Vibrio harveyi as a causative agent of mass mortalities of megalopa in the seed production of swimming crab Portunus trituberculatus

Outbreaks of mass mortalities among cultured megalopa of swimming crab (Portunus trituberculatus) occurred in a commercial hatchery during the spring of 2012 in Jiangsu province, P. R. China. Dominant bacteria were isolated and characterized by biochemical reactions, enzyme production, and sequencing analysis of the 16S rRNA, gyrB (DNA gyrase B subunit) genes, and two strains (DY1 and DY2) were selected as representative strains for virulence tests by immersion. The results showed that the characteristics of identified strains consist with Vibrio harveyi; the 16S rRNA and gyrB genes of the tested strains exhibited high similarity with V. harveyi; and the phylogenetic trees constructed using the neighbor-joining method based on 16S rRNA and gyrB genes. Two strains (DY1 and DY2) clustered with the V. harveyi and were supported by a higher bootstrap value, and two strains (DY1 and DY2) were found lethal to the healthy megalopa and juvenile crab. LD ₅₀ of DY1 and DY2 to megalopa and juvenile crab were 2.4 × 10⁶, 3.0 × 10⁶, 1.9 × 10⁶ and 2.5 × 10⁶ CFU/ml, respectively. The results confirmed that the diseased megalopa were infected by V. harveyi. In addition, we analyzed the tested strains (DY1 and DY2) by PCR for the presence of virulence genes that were known in V. harveyi, and the results showed that five virulence genes (luxR, toxR, vhhA, vhhB and pap6) were detected by a specific PCR assay, further supporting the assignment of isolates to V. harveyi and its pathogenicity. To our knowledge, this is the first report of V. harveyi as pathogenic bacteria of megalopa of swimming crab.     

Multilocus sequence analysis of Vibrionaceae isolated from farmed amberjack and the development of a multiplex PCR assay for the detection of pathogenic species

Since 2011, high mortality rates and symptoms consistent with vibriosis have been observed in farmed amberjack (Seriola dumerili) in Japan. To identify 41 strains isolated from diseased amberjack, a multilocus sequence analysis using nine concatenated genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S rRNA) was conducted. Twenty‐seven strains were identified as Vibrio harveyi, suggesting an epidemic of V. harveyi infection in amberjack farms. Other strains were identified as Vibrio anguillarum, Vibrio owensii and Photobacterium damselae subsp. damselae. To develop an efficient diagnostic method for vibriosis in amberjack, a multiplex PCR system was developed to identify V. anguillarum, V. harveyi and P. damselae subsp. damselae. The method successfully discriminated between these three bacterial species, with amplification products of 350 bp for V. anguillarum, 545 bp for V. harveyi and 887 bp for P. damselae subsp. damselae and can be used for diagnosis in aquaculture farms.     

Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene

Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 10² CFU mL^?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 10³ CFU g^?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.     

Microbiological characteristics of Vibrio scophthalmi isolates from diseased olive flounder Paralichthys olivaceus

In 2005, massive mortality occurred in olive flounder Paralichthys olivaceus farms in Korea, and five isolates were collected from diseased fish. In this study, microbiological and pathogenic characteristics of these isolates were studied. The isolates gave negative results in lysine and ornithine decarboxylase, ortho-nitrophenyl-??-galactoside, and citrate tests, and positive results in urease, esculinase, and nitrate reduction tests. The isolates produced acid from adipate, fructose, d-glucose, and maltose, and gave positive results in alkaline phosphatase, esterase lipase, leucine arylamidase, and naphthol-AS-BI-phosphohydrolase. According to genetic analysis, 16S rRNA gene sequences showed 98?C100?% identity with both Vibrio scophthalmi and V. ichthyoenteri. The dnaJ gene sequences presented a higher identity with V. scophthalmi than with V. ichthyoenteri. Thus, the isolates were identified as V. scophthalmi. Pathogenicity of the five isolates in olive flounder was different and LD₅₀ values were from 10⁶ to 10⁸?CFU/g fish. Symptoms included darkening of skin, hemorrhage of liver and intestine, ascites, and distended abdomen. Histopathological changes included hemopoiesis dilatation and epithelial hyaline droplets in kidney, macrophage infiltration and ellipsoid dilatation in spleen, vascular dilatation, submucosal edema, and serosa inflammation of intestine. Cumulative mortality was 25?% for fish singly infected by isolate A19008 or Streptococcus parauberis, and increased to 87.5?% in super-infection group with these two pathogens.     

Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene

Vibrio anguillarum is a major pathogenic bacterium that causes vibriosis and septicemia in fish and shellfish. In this study, we identified the groESL genes, which encode bacterial chaperonins, from V. anguillarum. The groE gene cluster consisted of a 291-bp groES gene, a 69-bp intergenic spacer region, and a 1,635-bp groEL gene order. Sequence analysis with the groESL gene of Vibrio species exhibited that the groEL gene was more species-specific and suitable than the groES gene for V. anguillarum detection. Owing to the difficulty in distinguishing V. anguillarum from the closely related V. ordalii, we compared the sequences of groEL from V. anguillarum and the groEL homolog hsp60 from V. ordalii, in order to design a primer set based on a region dissimilar between the two. PCR with the groEL primer set produced a clear 195-bp amplicon in six serotypes of V. anguillarum, whereas 23 Vibrio species of 39 samples, including V. ordalii, and 10 species of enteric bacteria gave no bands. PCR using the groEL primers also amplified a unique product from V. anguillarum-infected flounder and oyster tissues. These results demonstrate that the groEL-target PCR assay is a sensitive and species-specific tool for the detection of V. anguillarum infection.     

Occurrence of Vibrio cholerae and Vibrio parahaemolyticus among milkfish farms in Zanzibar

ABSTRACT

Fishing is among the main economic activities of the people of Zanzibar. Few fish dealers are transforming this sector into mariculture. Among the farmed fish is milkfish. Diseases are among the limiting factors in the development of the mariculture industry. Among other zoonotic diseases, vibriosis is caused by bacteria from the genus Vibrio. This study aimed to establish the occurrence of Vibrio cholerae and Vibrio parahaemolyticus among milkfish farms in Zanzibar. A total of 380 milkfish were sampled. Swabs were collected from gills, intestine, and kidney of each sampled milkfish. Preliminary identification of V. cholerae and V. parahaemolyticus was done by biochemical tests. PCR was run on 16S rRNA, outer membrane protein W, and collagenase genes to confirm Vibrio species, V. cholerae, and V. parahaemolyticus respectively. Almost one-third (32.1%) of all sampled milkfish were found to contain targeted Vibrio; 18% and 29.5% of the sampled milkfish were positive for V. cholerae and V. parahaemolyticus respectively.     

Detection of V. harveyi in shrimp postlarvae and hatchery tank water by the Most Probable Number technique with PCR

V. harveyi is the cause of serious disease in the shrimp industry in Thailand during cultivation. In this study, the gyrB gene of V. harveyi NICA, isolated from shrimp in Thailand, was sequenced. A pair of specific primers (A2B3) was designed that allowed amplification of a 363 bp gene fragment of V. harveyi. No cross reaction was detected in 17 other Vibrio species tested except for V. carchariae which is a synonym for V. harveyi. The possibility of using A2B3 for confirmation and enumeration of V. harveyi by PCR was demonstrated. Of 40 possible V. harveyi strains isolated from seafood on the basis of their growth on TCBS plates and biochemical reactions, 36 gave a reaction with the specific primers. The primers could detect V. harveyi at a level of as few as 15 cells/ml. The Most Probable Number (MPN) technique was applied to enumerate V. harveyi. We have demonstrated that when PCR was applied directly to the enrichment broth of shrimp artificially inoculated with V. harveyi, the MPN value was no different from the MPN value obtained using the standard technique with selective agar. This technique was employed to enumerate V. harveyi in postlarvae and hatchery tank water. V. harveyi were detected in 18 out of 21 postlarval samples and in 14 out of 21 tank water samples. The numbers of V. harveyi detected in postlarvae and water were 150-1.1 × 10⁸/g postlarvae and 7-4.6 × 10⁴/ml of water samples, respectively. Screening of postlarvae to reduce the high risk of V. harveyi contamination in cultivation ponds is suggested as a measure to prevent the catastrophic losses caused by V. harveyi disease.     

A novel multiplex PCR method for detecting virulent strains of Vibrio alginolyticus

The bacterial strains obtained from various origins were tested with the novel primers targeting the collagenase gene, ompK gene and toxR gene to establish a multiplex polymerase chain reaction (PCR) method. These primers successfully recognized all virulent strains of Vibrio alginolyticus, but the avirulent strains were not recognized by the multiplex PCR because of lack of the collagenase and toxR genes. In a 50 μL multiplex PCR mixture, the lowest detection limit is 8.8 × 10² cells of virulent strains of V. alginolyticus. The multiplex PCR method was successfully developed to identify virulent strains of V. alginolyticus, and provides a rapid, sensitive, specific and reliable technology for diagnosing virulent strains of V. alginolyticus. Therefore, the novel multiplex PCR in the present paper can be useful for any laboratory working with vibriosis detection of aquatic animals.     

Detection of Vibrio cholerae and Vibrio vulnificus by duplex PCR specific to the groEL gene

Vibrio cholerae and V. vulnificus are of major concern due to their effect on public health throughout the world. It is therefore imperative to identify a gene and method that are suitable for the accurate species-specific detection of these two species. A duplex polymerase chain reaction (PCR) assay was developed using two sets of primers targeting the groEL gene for the accurate simultaneous detection of V. cholerae and V. vulnificus. The nucleotide sequence of the groEL gene was compared with the sequences of other Vibrio and non-Vibrio species. The specificity of two primer sets for duplex PCR was checked using 24 Vibrio and 8 non-Vibrio species. The primer sets were found to be specific for these two species and could detect both of the target bacterial species without any ambiguity, even when comparing closely related species. For both species, the detection limit was 100 pg of purified genomic DNA. The duplex PCR showed high specificity and sensitivity for each species and was sufficient for the detection of V. cholerae and V. vulnificus from artificially infected shellfish tissue, flounder, and even inoculated seawater. This method is simple and cost-effective, and can be utilized for the simultaneous detection of both species, thus representing an effective tool for both epidemiologist and ecologist.     

Ingestion of food pellets containing Escherichia coli overexpressing the heat‐shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection

Feeding aquatic animals with bacterial encapsulated heat‐shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK‐DnaJ‐GrpE, the prokaryotic equivalents of Hsp70‐Hsp40‐Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g^?1 pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.     

Aerobic Bacterial Flora of Common Carp (Cyprinus carpio L.) Cultured in Earthen Ponds in Saudi Arabia

ABSTRACT

Quantitative and qualitative estimation of bacterial flora present in pond water, sediments, gills, and intestine of healthy common carp Cyprinus carpio cultured in Saudi Arabia were performed and identified to species level where possible. Mean total viable bacterial counts in pond water ranged from 1.2?±?2.9?×?10⁴ to 2.5?±?3.5?×?10⁵ cfu/mL; in sediments, 9.3?±?2.1?×?10⁷ to 2.7?±?3.5?×?10⁹ cfu/g; in gills filaments, 4.3?±?2.9?×?10⁶ to 1.6?±?3.9?×?10⁷ cfu/g; and in intestine, 8.7?±?4.1?×?10⁹ to 5.4?±?3.2?×?10¹⁰ cfu/g. Gram-negative rod-shaped bacteria dominated (76%) the populations. In total, 12 bacterial genera and 15 species were identified. Pond water and sediment bacteria had the reflection on bacterial composition of gills and intestine of carp. Intestinal bacteria showed more diversification in contrast to gill bacteria. Aeromonas hydrophila, Shewanella putrefaciens, Vibrio cholerae, Corynebacterium urealyticum, Vibrio vulnificus, Vibrio sp., and Staphylococcus sp. were the common bacteria in all the populations. In pond water and carp intestine, A. hydrophila, S. putrefaciens, V. cholerae, and C. urealyticum were the most dominant bacteria (prevalence ≥ 10%) where pond sediments and the carp gills experienced with more one dominant bacterium V. vulnificus. Only the A. hydrophila covered one fourth (25%) of the total bacterial populations.     

Occurrence of Listonella anguillarum in seed production environments of Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The present study was undertaken to investigate the distribution of Listonella anguillarum in the rearing water, fish and diets (rotifers) of Japanese flounder (Paralichthys olivaceus). A total of 793 isolates were obtained from the seed production environment of Japanese flounder and 175 out of them were identified as L. anguillarum by biochemical characterization, polymerase chain reaction (PCR) detection for VAH1 haemolysin gene and phylogenetic analysis of 16S ribosomal deoxyribonucleic acids (rDNA) sequences. These results strongly suggested that L. anguillarum is rapidly and accurately identified by the combination of incubation on thiosulphate–citrate–bile salt–sucrose agar at 35°C overnight and PCR detection for the VAH1 haemolysin gene. All flounder specimens and all rotifer samples harboured L. anguillarum at high densities of 6.9 × 10³–6.3 × 10⁵ colony forming units (CFU) g^?1 and 1.5 × 10⁴–2.3 × 10⁶ CFU g^?1, respectively, while as low as 5.0 × 10⁰–2.0 × 10¹ CFU mL^?1 of L. aguillarum were detected in only two of 11 seawater samples, even though no vibriosis occurred in larval and juvenile flounder of tanks. This fact strongly suggests that L. anguillarum is an inhabitant in the seed production environments of Japanese flounder.     

Pathogenicity comparison of high- and low-virulence strains of Vibrio scophthalmi in olive flounder Paralichthys olivaceus

Vibrio scophthalmi, a bacterial pathogen of olive flounder Paralichthys olivaceus, exhibits strain-dependent virulence. No information is available on the comparative pathogenicity of different strains of V. scophthalmi toward olive flounder. In this study, high- and low-virulence strains (HVS and LVS, respectively) were compared in terms of their pathogenic characteristics, including adhesion and survival, superoxide dismutase (SOD) activity, and extracellular products (ECP) of bacterial cells. The cell-mediated defense of macrophages from olive flounder against V. scophthalmi infection in vitro was also investigated. The results demonstrated that the SOD activity of the HVS was higher than that of the LVS. The number of viable cells of the HVS in serum increased by two log units after 18 h, whereas that of the LVS decreased. The number of cells of the HVS in skin mucus increased significantly while that of the LVS remained constant. The LD₅₀ values of the HVS and LVS ECP toward olive flounder were 10.14 and 15.99 μg protein/g fish, respectively. The ECP were positive for naphthol-AS-BI-phosphohydrolase, lipase, gelatinase, and leucine arylamidase. The extracellular O₂ ^? overflow and intracellular O₂ ^? concentration of macrophages induced by the HVS were lower than those induced by the LVS. Significantly more nitric oxide was produced by the HVS than by the LVS.     

Characterization of Functional furVs as a Biomarker for Detection of Aquatic Pathogen Vibrio splendidus

Vibrio splendidus is an important opportunistic pathogen that can infect a broad range of aquatic animals including Apostichopus japonicus, leading to skin ulceration syndrome. In this study, fur gene of V. splendidus (fur_Vs), the gene encoding ferric uptake regulator (Fur) of V. splendidus, was cloned and characterized. fur_Vs possessed the signature ammonia acids of Vibrio sp., and its mRNA level was regulated by iron and coelomic fluid of A. japonicus. While the amino acid sequence of the fur_Vs showed high homology with the compared Fur proteins of other Vibrio sp., the nucleotide sequence of fur_Vs differed greatly from that of the other fur genes from Vibrio sp., based on which a pair of specific primers VSFurRTF3 and VSFurRTR3, was designed. A distinct DNA band with a length of 223 bp can be amplified from V. splendidus with great specificity. The detection limits for V. splendidus in seawater, homogenized tissues of epidermis, muscles, and coelomic fluid from A. japonicus, was 1 × 10², 1 × 10⁵, 1 × 10³, and 1 × 10² CFU/mL, respectively. Moreover, the detection limit was as low as 8 × 10^?3 pg/μL when using DNA of V. splendidus as a template. Our results provide a simple and rapid polymerase chain reaction method for the sensitive and specific detection of V. splendidus.     

Oxidative stress and antioxidant enzymes activities in the African catfish,Clarias gariepinus,experimentally challenged with Escherichia coli and Vibrio fischeri

The impacts of bacterial infection on cultivated fish species, African catfish, were investigated using oxidative stress biomarkers [lipid peroxidation (LPO) and protein carbonylation] and the activities of important antioxidant/detoxifying enzymes [catalase and glutathione S-transferase (GST)]. Fish were inoculated via oral gavage with one of the following treatments: 1 × 10⁵ CFU/ml of Escherichia coli (EC1), 2 × 10⁵ CFU/ml of E. coli (EC2), 1 × 10⁵ CFU/ml of Vibrio fischeri (V1), 2 × 10⁵ CFU/ml of V. fischeri (V2), gavaged with distilled water and not gavaged. Fish were maintained in the laboratory for 7 days after the bacterial inoculation, and the levels of LPO, protein carbonylation, GST, and catalase activities were determined in the muscle, gills, and liver of fish. Fish inoculated with bacteria (either E. coli or V. fischeri) had a significant higher levels of tissue LPO, protein carbonylation, and GST activities in a tissue-specific pattern (liver > muscle > gills). This appears to be related with the levels of bacterial inoculation, with effects more pronounced in fish inoculated with either EC2 or V2. The catalase activity did not differ significantly between the inoculated and fish that were not inoculated. The results of this study indicate that bacterial inoculation could result in oxidative stress in fish, and liver has a higher rate of oxidative stress per mg tissue compared to the gills and the muscle.     

Antibacterial effect of Lactococcus lactis subsp. lactis on Artemia franciscana nauplii and Dicentrarchus labrax larvae against the fish pathogen Vibrio anguillarum

The antibacterial effect of Lactococcus lactis subsp. lactis against the fish pathogen Vibrio anguillarum was determined in Artemia franciscana nauplii and in European sea bass (Dicentrarchus labrax) larvae fed with ten pulses of nauplii enriched with L. lactis. The evaluation of the bactericidal activity of the extracellular products of L. lactis in vitro showed inhibition of growth of Vibrio (Listonella) anguillarum and Photobacterium damselae subsp. piscicida. The incorporation of L. lactis in Artemia nauplii did not affect their survival and offered protection in a challenge with V. anguillarum, significantly increasing LD₅₀. The administration of Artemia nauplii enriched with L. lactis for 48 h to sea bass larvae for five consecutive days had no adverse effect on survival of fish. In an in vivo challenge test with V. anguillarum using sea bass larvae, fish treated with nauplii enriched with the probiotic L. lactis showed significantly (P < 0.001) increased survival rates of 81 % compared with the untreated group of challenged fish (24 %). Our results indicate that L. lactis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

Skin‐injured Zebrafish,Danio rerio,are more Susceptible to Vibrio anguillarum Infection

Vibrio anguillarum, which is part of normal microflora on fish, is the causative agent of vibriosis in aquaculture. It is speculated that V. anguillarum does not affect the host in most situations, but can cause a severe disease once the host is compromised. In the study reported herein, skin‐injured and intestine‐injured zebrafish, Danio rerio, were established as a model to mimic the natural infection caused by V. anguillarum when fish suffered an injury to a mucosal surface. Our results showed the lethal dose to 50% of the population (LD₅₀) of skin‐injured zebrafish was 6.8 × 10³ colony‐forming unit (CFU)/mL, which was much lower than intestine‐injured zebrafish (1.9 × 10⁶ CFU/mL) or non‐injured zebrafish (5.5 × 10⁶ CFU/mL). With the quantitative polymerase chain reaction and immunohistochemical analysis, we found that V. anguillarum proliferated rapidly in the skin and muscle after the bacteria entered into the host via the skin injury. The bacteria were subsequently transported to the immune organs and then caused a systemic infection in the fish. However, mortality of skin‐injured zebrafish significantly decreased if the fish were allowed to heal. These results indicate that minimizing injury to the mucosal surfaces of fish, especially the skin, will reduce infections caused by V. anguillarum.     

Effects of dietary probiotic on the growth performance,non‐specific immunity and disease resistance of cobia,Rachycentron canadum

The present study was performed to investigate the effects of a commercially available probiotic product (compound probiotic) containing Bacillus subtilis 7.0 × 10⁹ CFU g^?1, Bacillus licheniformis 3.0 × 10⁹ CFU g^?1, Lactobacillus spp. 5.0 × 10⁸ CFU g^?1 and Arthrobacter spp. 1.0 × 10⁸ CFU g^?1 on the growth performance, non‐specific immunity and protection against Vibrio harveyi infection in cobia (Rachycentron canadum). Fish were fed diets containing six graded levels of compound probiotic (0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 g kg^?1) for 8 weeks. The results showed that the survival rate ranged from 81.1% to 84.4% with no significant difference among dietary treatments (P > 0.05) after feeding experiment. Dietary compound probiotic significantly increased the specific growth rate (SGR), serum lysozyme, alternative complement pathway (ACP) activity, phagocytosis percentage (PP) and respiratory burst activity of head‐kidney macrophages of cobia. Moreover, feeding of supplemented diets containing compound probiotic resulted in significantly lower mortality against the pathogens Vibrio harveyi compared with the control group. To elevate the growth and immune resistance ability of cobia, an optimal dose of dietary compound probiotic administration determined by second‐order polynomial regression analysis was 3.3 g kg^?1, on the basis of the SGR and mortality after challenge with V. harveyi.     

Effect of Customized Probiotics on the Physiological and Immunological Responses of Juvenile Western King Prawns (Penaeus latisulcatus Kishinouye, 1896) Challenged with Vibrio harveyi

Juvenile western king prawn P. latisulcatus were fed 10⁵ colony-forming units (CFU)/mL of two probiotics Pseudomonas synxantha and P. aeruginosa for 28 days. P. latisulcatus were then challenged with V. harveyi at 0 (control), 10³, 10⁵, and 10⁷ CFU/mL. During the seven days of challenge, disease resistance of the probiotic-fed prawns was compared with that of prawns not fed probiotics. The immunological responses of the prawns did not improve during the challenge period in terms of total haemocyte count, hyalinocyte, semi-ganulocyte, granulocyte, clotting time, bacteraemia, and intestinal bacterial load. Overall, when prawns were challenged with V. harveyi, the LT₅₀ values got shorter as V. harveyi concentration increased. LT₅₀ values of prawns fed probiotics were significantly longer (P < 0.05) than those not fed probiotics. At a V. harveyi concentration of 10³ CFU/mL, the 100% survival of the prawns fed probiotics was three times more likely than those of the prawns not fed probiotics.