Abundance, community structure and diversity of nitrifying bacterial enrichments from low and high saline brackishwater environments.

The study reports diversity in nitrifying microbial enrichments from low (0·5-5‰) and high (18-35‰) saline ecosystems. Microbial community profiling of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) enrichments was analysed by sequencing 16S rRNA and was processed using Mothur pipeline. The α-diversity indices showed the richness of nitrifying bacterial consortia from the high saline environment and were clustering based on the source of the sample. AOB and NOB enrichments from both the environments showed diverse lineages of phyla distributed in both groups with 38 and 34 phyla from low saline and 53 and 40 phyla in high saline sources, respectively. At class level, α- and γ-proteobacteria were found to be more dominant in both the enrichments. AOBs and NOBs in enrichments from low saline environments were dominated by Nitrosomonadaceae, Gallionellaceae (Nitrotoga sp.) and Ectothiorhodospiraceae and Nitrospira, respectively. Though Chromatiaceae were present in both AOB and NOB enrichments, Nitrosoglobus and Nitrosococcus dominated the AOBs while NOBs were dominated by uncultured genera, whereas Rhizobiales were found in both the enrichments. AOBs and NOBs in enrichments from high saline environments were dominated by Nitrospira-like AOBs, Nitrosomonas and Nitrosococcus genera, whereas ammonia-oxidizing archaea (AOA) group included Nitrosopumilus and Nitrososphaera genera comprising and Nitrospirae, respectively. The majority of the genera obtained in both the salinities were found to be either uncultured or unclassified groups. Results of the study suggest that the AOB and NOB consortia have unique and diverse microbes in each of the enrichments, capable of functioning in aquaculture systems practised at different salinities (0-60 ppt).

Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP) for the visual detection of Enterocytozoon hepatopenaei (EHP).

Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.

Characterization of quorum sensing system in Clostridium chauvoei.

Clostridium chauvoei causes fatal black quarter infection in cattle and buffaloes. The quorum sensing (QS) system, a bacterial cell to cell communication process, of the pathogen was characterized in the current study. The results indicated that C. chauvoei lacked luxS (autoinducer-2) based quorum sensing as detected by the sensor strain Vibrio harveyi BB170. This was supported by absence of luxS gene in C. chauvoei genome. However, the genomic analysis indicated the presence of agrBD system in all three genomes of C. chauvoei available at the NCBI database. The AgrD, which synthesizes QS messenger auto-inducing peptide, was a 44 amino acid protein which shared 59% identity and 75% similarity with AgrD of C. perfringens strain 13 and 56% identity (20% coverage) with Staphylococcus aureus N315. The functional cysteine amino acid was conserved in all the strains. The genomic organisation further suggests the presence of diguanylate cyclase, a gene responsible for synthesis of secondary messenger cyclic di-GMP, at 3' immediate downstream of agrD gene. The real time expression analysis for agrD gene indicated that expression was better at 37 °C (1.9-3.7 fold increase) compared to a higher temperature of 40 °C. However, stable expression was observed at different growth stages (log and early stationary phase) with 0.8-1.4 fold changes in expression pattern. The results indicate the presence of a constitutively expressed agrBD quorum sensing system in C. chauvoei.

Comparative efficacy of double-stranded RNAs targeting WSSV structural and nonstructural genes in controlling viral multiplication in Penaeus monodon.

RNA interference (RNAi) is a potential strategy to control shrimp viral diseases, including the white spot disease caused by White Spot Syndrome Virus (WSSV). Selection of genes for targeting is an important criterion. We have compared the efficacy of dsRNAs targeting structural (vp28 and vp281) and nonstructural genes (rr1 and dnapol) of WSSV in controlling viral multiplication in Penaeus monodon. Targeting the rr1 and vp28 genes provided better protection (93.3% and 90% survival respectively) compared to vp281 and dnapol in experimentally infected shrimp. Temporal transcriptional analysis of the corresponding genes and PCR-based diagnosis of WSSV in samples collected at different time points in the experiment supported this observation, thereby indicating that targeting a combination of rr1 and vp28 would be effective in limiting WSSV multiplication.

Delineating virulence of Vibrio campbellii: a predominant luminescent bacterial pathogen in Indian shrimp hatcheries.

Luminescent vibriosis is a major bacterial disease in shrimp hatcheries and causes up to 100% mortality in larval stages of penaeid shrimps. We investigated the virulence factors and genetic identity of 29 luminescent Vibrio isolates from Indian shrimp hatcheries and farms, which were earlier presumed as Vibrio harveyi. Haemolysin gene-based species-specific multiplex PCR and phylogenetic analysis of rpoD and toxR identified all the isolates as V. campbellii. The gene-specific PCR revealed the presence of virulence markers involved in quorum sensing (luxM, luxS, cqsA), motility (flaA, lafA), toxin (hly, chiA, serine protease, metalloprotease), and virulence regulators (toxR, luxR) in all the isolates. The deduced amino acid sequence analysis of virulence regulator ToxR suggested four variants, namely A123Q150 (AQ; 18.9%), P123Q150 (PQ; 54.1%), A123P150 (AP; 21.6%), and P123P150 (PP; 5.4% isolates) based on amino acid at 123rd (proline or alanine) and 150th (glutamine or proline) positions. A significantly higher level of the quorum-sensing signal, autoinducer-2 (AI-2, p = 2.2e-12), and significantly reduced protease activity (p = 1.6e-07) were recorded in AP variant, whereas an inverse trend was noticed in the Q150 variants AQ and PQ. The pathogenicity study in Penaeus (Litopenaeus) vannamei juveniles revealed that all the isolates of AQ were highly pathogenic with Cox proportional hazard ratio 15.1 to 32.4 compared to P150 variants; PP (5.4 to 6.3) or AP (7.3 to 14). The correlation matrix suggested that protease, a metalloprotease, was positively correlated with pathogenicity (p > 0.05) and negatively correlated (p < 0.05) with AI-2 and AI-1. The syntenic organization of toxS-toxR-htpG operon in V. campbellii was found to be similar to pathogenic V. cholerae suggesting a similar regulatory role. The present study emphasizes that V. campbellii is a predominant pathogen in Indian shrimp hatcheries, and ToxR plays a significant role as a virulence regulator in the quorum sensing-protease pathway. Further, the study suggests that the presence of glutamine at 150th position (Q150) in ToxR is crucial for the pathogenicity of V. campbellii.

Phylogenetic Relationship Among Brackishwater Vibrio Species.

Vibriosis is regarded as an important disease of penaeid shrimps affecting larvae in hatcheries. Among the Vibrio species, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio furnissii, Vibrio campbellii, Vibrio harveyi, Vibrio alginolyticus, and Vibrio anguillarum are often associated with diseases in finfish and shellfish of brackishwater ecosystem. Accurate species differentiating methods for the organisms present in an ecosystem are required for precise classification of the species and to take steps for their management. Conventional methods like 16s rRNA phylogeny and multilocus sequence typing (MLST) have often failed to correctly identify Vibrio species. This has necessitated a comprehensive investigation on methodologies available to distinguish Vibrio species associated with brackishwater aquaculture system. To achieve this, 35 whole genomes belonging to 7 Vibrio species were subjected to phylogenetic analysis based on 16s rRNA gene, MLST genes, single-copy orthologous genes, and single-nucleotide polymorphisms. In addition, genome-based similarity indices like average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) were computed as confirmatory tests to verify the phylogenetic relations. There were some misclassifications occurred regarding phylogenetic relations based on 16s rRNA genes and MLST genes, while phylogeny with single-copy orthologous genes produced accurate species-level clustering. Study reveals that the species identification based on whole genome-based estimates or genome-wide variants are more precise than the ones done with single or subset of genes.

Involvement of Enterobacter cloacae in the mortality of the fish, Mugil cephalus.

AIMS: To identify the causative agent of the mortality in the fish, Mugil cephalus, in Muttukadu lagoon. METHODS AND RESULTS: An enteric bacterium from the kidneys of moribund fish M. cephalus, was isolated and identified as Enterobacter cloacae (MK). Mugil cephalus was experimentally infected by this isolate and was re-isolated from the kidneys of the moribund fish. Enterobacter cloacae isolates from the lagoon water (MW1, MW2 and reference strain ATCC 13047) and the reference strain were not able to induce similar pathogenesis. The putative factor imparting pathogenicity to the MK isolate was identified as a cationic molecule, which migrated towards the cathode on agarose gel electrophoresis. CONCLUSIONS: The Ent. cloacae (MK) isolate harbouring a cationic factor was the causative agent for the mortality of M. cephalus, found in Muttukadu lagoon. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that human enteric bacteria MK which is considered as nonpathogenic to fish, may become pathogenic to fish when it harbours this cationic factor. This cationic factor is found to be pathogenic to the fish M. cephalus leading to mortality. It was also found to be pathogenic to mice. Therefore, the shuttling of Ent. cloacae, harbouring cationic factor, between human and fish may be of human health importance.

Loose shell syndrome of farmed Penaeus monodon in India is caused by a filterable agent.

Loose shell syndrome (LSS) of farmed black tiger shrimp Penaeus monodon has been reported from Indian shrimp farms since 1998 and is recognized as a major disease problem causing significant economic loss to the shrimp aquaculture sector. Unlike the rapid mortalities associated with viral pathogens such as white spot syndrome virus and yellow head virus, progression of LSS is gradual, leading to low-level progressive mortalities. The signs of LSS include a flaccid spongy abdomen due to muscular dystrophy, space between the exoskeleton and muscle, and a shrunken hepatopancreas. The feed conversion efficiency is reduced, and shrimp have poor meat quality, caused by impairment of the hepatopancreatic functions such as digestion and absorption as evidenced by the atrophy of the hepatopancreas. Histopathological investigations on LSS-affected shrimp showed shrinkage of extensor and flexor muscles with occasional hemocytic infiltration. The hepatopancreas showed inflammation of hepatopancreatic tubules with enlargement of intertubular spaces, hemocytic infiltration, and low levels of lipid reserves in the R cells. In advanced stages of LSS, many tubules were in highly necrotic condition with a sloughed epithelium, reflecting the dysfunction of the digestive gland. LSS could be induced in healthy tiger shrimp by challenge studies using membrane-filtered LSS-affected shrimp tissues, suggesting involvement of a filterable infectious agent.

Anti-oxidant status in embryonic, post-hatch and larval stages of Asian seabass (Lates calcarifer).

The concentrations of anti-oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and selenium-dependent glutathione peroxidase (SeGPx), and low molecular weight free-radical scavengers such as reduced glutathione (GSH) and ascorbic acid (vitamin C) were evaluated during the period from gastrulation (GS) to 25 days post-hatch (dph) in the larvae of Asian Seabass, Lates calcarifer. Oxidative damage due to lipid peroxidation (LPO) was also assessed, by evaluation of the formation of malondialdehyde (MDA). All the three anti-oxidant enzymes, SOD, CAT and GPx, showed high activities during gastrulation, suggesting an increased metabolic rate during the period of embryonic development. Though the SOD activity apparently decreased progressively during 3-20 dph of larval development, the difference was not significant. CAT showed high activity during gastrulation and remained constant up to 3 dph, suggesting an increased need to metabolise hydrogen peroxide (H2O2) and organic peroxides. In contrast, SeGPx activity increased progressively from 5 dph to 25 dph during larval development, indicating an increased need to detoxify lipid peroxides. This is evident from the observation of increased lipid peroxidation from 10 dph to 25 dph during larval development. GSH levels were low at gastrulation, indicating increased metabolic rate and formation of lipid radicals during this period, corresponding to the decrease in the level of ascorbic acid, which is consumed for regeneration of GSH.

Draft Genome Sequence of Vibrio parahaemolyticus Strain VP14, Isolated from a Penaeus vannamei Culture Farm.

Here, we report the draft genome sequence of an isolate of Vibrio parahaemolyticus, VP14, recovered from the gut of Penaeus vannamei shrimp farmed in southern India. The genome of VP14 comprised 5,224,046 bp with a GC content of 45.3% and contained 5,326 genes, including 4,972 coding sequences.

Draft Genome Sequence of the Luminescent Strain Vibrio campbellii LB102, Isolated from a Black Tiger Shrimp (Penaeus monodon) Broodstock Rearing System.

We report here the genome sequence of Vibrio campbellii LB102, isolated from the broodstock rearing system of a shrimp hatchery in India. Sequence analysis revealed the presence of effector toxins of the type III (YopT, sharing 39% identity with Yersinia pestis) and type VI (VgrG-3 and hemolysin coregulated protein of V. cholerae) secretion systems.

Development of antiviral gene therapy for Monodon baculovirus using dsRNA loaded chitosan-dextran sulfate nanocapsule delivery system in Penaeus monodon post-larvae.

In the present study, a suitable carrier system was developed for the delivery of dsRNA into Penaeus monodon (P. monodon) post larvae to silence the Monodon baculovirus (MBV) structural gene of p74. The carrier system was developed by layer by layer adsorption of oppositely charged chitosan-dextran sulfate, on charged silica nanoparticles. The silica template was removedto produce multilayered hollow nanocapsules (CS-DS) that were utilized for dsRNA loading at an alkaline pH. The capsule's surface was modified by conjugating with shrimp feed for enhanced cellular uptake. In vivo cellular uptake of CS-DS/FITC loaded nanocapsules conjugated with feed was studied after oral administration into post-larvae. The results revealed that the encapsulated FITC was effectively delivered and exhibited a sustained release into the cytoplasm of shrimp post-larvae. The MBV challenge study for structural gene p74was conducted after 3-25 days of post infection (dpi) with respective CS-DS/dsRNA coated with feed. The results showed a significant survival rate of 86.63% and effective gene silencing in P. monodon. Our findings indicated that the delivery of dsRNA using shrimp feed coatedCS-DSnanocapsules could be a novel approach to prevent viral infections in shrimp.

Polychaete worms--a vector for white spot syndrome virus (WSSV).

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.

Optimization of Culture Conditions for Mass Production of the Probiotics Pseudomonas MCCB 102 and 103 Antagonistic to Pathogenic Vibrios in Aquaculture.

Rapid growth of shrimp farming industry is affected by the recurrence of diverse diseases, among which vibriosis is predominant. Eco-friendly disease management strategy by the application of antagonistic probiotics is widely accepted. In the present study, culture conditions of antagonistic probiotics, Pseudomonas MCCB 102 and 103, were optimized to enhance their biomass production and antagonistic activity against the shrimp pathogen V. harveyi MCCB 111. Primarily, one-dimensional screening was carried out to fix the optimum range of sodium chloride concentration, pH and temperature. The second step optimization was done using a full-factorial central composite design of response surface methodology. As per the model, 12.9 g/L sodium chloride and pH 6.5 for Pseudomonas MCCB 102, and 5 g/L sodium chloride and pH 7 for Pseudomonas MCCB 103 were found to be ideal to maximize antagonistic activity. However, optimum temperature was the same (25 °C) for both isolates. Finally, the models were experimentally validated for enhanced biomass production and antagonistic activity. The optima for biomass and antagonistic activity were more or less the same, suggesting the possible influence of biomass on antagonistic activity.

Tangential flow ultrafiltration for detection of white spot syndrome virus (WSSV) in shrimp pond water.

Water represents the most important component in the white spot syndrome virus (WSSV) transmission pathway in aquaculture, yet there is very little information. Detection of viruses in water is a challenge, since their counts will often be too low to be detected by available methods such as polymerase chain reaction (PCR). In order to overcome this difficulty, viruses in water have to be concentrated from large volumes of water prior to detection. In this study, a total of 19 water samples from aquaculture ecosystem comprising 3 creeks, 10 shrimp culture ponds, 3 shrimp broodstock tanks and 2 larval rearing tanks of shrimp hatcheries and a sample from a hatchery effluent treatment tank were subjected to concentration of viruses by ultrafiltration (UF) using tangential flow filtration (TFF). Twenty to 100l of water from these sources was concentrated to a final volume of 100mL (200-1000 fold). The efficiency of recovery of WSSV by TFF ranged from 7.5 to 89.61%. WSSV could be successfully detected by PCR in the viral concentrates obtained from water samples of three shrimp culture ponds, one each of the shrimp broodstock tank, larval rearing tank, and the shrimp hatchery effluent treatment tank with WSSV copy numbers ranging from 6 to 157mL(-1) by quantitative real time PCR. The ultrafiltration virus concentration technique enables efficient detection of shrimp viral pathogens in water from aquaculture facilities. It could be used as an important tool to understand the efficacy of biosecurity protocols adopted in the aquaculture facility and to carry out epidemiological investigations of aquatic viral pathogens.

Production of siderophores & effect of iron restriction on the protein profiles of Aeromonas species isolated from water & patients suffering from acute diarrhoeal disease.

Among 48 strains of Aeromonas species, 21 isolates from patients suffering from acute diarrhoea and 27 from metropolitan water samples grown under iron-restricted conditions, 45 strains produced siderophores. Forty one of the 46 strains tested produced siderophores on chrome azurol S (CAS) agar, while 43 isolates did so when the culture supernatants of the bacterial isolates grown in minimal medium were assayed with chrome azurol S assay solution. The whole cell protein profiles of A. hydrophila strains grown under iron restricted conditions expressed new proteins that were not detected in those cultured in iron rich conditions. Five high molecular weight proteins ranging from 70 to 96 kDa were distinctly absent in cultures grown in the presence of iron, indicating their role in iron acquisition by the aeromonads.

Biochemical characteristics & secretory activity of Aeromonas species isolated from children with gastroenteritis in Chennai.

An attempt was made to delineate the phenotypic markers for the detection of enterotoxigenic strains of Aeromonas. Eighteen Aeromonas species comprising one isolate of A. hydrophila, six isolates of A. sobria and 11 isolates of A. caviae were obtained from 379 children suffering from acute diarrhoea in Chennai. Nine of these isolates inclusive of three A. sobria and six A. caviae were found to produce secretory response in vitro in the rabbit intestinal mucosa mounted in the Ussing chambers as revealed by significant increases in the short circuit current. Eleven strains hydrolysed aesculin, 8 fermented arabinose, 6 produced acetyl methyl carbinol, 14 produced lysine decarboxylase, 3 fermented salicin, 9 produced beta-haemolysin, 9 produced CAMP-like factor and only two isolates took up congo red dye. None of these phenotypic traits were found to correlate with the in vitro secretory activity.

Incidence & enteropathogenicity of Aeromonas spp in children suffering from acute diarrhoea in Chennai.

A total of 200 stool samples from children below 10 yr suffering from diarrhoea were screened for enteric pathogens with special interest on Aeromonas. Aeromonas spp were isolated from 6.5 per cent of the patients, comprising 4 per cent A. hydrophila, 2 per cent A. sobria and 0.5 per cent A. caviae. Among the 13 isolates obtained, 10 isolates produced enterotoxin in ligated rabbit ileal loops, and 11 produced cytotoxin in HEp 2 cells. Many of the Aeromonas isolates exhibited resistance to commonly used antibiotics such as trimethoprim, sulphdiazine, chloramphenicol and tetracycline. None of the stool samples obtained from 52 age matched control children yielded Aeromonas species. Four isolates of Salmonella typhi, 7 of S. paratyphi A, 6 of Shigella flexneri, 4 of Sh. dysenteriae and 3 isolates of Vibrio cholerae (Ogawa) were also recovered during the study. Among the samples analyzed, one from a 7 yr old female patient, had A. hydrophila with S. paratyphi A. The results of this study indicate that drug resistant enteropathogenic Aeromonas is also an important etiological agent of childhood diarrhoea in Chennai.

Occurrence of haemolytic & cytotoxic Aeromonas species in domestic water supplies in Chennai.

A study on the occurrence of Aeromonas species in the domestic water supplies in Chennai showed that as much as 37.9 per cent of the water samples analyzed from various sources harbored Aeromonas spp. Majority of the isolates belonged to Aeromonas sobria (13.7%), A. caviae (11.6%) and A. hydrophila (9.5%). Among the 37 metropolitan water samples analyzed, 11 samples yielded Aeromonas spp. inclusive of three isolates of A. hydrophila, four of A. sobria and two isolates each of A. caviae and A. jandaei. From a total of 28 bore well water samples analyzed, Aeromonas spp. were recovered from 15 samples, comprising five isolates of A. hydrophila, six of A. sobria and four isolates of A. caviae. Aeromonas spp. inclusive of one isolate of A. hydrophila, five of A. caviae, three of A. sobria and one isolate of A. veronii were isolated from 10 of the 30 water packets of various commercial brands sold in Chennai. Of a total of 36 isolates obtained, 32 (89%) produced beta-haemolysin with the titres ranging from 2-32 and 20 isolates (56%) were cytotoxic to vero cell monolayers. All the Aeromonas isolates were resistant to ampicillin and polymyxin B. All A. hydrophila and A. caviae isolates were also resistant to cephalothin and erythromycin and 83.3 per cent of Aeromonas isolates were resistant to erythromycin. Aeromonads resistant to tetracycline, gentamycin, co-trimoxazole and nalidixic acid appear to be emerging. The study revealed that Aeromonas spp. occur in the potable and domestic water supplies and even in the chlorinated water supplies in Chennai city, which are potentially enteropathogenic and hence may be hazardous to public health. In view of these findings drinking and domestic water quality standards need to be re-evaluated.

Typing of Aeromonas isolates from children with diarrhoea & water samples by randomly amplified polymorphic DNA polymerase chain reaction & whole cell protein fingerprinting.

BACKGROUND & OBJECTIVES: Aeromonas spp. are water-borne organisms, often associated with childhood diarrhoea. The present study was conducted to examine the epidemiological relationship among the Aeromonas spp. isolated from water and children with acute diarrhoea in Chennai. METHODS: Thirty six Aeromonas isolates inclusive of 16 from children with diarrhoea, 15 from domestic water samples and 5 reference strains were studied by randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Twenty eight Aeromonas isolates, 15 from children with diarrhoea, 10 from domestic water samples and three reference strains were analysed by SDS-PAGE for their whole cell protein profiles. RESULTS: The 36 Aeromonas isolates examined by RAPD-PCR generated RAPD fingerprints with majority of the bands ranging from about 250 to 2800 bp. The RAPD fingerprints did not correspond with the phenospecies and varied greatly among the strains within the phenospecies. Cluster analysis revealed two major groups at 75 per cent hierarchical level, comprising 18 Aeromonas isolates, mainly recovered from domestic water samples, while the clinical isolates were scattered in different hierarchical levels in the dendrogram. The whole cell protein fingerprints examined by SDS-PAGE did not correspond with the phenospecies. Only four isolates of A. caviae were found to produce similar protein fingerprints allowing them to form a cluster at about 90 per cent hierarchical level, while the rest of the isolates were scattered at various hierarchical levels in the dendrogram. INTERPRETATION & CONCLUSIONS: In the present study, RAPD fingerprinting was found to be useful in distinguishing Aeromonas isolates recovered from clinical and domestic water supplies. However, RAPD-PCR could not distinguish the phenospecies of the genus Aeromonas. Whole cell protein fingerprinting and cluster analysis could neither differentiate isolates from clinical and domestic water sources nor the phenospecies of the genus Aeromonas.