and P6I), Staphylococcus spp. (P65 and P66), Arthrobacter spp. (P131, P161and PI8I) and also by Gram negative antagonistic strains such as Pseudomonas spp. (P37, P38 and P72) and Enterobacter sp. (P36). The inhibitory activity of the producer strains was observed by the growth inhibition of test organisms in the confluent area. Pseudomonas aeruginosa P72 effected the maximum inhibition against the test strains and the inhibitory zone size exhibited by the test strains ranged from 2 to 31 mm. The inhibitory zone sizes ranging from 1 to 35 mm were exhibited by the test strains against Arthrobacter sp. P 13l. Depending on the wide spectrum of inhibitory activity el ucidated by Pseudomonas aeruginosa P72 and Arthrobacter sp. P 131, they were considered to be the most potent antagonistic strains. Further, 9 out of 18 pathogenic test strains, viz., Pseudomonas aeruginosa CNMC isolated from patient suffering from acute pneumoniae; Escherichia coli CNMC from human stool; Edwardsiella tarda SDDL isolated from diseased fish in Chennai; Staphylococcus aureus ATCC 12598 FCRI; Vibrio cholerae FCRI; Enterohaemorrhagic Escherichia coli FCRI; Salmonella Typhi FCRI; Aeromonas sp. AAH and Staphylococcus aureus FPT have shown zone size >10 mm against Pseudomonas aeruginosa P72 (Table 4.3). Arthrobacter sp. P131 was effective against only seven pathogenic test strains, which showed zone size> I 0 mm. Moreover, the test strains produced zone size ranging from 1 to 37 mm against Bacillus sp. P35 and among them only nine test strains produced zone size >5 mm. 61
Results
Likewise, the test strains produced zone size ranging from 2 to 35 mm against Bacillus sp. P39 and among them only six test strains produced zone size >5 mm. Similarly, the test strains exhibited zone size varying from 1 to 35 mm, 2 to 29 mm, 2.5 to 7.5mm respectively against Bacillus sp. P55, Bacillus sp. P92, Bacillus sp. PI 01 and Bacillus sp. P251. Inhibition zone size produced by the test strains against Arthrobacter sp. P 161 and P 181 were in the range of 1 - 8 mm. On the basis of the broad spectrum of inhibitory activity, Pseudomonas aeruginosa P72 was, therefore, chosen as a potent producer strain for further characterization.
4.5. Antibacterial spectrum of Pseudomonas aeruginosa P72 against human and fish pathogens A potent antagonistic bacterial strain, Pseudomonas aeruginosa P72 was chosen
among the 41 antagonistic bacterial strains on the basis of the broad spectrum of activity and zone of inhibition (>5 mm) produced against the 18 test strains by cross streaking method are presented in Tables 4.4, 4.5, Plate 1(4,5) and Plate 2(6). In parallel streaking method, it was observed that the growth of Pseudomonas aeruginosa CNMC; Escherichia coli CNMC, Edwardsiella tarda SDDL; Staphylococcus aureus ATCC 12598 Vibrio cholerae FCRI; Enterohaemorrhagic Escherichia coli FCRI; Salmonella Typhi FCRI; Aeromonas sp. AAH; Pseudomonas sp. FPT; Bacillus pumilus FPT; Bacillus amyloliquefaciens FPT, Staphylococcus aureus S2, FPT; Escherichia coli FPT; p-haemolytic Streptococcus sp. AAH; p- haemolytic Aeromonas hydrophila AAH was totally inhibited; whereas Klebsiella pneumoniae CNMC and Staphylococcus aureus CNMC were only slightly inhibited by Pseudomonas aeruginosa P72 strain and represented in Table 4.5, Plate 3(15) and Plate 4(16,17,18,19,20,21 and 22).
4.6. Biochemical characterisation of the antagonistic and indicator bacterial strains The results of the biochemical characterisation of the potent antagonistic bacterial strain Pseudomonas aeruginosa P72 and indicator bacterial strain Brevibacillus brevis 123 strains and those of standard reactions of respective bacteria are presented in Table 4.6.
62
Results
4.7. Characteristics of potential antagonistic bacterium Pseudomonas aeruginosa P72 4.7.1. Qualitative biofilm formation on glass surface by Christensen test Pseudomonas aeruginosa P72 produced a very strong visible biofilm on test tube walls, which recorded a score of 4 for the amount of biofilm produced and presented in Table 4.7; Plate 8 (30A and 30B). 4.7.2. Haemolytic assay The antagonistic strain Pseudomonas aeruginosa P72 was a non-haemolytic or gamma haemolytic (y-haemolysis) as the sheep blood agar under and around the colony remain unchanged after inoculating the bacterium and presented in Table 4.7; Plate 8 (3 1). 4.7.3. Enzymatic activity The proteinase, amylase, lipase and phospholipase activities of Pseudomonas aeruginosa P72 as determined on gelatin agar, starch agar, tributyrin agar, egg yolk emulsion agar, respectively were recorded to be positive and presented in Table 4.7 and Plate 8 (32). 4.7.4. Growth of Pseudomonas aeruginosa P72 at different temperatures The growth was measured by observing the turbidity of the broth inoculated with the bacterium. Dense growth of Pseudomonas aeruginosa P72 was observed when incubated at 22°C, 32°C and 45°C. The bacterium was unable to grow at 4°C and 65°C (Table 4.7). 4.7.5. Growth of Pseudomonas aeruginosa P72 in nutrient broth with different pH The turbidity of the nutrient broth was measured to ascertain the growth of the bacterium in different pH range (Table 4.7). Profuse growth of Pseudomonas aeruginosa P72 was observed in the test tubes containing nutrient broth with pH 5.2, 6.0, 6.8, 7.6, 8.4, 9.2 and 10.0. Feeble growth was observed at pH 4.4. The bacterium was unable to grow at pH 3.6 and below.
63
Results
4.7.6. Growth of Pseudomonas aeruginosa P72 in 6.5% salt concentration The Pseudomonas aeruginosa P72 was proved to be a salt tolerant bacterium as it was able to grow in nutrient broth with 6.5% sodium chloride concentration (Table 4.7). 4.7.7. Bile tolerance of Pseudomonas aeruginosa P72 The Pseudomonas aeruginosa P72 was proved to be a bile tolerant bacterium as it was able to grow profusely on McConkey agar plate containing 5% bile salt (Table 4.7). 4.7.8. Pathogenicity of Pseudomonas aeruginosa P72 and LDso value The pathogenicity of Pseudomonas aeruginosa P72 was tested on Labeo rohita fingerlings and the LDso value was calculated as >5.65x 10 10 cful fish (Table 4.13). 4.8. Antibiotic sensitivity testing by agar-disc diffusion assay A total of 18 test bacterial strains of the genera Klebsiella, Pseudomonas,
Staphylococcus, Escherichia, Edwardsiella, Vibrio, Salmonella, Aeromonas, Bacillus, Streptococcus from diseased human and fish samples (Table 4.8) and Pseudomonas aeruginosa P72 (Table 4.7) were subjected to antibiogram against six antibiotics, viz., oxytetracycline, chloramphenicol, gentamycin, nitrofurantoin, ciprofloxacin and cotrimoxazole. The human and fish bacterial pathogens were sensitive to gentamycin followed by ciprofloxacin and chloramphenicol (Table 4.8 and Fig. 4). Out of the three fish pathogens
P - haemolytic Aeromonas hydrophila-HS 3 AAH
was resistant to co-
trimoxazole and Edwardsiella tarda SDDL was resistant to oxytetracycline and cotrimoxazole; whereas in case of human pathogens 8 were found resistant to cotrimoxazole, 7 were found to be resistant to oxytetracycline, 4 were resistant to nitrofurantoin and one each was resistant to chloramphenicol and ciprofloxacin. The antagonistic
bacterium
Pseudomonas
a eruginosa
P72
was
sensitive
to
chloramphenicol, ciprofloxacin and gentamycin and resistant to co-trimoxazole, nitrofurantoin and oxytetracycline (Table 4.7).
64
Results
4.9. In-vitro inhibitory activity of whole cell and spent medium ethyl acetate
extract of Pseudomonas aeruginosa P72 by agar disc diffusion assay The crude extracts of the antibacterial substance from the whole cells and spent medium were extracted separately with ethyl acetate and both of them displayed inhibitory activity against the test organisms (Table 4.9). Out of the total 6 Gram positive strains, the growth of Bacillus amyloliquefaciens FPT was maximum inhibited by the crude extracts of Pseudomonas aeruginosa P72 followed by
Staphylococcus aureus FPTI, Staphylococcus aureus FPT2, Staphylococcus aureus ATCC 12598, Staphylococcus aureus CNMC and Bacillus pumilus FPT. The crude extracts of Pseudomonas aeruginosa P72 from spent media effected larger zone of inhibition against the Gram negative bacterial pathogens such as Pseudomonas sp. FPT, Pseudomonas aeruginosa CNMC, Vibrio cholerae FCRI, Salmonella Typhi FCRI and Escherichia coli CNMC and the zone size ranged from 6 to 12 mm.
Pseudomonas aeruginosa P72 proved it's inhibitory activity against Gram positive fish pathogens such as ~ -haemolytic Streptococcus sp. AAH, isolated from diseased Nile Tilapia, Oreochromis niloticus and Gram negative fish pathogens such as
Aeromonas hydrophila HS 3 AAH, Aeromonas sp. AAH and Edwardsiella tarda SDDL. It effected the largest zone of inhibition (10 - 16 mm) against Aeromonas sp. AAH. In all the cases, the ethyl acetate extract of the spent medium produced the larger zone of inhibition as compared to the whole cell extract (Table 4.9, Plate 2 (7, 8, 9 and 10) and Plate 3 (II, 12, 13 and 14».
4.10. Molecular and phylogenetic characterization of Pseudomonas aeruginosa P72
The identity of the antagonistic bacterial strain Pseudomonas aeruginosa P72 was further confirmed by molecular technique, 16S rONA analysis. The assay involved isolation, amplification and sequencing of the gene coding for the 16S rONA, i.e., the 1.5 kbp 16S rONA from bacterium.
4.10.1. 16S rRNA analysis and nucJeotide sequences accession number
In ).5% agarose gel electrophoresis peR amplification. depicted Ii, F'
19.6.
1500 bp band (Fig. 5) was obtained by sequence of Ps('udo mon The chromatogram " , as aenlginosQ F o furward ARP72 is 65 and reverse s
Consensus
equences of
Results
Pseudomonas aeruginosa FARP72 was presented in Figs. 7.1 and 7.2. BLAST analysis (Fig. 8) of the sequence data and phylogenetic tree (Fig. 10) revealed that the strain Pseudomonas aeruginosa P72 showed closest similarity with two strains of
Pseudomonas aeruginosa (Accession no KC776528 and EU221384). Multiple sequence analysis were done and presented in Fig.9. Homogeneity of 16S rDNA gene sequence of Pseudomonas aeruginosa FARP72 to other Pseudomonas spp. is presented in Table 4.10. The 16S rONA sequences of isolate have been deposited in the National Center for Biotechnology Information (NCB!) GenBank database, USA under the accession number KC570343 and designated as Pseudomonas aeruginosa FARP72. Pair wise analysis of 16S rDNA gene of Pseudomonas aeruginosa FARP72 (Accession number KC570343) with Pseudomonas aeruginosa KA VKOI (Accession number KC 119195) is represented in Fig. 11.
4.10.2. Fatty Acid Methyl Esters (FAME) Analysis Cellular fatty acids analysis by using gas chromatography of fatty acid methyl esters (FAME) have been used as the second finger print for the identification of bacteria and to interpret the results with the results of 16S rONA analysis and nucleotide sequencing. Table 4.11 showed peaks corresponding to the fatty acids identified through FAME analysis of bacterial sample FARP72. The FAME analysis identified strain FARP72 as Pseudomonas aeruginosa with similarity index 0.700 and the bacterial compositions were also reported (Fig. 12 and Table 4.11). 4.11. Scanning electron microscopic observation of Pseudomonas aeruginosa P72
Pseudomonas aeruginosa strain P72 colonies were examined by scanning electron microscope and the results were represented in Plate 9 (34, 35). Under 8000X magnification, the clear rod like morphology of the bacteria was observed in the fixed material.
4.12. Isolation
d an partial characterization of ethyl acetate Pseudomonas aeruginosa P72 ·40 J 2. J. Identification and ch
'. aracteruatJOn of th
extract of
e metabolites From TlC resuJrs fr ' . , actIons Were "olnb' fc f ... rncd 5.65xI01O cells/ fish (Table 4.13). Samples from the affected parts of the body of freshly dead fish yielded translucent and colourless colonies initially on Pseudomonas isolation agar (Hi-Media, Mumbai, India) plates with 24 h of incubation at 30±2°C, and up on subsequent incubation, it yielded diffusible brown pigmentation surrounding the colonies. The isolated bacterium was identified as Pseudomonas aeruginosa by standard biochemical tests. 4.13.2 Challenge test with pathogenic p-haemolytic Aeromonas hydrophila NI0P Clinical signs, behaviour and mortalities of the challenged fish were recorded after injection. Lethargy, abnormal behaviour, wandering around comers and erect swimming Were the first s· . b . observed . 1 . - Igns 0 served In challenged Labeo rohita. Wit lin , h Pt' . os -Ifljectio All I~~'drophila N p . n. 10 fish died where n h I '
The first mortality
were II 1'- aemOIJt .1
[email protected]"JJdC/lerOmOn'lIC' Out of J0 cell Iii UIJ celiS/fish . . challenged fi h . S Ish in 24 h f i oc WIthm 24 h IS dIed in 0 n U1ated· . All the fi the stOck . l11)ectlOn' WIth 6 Ish in h. 1110c 1 ' cellS/fish in. .J 7.,( Jas cel/Slr h t Ird stock, . Uated with 6 OCUlated stOck IS died "'ilh' ContaIning 10 f .37X109 recorded h\'o ID 48 h POst_.. Ish, which mOrtalities It lll]ection wcr~ a er 3 rd d and 6 37 ay N . x'107 Whereas 9
10
post-'· .
b
.
0
men"j' Cl Jtles
It,
""ere
Results
noticed in stocks which were used as positive control and negative control. After 15 days observation, 100% fish mortalities were observed in the stocks inoculated with 8
6.37x10 10 cells/fish, 6.37x109 cells/fish and 6.37x10 cells/fish. Twenty percent (20%) mortalities were recorded in the stock where fish were inoculated with ~-haemolytic
Aeromonas hydrophila N lOP @ 6.37x 107 cells/fish. The lethal dose at which 50% of the experimental population die (LDso) was calculated on the basis of mortalities in all test concentrations over a period of 15 days (Annexure 3.2). The LDso value of ~ 8
haemolytic Aeromonas hydrophila N lOP was calculated as 2.37x 10 cells/fish (Table 4.13).
4.13.3. Pathogenicity test by cohabitation challenge The results of the mortalities in Labeo rohita challenged with Aeromonas
hydrophila N lOP and Pseudomonas aeruginosa P72 at different concentrations in cohabitation challenge are represented in Fig. 14. Clinical signs, behaviour and mortalities of the fish were recorded daily up to 30 days after challenge and cohabitation. Lethargy, abnormal behaviour, wandering around comers and vertical swimming were the first signs observed in challenged Labeo rohita. About 75% of the fish treated with Aeromonas hydrophila N lOP suspension containing 1x 105 cells/ml died and only 30% mortalities were observed in fish challenged with Pseudomonas 7
aeruginosa P72 at a level of 1x 10 cells/ml. Significant difference existed in the mortalities of L.
rohita challenged with Aeromonas hydrophila N lOP and
Pseudomonas aeruginosa P72 (t = 0.002; df: 2; P
