Arbitrarily primed PCR to type Vibrio spp. pathogenic for shrimp.
A molecular typing study on Vibrio strains implicated in shrimp disease outbreaks in New Caledonia and Japan was conducted by using AP-PCR (arbitrarily primed PCR). It allowed rapid identification of isolates at the genospecies level and studies of infraspecific population structures of epidemiological interest. Clusters identified within the species Vibrio penaeicida were related to their area of origin, allowing discrimination between Japanese and New Caledonian isolates, as well as between those from two different bays in New Caledonia separated by only 50 km. Other subclusters of New Caledonian V. penaeicida isolates could be identified, but it was not possible to link those differences to accurate epidemiological features. This contribution of AP-PCR to the study of vibriosis in penaeid shrimps demonstrates its high discriminating power and the relevance of the epidemiological information provided. This approach would contribute to better knowledge of the ecology of Vibrio spp. and their implication in shrimp disease in aquaculture. (+info
Natural mass infection by heterophyid metacercariae in aquacultured Japanese eel in Taiwan.
A natural mass infection of heterophyid metacercariae in aquacultured Japanese eel Anguilla japonica in Taiwan was observed. Of the 28,000 adult eels in 2 ponds, about 25,000 (90%) showed swollen, cloudy and white eyes. Although morbidity was about 90%, there was no mortality among the affected eels. Histopathological sections showed edema and hemorrhage of the eye. Numerous metacercariae were observed in the muscle tissues around the eyeball, the subcutaneous tissue and even in the cartilage. Of the 6 eels digested with artificial gastric juice, all were found to contain metacercariae in their muscle tissues. The average number of metacercariae recovered from the 6 eels was 1219, with a range of 50 to 3762. These metacercariae, when fed orally to immunodeficient (scid) mice, developed into adult worms which were identified as Procerovum cheni Hsu 1950. The naturally infected eels were transferred to a new pond without snails and their eye lesions were not apparent anymore after 2 wk. In a follow-up investigation, 19 of 20 apparently healthy eels in a nearby aquaculture farm were found to harbour metacercariae in their muscles. However, the number of the metacercariae ranged from 1 to 14, with an average of 4.21. This is the first report of heterophyid metacercariae causing mass morbidity in aquacultured eels. (+info
Isolation and characterization of Vibrio parahaemolyticus causing infection in Iberian toothcarp Aphanius iberus.
High mortality among laboratory cultured Iberian toothcarp Aphanius iberus occurred in February 1997 in Valencia (Spain). The main signs of the disease were external haemorrhage and tail rot. Bacteria isolated from internal organs of infected fish were biochemically homogeneous and identified as Vibrio parahaemolyticus. The bacteria were haemolytic against erythrocytes from eel Anguilla anguilla, amberjack Seriola dumerili, toothcarp A. iberus and humans, and were Kanagawa-phenomenon-negative. Infectivity tests showed that the virulence for A. iberus was dependent on salinity. Finally, all strains were virulent for amberjack and eel. (+info
A yellow head virus gene probe: nucleotide sequence and application for in situ hybridization.
A portion of the genome of yellow head virus (YHV) of penaeid shrimp was cloned and the cDNA fragment (1161 bp) was designated clone 3-27. The fragment was labeled with digoxigenin and hybridized in situ to tissue sections of YHV-infected Penaeus vannamei. Positively reacting tissues included those of the lymphoid organ, cuticular epithelium, and gills. In addition, connective tissue of hepatopancreas, heart, antennal gland, hematopoietic organ, nerve tract, midgut cecum and muscle reacted to the probe. The probe was highly specific since it hybridized only to tissues from YHV-infected shrimp. It did not react to those of uninfected shrimp or shrimp infected with WSSV (white spot syndrome virus), IHHNV (infectious hypodermal and hematopoietic necrosis virus), or TSV (Taura syndrome virus). The clone was sequenced, and primers were synthesized for rapid detection of YHV in hemolymph using RT-PCR (reverse transcription-polymerase chain reaction). The strand that constituted the viral sequence in the cDNA was also determined via RT-PCR and in situ hybridization with a single-stranded RNA (ssRNA) probe. (+info
Indole-positive Vibrio vulnificus isolated from disease outbreaks on a Danish eel farm.
Vibrio vulnificus was isolated in 1996 from 2 disease outbreaks on a Danish eel farm which used brackish water. A characteristic clinical sign was extensive, deep muscle necrosis in the head region. V. vulnificus was isolated from kidney, mucus, spleen, gill and intestine of diseased eels. Thirty-two isolates were examined phenotypically and serologically for pathogenicity to eels and for correlation to ribotype and plasmid profile. Biochemically, the isolates showed properties similar to those described previously for eel-pathogenic strains of V. vulnificus, with the exception of indole production. Virulence was evaluated by LD50 (the 50% lethal dose), which ranged from < 9.4 x 10(3) to 2.3 x 10(5) CFU (colony-forming units) per fish. The isolates which were lethal for eels showed identical ribotypes and serotypes. A relationship between certain plasmids and virulence was not found. A serotyping system based on lipopolysaccharide (LPS)-associated O antigen type and on carbohydrate capsule antigens showed that the eel-virulent isolates shared a common LPS-based homogeneous O serogroup and a capsule antigen. V. vulnificus serovar O4 and capsule type 9 was identical serologically to the Japanese isolate ATCC 33149 and was the agent responsible for the disease outbreaks that occurred on the Danish eel farm. Despite absence of antibiotic resistance, treatment had little effect and disease reoccurred. (+info
Vibrios associated with Litopenaeus vannamei larvae, postlarvae, broodstock, and hatchery probionts.
Several bacteriological surveys were performed from 1994 to 1996 at different Litopenaeus vannamei hatcheries (in Ecuador) and shrimp farms (in Mexico). Samples were taken from routine productions of healthy and diseased L. vannamei larvae, postlarvae, and their culture environment and from healthy and diseased juveniles and broodstock. In Ecuador, the dominant bacterial flora associated with shrimp larvae showing symptoms of zoea 2 syndrome, mysis mold syndrome, and bolitas syndrome has been determined. Strains were characterized by Biolog metabolic fingerprinting and identified by comparison to a database of 850 Vibrio type and reference strains. A selection of strains was further genotypically fine typed by AFLP. Vibrio alginolyticus is predominantly present in all larval stages and is associated with healthy nauplius and zoea stages. AFLP genetic fingerprinting shows high genetic heterogeneity among V. alginolyticus strains, and the results suggest that putative probiotic and pathogenic strains each have specific genotypes. V. alginolyticus was found to be associated with larvae with the zoea 2 syndrome and the mysis mold syndrome, while different Vibrio species (V. alginolyticus and V. harveyi) are associated with the bolitas syndrome. V. harveyi is associated with diseased postlarvae, juveniles, and broodstock. The identities of the strains identified as V. harveyi by the Biolog system could not be unambiguously confirmed by AFLP genomic fingerprinting. Vibrio strain STD3-988 and one unidentified strain (STD3-959) are suspected pathogens of only juvenile and adult stages. V. parahaemolyticus, Photobacterium damselae, and V. mimicus are associated with juvenile and adult stages. (+info
Gill lamellar pillar cell necrosis, a new birnavirus disease in Japanese eels.
Since the late 1980s, a birnaviral gill disease has been occurring in Japanese eels Anguilla japonica reared in warmwater ponds in western regions in Japan. Diseased eels mostly displayed marked formations of aneurysmal hematomas within gill lamellae and high mortalities. Histological examination revealed necrosis of pillar cells and subsequent aggregation of erythrocytes inside the lamellar capillaries, and proliferation of interlamellar epithelia onto the lamellae. Gastric gland cells were also necrotized. Electron microscopy revealed birnavirus infection in lamellar pillar cells. The causative birnavirus was isolated and cultured in fish cell lines and was found to be related to an infectious pancreatic necrosis virus (IPNV) Sp serotype by neutralization tests. The viral pathogenicity was confirmed by the results of histopathological examinations and infectivity experiments. (+info
Routes of entry of Piscirickettsia salmonis in rainbow trout Oncorhynchus mykiss.
Since 1989, Piscirickettsia salmonis, the causal agent of piscirickettsiosis, has killed millions of farmed salmonids each year in southern Chile. The portal of entry for the pathogen was investigated by use of selected experimental infections in juvenile rainbow trout (12 g). The methods used were intraperitoneal injection, subcutaneous injection, patch contact on skin, patch contact on gills, intestinal intubation and gastric intubation. Cumulative mortalities at Day 33 post-inoculation were 98, 100, 52, 24, 24, and 2%, respectively. It was shown that intact skin and gills could be penetrated by P. salmonis. The high mortality obtained in subcutaneously injected fish indicated that skin injuries could facilitate the invasion of this pathogen. Results suggested that the main entry sites are through the skin and gills and that the oral route may not be the normal method by which P. salmonis initiates infection of salmonids. (+info