Isolation and Antimicrobial Resistance of Staphylococcus spp. in Freshwater Fish and Greek Marketplaces

The presence and antibiotic resistance of Staphylococcus spp. in freshwater fish and the environment of fish markets of Northern Greece were investigated. A total of 269 samples were examined, consisting of 71 rainbow trout (Oncorhynchus mykiss), 65 gibel carp (Carassius gibelio), and 133 environmental samples swabbed from various surfaces at fish markets.Staphylococcus spp. was isolated from 27% of the samples, and 16%, 9%, 0.7%, 0.4% and 0.4% were found to be positive for S. aureus, S. epidermidis, S. warneri, S. hominis-hominis, and S. haemolyticus, respectively. All isolates were examined for their susceptibility to 20 antibiotics. None of the S. aureus isolates were resistant to oxacillin and glycopeptides. Three S. epidermidis, two S. Warneri, and one S. haemolyticus isolates were resistant to oxacillin (methicillin resistant Staphylococcus, MRS). All isolates, except one, showed resistance to as many as 15 antibiotics. The population of Staphylococcus spp. in fish did not exceed 1.0 log CFU/g. Freshwater fish and the environments of retail fish markets were found to harbor multi-drug resistant staphylococci. Whether these findings present a real health risk for humans and to what extent needs to be evaluated.     

广西卵形鲳鲹海豚链球菌基因分型、耐药谱型以及毒力基因检测

为了解近年来广西卵形鲳鲹海豚链球菌流行菌株的基因型信息以及菌株间的差异,对2015—2016年从广西地区7个养殖场的患病卵形鲳鲹体内分离得到的17株海豚链球菌菌株分别进行了基因型分析、耐药谱测定以及毒力基因检测。采用随机扩增多态性DNA标记技术(RAPD)和基因组重复序列PCR(rep-PCR)分析其基因型。结果显示,RAPD和rep-PCR指纹图谱结果一致,17株海豚链球菌可分为2种基因型。对海豚链球菌7种主要的毒力相关基因特异PCR检测,所有菌株均为sim A+scp I+pdi+sag A+cps D+pgm A+cfi+毒力基因型,表明这2种基因型的海豚链球菌似乎均为毒力较强的菌株。采用K-B法进行了20种抗生素敏感实验分析其耐药谱,结果表明归于基因1型的菌株耐药谱为AZT,基因2型菌株则出现3种相似的耐药谱,分别为SIZ/T/S/PEN/AZT/SPE/CAZ/PB、SIZ/T/S/PEN/AZT/SPE/CAZ/CRO和SIZ/T/S/PEN/AZT/SPE/CAZ/PB/RIF,证实基因型相同的菌株耐药谱型也相似,2种基因型的菌株耐药谱型差异显著,因此基因型和耐药谱型存在相关性。此结果为卵形鲳鲹海豚链球菌病流行病学研究、疫苗研制以及疫病监测提供理论依据。     

Occurrence of multiple antibiotic resistance and genotypic characterization in Edwardsiella tarda isolated from cage‐cultured hybrid red tilapia (Oreochromis sp.) in the Ping River,Northern Thailand

Edwardsiella tarda is a pathogen that causes edwardsiellosis in aquatic animals. The emergence of multiple antibiotic‐resistant strains makes antibiotic treatment difficult. This study aimed to investigate the antibiotic susceptibility patterns and the genotypic characterization of E. tarda isolated from cage‐cultured red tilapia in Thailand. A total of 30 isolates were identified as E. tarda using biochemical and molecular analysis. The disc diffusion method for testing antibiotic susceptibility showed all the isolates were resistant to colistin sulphate and oxolinic acid. High levels of resistance to amoxicillin, ampicillin, ceftazidime, oxytetracycline and sulphamethoxazole/trimethoprim were observed as well. The multiple antibiotic resistance index ranged from 0.25 to 0.92, indicating that these isolates had been exposed to high risk sources of contamination where antibiotics were commonly used. All the isolates carried the bla_TEM gene based on polymerase chain reaction (PCR). The tetA and sul3 genes were detected in 90% (27/30) and 26.7% (8/30) of the isolates respectively. Nine different genetic groups of isolates were obtained using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC‐PCR). A correlation between genetic types and multiple antibiotic‐resistant patterns was found. These results highlight the potential risks of multiple antibiotic‐resistant isolates for humans and the environment.     

Quantitative and Qualitative Aspects of Bacterial Communities Associated with Cultures of Chlorella minutissima

Microbiological conditions in 200 L cultures of microalgae Chlorella minutissima in polyethylene sleeves were examined. The influence of addition of antibiotic (nitrofurantoin) was studied. Samples were taken 2, 5, 9, and 14 d after inoculation and were spread on solid Zobell medium and on thiosulfate citrate bile salts sucrose agar for the enumeration of total numbers of bacteria and numbers of presumptive Vibrio bacteria, respectively. Microalgae grew well in the cultures and high numbers of culturable bacteria (about 10⁷ bacteria/mL) were present in all microalgae cultures. Addition of antibiotic in 200‐L cultures of C. minutissima did not result in a significant decrease of total counts of bacteria per unit volume (P > 0.05), while the numbers of presumptive Vibrio were higher in cultures added antibiotic compared with the control treatment (P < 0.05). The numbers of presumptive Vibrio per unit volume and the percentage of fast‐growing bacteria were higher in microalgae supernatant than in noncentrifuged samples. Representative colonies were taken from all samples and in total 649 bacterial strains were isolated during this study. A range of phylotypes was identified by amplification and sequencing of the 16S rDNA gene and phylogenetic trees were constructed. Several members of the Roseobacter clade were dominant among the culturable isolates.     

Feed management for Pacific white shrimp Litopenaeus vannamei under semi‐intensive conditions in tanks and ponds

To improve feed management strategies for the semi‐intensive culture of Litopenaeus vannamei, outdoor tank and pond trials were conducted. In the tank trial, shrimp (35 shrimp/m², n = 4) were offered feed for 6 weeks based on a standard feeding protocol (SFP, designed as T100) with five variations (T80:90:100, T90, T90:100, T100:110, T110) of this protocol produced by varying the feed inputs and expressing the treatments as a percentage. Results demonstrated no significant differences in survival rate and feed conversion ratio (FCR) among treatments. The mean final weight and final biomass in the treatments T100:110 and T110 were significantly higher than those in treatment T90 but were not different from the other treatments. In the pond trial, juvenile shrimp (28 shrimp/m², n = 4) were stocked into twelve 0.1‐ha ponds over a 16‐week period. Three feeding protocols were evaluated including a SFP, a 10% reduction in the SFP (SFP:90), and a variable feed input (SFP:80:90:100), which included 80% SFP at week 4th–8th, 90% SFP from week 9th–12th and 100% SFP for week 12th–16th. There were no significant differences in growth performance and economic return among treatments. Based on previous studies, in which higher feed inputs were evaluated, and results of this study, it does not appear to be economically beneficial to use high feed inputs. Instead, feed input could be either applied at a standard ration to optimize growth and economic return or at restricted rations to reduce FCR (feed cost) albeit at the expense of some growth.     

Outbreaks of Streptococcosis associated with Streptococcus iniae in Siberian sturgeon (Acipenser baerii) in China

Streptococcus iniae has emerged as an important fish pathogen over the past few decades causing high losses in aquaculture farms all over the world. At least 27 species of fish have been documented to be infected by S. iniae, including cultured and wild populations. In August and October 2013, a serious infectious disease characterized by body ulcer, internal organs haemorrhages and nodules showing on epicardium occurred on the Acipenser baerii farms in Ya'an country, China. Histological examination revealed a multisystemic, necrotising inflammatory response that was particularly marked in liver, kidney, heart and brain. Mass mortality (>40%) was observed in infected fish and two Gram‐positive cocci (Ab130920 and Ab131025) were obtained from kidneys and livers of diseased fish. Experimental infections with these two isolates resulted in marked symptoms in the sturgeons similar to those observed in natural outbreaks, and the LD₅₀ values of the two isolates were 5.1 × 10⁵ and 6.4 × 10⁵ cfu per fish respectively. The two microorganisms were identified as S. iniae through physiological and biochemical tests, 16S rRNA and lctO gene sequence analysis. Both two isolates showed a similar antibiotic susceptibility, which were sensitive to ampicillin, amoxicillin, cefazolin, amikacin, deoxycycline, florfenicol, azithromycin, ciprofloxacin, vancomycin and resistant to streptomycin, gentamicin, kanamycin, norfloxacin and sinomin (SMZ/TMP). Multiplex PCR assay for virulence genes showed both isolates possessed six main virulence genes: simA, scpI, pdi, pgm, cpsD and sagA genes. These results indicated that S. iniae could act as a pathogen of farmed A. baerii. This is the first report of S. iniae infection associated with mass mortality in A. baerii.     

Prevalence and antimicrobial resistance of vibrios of human health significance in inland saline aquaculture areas

This study was conducted to evaluate the prevalence, potential pathogenicity and antimicrobial resistance of Vibrio isolates from 65 soil/water/fish samples collected from inland saline aquaculture areas. Depending on the sample type, presumptive Vibrio counts ranged from 2.50 to 6.16 log₁₀ CFU/ml (or/g). Among the 119 confirmed Vibrio isolates, Vibrio cholerae was found to most dominant (91.6%) and it was detected in all the samples from inland saline areas. Seven other Vibrio spp. including Vibrio parahaemolyticus and Vibrio vulnificus were also detected. Except one O139 serotype, rest of the V. cholerae isolates were found belonging to non‐O1/non‐O139 serogroups. None of the V. cholerae isolate was found positive for ctx gene. Antimicrobial susceptibility testing against 7 commonly used antibiotics revealed highest resistance (50.4%) against ampicillin. Very high intermediate resistance (87.4%) was also observed against erythromycin. Contrary to previous studies, high susceptibility (>70%) to chloramphenicol, nalidixic acid, tetracycline and trimethoprim was observed in Vibrio isolates obtained in present study. Almost 20% of Vibrio isolates were resistant to two or more antibiotic classes with multiple antibiotic resistance (MAR) index value of ≥0.28. Presence of V. cholerae isolates with very high MAR index value of 0.85 also suggested that these multidrug‐resistant environment isolates could serve as reservoir of antibiotic‐resistant genes in aquatic systems. The presence of multiple drug resistance vibrios in emerging inland saline aquaculture systems emphasizes the need for their routine monitoring for developing the risk assessment and mitigation strategies.     

Antibiotic resistance and repetitive‐element PCR fingerprinting in Aeromonas veronii isolates

This study evaluated antibiotic resistance and the related genes in total 47 Aeromonas veronii isolates from pet fish, eel (Anguilla japonica) and koi (Cyprinus carpio) in Korea. In comparison with the antibiotic susceptibilities of isolates from eel and koi, those of pet fish were more resistant to ceftiofur, aminoglycosides, tetracycline and nitrofurantoin. And isolates from pet fish showed high prevalences of class 1 integron, quinolones and tetracycline resistance determinants than those from eel and koi. Repetitive‐element palindromic PCR (rep‐PCR) showed larger diversities among A. veronii isolates. Collectively, pet fish may be a reservoir for multiple clones of A. veronii involved in antibiotic resistance. In this aspect, imported fish in the aquaculture trade should be steadily and continually screened for bacterial antibiotic resistance and related genes.     

First identification and characterization of Streptococcus iniae obtained from tilapia (Oreochromis aureus) farmed in Mexico

This is the first study to isolate, identify and characterize Streptococcus iniae as the causative disease agent in two tilapia (Oreochromis aureus) populations. The populations were geographically isolated, of distinct origins, and did not share water sources. Affected fish showed various external (e.g., exophthalmia and cachexia, among others) and internal (e.g., granulomatous septicaemia and interstitial nephritis, among others) signs. All internal organ samples produced pure cultures, two of which (one from each farm, termed S‐1 and S‐2) were subjected to biochemical, PCR and 16S rRNA sequencing (99.5% similarity) analyses, confirming S. iniae identification. The two isolates presented genetic homogeneity regardless of technique (i.e., RAPD, REP‐PCR and ERIC‐PCR analyses). Pathogenic potentials were assessed through intraperitoneal injection challenges in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). Rainbow trout mortalities were respectively 40% and 70% at 10⁴ and 10⁶ CFU per fish with the S‐1 isolate, while 100% mortality rates were recorded in zebrafish at 10² and 10⁴ CFU per fish with the S‐2 isolate. The obtained data clearly indicate a relationship between intensified aquaculture activities in Mexico and new disease appearances. Future studies should establish clinical significances for the tilapia industry.     

Genetic diversity and antimicrobial resistance of Flavobacterium psychrophilum isolated from cultured rainbow trout,Onchorynchus mykiss (Walbaum), in Spain

Flavobacterium psychrophilum is the causative agent of bacterial cold‐water disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. These diseases are a major problem in the aquaculture industry in Spain, and a better understanding of the epidemiology of F. psychrophilum isolates is necessary to improve management strategies. In this study, to investigate genetic variability of this bacterium, pulsed‐field gel electrophoresis after DNA digestion with endonuclease StuI, plasmid profiling analysis and antimicrobial susceptibility testing were undertaken with 25 isolates of F. psychrophilum from Spain. These isolates were classified into 17 patterns by PFGE analysis, which were grouped into four clusters and seven independent branches. Twenty isolates (80%) possessed plasmids of 3.5 kb (n = 13) or 5.5 kb (n = 7). No plasmids were associated with antibiotic resistance to oxytetracycline (OTC) or florfenicol (FLO). Twenty isolates (80%) had minimum inhibitory concentrations (MICs) to OTC of between 2.4 and 9.7 μg mL^?1, and all isolates were susceptible to FLO. A relationship between the origin of the isolates and PFGE genotypes was found. Plasmid profile typing correlated with PFGE profile typing, whereas no correlation was found between antimicrobial susceptibility testing and PFGE profiles. These results suggest that the population of F. psychrophilum with pathogenic potential in northern Spain is quite heterogeneous.     

Control of Bacterial Pathogens by Liquid Smoke and Sodium Lactate During Processing of Cold-Smoked and Dried Salmon Strips

Abstract

Liquid smoke and sodium lactate (NaL) were evaluated as inhibitors to control microbial growth in Alaskan native-style smoked and dried salmon strips. Brined salmon strips were dipped in aqueous solutions of liquid smoke (25%, 50%, 75%, 100%) or NaL (20%, 40%) and then inoculated with 3000 CFU of Staphylococcus aureus/100 g sample prior to drying in a laboratory smoke house without application of smoke, at 30°C for 5 days. S. aureus populations increased to 10⁵/g in non-dipped control samples, 4.5 × 10³/g in 20%, and 4.6 × 10²/g in 40% NaL samples. Liquid smoke was not effective at a concentration of 25%, but reduced S. aureus levels below detectable limits when used at higher concentrations. When the additives were tested by a processor in Alaska, aerobic populations were less than 30 CFU/g with 30% NaL and 50% liquid smoke and the water activity (a_w) of these finished products was below 0.85, a level that would inhibit further microbial growth. Processors should test their products for microbiological populations, WPS and a_w until they can show that their process is adequate to control bacterial pathogens.     

What drives changes in the virulence and antibiotic resistance of Vibrio harveyi in the South China Sea?

To understand the driving environmental factors in changes of bacterial virulence and antibiotic resistance, we determined the prevalence, antibiotic resistance and antibiotic resistance and virulence genes of Vibrio harveyi isolated from diseased marine fish in south coastal China. We isolated 2, 52 and 53 V. harveyi strains from Fujian, Hainan and Guangdong, respectively, and identified them by multilocus sequence analysis of 16S rRNA-toxR_Vh-rctB. Nine typical virulence genes were represented at a higher average in Hainan (7.39 ± 0.24) than in Guangdong (6.91 ± 0.28). Five atypical virulence genes were detected in some isolates. In particular, flaC and vvh were detected in more than 60% of isolates. Their average number was significantly higher in Hainan (2.30 ± 0.20) than in Guangdong (1.70 ± 0.10). Multidrug resistance was widespread with an average resistance to 4.57 ± 0.18 of 15 antibiotics. Both the average number of antibiotic resistance and antibiotic resistance genes were higher in Hainan (5.25 ± 0.27 and 1.11 ± 0.15, respectively) than in Guangdong (3.87 ± 0.21 and 0.75 ± 0.10, respectively). This study demonstrated that there were more virulence genes and greater drug resistance in Hainan than in Guangdong, suggesting that warmer temperature and antibiotics pollutants probably enhance antibiotic resistance and bacterial infection.     

Antibacterial activity of oxytetracycline on microbial ecology of Nile tilapia (Oreochromis niloticus) gastrointestinal tract under laboratory condition

The antibacterial effect of oxytetracycline (OTC) on bacterial load, pathogen and possible development of antibiotic resistance in intestinal flora of apparently healthy Nile tilapia (Oreochromis niloticus) was investigated under laboratory condition. OTC, a broad spectrum approved antibiotic widely used in aquaculture, was fed to Nile tilapia through medicated diet at a rate of 2 g/ kg of feed for consecutive 7 days in treatment 1 (T₁) and 30 days in treatment 2 (T₂). Changes in physico‐chemical parameters of water were also recorded where pH and dissolved oxygen influenced bacterial load and coliform count respectively. At the start of the experiment, initial bacterial load in fish intestine was 1.61 ± 2.25 × 10¹⁰ cfu/g, which after OTC treatment decreased significantly (p < .05) to 3.06 ± 2.08 × 10⁸ cfu/g on day 7 and 3.45 ± 4.46 × 10⁷ cfu/g on day 30 when compared with the control group. Among the 10 bacterial genera identified in the intestine, 4 potential public health concern bacteria viz., Salmonella, Escherichia, Enterobacter and Staphylococcaus were predominant. There was a sharp increase in bacterial load on day 18 in T₂ that may be attributed to the development of antibiotic resistance in intestinal bacteria. Antibiotic susceptibility test for isolates against seven antibiotics: amoxycillin, ciprofloxacin, co‐trimoxazole, erythromycin, gentamicin, nitrofurantoin and tetracycline using disc diffusion method revealed significantly increased resistance of Gram‐negative rods (p < .05) that possibly caused increased frequency of OTC‐resistant microorganisms. In conclusion, short‐ and long‐term exposure to OTC treatment affected the distribution of bacterial genera including pathogens in the Nile tilapia gastrointestinal tract and concomitantly influenced their antimicrobial resistance.     

Isolation and identification of fish pathogen Edwardsiella tarda from mariculture in China

The causative agent was isolated from diseased turbots (Scophthalmus maximus) stricken by a high‐mortality outbreak of bacterial septicaemia occurring in a mariculture farm in Yantai, a northern coastal city of China. Seven pure isolates, namely EH‐15, EH‐103, EH‐107, EH‐202, EH‐203, EH‐305 and EH‐306, belonged to Edwardsiella tarda. The phenotypic features of the cultures were analysed extensively. Three of the isolates showed high 16S rDNA sequence similarities with E. tarda sequence (GenBank accession no. EF467289). However, unlike the E. tarda ATCC 15947, all the isolates, except EH‐15, contained a novel large plasmid sized about 23.7 kb. Furthermore, pathogenicity of the isolates was addressed by experimental challenges with fish models. The isolates exhibited strong virulence to swordtail fish with LD₅₀ ranging between 3.8 × 10³ and 3.8 × 10⁵ CFU g^?1, and EH‐202 displaying the lowest LD₅₀ value among them. Antibiotic susceptibilities of E. tarda isolates were assayed. Compared with E. tarda ATCC 15947, the isolates displayed strong resistance to chloramphenicol, and the probable dominant chloramphenicol resistance determinant was cat III. Depicting the main biological properties of turbot‐borne E. tarda strains in China, the study provided useful information for further unveiling their pathogenic mechanisms.     

Antimicrobial susceptibility and enrofloxacin resistance of streptococcal bacteria from farmed Nile tilapia,Oreochromis niloticus (Linnaeus 1758) in Thailand

This study examined the antimicrobial susceptibility and mutation(s) in quinolone resistance‐determining regions (QRDRs) in streptococcal pathogens isolated from farmed Nile tilapia Oreochromis niloticus in Thailand. Surveillance of antimicrobial susceptibility in tilapia streptococcal pathogens reveals that Streptococcus agalactiae (n = 97) and Streptococcus iniae (n = 3) from diseased tilapia were susceptible to amoxicillin, florfenicol, sulfamethoxazole/trimethoprim and sulfadimethoxine/ormetoprim, however, only 78 isolates were susceptible to enrofloxacin. Twenty‐two enrofloxacin‐resistant S. agalactiae isolates were further examined for mutations in the QRDRs of gyrA, gyrB, parC and parE genes. Twenty isolates had single base pair changed in the gyrA sequence, C‐242‐T. Point mutations in gyrB, GC‐1135, 1136‐AA and T‐1466‐G, were identified in one isolate. All resistant isolates harboured a mutation in the parC gene, C‐236‐A, while no mutations were observed in the parE gene. The study represented mutations of gyrA and parC genes as marked modification of the enrofloxacin‐resistant S. agalactiae from farmed tilapia. This study is a primary report of the QRDRs mutations associated with fluoroquinolone resistance from streptococcal pathogen in the cultured fish. The phenotypic and genotypic characterization of enrofloxacin resistance S. agalactiae evident in this study has led to an improved regulation of antimicrobial use in Thai aquaculture.     

Induction and characterization of a lysogenic bacteriophage of Lactococcus garvieae isolated from marine fish species

This study investigated the presence of prophages in Lactococcus garvieae isolated from several marine fish species in Japan. Representative strains of 16 bacterial genotypes (S1–S16) selected from more than 400 L. garvieae isolates were used to induce lysogenic bacteriophages. These strains were treated with 500 ng mL^?1 freshly prepared mitomycin C. A cross‐spotting assay was performed to validate the lysogenic and indicator strains. The lysogenic strains were selected for isolation and concentration of the phages. Phage DNA was digested with EcoRI for biased sinusoidal field gel electrophoresis analysis. Polymerase chain reaction (PCR) was used to detect integrated prophage DNA. Of the 16 representative bacterial genotypes, 12 strains integrated prophages as indicated by the PCR assay, and 10 phages were detected and isolated using two indicator bacterial strains. Analysis of genomic DNA showed that these phages were homologous and named as PLgT‐1. Transmission electron microscopy revealed that the morphology of PLgT‐1 was consistent with the virus family Siphoviridae. PCR analysis of the prophage DNA revealed that all of the S1 genotype strains were lysogenic (30/30), but none of the S16 genotype strains were lysogenic (0/30). This is the first study to investigate lysogenic bacteriophages from L. garvieae.     

High prevalence of antimicrobial resistance in Escherichia coli,Salmonella spp. and Staphylococcus aureus isolated from fish samples in India

Antibiotic resistance, driven by inappropriate use of antimicrobial agents for fish farming, is an expanding global health threat. Demand of animal protein for human consumption is entraining at remarkable pace which in turn, upturning the non‐judicial use of life‐saving drugs in these modern animal production practices at an incautious rate. Hence, study was devised to observe the antimicrobial resistance (AMR) pattern of human prominence bacteria, against drugs of critically and highly importance of both human and veterinary medicine, circulating over the fish farming in India. One hundred sixty fish faecal samples were obtained from distinct geographical locales of Rajasthan, India. Overall, 202 isolates comprising of Escherichia coli (81, 51%), Salmonella spp. (72, 45%) and Staphylococcus aureus (49, 31%) were isolated to screen for resistance. Antimicrobial resistance has been analysed using broth microdilution method. In case of E. coli and Salmonella spp., highest resistance (>95%) was observed against streptomycin while for S. aureus, it was observed against trimethoprim, along with high resistance to other antibiotics. Majority of the isolates were found to be resistant to more than three antibiotics implying multi‐drug resistance (>89%) relative to the critical and highly important antibiotics. The high AMR is a reflection of misuse of these agents in the fish farming, which is a concern of serious health issues. Our findings persuade the prudent use of antimicrobial agents in fish farming and other aquaculture settings that may assist in drafting of the new policies for the judicial use of these life‐saving drugs in these sectors.     

The effect of dietary sodium chloride on some osmoregulatory parameters of the teleost, Oreochromis niloticus, after transfer from freshwater to seawater

The aim of this work was to determine the effects of supplemental dietary sodium chloride on salt water acclimation of tilapia Oreochromis niloticus. Fish were fed a basal diet supplemented with NaCl (8%) during three weeks in fresh water (FW) and then transferred to salt water (SW) at 15 and 20. Changes in plasma osmolality, chloride ion concentration (Cl^–), plasma level of cortisol and gill Na⁺, K⁺-ATPase activity were measured at 6, 12, 24, 48, 72 and 168 h after transfer to 15SW, while the higher strength SW group (20) was only monitored up to 24 h. Morphological changes in the gill mitochondria-rich (MR) cells were examined in relation to environmental salinity. The changes associated with dietary NaCl were sporadic and of small magnitude. The plasma osmolality and Cl^– increased immediately after transfer up to 12–24 h, but fish fed dietary salt (S) showed lower values than the control group (C). The S group showed higher plasma levels of cortisol than the control, which maintained its initial levels during the experiment. Gill Na⁺, K⁺-ATPase activity of the S group began to increase in the first hours after transfer, reaching maximum at 12 h and returned to basal level at 24 h, while the control group maintained basal levels. The differences between gill Na⁺, K⁺-ATPase activity of S and C fish were significant (p < 0.05) at 12 h. Transmission electron microscopy (TEM) revealed that MR cells in SW show more mitochondria and a more developed tubular system arising from the basolateral membrane. The MR cells of both groups frequently formed a multicellular complex in SW, consisting of a main MR and one or more accessory cells. Such complexes are rarely observed in FW. Some MR cells of fish fed supplemented dietary salt displayed convex apical membrane in FW.     

Vibrio parahaemolyticus Associated with Mass Mortality of Postlarval Abalone,Haliotis diversicolor supertexta (L.), in Sanya,China

Outbreaks of mass mortality in postlarval abalone, Haliotis diversicolor supertexta (L.), have swept across south China since 2002 and in turn have resulted in many abalone farms closing. Twenty‐five representative bacterial isolates were isolated from a sample of five diseased postlarval abalone, taken 15 d postfertilization during an outbreak of postlarval disease in Sanya, Hainan Province, China in October 2004. A dominant isolate, referred to as Strain 6, was found to be highly virulent to postlarvae in an experimental challenge test, with a 50% lethal dose (LD₅₀) value of 3.2 × 10⁴ colony forming units (CFU)/mL, while six of the other isolates were weakly virulent with LD₅₀ values ranging from 1 × 10⁶ to 1 × 10⁷ CFU/mL, and the remaining 18 isolates were classified as avirulent with LD₅₀ values greater than 1 × 10⁸ CFU/mL. Using both an API 20E kit and 16S recombinant DNA sequence analysis, Strain 6 was shown to be Vibrio parahaemolyticus. It was sensitive to 4 and intermediately sensitive to 5 of the 16 antibiotics used when screening the antibiotic sensitivities of the bacterium. Extracellular products (ECPs) prepared from the bacterium were lethal to postlarvae when used in a toxicity test at a concentration of 3.77 mg protein/mL, and complete liquefaction of postlarvae tissues occurred within 24 h postexposure. Results from this study implicate V. parahaemolyticus as the pathogen involved in the disease outbreaks in postlarval abalone in Sanya and show that the ECPs may be involved in the pathogenesis of the disease.     

Molecular characterization and pathogenicity of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis sp.) in Taiwan

Francisella noatunensis subsp. orientalis is a causative agent of systemic granulomatous disease in tilapia. The present study was designed to understand the genetic and phenotypic diversities among Taiwanese Fno isolates obtained from tilapia (n = 17) and green Texas cichlid (Herichthys cyanoguttatus) (n = 1). The enzymatic profiles of the isolates were studied using the API ZYM system. Phylogenetic tree analysis of the 16S rRNA and housekeeping gene and pulsed‐field gel electrophoresis (PFGE) were carried out to determine the genotypic characters of all isolates. The phylogenetic tree showed similarity of 99%–100% nucleotide sequences of 16S rRNA and housekeeping genes compared to the Fno references genes from GenBank database. Comparatively, the results revealed an identical profile of enzymatic and PFGE pattern which was distincted from that of F. philomiragia. To understand the pathogenicity, the isolates were intraperitoneal injected to tilapia the gross lesions were observed concomitant with natural outbreak. Median lethal dose upon Nile tilapia and red tilapia were 9.06 × 10³ CFU/fish and 2.08 × 10² CFU/fish, respectively. Thus, our data provide understanding the epidemiology of Taiwanese Fno isolates, and help in development of future control and prevention.