Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 °C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10⁴ cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 °C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection.The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.     

Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin, hemolysin, and extracellular serine protease (i.e., aerA, hlyA, and ahpA, respectively). These genes were investigated using polymerase chain reaction (PCR) with specific primers for each gene. And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed. PCR results demonstrated that the distribution patterns of aerA, hlyA, and ahpA were different in these strains. 6/9 of A. hydrophila strains were aerA positive, 8/9 of strains hlyA positive, 7/9 of strains ahpA positive, respectively. However, the assay for pathogenesis showed that two strains (A. hydrophila XS91-4-1 and C2) were strong virulent, two strains (A. hydrophila ST78-3-3 and 58-20-9) avirulent and the rest middle virulent was to the fish. In conclusion, there are significant correlation between the distribution pattern of the three virulence genes and the pathogenicity to Carrassius auratus ibebio. All strong virulent A. hydrophila strains were aerA ⁺ hlyA ⁺ ahpA ⁺ genotype, and all aerA ⁺ hlyA ⁺ ahpA ⁺ strains were virulent. Strains with the genotype of aerA ⁻ hlyA ⁻ ahpA ⁺ have middle pathogenicity. In the present study, we found for the first time that all A. hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio. Additionally, there was a positive correlation between the virulence of A. hydrophila and the presence of aerA and ahpA. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2006, 45(1): 82–85 [译自: 中山大学学报 (自然科学版)]     

The effect of Euglena viridis on immune response of rohu, Labeo rohita (Ham.)

The study evaluated the effect of dietary doses of Euglena viridis on the immune response and disease resistance of Labeo rohita fingerlings against infection with the bacterial pathogen Aeromonas hydrophila. L. rohita fingerlings were fed with diet containing 0 (Control), 0.1 g, 0.5 g, 1.0 g Euglena powder kg⁻¹ dry diet for 90 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined after 30, 60 and 90 days of feeding. Fish were challenged with A. hydrophila 90 days post-feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with Euglena showed increased levels of superoxide anion production, lysozyme, serum bactericidal activity, serum protein and albumin (P < 0.05) compared with the control group. Following challenge with A. hydrophila less survivability was observed in the control group (56.65%) than the group fed the experimental diets. The group fed 0.5 g Euglena kg⁻¹ dry diet showed the highest percentage survival (75%). These results indicate that Euglena stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.     

Molecular evidence for species level divergence in African Nile Crocodiles Crocodylus niloticus (Laurenti, 1786)

Relationships of the newly discovered dwarf crocodiles from Mauritania were inferred from mitochondrial 12S sequences. Specimens from 13 different Crocodylus niloticus populations (from East Africa, West Africa and Madagascar) were compared. Additional representatives of the genus Crocodylus (one from Africa and one from Australia), the African genus Osteolaemus and the South American alligatorid Paleosuchus palpebrosus (as outgroup) were included in the analysis. Maximum-likelihood and Bayesian analyses yielded relationships that were strikingly different from currently prevailing phylogenetic hypotheses. Both analyses consistently revealed two groups, one consisting of the monophyletic West- and Central African populations and the other of a paraphyletic group containing the East African and Madagascan populations. High genetic divergence between those groups indicates separation on the species level. Furthermore ‘C’ cataphractus is clearly shown not to be a member of the genus Crocodylus. The resulting nomenclatural changes are discussed. To cite this article: A. Schmitz et al., C. R. Palevol 2 (2003).     

Muscle growth in Nile tilapia (Oreochromis niloticus): histochemical,ultrastructural and morphometric study

Muscle growth in Nile tilapia (Oreochromis niloticus) was studied focusing on histochemical, ultrastructural, and morphometric characteristics of muscle fibers. Based on body length (cm), we studied four groups: G1=1.36+/-0.09, G2=3.38+/-0.44, G3=8.90+/-1.47, and G4=28.30+/-3.29 (mean+/-S.D.). All groups showed intense reaction to NADH-TR in subdermal fibers and weak or no reaction in deep layer fibers. In G3 and G4, an intermediate layer was also observed with fibers presenting weak reaction; in G4, groups of fibers with intense reaction were observed in the subdermal region. The myosin ATPase (m-ATPase) activities were acid-stable and alkali-labile in subdermal fibers; most deep layer fibers were alkali-stable and acid-labile. Intermediate fibers were acid-labile and alkali-stable. Two fiber populations were observed near deep muscle layer: one large presenting weak acid- and alkali-stable and the other small alkali-stable.During growth, muscle fiber hypertrophy was more evident in intermediate and white fibers for G3 and G4. However, in these groups, the presence of fiber diameters < or =21 microm suggested that there is still substantial fiber recruitment, confirmed by ultrastructural study, but hypertrophy is the main mechanism contributing to increase in muscular mass.     

Lymphoid apoptosis in Edwardsiella tarda septicemia in tilapia, Oreochromis niloticus

The present study revealed a relationship between the kinetic change of apoptosis and the inflammatory response during experimental intraperitoneal infection with Edwardsiella tarda as a septicemic model. The morphological changes of apoptotic cells including cellular shrinkage, condensed nuclear chromatin, nuclear fragmentation and membrane blebbing were detected by light and transmission electron microscopy. TUNEL and agarose gel electrophoresis confirmed the fragmentation of DNA in the apoptotic cells. Apoptosis was highly detected in lymphoid organs prior to the inflammatory process and gradually decreased after an extensive inflammatory response. Apoptosis in thymus and spleen was extensive and an in vitro study revealed that lymphocytes were the major cell population which underwent apoptosis. The result suggests that E. tarda-induced systemic immunosuppression via lymphocyte apoptosis as determined by suppression of the systemic inflammatory response during an initial step of generalized septicemia.     

Growth and IGF-I response of juvenile Nile tilapia (Oreochromis niloticus) to changes in water temperature and dietary protein level

Water temperature and dietary protein level play an important role in influencing the growth and insulin-like growth factor I (IGF-I) in Nile tilapia juveniles. The combined effect of temperature (20–34 °C) and dietary protein level (25–50%) on the specific growth rate (SGR), feed efficiency (FE), serum IGF-I level and hepatic IGF-I mRNA level was examined under laboratory conditions by employing central composite design and response surface method. Results showed that the linear effects of temperature and dietary protein level on the SGR, FE, serum IGF-I and hepatic IGF-I mRNA level were significant (P<0.05); the quadratic effects of temperature and dietary protein level on the FE and serum IGF-I were significant (P<0.05). The interaction of temperature and dietary protein level on the FE, serum IGF-I and hepatic IGF-I mRNA level all proved significant (P<0.05). The optimal temperature/dietary protein level combination was determined, i.e., 29.9 °C/40.3%, at which the greatest SGR (2.748%/d) and FE (0.775) were simultaneously arrived. Both SGR and FE were linearly correlated with serum IGF-I or hepatic IGF-I mRNA level. These results suggested that optimum combination of temperature and dietary protein level would enhance tilapia growth efficiency and IGF-I would regulate growth and FE.     

The blood-testis barrier and its breakdown following immunization to testis material in the Nile tilapia,Oreochromis niloticus

Summary The blood-testis barrier and its changes following immunization to testis material, were investigated by light- and electron microscopy in a teleost fish, the Nile tilapia Oreochromis niloticus, using horseradish peroxidase and bovine serum albumin as tracers. In the normal testis, histochemistry using horseradish peroxidase revealed that a barrier composed of junctional complexes connecting adjacent Sertoli cells existed around the central lumina of the seminal lobules, and also around the germ-cell cysts containing spermatids at the middle or late phase of chromatin condensation. By contrast, bovine serum albumin was prevented from passing through the basement membrane and could not penetrate any of the spermatogenetic cysts, indicating that the basement membrane may be an ion-selective barrier. In tilapia immunized with allogeneic testis homogenate emulsified in Freund's complete adjuvant, bovine serum albumin could penetrate the spermatogenetic cysts, and horseradish peroxidase was able to pass through the intercellular spaces between Sertoli cells to the region nearer the seminal lobule lumen, due to the junctional complexes becoming loosened. The results suggest that the blood-testis barrier, both junctional complexes and the basement membrane, are broken down during immune responses.     

Susceptibility of two fishes (Oreochromis niloticus and Cyprinodon variegatus) to Pfiesteria shumwayae and its associated toxin: Influence of salinity

Researchers examining the mechanisms of ichthyotoxicity of Pfiesteria shumwayae have come to different conclusions about the role of toxin in this process. Some attribute fish mortality solely to direct attack by these pedunculate dinoflagellates on exposed fish tissue while others have provided evidence for a role of a soluble toxin. Detection of toxin, especially in low concentrations, is a function of the sensitivity of the selected bioassay methods and the various groups addressing this question have utilized different methods. One notable difference in fish bioassay methods utilized to detect Pfiesteria-associated toxin (PfTx) is the species of fish tested. Studies that have not detected PfTx in bioassays generally have used Cyprinodon variegatus (sheepshead minnow) as the test fish while those that have detected toxin generally used Oreochromis spp. (Tilapia). In this study response of these two fish species was compared to determine their relative sensitivity to physical attack by P. shumwayae and to PfTx. The results indicate that Oreochromis niloticus is more susceptible to P. shumwayae and its associated toxin than C. variegatus and implicate differences in the ability these species to osmoregulate as a contributing factor for this phenomenon. Salinity stress enhanced susceptibility of O. niloticus to PfTx and thus improved the sensitivity of the bioassay. The observation that salinity stress enhances toxicity to O. niloticus provides additional information regarding the mechanism of PfTx toxicity although the conditions utilized are not representative of the natural habitat of these freshwater fish.     

A place preference test in the fish Nile tilapia

The Nile tilapia fish (Oreochromis niloticus) has a high potential to be used as a model in neuroscience studies. In the present study, the preference of the Nile tilapia between a gravel-enriched (GEE), a shelter-enriched (SEE) or a non-enriched (NEE) environment was determined, for developing a place preference model. Nile tilapia had an initial preference for GEE, but after 1 day of observation, the fish stabilized their frequency of visits among compartments. Hence, any stimulus motivating tilapia increase in compartment visiting indicates a positively reinforcing effect. This feature is very useful for the development of new behavioural paradigms for fish in tests using environmental discrimination, such as the conditioning place preference test.     

The influence of social status on the rate of growth, eye color pattern and insulin-like growth factor-I gene expression in Nile tilapia, Oreochromis niloticus

Many aspects of teleost physiology are subject to regulation by social interactions. To evaluate the relationship of social status with growth, eye color pattern and hepatic Insulin-like Growth Factor-I (IGF-I) mRNA expression, 30 Oreochromis niloticus were isolated for 10 days and were used in a social pair study. Results revealed that growth of both dominant (except 1 day after social interaction) and subordinate individuals was suppressed, but growth suppression was greater in the subordinates. The dominant fish completely inhibited the feeding of the subordinate individuals during and 1 day after they were introduced into the aquaria together. After that, a pattern of highly aggressive attacks by dominant fish only partially inhibited feeding by the subordinates. Differential alterations in growth rate between dominants and subordinates were attributed more to behavioral changes (i.e., feeding) as transduced by physiological regulators (i.e., IGF-I level and possibly serotonin and/or neuropeptide Y) but may also be due to changes in metabolism. The fish's relative position in the social hierarchy consistently influenced the levels of IGF-I mRNA in the liver and the eye color pattern. Lower social status depressed hepatic IGF-I levels while dominant status stimulated hepatic IGF-I production, possibly in response to inhibition of somatostatin release in the hypothalamus, leading to greater secretion of pituitary growth hormone (GH). A significant positive association was detected between the IGF-I mRNA expression of the dominant fish and the level of aggression (number of attacks) during the encounter. Social status also influenced the eye color pattern of the fish. During aggressive interactions, most of the fish (22 out of 24) displayed decreased eye darkening. At the later part of the encounter, all subsequent subordinates displayed eye-darkening patterns which acted as a social signal announcing social submission. After the encounter dominant fish had paler eye color pattern than subordinates.     

Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.     

Effect of water temperature,salinity, and their interaction on growth,plasma osmolality,and gill Na,K-ATPase activity in juvenile GIFT tilapia Oreochromis niloticus (L.)

We used a central composite rotatable experimental design and response surface methodology to evaluate the effects of temperature (18–37 °C), salinity (0–20‰), and their interaction on specific growth rate (SGR), feed efficiency (FE), plasma osmolality, and gill Na⁺, K⁺-ATPase activity in GIFT tilapia juveniles. The linear and quadratic effects of temperature and salinity on SGR, plasma osmolality, and gill Na⁺, K⁺-ATPase activity were statistically significant (P<0.05). The interactive effects of temperature and salinity on plasma osmolality were significant (P<0.05). In contrast, the interaction term was not significant for SGR, FE, and gill Na⁺, K⁺-ATPase activity ⁽P>0.05). The regression equations for SGR, FE, plasma osmolality, and gill Na⁺, K⁺-ATPase activity against the two factors of interest had coefficients of determination of 0.944, 0.984, 0.966, and 0.960, respectively (P<0.01). The optimal temperature/salinity combination was 28.9 °C/7.8‰ at which SGR (2.26% d¹) and FE (0.82) were highest. These values correspond to the optimal temperature/salinity combination (29.1 °C/7.5‰) and the lowest plasma osmolality (348.38 mOsmol kg⁻¹) and gill Na⁺, K⁺-ATPase activity (1.31 µmol Pi. h⁻¹ g⁻¹ protein), and resulted in an energy-saving effect on osmoregulation, which promoted growth.     

Genetic characterization of pAsa6, a new plasmid from Aeromonas salmonicida subsp. salmonicida that encodes a type III effector protein AopH homolog

A new plasmid designated pAsa6 from an Aeromonas salmonicida subsp. salmonicida strain isolated from diseased turbot has been characterized. pAsa6 consists of 18536 bp, has a G+C content of 53.8% and encodes 20 predicted open-reading frames (ORFs). Eight ORFs showed homology to transposases, of which six are complete and two are partial IS sequences. Two ORFs showed homology to replication proteins, and six ORFs showed homology to hypothetical proteins. Two ORFs are truncated homologs of putative A. salmonicida sulfatases. Two genes, aopH and sycH encode homologs of an effector protein for which a role in fish colonization by A. salmonicida has been previously reported, and its chaperone, respectively. The results of filter conjugation experiments suggested that pAsa6 is not mobilizable, as it failed to be conjugally-transferred to several species of marine bacteria tested. All the ORFs of pAsa6 with the exception of four copies of a IS1 transposase gene, have a counterpart in the recently sequenced 155-kb A. salmonicida plasmid pAsa5, suggesting either that pAsa6 is a derivative of pAsa5, or that pAsa5 is the result of the fusion of a pAsa6-like plasmid and a larger plasmid of ca. 135-kb. The pAsa6-encoded repA and aopH genes could be PCR-amplified from strains lacking pAsa6, suggesting presence of a large, possibly pAsa5-like plasmid that was not detected on agarose gels, or the existence of chromosome-integrated plasmid sequences. This study demonstrates that genomic locations for the aopH gene different to pAsa5 or pAsa5-like plasmids exist in A. salmonicida.     

Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts

This study reports the effect of aqueous, ethanol and methanol triherbal solvent extract from Azadirachta indica, Ocimum sanctum and Curcuma longa on innate immune mechanisms such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and disease resistance in goldfish (Carassius auratus) against Aeromonas hydrophila. Fish were intraperitoneally injected with different doses of 0, 5, 50 and 100 mg kg⁻¹ body weight of each triherbal solvent extracts. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) and innate immune response was assessed on week 1, 2 and 4 by challenging with live A. hydrophila (1 × 10⁷ cells ml⁻¹). All the chosen innate immune parameters were enhanced in the ethanol and methanol triherbal solvent extract treatment after week 2. However, the aqueous triherbal extract was enhanced only after week 4. The ethanol and methanol triherbal solvent extracts administration preceding the challenge with live A. hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in RPS values. The study indicates that all the doses of ethanol or methanol triberbal solvent extracts could be positively influence the immune response and protect the heath status of goldfish against A. hydrophila infection.     

Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections

The probiotic activity of two bacteria (Bacillus subtilis and Lactobacillus acidophilus) was evaluated by its effect on the immune response of Nile tilapia (Oreochromis niloticus), beside its protective effect against challenge infections. Furthermore, their in-vitro inhibitory activity was evaluated. The in-vitro antimicrobial assay showed that Bacillus subtilis and Lactobacillus acidophilus inhibited the growth of A. hydrophila. The B. subtilis inhibited the development of P. fluorescens while L. acidophilus inhibited the growth of Strept. iniae. The B. subtilis and L. acidophilus proved harmless when injected in the O. niloticus. The feed, containing a mixture of B. subtilis and L. acidophilus or B. subtilis alone, showed significantly greater numbers of viable cells than feed containing L. acidophilus only after 1, 2, 3 and 4 weeks of storage at 4 degrees C and 25 degrees C. The survival rate and the body-weight gain were significantly increased in the fish given B. subtilis and L. acidophilus for one and two months after application. The hematocrit values showed a significant increase in the group that received the mixture of B. subtilis and L. acidophilus compared with the control group. The nitroblue tetrazolium (NBT) assay, neutrophil adherence and lysozyme activity, showed a significant increase in all the probiotic-treated groups after 1 and 2 months of feeding, when compared with the untreated control group. The serum bactericidal activity was high in the group that was given a mixture of the two bacteria. The relative level of protection (RLP) was significantly higher against A. hydrophila, in the bacterial mixture treated group and against P. fluorescens in the L. acidophilus treated group, after one month of the feeding trial. A significantly higher RLP, against A. hydrophila or P. fluorescens, was noticed after 2 months of the feeding trial in the group given a mixture of the two bacteria, and against Strept. iniae in the group fed a diet containing L. acidophilus.