Isolation and characterization of Edwardsiella ictaluri strains as pathogens from diseased yellow catfish Pelteobagrus fulvidraco (Richardson) cultured in China

Yellow catfish Pelteobagrus fulvidraco (Richardson) is a commercially important fish generally distributed in Southeast Asian countries. The well-known aetiological agent of enteric septicaemia of catfish, Edwardsiella ictaluri, was isolated from diseased yellow catfish P. fulvidraco (Richardson) reared at two commercial fisheries in China. The economic losses due to the high mortalities (about 50%) caused by this bacterium have been increasing annually. The affected fish presented two different, typical symptoms: pale gills, slight exophthalmia and a 'hole in the head', and haemorrhage on the opercula, in the skin under the jaw, creating a 'hole under the jaw'. These diseases were found frequently in cultured yellow catfish throughout China. The isolates from both outbreaks were all Gram negative, facultatively anaerobic and short rod. Morphological and biochemical tests and phylogenetic analysis based on the 16S rDNA sequences all strongly indicated that these yellow catfish isolates were highly identical to the known E. ictaluri. In addition, the isolates possessed the typical plasmid profile of E. ictaluri. Experimental infection assays were conducted and pathogenicity (by an intraperitoneal injection) was demonstrated in yellow catfish and channel catfish Ictalurus punctatus. The results showed that yellow catfish isolates were quite conservative pheno-typically and genetically, and were able to cause two different, typical symptoms in this fish under unknown conditions and mechanism

First case of Aeromonas schubertii infection in the freshwater cultured snakehead fish, Ophiocephalus argus (Cantor), in China

An epizootic in snakehead fish, Ophiocephalus argus, in earthen ponds in Xianning, Hubei Province, central China, from June to August 2009 was found to be caused by Aeromonas schubertii. The cumulative mortality within 40 days was 45%, and the diseased fish were 18 months old and 3545 cm in length. Multiple, ivory-white, firm nodules, 0.51 mm in diameter, were scattered throughout the kidney. Blood clots, 35 mm in diameter, were found in the liver. This is a disease frequently found in cultured snakehead throughout China. Isolated bacteria were Gram negative, facultatively anaerobic, motile, short rod-shaped, with a length of 0.31.0 mu m. Morphological and biochemical tests, as well as phylogenetic analysis derived from 16S rRNA, gyrB, rpoD and dnaJ gene sequencing all strongly indicated that these snakehead isolates are identical to A.similar to chubertii. In addition, the isolates possessed two plasmids: 5.0 kb and 10.0 kb. Antibiotic sensitivity testing of the isolates was carried out by the standard KirbyBauer disc diffusion method. Experimental infection assays were conducted, and pathogenicity (by intraperitoneal injection) was demonstrated in snakehead fingerlings and zebrafish, Brachydanio rerio (Hamilton).An epizootic in snakehead fish, Ophiocephalus argus, in earthen ponds in Xianning, Hubei Province, central China, from June to August 2009 was found to be caused by Aeromonas schubertii. The cumulative mortality within 40 days was 45%, and the diseased fish were 18 months old and 3545 cm in length. Multiple, ivory-white, firm nodules, 0.51 mm in diameter, were scattered throughout the kidney. Blood clots, 35 mm in diameter, were found in the liver. This is a disease frequently found in cultured snakehead throughout China. Isolated bacteria were Gram negative, facultatively anaerobic, motile, short rod-shaped, with a length of 0.31.0 mu m. Morphological and biochemical tests, as well as phylogenetic analysis derived from 16S rRNA, gyrB, rpoD and dnaJ gene sequencing all strongly indicated that these snakehead isolates are identical to A.similar to chubertii. In addition, the isolates possessed two plasmids: 5.0 kb and 10.0 kb. Antibiotic sensitivity testing of the isolates was carried out by the standard KirbyBauer disc diffusion method. Experimental infection assays were conducted, and pathogenicity (by intraperitoneal injection) was demonstrated in snakehead fingerlings and zebrafish, Brachydanio rerio (Hamilton)

Isolation and characterization of Streptococcus dysgalactiae from diseased Acipenser schrenckii

Streptococcosis became an increasingly significant health problem in intensive aquaculture in China. Fifteen strains of Gram-positive, chain-forming coccus were isolated from moribund Amur sturgeon, Acipenser schrenckii, fanned with high density in central China. The coccoid microorganism was identified as Streptococcus dysgalactiae by means of physiological. biochemical properties and molecular analysis; furthermore, this coccus was confirmed as pathogen of sturgeon by challenge experiments and its infection potential on the cyprinid was also evaluated. To our knowledge, this was the first report of S. dysgalactiae linked to diseased A. schrenckii. (C) 2009 Elsevier B.V. All rights reserved.Streptococcosis became an increasingly significant health problem in intensive aquaculture in China. Fifteen strains of Gram-positive, chain-forming coccus were isolated from moribund Amur sturgeon, Acipenser schrenckii, fanned with high density in central China. The coccoid microorganism was identified as Streptococcus dysgalactiae by means of physiological. biochemical properties and molecular analysis; furthermore, this coccus was confirmed as pathogen of sturgeon by challenge experiments and its infection potential on the cyprinid was also evaluated. To our knowledge, this was the first report of S. dysgalactiae linked to diseased A. schrenckii. (C) 2009 Elsevier B.V. All rights reserved

First description of a novel Weissella species as an opportunistic pathogen for rainbow trout Oncorhynchus mykiss (Walbaum) in China

Six strains of Gram-positive, catalase-negative, non-motile, irregular short rod-shaped Weissella bacteria, with width and length of 0.5-0.6 and 1.2-2.7 mu m were isolated from diseased rainbow trout Oncorhynchus mykiss (Walbaum) in winter of 2007 at a commercial fishery in Jingmen, Hubei province, China. The diseased rainbow trout exhibited hemorrhage in eyes, anal region, intestine and abdomen wall, petechia of liver, some fish with hydrocele in stomach. Six isolates had identical biochemical reactions, phylogenetic analysis of 16S rDNA sequences, amplified ribosomal DNA restriction analysis (ARDRA), enzymatic profile analysis and antimicrobial susceptibility results, indicating as a single clonal outbreak. But all were different from any other validated twelve Weissella species in the term of physiological and biochemical characters. It is indicated that isolates are phylogenetically closer to Weissella halotolerans, Weissella viridescens and Weissella minor on 16S rDNA phylogenetic analysis result, than to W halotolerans and W viridescens on the result of ARDRA study and enzymatic profile analysis. Antimicrobial susceptibility testing was used to scan effective drugs for the therapy of this disease. Experimental infection assays with one isolate were conducted and pathogenicity (by intraperitoneal injection) was demonstrated in rainbow trout O. mykiss (Walbaum) and crucian carp (Carassius auratus gibelio) fingerlings. Because no Weissella was detected in fish feedstuffs and pond water, the source of this pathogen remains unknown, and Weissella isolates were regarded as an opportunistic pathogen for rainbow trout. This is the first report of Weissella strains which can cause disease of cultured fish in the world. (C) 2009 Elsevier B.V. All rights reserved.Six strains of Gram-positive, catalase-negative, non-motile, irregular short rod-shaped Weissella bacteria, with width and length of 0.5-0.6 and 1.2-2.7 mu m were isolated from diseased rainbow trout Oncorhynchus mykiss (Walbaum) in winter of 2007 at a commercial fishery in Jingmen, Hubei province, China. The diseased rainbow trout exhibited hemorrhage in eyes, anal region, intestine and abdomen wall, petechia of liver, some fish with hydrocele in stomach. Six isolates had identical biochemical reactions, phylogenetic analysis of 16S rDNA sequences, amplified ribosomal DNA restriction analysis (ARDRA), enzymatic profile analysis and antimicrobial susceptibility results, indicating as a single clonal outbreak. But all were different from any other validated twelve Weissella species in the term of physiological and biochemical characters. It is indicated that isolates are phylogenetically closer to Weissella halotolerans, Weissella viridescens and Weissella minor on 16S rDNA phylogenetic analysis result, than to W halotolerans and W viridescens on the result of ARDRA study and enzymatic profile analysis. Antimicrobial susceptibility testing was used to scan effective drugs for the therapy of this disease. Experimental infection assays with one isolate were conducted and pathogenicity (by intraperitoneal injection) was demonstrated in rainbow trout O. mykiss (Walbaum) and crucian carp (Carassius auratus gibelio) fingerlings. Because no Weissella was detected in fish feedstuffs and pond water, the source of this pathogen remains unknown, and Weissella isolates were regarded as an opportunistic pathogen for rainbow trout. This is the first report of Weissella strains which can cause disease of cultured fish in the world. (C) 2009 Elsevier B.V. All rights reserved

Mixed mycobacterial infections in farmed sturgeons

Based on microbiological and histopathological examinations and DNA sequencing, several outbreaks of mycobacteriosis in the reared sturgeons, including Chinese sturgeon (Acipenser sinensis Gray) and Amur sturgeon (Acipenser schrencki), were identified during 2009 to 2010. Forty-nine isolates of non-tuberculous mycobacteria(NTM)were isolated from 19 diseased sturgeons. In total, seven species of Mycobacterium were identified, namely, Mycobacterium chelonae, Mycobacterium marinum, Mycobacterium gordonae, Mycobacterium fortuitum, Mycobacterium szulgai, Mycobacterium arupense and Mycobacterium porcinum. Among them, M. marinum was found to be more prevalent (89.5%) compared with the other mycobacterial species. When two molecular biological methods, PCR-DGGE (denaturing gradient gel electrophoresis) analysis and rpoB gene library sequencing, were used to analyse the mycobacterial DNAs extracted from the diseased fish tissues, mixed infections of two or three mycobacterial species were found being the predominant infection form (94.7%) in sturgeon mycobacteriosis. M. marinum was the only one species that caused sturgeon mycobacteriosis alone. Virulence assay showed that M. marinum possessed stronger pathogenicity to zebrafish killing 100% of fish in 28days at 10(3) cfu/fish than the other species. These results suggested that M. marinum is the major pathogenic bacteria in sturgeon mycobacteriosis. To the best of our knowledge, this study is the first report on mycobacteriosis in farmed Chinese and Amur sturgeons as well as the first isolation of M. porcinum and M. arupense from fish

Humoral immune responses of the grouper Eninephelus akaara against the microsporidium Glugea epinephelusis

The humoral immune responses of grouper Epinephelus akaara to a natural infection with Glugea epinephelusis was studied by ELISA utilizing intact mature spores as the coated antigen. Results showed that a specific humoral immune response was elicited, but the intensity of infection (in terms of the number of cysts) was not related to the antibody level in naturally infected hosts. The differences in the antigenicity of intact mature spores and soluble spore proteins derived from cracked mature spores were also analyzed. Results suggested that similar antigen epitopes existed between the 2 groups. Additionally, antigen component patterns and the distribution of antigen with immunogenicity were investigated by using the western blot and the immunofluorescent antibody technique (IFAT). The new parasitic microsporidium has specific polypeptide patterns comparable to the reported fish microsporidians. The main antigenic substances are concentrated on the surface of spores, and are mostly located on the anterior and posterior end of the spore bodies. Most surface components of the G. epinephelusis spores are soluble, The potential role of the surface components in initiating infection was also discussed.The humoral immune responses of grouper Epinephelus akaara to a natural infection with Glugea epinephelusis was studied by ELISA utilizing intact mature spores as the coated antigen. Results showed that a specific humoral immune response was elicited, but the intensity of infection (in terms of the number of cysts) was not related to the antibody level in naturally infected hosts. The differences in the antigenicity of intact mature spores and soluble spore proteins derived from cracked mature spores were also analyzed. Results suggested that similar antigen epitopes existed between the 2 groups. Additionally, antigen component patterns and the distribution of antigen with immunogenicity were investigated by using the western blot and the immunofluorescent antibody technique (IFAT). The new parasitic microsporidium has specific polypeptide patterns comparable to the reported fish microsporidians. The main antigenic substances are concentrated on the surface of spores, and are mostly located on the anterior and posterior end of the spore bodies. Most surface components of the G. epinephelusis spores are soluble, The potential role of the surface components in initiating infection was also discussed

Molecular characterization of Streptococcus agalactiae in diseased farmed tilapia in China

A severe outbreak of streptococcosis in cultured tilapia (Oreochromis niloticus) caused by Streptococcus agalactiae (Group B streptococcus, GBS) has occurred annually in Southern China during the past years, causing significant economic losses. Little is known about the genetic characteristics of the tilapia S. agalactiae that is prevalent in these areas. A total of 51 GBS isolates from tilapia were collected from 27 farms located in distinct geographic regions. The genetic characteristics of GBS were analyzed by MLST, MLVA, serotyping, and PCR screening of mobile genetic elements, genetic marker, and virulence-related genes. The results showed that all of S. agalactiae tilapia isolates have the genotype, Ia-ST7-bac-bca-fbsA-sip-cfb-IS1381-IS861-GBSil-ISSag2, which suggests a low level of genetic diversity and sharing of a recent and similar origin and a low level of genetic differentiation under similar environment selective pressures. Compared with the control, significant differences were detected between the tilapia strains and the S. agalactiae strains of human and bovine origin. Phage typing successfully differentiated all 51 tilapia GBS isolates into two distinct molecular types (type A and B). Interestingly, the phage type of the tilapia GBS isolates was also observed to shift from type A to type B, with the year 2010 as the turning point. Type B GBS isolates are currently prevalent in tilapia populations in China, and should thus be the target strains to detect and develop prophylactic-therapeutic measures. The genetic data obtained in the present study will be helpful in the determination of the epidemiology of tilapia streptococcosis internationally. (C) 2013 Elsevier B. V. All rights reserved.A severe outbreak of streptococcosis in cultured tilapia (Oreochromis niloticus) caused by Streptococcus agalactiae (Group B streptococcus, GBS) has occurred annually in Southern China during the past years, causing significant economic losses. Little is known about the genetic characteristics of the tilapia S. agalactiae that is prevalent in these areas. A total of 51 GBS isolates from tilapia were collected from 27 farms located in distinct geographic regions. The genetic characteristics of GBS were analyzed by MLST, MLVA, serotyping, and PCR screening of mobile genetic elements, genetic marker, and virulence-related genes. The results showed that all of S. agalactiae tilapia isolates have the genotype, Ia-ST7-bac-bca-fbsA-sip-cfb-IS1381-IS861-GBSil-ISSag2, which suggests a low level of genetic diversity and sharing of a recent and similar origin and a low level of genetic differentiation under similar environment selective pressures. Compared with the control, significant differences were detected between the tilapia strains and the S. agalactiae strains of human and bovine origin. Phage typing successfully differentiated all 51 tilapia GBS isolates into two distinct molecular types (type A and B). Interestingly, the phage type of the tilapia GBS isolates was also observed to shift from type A to type B, with the year 2010 as the turning point. Type B GBS isolates are currently prevalent in tilapia populations in China, and should thus be the target strains to detect and develop prophylactic-therapeutic measures. The genetic data obtained in the present study will be helpful in the determination of the epidemiology of tilapia streptococcosis internationally. (C) 2013 Elsevier B. V. All rights reserved