Screening and evaluation of probiotics as a biocontrol agent against pathogenic Vibrios in marine aquaculture

AIMS: The present work aims at finding potential probionts from marine sources as a biocontrol agent against pathogenic Vibrio species in shrimp larval culture. METHODS AND RESULTS: A total of 109 bacterial strains were isolated from seawater, sediment and marine fish-gut samples, and were screened for their antagonistic activity against Vibrio species. Three strains (Q, Q1 and M) isolated from the marine sediment were found antagonistic against Vibrio strains. Based on 16S ribosomal DNA gene sequence analysis, the strain Q was identified as Paenibacillus spp. (EF012164); Q1 as Bacillus cereus (DQ915582); and the M as Paenibacillus polymyxa (DQ915580). Further, the two bacterial species, Paenibacillus spp. and B. cereus were challenged separately at two different concentrations of 10(4) and 10(5) CFU ml(-1) for probiotic activity in the postlarvae of Penaeus monodon against pathogenic Vibrio harveyi and Vibrio spp. CONCLUSIONS: The present study identified the probiotic activity of Paenibacillus spp., B. cereus and Pa. polymyxa against the pathogenic Vibrios in the postlarvae of P. monodon. SIGNIFICANCE AND IMPACT OF THE STUDY: In vivo study reveals that the marine bacterial species can be used as probionts against pathogenic Vibrios in shrimp larval culture practices.     

Inhibitory effects of Bacillus probionts on growth and toxin production of Vibrio harveyi pathogens of shrimp

Aim:  To investigate the effects of Bacillus subtilis , Bacillus licheniformis and Bacillus megaterium in terms of toxin and growth of pathogenic Vibrio harveyi .
Methods and Results:  Three Bacillus probionts were isolated from probiotic BZT aquaculture and identified using a 16S rDNA sequence. Growth inhibition assay showed that supernatants from the 24-h culture of three Bacillus species were able to inhibit the growth of V. harveyi (LMG 4044); B. subtilis was the most effective based on the well diffusion method. Results of a liquid culture model showed that B. subtilis was also widely effective in inhibiting three strains of V. harveyi (isolated from Thailand, the Philippines and LMG 4044), and that both B. licheniformis and B. megaterium inhibit the growth of V. harveyi isolated from the Philippines. Moreover, a haemolytic activity assay demonstrated that V. harveyi (IFO 15634) was significantly decreased by the addition of B. licheniformis or B. megaterium supernatant.
Conclusions:  Bacillus subtilis inhibited Vibrio growth, and both B. licheniformis and B. megaterium suppressed haemolytic activity in Vibrio .
Significance and Impact of the Study:  The cell-free supernatants produced by Bacillus probionts inhibit Vibrio disease, and Bacillus probionts might have an influence on Vibrio cell-to-cell communications.     

Effect of probiotics on bacterial population and health status of shrimp in culture pond ecosystem

The artificially manufactured probiotics having beneficial bacteria, Bacillus spp. was applied regularly in a modified extensive shrimp (Penaeus monodon) culture pond, located on the bank of Vellar estuary, Parangipettai. The populations of total heterotrophic bacteria (THB), beneficial bacteria (Bacillus spp.) and pathogenic bacteria (vibrios) were monitored in water and sediment of the pond. The results were compared with a control pond, situated in the same location having same water spread area, stocking density, species managed with same technologies and optimum environmental parameters in which no probiotic was applied. The populations of THB and Bacillus spp. in the experimental pond increased and the vibrios decreased after each application of probiotics. But the result of the control pond showed an increasing trend of the populations of THB, Bacillus spp. and vibrios towards days of culture. The control pond had lower levels of THB and Bacillus spp. and higher levels of vibrios than the probiotic applied (experimental) pond. Also the probiotics maintained optimum transparency and low organic load in the experimental pond as compared to control. In general, water and sediment had almost equal number of Bacillus spp. and vibrios, but sediment had higher THB load than water. The applications of probiotics lesser pathogenic vibrios and enhance beneficial bacilli in the culture leading to improved water quality, promoted growth and survival rates and increased the health status of the shrimp without stress and disease outbreaks. Thus the application of probiotics could lead to disease-free and profitable shrimp culture operations which will be helpful for shrimp farmers as most of them are now-a-days severely affected by microbial diseases.     

Enhanced cellular immunity in shrimp (Litopenaeus vannamei) after 'vaccination'

It has long been viewed that invertebrates rely exclusively upon a wide variety of innate mechanisms for protection from disease and parasite invasion and lack any specific acquired immune mechanisms comparable to those of vertebrates. Recent findings, however, suggest certain invertebrates may be able to mount some form of specific immunity, termed 'specific immune priming', although the mechanism of this is not fully understood (see Textbox S1). In our initial experiments, either formalin-inactivated Vibrio harveyi or sterile saline were injected into the main body cavity (haemocoel) of juvenile shrimp (Litopenaeus vannamei). Haemocytes (blood cells) from V. harveyi-injected shrimp were collected 7 days later and incubated with a 1:1 mix of V. harveyi and an unrelated gram positive bacterium, Bacillus subtilis. Haemocytes from 'vaccinated' shrimp showed elevated levels of phagocytosis of V. harveyi, but not B. subtilis, compared with those from saline-injected (non-immunised) animals. The increased phagocytic activity was characterised by a significant increase in the percentage of phagocytic cells. When shrimp were injected with B. subtilis rather than vibrio, there was no significant increase in the phagocytic activity of haemocytes from these animals in comparison to the non-immunised (saline injected) controls. Whole haemolymph (blood) from either 'immunised' or non-immunised' shrimp was shown to display innate humoral antibacterial activity against V. harveyi that was absent against B. subtilis. However, there was no difference in the potency of antibacterial activity between V. harveyi-injected shrimp and control (saline injected) animals showing that 'vaccination' has no effect on this component of the shrimp's immune system. These results imply that the cellular immune system of shrimp, particularly phagocytosis, is capable of a degree of specificity and shows the phenomenon of 'immune priming' reported by other workers. However, in agreement with other studies, this phenomenon is not universal to all potential pathogens.     

Improvement in the growth performance of white shrimp, Litopenaeus vannamei, by a protease-producing probiotic, Bacillus subtilis E20, from natto

Aims:  To isolate and identify a benefic bacterium, Bacillus subtilis E20, from natto (fermented soybeans), and incorporate it into shrimp feed to promote shrimp growth performance.
Methods and Results:  A protease-producing bacterium, E20, isolated from natto was identified as B. subtilis by an API 50 CHB kit and the 16S rDNA sequence. B. subtilis E20 was able to grow at a broad range of temperatures (10–50°C), pH values (5–10), and NaCl levels (0–9%). The best culture conditions for B. subtilis E20 to produce the protease were 40°C, a pH of 6–8 and 0% NaCl. No shrimp died after being injected with B. subtilis E20 [up to 10⁹ colony-forming units (CFU) per shrimp]. Bacillus subtilis E20 was incorporated in diets at the levels of 0 (control), 10⁶, 10⁷, and 10⁸ CFU kg⁻¹ for shrimp grow-out culture, and results showed that after feeding on B. subtilis E20-containing diets (10⁸ CFU kg⁻¹ of diet), shrimp had excellent growth performance and production compared to the control because protease activities in the digestive tract were improved by B. subtilis E20.
Conclusions:  Bacillus subtilis E20 isolated from natto is a great protease producer and is able to improve shrimp growth performance through increasing the digestibility of food.
Significance and Impact of the Study:  Results suggest that B. subtilis E20 is a potential candidate for use as a probiotic to improve shrimp growth performance, and consequently reduce feed costs.     

Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei

We studied the effect of two probiotic Bacillus subtilis strains on the growth performance, digestive enzyme activity, immune gene expression and disease resistance of juvenile white shrimp (Litopenaeus vannamei). A mixture of two probiotic strains, L10 and G1 in equal proportions, was administered at two different doses 10(5) (BM5) and 10(8)?(BM8)?CFU?g(-1) feed to shrimp for eight weeks. In comparison to untreated control group, final weight, weight gain and digestive enzyme activity were significantly greater in shrimp fed BM5 and BM8 diets. Significant differences for specific growth rate (SGR) and survival were recorded in shrimp fed BM8 diet as compared with the control; however, no significant differences were recorded for food conversion ratio (FCR) among all the experimental groups. Eight weeks after the start of the feeding period, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 63.3%, whereas cumulative mortality of the shrimp that had been given probiotics was 20.0% with BM8 and 33.3% with BM5. Subsequently, real-time PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was significantly up-regulated (P?相似文献   

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum)

AIM: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. METHODS AND RESULTS: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 10(7) cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and alpha1-antiprotease and lymphocyte populations. CONCLUSIONS: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.     

Enhanced immune defences in Pacific white shrimp (Litopenaeus vannamei) post-exposure to a vibrio vaccine

This study was conducted to determine if exposure of shrimp, Litopenaeus vannamei, to a commercial anti-vibrio vaccine caused changes in antibacterial and cellular (phagocytosis) defences. Shrimp post-larvae were administered either Vibromax™ vaccine or a blank preparation. Whole body homogenates were prepared before (day 0), during (day 10) and after (day 20) vaccination and incubated with a selection of pathogenic vibrios. Homogenate from day 0 animals showed natural antibacterial activity towards Vibrioanguillarum which was significantly enhanced for bacteria-exposed shrimp at 10 days post-challenge. This effect of the vaccine was short-term in its duration. No antibacterial activity was observed in day 0 shrimp homogenate against Vibrio alginolyticus but it was significantly enhanced for both vaccinated and blank-vaccinated shrimp by day 10. No natural or inducible antibacterial activity was observed against Vibrio harveyi at 0, 10 or 20 days post-challenge. To determine if prior exposure of shrimp to inactivated vibrios results in elevated hemocyte phagocytic activity, juveniles were injected with either a mixture of formalin-inactivated vibrios or saline. Hemocyte monolayers made from these shrimp were overlaid with a 1:1 mix of Bacillus subtilis and these vibrios. Hemocytes from vibrio-exposed animals showed elevated levels of internalised vibrios compared with those from the saline injected group. These studies show selectively enhanced cellular defences of shrimp following ‘vaccination’.     

Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish

AIMS: To isolate, select and evaluate Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. METHODS AND RESULTS: Natural isolates obtained from mud sediment and Cyprinus carpio were purified and assessed in vitro for efficacy based on the inhibition of growth of pathogenic Aeromonas hydrophila and the decrease in concentrations of ammonium, nitrite, nitrate and phosphate ions. Based on suitability to predefined characteristics, the isolates B001, B002 and B003 were selected and evaluated in vitro in the presence of Aer. hydrophila and in a preliminary in vivo trial with C. carpio. The inhibitory effect on pathogen growth and the decrease in concentrations of waste ions was demonstrated. Based on 16S RNA sequence homology, the isolates were identified as Bacillus subtilis, Bacillus cereus and Bacillus licheniformis, respectively. Isolate B002 did not contain the anthrax virulence plasmids pOX1, pOX2 or the B. cereus enterotoxin. CONCLUSIONS: Selected isolates effected synergistic reduction in pathogen load and the concentrations of waste ions in vitro and in vivo and are safe for use in ornamental aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: A new approach for assessment of biological agents was demonstrated and has yielded putative isolates for development into aquaculture products.     

Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple

AIM: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea. METHODS AND RESULTS: GA1 was first tested for its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance. The potential of strain GA1 to reduce post-harvest infection caused by B. cinerea was tested on apples by treating artificially wounded fruits with endospore suspensions. Strain GA1 was very effective at reducing disease incidence during the first 5 days following pathogen inoculation and a 80% protection level was maintained over the next 10 days. Treatment of fruits with an extract of GA1 culture supernatant also exerted a strong preventive effect on the development of grey mould. Further analysis of this extract revealed that strain GA1 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families. A strong evidence for the involvement of such compounds in disease reduction arose from the recovery of fengycins from protected fruit sites colonized by bacterial cells. CONCLUSIONS: The results presented here demonstrate that, despite unfavourable pH, B. subtilis endospores inoculated on apple pulp can readily germinate allowing significant cell populations to establish and efficient in vivo synthesis of lipopeptides which could be related to grey mould reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: This work enables for the first time to correlate the strong protective effect of a particular B. subtilis strain against grey mould with in situ production of fengycins in infected sites of apple fruits.     

N-acyl homoserine lactone-degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures

Three bacterial enrichment cultures (ECs) were isolated from the digestive tract of Pacific white shrimp Penaeus vannamei, by growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL) molecules. The ECs, characterized by denaturing gradient gel electrophoresis analysis and subsequent rRNA sequencing, degraded AHL molecules in the degradation assays. Apparently, the resting cells of the ECs also degraded one of the three types of quorum-sensing signal molecules produced by Vibrio harveyi in vitro [i.e. harveyi autoinducer 1 (HAI-1)]. The most efficient AHL-degrading ECs, EC5, was tested in Brachionus experiments. EC5 degraded the V. harveyi HAI-1 autoinducer in vivo, neutralizing the negative effect of V. harveyi autoinducer 2 (AI-2) mutant, in which only the HAI-1- and CAI-1-mediated components of the quorum-sensing system are functional on the growth of Brachionus. This suggests that EC5 interferes with HAI-1-regulated metabolism in V. harveyi. These AHL-degrading ECs need to be tested in other aquatic systems for their probiotic properties, preferably in combination with specific AI-2-degrading bacteria.     

Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds

AIMS: A bacterial strain producing antifungal compounds active against the plant pathogenic fungi Fusarium, Rhizoctonia and Sclerotinia has been characterized and shown to control Rhizoctonia root rot of soya bean. METHODS AND RESULTS: The metabolites excreted by Bacillus BNM 122 remained active after autoclaving, were resistant over a wide pH range and to hydrolytic enzymes. By (1)H-NMR and thin-layer chromatography analyses surfactin and iturin-like compounds were partially identified. Moreover, soya bean seeds bacterization with BNM 122 in a compost-based formulation was as effective controlling Rhizoctonia solani as pentachloronitrobenzene. According to its 16S rDNA sequence BNM 122 was closely related to Bacillus amyloliquefaciens and Bacillus subtilis. PCR analysis of the 16S-23S rRNA intergenic spacer region and repetitive sequence-based PCR (rep-PCR) genomic fingerprinting revealed a close genetic relationship to B. amyloliquefaciens. However, by physiological characterization using API tests, this strain resembled more B. subtilis. CONCLUSIONS: This is the first report describing the co-production of surfactin and iturin-like compounds by a putative strain of B. amyloliquefaciens. The synergistic effect of both lipopetides is a remarkable trait for a candidate biocontrol agent. SIGNIFICANCE AND IMPACT OF THE STUDY: This kind of research has relevance in order to minimize the use of synthetic fungicides and surfactants, contributing to the preservation of the environment.     

Study on the relationship of protease production and luminescence in Vibrio harveyi

AIMS: To demonstrate that Vibrio harveyi produces various types of toxins and how the production of those toxins is related with luminescence. METHODS AND RESULTS: Luminescence and toxicity of eight V. harveyi were evaluated. We demonstrated that all V. harveyi emitting luminescence were isolated from marine organisms and also showed that they were highly pathogenic when compared with culture collection V. harveyi based on cytotoxic assay test. On the contrary, V. harveyi isolated from shrimp farm showed no luminescence but showed high pathogenicity based on toxicity test. The effect of protease inhibitors on pathogenicity and luminescence was also investigated. We demonstrated that light emission of pathogenic V. harveyi remarkably decreased after addition of protease inhibitor. Furthermore, extracellular proteins from cell-free culture supernatant of luminescent and nonluminescent V. harveyi were compared using SDS-PAGE analysis. Results showed that there were differences in molecular weight and amount of proteins. CONCLUSIONS: Vibrio harveyi parasiting marine organisms have both luminescence and pathogenicity. Based on this study, luminescence and protease toxin activity in V. harveyi are related. Moreover, this paper clarified that V. harveyi produces various types of toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study demonstrated that V. harveyi produces two kinds of toxins, haemolysin and protease toxin. It may be clear roots of V. harveyi toxin.     

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis 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.     

Phenotypic and molecular typing of Vibrio harveyi isolates and their pathogenicity to tiger shrimp larvae

AIMS: The objective of the present study was to identify the biotype(s) and molecular type(s) of Vibrio harveyi associated with pathogenicity in tiger shrimp (Penaeus monodon) larvae. METHODS AND RESULTS: Five luminescent and four nonluminescent V. harveyi isolates were subjected to phenotyping and random amplified polymorphic DNA (RAPD) fingerprinting, and pathogenicity testing to P. monodon mysis. Four isolates induced 34-41% mortality of P. monodon mysis when challenged at the rate of 10(6) CFU ml(-1) within 60 h. Sucrose-fermenting biotypes of V. harveyi appeared to be associated with pathogenicity to larval shrimp. Higher temperature and salinity appeared to play a role on the onset of vibriosis and mortality in the challenged larval shrimp. Pathogenic isolates of V. harveyi could be demarcated as revealed by their clustering in the dendrogram constructed based on the RAPD fingerprints. CONCLUSIONS: Nonluminescent V. harveyi also appear to be important aetiological agents of vibriosis of shrimp larvae. Sucrose-fermenting biotypes are likely to be pathogenic. High temperature may trigger onset of vibriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Biotyping of V. harveyi isolates and looking for traits, such as ability to ferment sucrose may be helpful in identifying the pathogenic forms, and such approach requires to be investigated further with larger number of isolates.     

Characterization of Bacillus probiotics available for human use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Partial characterization of polyfermenticin SCD, a newly identified bacteriocin of Bacillus polyfermenticus

AIMS: To characterize polyfermenticin SCD, a newly identified bacteriocin of Bacillus polyfermenticus SCD. METHODS AND RESULTS: Bacillus polyfermenticus SCD was identified as a bacteriocin producer with a bactericidal activity against Bacillus subtilis IFO 12113. Polyfermenticin SCD, named tentatively as the bacteriocin produced by B. polyfermenticus SCD, showed a narrow spectrum of activity against Gram-positive and Gram-negative bacteria, a yeast and moulds. Production of polyfermenticin SCD in a 5 l jar fermenter followed typical kinetics of primary metabolite synthesis. The antibacterial activity of polyfermenticin SCD on sensitive indicator cells disappeared completely by treatment with proteinase K, which indicates its proteinaceous nature. Polyfermenticin SCD seemed to be very stable throughout the pH range of 2.0 to 9.0, and it was relatively heat labile compared with other bacteriocins. Direct detection of polyfermenticin SCD activity on SDS-PAGE suggested that it had an apparent molecular mass of about 14.3 kDa. CONCLUSIONS: Bacillus polyfermenticus SCD produced relatively heat-labile polyfermenticin SCD with a narrow spectrum of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus polyfermenticus SCD is a commercial probiotic which has been used for the treatment of long-term intestinal disorders. New findings on polyfermenticin SCD will be valuable in the evaluation of commercial probiotics. Polyfermenticin SCD can be used to control Bacillus spoilage organisms as a biological control agent.     

Vibrio parahaemolyticus and V. harveyi cause detachment of the epithelium from the midgut trunk of the penaeid shrimp Sicyonia ingentis

Shrimp Sicyonia ingentis were either injected with Vibrio parahaemolyticus (10(4) CFU) or V. harveyi (10(6) CFU) or immersed in ASW containing either species at 10(5) CFU ml(-1). These densities were shown in preliminary experiments to kill approximately half the population by 7 d. On Day 7, surviving shrimp were classified as either diseased or apparently healthy, and their midgut trunks (MGT) were examined by light and electron microscopy. All shrimp immersed in ASW containing either species of Vibrio showed detachment of the epithelium in the MGT. In shrimp injected with either species of Vibrio, epithelial detachment was common in diseased shrimp but not in apparently healthy animals. Experiments with live shrimp were supported by in vitro experiments where MGTs were removed, tied off at both ends, and injected with either pathogenic bacteria (V. parahaemolyticus or V. harveyi), non-pathogenic bacteria (Bacillus subtilis or Escherichia coli), or ASW. After 2 h incubations in ASW at 15 degrees C, the MGTs were processed and examined. The epithelium consistently detached from isolated MGTs injected with either species of Vibrio, but not from MGTs injected with non-pathogenic bacteria or ASW. Because the MGT epithelium secretes the peritrophic membrane, loss of the epithelium eliminates 2 layers that may restrict penetration of ingested pathogens into the shrimp body and may disrupt the osmoregulatory function of the MGT. A second finding was that fixed, large-granule hemocytes associated with the basal lamina degranulated in the presence of the 2 species of Vibrio, but not with the non-pathogenic bacteria or ASW. These blood cells may help fight specific bacteria penetrating the MGT.     

Inhibitory Activity of Probiotic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> UTM 126 Against <Emphasis Type="Italic">Vibrio</Emphasis> Species Confers Protection Against Vibriosis in Juvenile Shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>)

The bacterial strain Bacillus subtilis UTM 126 produced antimicrobial activity against pathogenic Vibrio species, including V. alginolyticus, V. parahaemolyticus, and V. harveyi. The probiotic effect of B. subtilis was tested by feeding juvenile shrimp (Litopenaeus vannamei) food supplemented with B. subtilis (10⁵ CFU/g) for 28 days before an immersion challenge with V. harveyi at 10⁵ CFU/mL for 24 h. The treatment with B. subtilis UTM 126 decreased final mortality to 18.25%, compared with 51.75% in the control group. Bacillus subtilis UTM 126 has potential applications for controlling pathogenic V. harveyi in shrimp aquaculture.     

Synthesis of organometallic-based biologically active compounds: In vitro antibacterial, antifungal and cytotoxic properties of some sulfonamide incorporated ferrocences

Sulfonamides incorporated ferrocene (SIF) have been synthesized by the condensation reaction of sulfonamides (sulfanilamide, sulfathiazole or sulfamethaxazole) with 1,1'-diacetylferrocene. The synthesized compounds (SIF(1)-SIF(4)) have been characterized by their physical, spectral and analytical properties and have been screened for their in vitro antibacterial properties against pathogenic bacterial strains e.g., Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis Staphylococcus aureus and Salmonella typhi and for antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata using Agar-well diffusion method. Most of the compounds showed good antibacterial activity whereas, all the compounds exhibited significant antifungal activity. Brine shrimp bioassay was also carried out for in vitro cytotoxic properties against Artemia salina.