Occurrence and characterization of Aeromonas spp. in mussels from the Adriatic Sea.

Samples of mussels (Mytilus galloprovincialis) were collected from natural beds on the Adriatic Sea in front of Ancona Province (Marches, Central Italy) officially recognized for human consumption. The mussels were analyzed to evaluate the prevalence of Aeromonas spp. pathogenic strains. Out of 144 samples, 32 Aeromonas strains were isolated and 12 showed virulence and enterophatogenicity on mice. Isolates were also examined to check their sensitivity to different antimicrobial agents. All isolates were sensitive to imepenem, neomycin, ciprofloxacin, tetracycline, oxytetracycline, doxycicline, chloramphenicol, polymyxin B, and colistin sulfate, but resistant to penicillin, carbenicillin, cephalotin, sulfamethoxazole, lincomycin, novobiocin, and rifampicin. Multiple resistance to antimicrobial agents with different mechanism of action was observed. Owing to the high occurrence of pathogenic Aeromonas strains, the risk of developing human infections by eating raw or undercooked mussels is envisaged.

Isolation of Vagococcus salmoninarum in rainbow trout, Oncorhynchus mykiss (Walbaum), broodstocks: characterization of the pathogen.

Coldwater 'streptococcosis', caused by Vagococcus salmoninarum, is an emerging disease of rainbow trout in the European Union, causing mortality rates up to 50% in broodstock during the spawning period, with water temperature of 10-12 degrees C. A study to determine the presence and role of this bacterium was undertaken using classical bacteriological techniques confirmed with polymerase chain reaction. This is the first report of isolation of V salmoninarum in relation to outbreaks of mortality in a rainbow trout farm devoted exclusively to broodstock rearing in Spain. A total of 10 isolates of V salmoninarum were characterized by their morphological, cultural, physiological, biochemical and enzymatic traits. Some differences were observed in parameters such as growth on MacConkey agar, H2S production, acid production from starch, and some other minor variations. Isolates were sensitive to erythromycin and oxytetracycline tested in vitro, but treatments conducted in the field were ineffective. An attempt at vaccination did not provide encouraging results.

Streptococcus iniae type II infections in rainbow trout Oncorhynchus mykiss.

Clinical and pathological findings (anorexia, hemorrhage, lethargy, loss of orientation and exophthalmia) indicated that Streptococcus iniae type II is responsible for a fatal disease in rainbow trout. Histopathological findings revealed that S. iniae type II produces a systemic disease, including a diffuse necrotizing myositis. The distribution of viable bacteria in infected tissues substantiated the pathological findings, confirming that S. iniae type II is responsible for a generalized septic disease of rainbow trout.

Emerging pathologies in aquaculture: effects on production and food safety.

Infectious diseases represent a limiting factor for the further development of Italian aquaculture. The recent introduction and spreading of new pathogens, along with the global climatic change, has contributed to a considerable decrease in trout production. Emerging pathologies in rainbow trout culture include viral diseases, e.g. infectious haematopoietic necrosis (IHN), bacterial diseases, such as lactococcosis and visceral flavobacteriosis, and parasitical diseases, e.g. proliferative kidney disease (PKD). Higher mortality rates in trout fry and fingerlings are generally induced by visceral flavobacteriosis and IHN, while the main losses in large trout during the warm season are due to lactococcosis and PKD. Mariculture has at present a better sanitary status compared to trout culture, but a rapid dissemination of pathogens, including zoonosis agents, is envisaged also for seabass and seabream. Emerging pathologies in sea bass include VNN, pseudotuberculosis, streptococcosis and tuberculosis. Seabream is much more resistant and is mainly affected by novel Vibrio infections and enteromyxidiosis. A good sanitary management of fish farms is essential for avoiding or limiting losses caused by emerging pathologies. Transmission of zoonosis agents to man, through the consumption of cultured fish, is very remote in Italy. On the contrary, transmission of Streptococcus iniae, Vibrio vulnificus and Mycobacterium marinum by means of improper manipulation of infected fish, could represent a potential hazard for fish farmers and fish processors, as well as for people preparing fish meals.

Lactococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, but different diseases.

Clinical and macroscopic findings (anorexia, lethargy, loss of orientation and exophthalmia) indicate that Streptococcus iniae and Lactococcus garvieae infections of trout share some common features, but histopathology reveals notable differences between the 2 diseases. Meningitis and panophthalmitis are the main lesions among S. iniae infected trout, whereas L. garvieae infection results in a hyperacute systemic disease. Differences in the LD50s of the 2 pathogens and the sudden onset of signs and death correlate with the histopathological findings, indicating the severity of L. garvieae infection of trout.

Biodiversity of Lactococcus garvieae strains isolated from fish in Europe, Asia, and Australia.

Lactococcus garvieae (junior synonym, Enterococcus seriolicida) is a major pathogen of fish, producing fatal septicemia among fish species living in very diverse environments. The phenotypic traits of L. garvieae strains collected from three different continents (Asia, Europe, and Australia) indicated phenotypic heterogeneity. On the basis of the acidification of D-tagatose and sucrose, three biotypes were defined. DNA relatedness values and a specific PCR assay showed that all the biotypes belonged to the same genospecies, L. garvieae. All of the L. garvieae strains were serotyped as Lancefield group N. Ribotyping proved that one clone was found both in Japan, where it probably originated, and in Italy, where it was probably imported. PCR of environmental samples did not reveal the source of the contamination of the fish in Italy. Specific clones (ribotypes) were found in outbreaks in Spain and in Italy. The L. garvieae reference strain, isolated in the United Kingdom from a cow, belonged to a unique ribotype. L. garvieae is a rising zoonotic agent. The biotyping scheme, the ribotyping analysis, and the PCR assay described in this work allowed the proper identification of L. garvieae and the description of the origin and of the source of contamination of strains involved in outbreaks or in sporadic cases.

Identification of Lactococcus garvieae by PCR.

Lactococcus garvieae junior synonym Enterococcus seriolicida) is an emerging zoonotic agent isolated from economically important fish (rainbow trout and yellowtail), from cattle, and from humans. Clindamycin susceptibility is the only phenotypic test which can differentiate L. garvieae from Lactococcus lactis, another emerging agent in humans. A PCR assay for the identification of L. garvieae was developed and resulted in an amplified fragment of 1,100 bp in size. The PCR assay was shown to be specific to L. garvieae. The PCR assay was positive for all the L. garvieae strains tested, which originated from three different continents (Asia, Australia, and Europe). The PCR assay was negative for the phenotypically similar L. lactis and for all the other fish pathogens tested, including Streptococcus iniae and Aeromonas salmonicida. The PCR assay was applied to plasma obtained from diseased animals and was found sensitive enough to detect bacteria from 1 microl of plasma. The PCR assay that was developed is the only practical test besides the clindamycin test which can specifically identify the zoonotic agent L. garvieae and which can differentiate it from L. lactis.

Immunization with bacterial antigens: infections with streptococci and related organisms.

Streptococcal infections of fish have been reported from various parts of the world, including the Far East, the United States, South Africa, Australia, Israel and Europe. Classification of Gram-positive cocci (DNA-DNA hybridization studies coupled with 165 sequencing) has shown that at least five different defined species are pathogenic to fish, e.g. Streptococcus iniae (syn. S. shilot), Streptococcus difficile, Lactococcus garvieae (syn. Enterococcus seriolicida), Lactococcus piscium and Vagococcus salmoninarum. "Streptococcosis" of fish should therefore be regarded as a complex of similar diseases caused by different genera and species of Gram-positive cocci, each capable of inducing CNS damage, as well as various degrees of multisystem organ involvement. Panophthalmitis ("pop-eye") and meningitis/meningoencephalitis are the sole findings in trout infected by S. iniae and in tilapines infected by S. difficile. In contrast, L. garvieae-infected trout bear a systemic hyperacute infection with diffuse haemorrhages. Therapeutic measures are generally ineffective. Development of vaccines is therefore essential to control these diseases. In our studies, trout were vaccinated intraperitoneally with whole-cell formalin-inactivated S. iniae and L. garvieae and tilapines with whole-cell formalin-inactivated and acellular S. difficile extract. Under laboratory conditions, S. difficile-vaccinated tilapines were protected against a challenge of 100 LD50s. Protection was correlated with the development of specific agglutinins. Western blot analysis supported the hypothesis that only a few proteins act as protective antigens. S. iniae autovaccines were effective in preventing the disease in rainbow trout in Israel. Under field conditions, fish vaccinated at 50 g were protected for over four months. The qualitative analysis of the humoral response indicated that specific antibodies are directed against a few protein moieties. The fact that passive transfer of antibodies protected fish from experimental infection suggests that the basic mechanism of protection is antibody mediated. L. garvieae autovaccines developed for Italian trout farming were found to elicit a response similar to that of S. iniae. Despite the high virulence of L. garvieae (LD50 of 6 x 10(1) CFU/fish, compared with 3 x 10(4) CFU/fish of S. iniae), the protection against the experimentally induced disease lasted for five months under laboratory conditions, with survival rates of 80-90%. A single injection of the vaccine (0.1 ml/fish) resulted in specific antibody production detectable for six months. In the field, protection rates of 70-80% were obtained for a period of three months, in fish of 200-300 g reared at water temperatures of 18-21 degrees C.

Enterococcus seriolicida is a junior synonym of Lactococcus garvieae, a causative agent of septicemia and meningoencephalitis in fish.

The reference strains of Enterococcus seriolicida (ATCC 49156T) (T = type strain) and of Lactococcus garvieae (ATCC 43921T) and 30 field strains of Gram-positive cocci isolated from diseased rainbow trout in Italy were found to be phenotypically (API 20 STREPT and API 50 CH) and genetically (DNA-DNA hybridization) similar. The high DNA-DNA homologies (70-100%) and the low delta Tme (less than 1.1 degrees C) among these strains showed that Enterococcus seriolicida and Lactococcus garvieae are synonyms, describing a single bacterial species. E. seriolicida strains should be classified as L. garvieae, which must be considered as a major pathogen of freshwater and salt water fish with a world-wide distribution.

Identification of the newly described Mycobacterium poriferae from tuberculous lesions of snakehead fish (Channa striatus).

A mycobacterium isolated form a cultured snakehead with nodular lesions was identified on the basis of high performance liquid chromatography (HPLC) profile of cell wall mycolic acids, and confirmed by conventional tests, as Mycobacterium poriferae, a species previously isolated only from a marine sponge. The profiles of M. poriferae, Mycobacterium aurum and Mycobacterium parafortuitum are here reported for the first time.