Isolation ofVibrio parahaemolyticus andVibrio cholerae (Non-O1 and O139) from moribund shrimp (Penaeus monodon) and experimental challenge study against post larvae and juveniles

Outbreak ofVibrio infection was reported from a shrimp farm near Chennai, south India. Both green and yellowVibrio were isolated from disease outbreak ofPenaeus monodon (Milne Edwards) culture farm and biochemically confirmed asVibrio parahaemolyticus andVibrio cholerae. Randomly selectedV. parahaemolyticus (VP1) andV. cholerae (VC1) were reconfirmed by partial 16S rRNA gene analysis. Both were tested against post larvae and juveniles ofP. monodon by bath challenge test and intramuscular injection respectively. The study revealed that VP1 is more virulent than VC1 isolated from same source againstP. monodon post larvae and juveniles. Phylogenetic tree based on comparison of partial 16S rRNA gene analysis revealed close relationship of VP1 with other shrimp pathogens likeVibrio harveyi andVibrio alginolyticus. There might be some close relationship for disease development among all these strains.     

Temperature-Dependent Inhibition of Opportunistic Vibrio Pathogens by Native Coral Commensal Bacteria

Bacteria living within the surface mucus layer of corals compete for nutrients and space. A number of stresses affect the outcome of this competition. The interactions between native microorganisms and opportunistic pathogens largely determine the coral holobiont's overall health and fitness. In this study, we tested the hypothesis that commensal bacteria isolated from the mucus layer of a healthy elkhorn coral, Acropora palmata, are capable of inhibition of opportunistic pathogens, Vibrio shiloi AK1 and Vibrio coralliilyticus. These vibrios are known to cause disease in corals and their virulence is temperature dependent. Elevated temperature (30 °C) increased the cell numbers of one commensal and both Vibrio pathogens in monocultures. We further tested the hypothesis that elevated temperature favors pathogenic organisms by simultaneously increasing the fitness of vibrios and decreasing the fitness of commensals by measuring growth of each species within a co-culture over the course of 1 week. In competition experiments between vibrios and commensals, the proportion of Vibrio spp. increased significantly under elevated temperature. We finished by investigating several temperature–dependent mechanisms that could influence co-culture differences via changes in competitive fitness. The ability of Vibrio spp. to utilize glycoproteins found in A. palmata mucus increased or remained stable when exposed to elevated temperature, while commensals' tended to decrease utilization. In both vibrios and commensals, protease activity increased at 30 °C, while chiA expression increased under elevated temperatures for Vibrio spp. These results provide insight into potential mechanisms through which elevated temperature may select for pathogenic bacterial dominance and lead to disease or a decrease in coral fitness.     

Characterization of Vibrios Diversity in the Mucus of the Polychaete Myxicola infundibulum (Annellida,Polichaeta)

Vibrios are among the most abundant culturable microbes in aquatic environments. They can be either free-living in the water column or associated with several marine organisms as mutualists, saprophytes, or parasites. In the present study we analysed vibrios abundance and diversity in the mucus of the polychaete Myxicola infundibulum, complementing culture-based with molecular methods. Vibrios reached 4.6?×?10³ CFU mL^?1 thus representing a conspicuous component of the heterotrophic culturable bacteria. In addition, luminous vibrios accounted for about 60 % of the total culturable vibrios in the mucus. The isolates were assigned to: Vibrio gigantis, Vibrio fischeri, Vibrio jasicida, Vibrio crassostreae, Vibrio kanaloae, and Vibrio xuii. Two Vibrio isolates (MI-13 and MI-15) may belong to a new species. We also tested the ability of the Vibrio isolates to grow on M. infundibulum mucus as the sole carbon source. All strains showed appreciable growth in the presence of mucus, leading us to conclude that this matrix, which is abundant and covers the animal entirely, may represent a microcosm and a food source for some bacteria, playing a crucial role in the structuring of a mucus-associated beneficial microbial community. Moreover, the trophic relationship between vibrios and M. infundibulum mucus could be enhanced by the protection that mucus offers to vibrios. The results of this study represent a contribution to the growing evidence for complex and dynamic invertebrate-microbe associations present in nature and highlight the importance of exploring relationships that Vibrio species establish with marine invertebrates.     

Use of formic acid to control vibriosis in shrimp aquaculture

Luminous vibriosis is a shrimp disease that causes major economic losses in shrimp industry as a result of massive shrimp kills due to bacterial infection caused by Vibrio species. Use of antibiotics to control Vibrio in shrimp aquaculture is not allowed in the United States and so it is necessary to develop an alternative pathogen control method for shrimp production. Short-chain fatty acids have been used as food preservatives for a long time. Organic acids are commonly added in feeds in animal production, such as chicken, pig, and cattle. In this study, growth inhibition effects of formic acid on five selected Vibrio species, namely Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio parahaemolyticus and Vibrio vulnificus were studied. The Vibrio bacteria were grown on both solid and liquid media using Muller-Hinton agar and alkaline peptone water, respectively, with various concentrations of formic acid. Bacterial growth was monitored in the liquid media using optical density method. The results showed significant inhibition of growth of all five Vibrio species by formic acid at low concentration. The effective concentration (EC₅₀) values were calculated for all five Vibrio species, which were less than 0.039% of formic acid. The results are encouraging to supplement formic acid in the shrimp feed as a control mechanism to reduce Vibrio outbreak in shrimp aquaculture system.     

Lactose utilization byVibrio vulnificus

The ability of wild-type strains ofVibrio vulnificus to utilize lactose as a sole source of carbon and energy and produce acid in lactose-containing media is associated with the appearance of spontaneous lactose-utilizing mutants. These contain increased activities of an enzyme able to hydrolyzeo-nitrophenyl--d-galactoside as well as lactose. This activity is constitutive in some mutants and inducible by both lactose and isopropyl--d-thiogalactoside in others. A limited survey of otherVibrio species indicates thatV. pelagius also can acquire, by mutation, the ability to grow on and make acid from lactose. No immunological cross-reaction was detected between the enzymes fromVibrio and the -galactosidases ofEscherichia coli andKlebsiella.     

Association of a luminous Vibrio sp., taxonomically related to Vibrio harveyi, with Clytia linearis (Thornely, 1900) (Hydrozoa, Cnidaria)

A previously unknown association between a luminous Vibrio sp., taxonomically related to the species Vibrio harveyi and a common member of the shallow/mid water communities of the Mediterranean Sea, the hydrozoan Clytia linearis is described. All the specimens of C. linearis observed under blue light excitation showed both a natural luminescence appearing as a series of fine dots due to clytin, and a clear fluorescence on the external side of the perisarc around the colonies due to the presence of luminous bacteria. Luminous bacteria were isolated from the surface of C. linearis, their phenotypic characterization as isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a Vibrio sp. taxonomically related to V. harveyi. The association of the V. harveyi-related species with C. linearis, as already suggested for another hydroid, Aglaophenia octodonta, could be explained with the activity of these bacteria of feeding on the chitinous structures present in these hydroids. Moreover, the adhesion of the luminous bacterium (here referred to as Vibrio sp. CL1) on C. linearis may contribute to the survival of this Vibrio species in the marine environment providing a suitable growth habitat.     

Occurrence, Diversity, and Pathogenicity of Halophilic Vibrio spp. and Non-O1 Vibrio cholerae from Estuarine Waters along the Italian Adriatic Coast

The occurrence, diversity, and pathogenicity of Vibrio spp. were investigated in two estuaries along the Italian Adriatic coast. Vibrio alginolyticus was the predominant species, followed by Vibrio parahaemolyticus, non-O1 Vibrio cholerae, and Vibrio vulnificus. By using a biochemical fingerprinting method, all isolates were grouped into nine phenotypes with similarity levels of 75 to 97.5%. The production of toxins capable of causing cytoskeleton-dependent changes was detected in a large number of Vibrio strains. These findings indicate a significant presence of potentially pathogenic Vibrio strains along the Adriatic coast.     

Nosema whitei,a microsporidan pathogen of some species ofTribolium V. Comparative pathogenicity and host range

A dose-mortality technique was used to compare the pathogenicity ofNosema whitei for 4 stored products pests.Tribolium castaneum, T. confusum andOryzaephilus surinamensis were similar in susceptibility, whileT. anaphe was at least 100 times more susceptible. A possible explanation for this difference is thatT. anaphe has a much longer developmental time (28 days at 25°C) than the other 3 species (20 days at 25°C). The host range was studied in another 8 species from 2 orders.T. brevicornis, T. destructor, andT. madens were all susceptible while the other species tested (includingTenebrio molitor, Palorus ratzburgi andPlodia interpunctella) were refractile to infection.     

Vibrio parahaemolyticus andVibrio alginolyticus: Short generation-time marine bacteria

The growth rates of 30 different strains ofVibrio parahaemolyticus andVibrio alginolyticus at 37°C was determined. Each species consists of two major groups, one having a short generation time (12–14 min) and one with a longer generation time (20–25 min). The diversity in generation times of different strains belonging to the same species is discussed. The effect of temperature, salt, and nutrient concentrations on the growth rate of oneV. alginolyticus strain (NCMB 1803) was studied. The most striking is the effect of the temperature; at 39°C the generation time is 10–11 min, while at 21°C it is 60 min. The heat of activation for growth calculating from such data is 22,580 kcal/mole/hr^?1. The ecological significance of these results is discussed.     

Administration of Probiotics Improves the Brine Shrimp Production and Prevents Detrimental Effects of Pathogenic <Emphasis Type="Italic">Vibrio</Emphasis> Species

In this study, we evaluated a consortium of probiotic bacteria as an environmentally-friendly strategy for controlling pathogenic Vibrio species during the brine shrimp incubation period. Probiotic strains were initially selected on basis of (i) their ability to colonize the cyst surfaces, (ii) their absence of cross-inhibitory effects, and (iii) no detrimental effect on cyst hatching. The cysts and nauplius surfaces were immediately colonized after the application of selected probiotic strains, without detrimental effects on survival. Ten probiotic strains were mixed at similar proportions (probiotic consortium) and evaluated at different concentrations into brine shrimp cultures during incubation and early stages of development. Subsequently, these cultures were challenged with Vibrio parahaemolyticus and Vibrio harveyi. The probiotic consortium was effective to reduce the abundance of pathogenic Vibrio species and to prevent the mortality during Vibrio challenges; however, its effect was concentration-dependent and was successful at a starting concentration of 1.8?×?10⁶ CFU/ml. Our results suggest that this probiotic consortium offers an alternative to antimicrobial agents routinely used to reduce the incidence and prevalence of pathogenic Vibrio species in brine shrimp production.     

Phenotypic characterization ofPhotobacterium logei (sp. nov.), a species related toP. fischeri

Ten luminous marine bacteria having the diagnostic traits ofPhotobacterium fischeri but differing from this species in their ability to grow at 4°C have been subjected to a more extensive phenotypic characterization. The results indicated a high phenotypic similarity toP. fischeri; the major additional diagnostic trait was their inability to grow at 30°C. In these properties the isolates resembled a previously characterized nonluminous strain, ATCC 15382, which had a DNA homology of about 40% toP. fischeri. Immunological comparisons of the glutamine synthetases and Fe-superoxide dismutases of these strains as well as additional properties that are discussed indicated that the ten luminous isolates and strain ATCC 15382 constituted a new species, which we have designatedPhotobacterium logei sp. nov. (type strain 584, ATCC 29985).     

Abundance and Distribution of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus Following a Major Freshwater Intrusion into the Mississippi Sound

In response to a major influx of freshwater to the Mississippi Sound following the opening of the Bonnet Carre Spillway, water samples were collected from three sites along the Mississippi shoreline to assess the impact of altered salinity on three pathogenic Vibrio species. Salinity readings across the affected area during the 2011 sample period ranged from 1.4 to 12.9 ppt (mean?=?7.0) and for the 2012 sample period from 14.1 to 23.6 ppt (mean?=?19.8). Analyses of the data collected in 2011 showed a reduction in densities of Vibrio parahaemolyticus and Vibrio vulnificus with a concurrent increase of Vibrio cholerae numbers, with V. cholerae becoming the only Vibrio detected once salinity readings dropped to 6 ppt. Follow-up samples taken in 2012 after recovery of the salinity in the sound showed that the relative densities of the three pathogenic vibrios had reverted back to normal levels. This study shows that although the spillway was open but a few weeks and the effects were therefore time limited, the Mississippi River water had a profound, if temporary, effect on Vibrio ecology in the Mississippi Sound.     

Metarhizium spp. isolates from madagascar: Morphology and effect of high temperature on growth and infectivity to the migratory locust,Locusta migratoria

Five strains ofMetarhizium anisopliae (Metsch.) Sorokin and one strain ofMetarhizium flavoviride Gams &; Rozsypal originally isolated in Madagascar were studied. Measurements of conidia and, for the first time, also of blastospores produced in a liquid medium were used for species and variety determination. Blastospores ofM. flavoviride were more homogenous in their size than those ofM. anisopliae. Growth at high temperatures between 25° and 40°C showed that 4 isolates ofM. anisopliae grew at 36°C andM. flavoviride grew at 38°C. Using alternating day/night temperatures (8/16 h) the three strains tested could also tolerate 40°/25°C. In bioassays, fiveMetarhizium spp. isolates were tested against third and fourth instar larvae ofLocusta migratoria (L.) at two alternating day/night temperatures of 30°/25°C and 36°/25°C. In the cooler regime, all strains caused a mortality of 50% within 5.9 to 8.5 days (median lethal time), while in the 36°/25°C treatment only the thermophilicM. flavoviride and oneM. anisopliae strain isolated from a soil sample gave comparable results with median lethal times of 6.8 and 7.3 days, respectively.     

<Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and<Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>: Short generation-time marine bacteria

The growth rates of 30 different strains ofVibrio parahaemolyticus andVibrio alginolyticus at 37°C was determined. Each species consists of two major groups, one having a short generation time (12–14 min) and one with a longer generation time (20–25 min). The diversity in generation times of different strains belonging to the same species is discussed. The effect of temperature, salt, and nutrient concentrations on the growth rate of oneV. alginolyticus strain (NCMB 1803) was studied. The most striking is the effect of the temperature; at 39°C the generation time is 10–11 min, while at 21°C it is 60 min. The heat of activation for growth calculating from such data is 22,580 kcal/mole/hr⁻¹. The ecological significance of these results is discussed.     

Temperatures lethal for citrus blackfly parasites,Encarsia opulenta andE. Smithi [Hym.: Aphelinidae]

Seasonally acclimatized adult and immature parasites of the citrus blackfly (CBF),Aleurocanthus woglumi Ashby, were exposed to high or low temperature extremes for 3 h periods. Death of all summer adults ofEncarsia opulenta Silvestri andE. smithi Silvestri occurred between 35° and 40°C. Within CBF hosts,E. opulenta were not able to emerge when temperatures reached between 45° and 50°C. In winter experiments adults of bothEncarsia species succumbed between −5° and −10°C. In a comparison of the 2 seasonal tests, a higher percentage ofE. smithi adults were able to survive both higher and lower temperatures thanE. opulenta, but the main interspecific difference was the ability ofE. opulenta within CBF to survive −10° to − 15°C whileE. smithi did not. Limited data forAmitus hesperidum Silvestri [Hym.: Platygasteridae] indicated that the immatures survived better at low, and not as well at high, temperatures as either species ofEncarsia. Florida Agricultural Experiment Station Journal Series # 5549.     

Antibacterial activities of marine epibiotic bacteria isolated from brown algae of Japan

One hundred and sixteen epibiotic bacteria were isolated from the surface of nine species of brown algaeSargassum serratifolium, Sargassum fusiforme, Sargassum filicinum, Padina arborescens, Undaria pinnatifida, Petalonia fascia, Colpomenia sinuosa, Scytosiphon lomentaria andEcklonia cava which were collected at Awaji Island, Japan. Primary screening results using disc-diffusion assay revealed that, among the bacteria isolated 20% of epibiotic bacteria exhibited antibacterial activity. Among them, 10 bacteria which showed high antibacterial activity were further studied for their ability against (i) a set of fouling bacteria isolated from marine natural biofilm, (ii) some luminescentVibrio andPhotobacterium species and (iii) a panel of pathogenic bacteria. In general, inhibitory activities were high or moderate against fouling bacteria,Vibrio andPhotobacterium species, while they were found to be low against pathogenic bacteria tested. The phylogenetic analysis using 16S rRNA sequencing revealed that all of the bacteria with high antibacterial activity showed a close affiliation with genusBacillus. This result suggested that the genusBacillus is efficient producers of of antibacterial compounds and these epibiotic bacteria isolated are highly successful colonizers on macroalgal surfaces.     

Population Dynamics of Vibrio and Pseudomonas Species Isolated from Farmed Tasmanian Atlantic Salmon (Salmo salar L.): A Seasonal Study

Vibrio and Pseudomonas species have been shown to be part of the normal microbiota of Atlantic salmon (Salmo salar L.), with some strains causing disease in fish. The factors affecting their prevalence and persistence in the salmon gut, however, have not been well studied. In this study, we collected 340 Vibrio and 150 Pseudomonas isolates from the hindgut of farmed Tasmanian Atlantic salmon, fed with two commercially available diets. Samples were collected every 6–8 weeks between July 2011 and May 2012. Isolates from selective agar were initially identified using biochemical tests and confirmed using genus-specific primers and 16S ribosomal RNA (16S rRNA) sequencing. Random amplified polymorphic DNA (RAPD) PCR was used to type both Pseudomonas and Vibrio; the latter was further typed using a biochemical fingerprinting method (PhP-RV plates). We observed low species diversity with strains comprising Vibrio ichthyoenteri/Vibrio scophthalmi, Vibrio crassostreae/Vibrio splendidus, Aliivibrio finisterrensis, Photobacterium phosphoreum and Pseudomonas fragi. Out of 340 Vibrio isolates, 238 (70 %) belonged to 21 clonal types and were found predominantly during summer when water temperatures reached 15 to 21 °C. Of these, the four major clonal types were found in multiple samples (70 %). P. fragi, on the other hand, was only found during the colder water temperatures and belonged to 18 clonal types. The presence of both groups of bacteria and their clonal types were independent of the fish diets used, suggesting that the water temperature was the main factor of the prevalence and persistence of these bacteria in the gut of Atlantic salmon.     

High diversity of Vibrio spp. associated with different ecological niches in a marine aquaria system and description of Vibrio aquimaris sp. nov

The aim of the study was to characterise the diversity and niche-specific colonization of Vibrio spp. in a marine aquaria system by a cultivation-dependent approach. A total of 53 Vibrio spp. isolates were cultured from different ecological niches in a marine aquarium including microplastic (MP) and sandy sediment particles (12 weeks after added sterile to the system), detritus, and the surrounding aquarium water. Based on the 16S rRNA gene sequence phylogeny and multilocus sequence analysis (MLSA) the isolates were assigned to seven different phylotypes. Six phylotypes were identified by high probability to the species level. The highest phylotype diversity was cultured from detritus and water (six out of seven phylotypes), while only two phylotypes were cultured from MP and sediment particles. Genomic fingerprinting indicated an even higher genetic diversity of Vibrio spp. at the strain (genotype) level. Again, the highest diversity of genotypes was recovered from detritus and water while only few partially particle-type specific genotypes were cultured from MP and sediment particles. Phylotype V-2 formed an independent branch in the MLSA tree and could not be assigned to a described Vibrio species. Isolates of this phylotype showed highest 16S rRNA gene sequence similarity to type strains of Vibrio japonicus (98.5%) and Vibrio caribbeanicus (98.4%). A representative isolate, strain THAF100^T, was characterised by a polyphasic taxonomic approach and Vibrio aquimaris sp. nov., with strain THAF100^T (=DSM 109633^T = LMG 31434^T = CIP 111709^T) as type strain, is proposed as novel species.     

Comparative biological studies on two species of predatory beetles of the genusCybocephalus [Col.: Cybocephalidae]

The development, reproduction and longevity ofCybocephalus micans Reitter andC. nigriceps nigriceps (Sahlberg) were studied under controlled laboratory conditions. The data obtained explain the distribution of the 2 predators in different climatic regions of Israel. Under constant temperatures ranging between 16° and 36° C, the duration of development of the egg. larva and pupa of the 2 species became shorter as the temperature increased. For each developmental stage, at each temperature tested, the mean duration of development ofC. n. nigriceps was higher than that ofC. micans. The average time needed for completion of a generation at 28° C was about 39 days forC. micans, as compared with 56 days forC. n. nigriceps. Progeny production of both cybocephalid species was higher and adult longevity was longer at 28° C than at 32° C.