Seasonal Dynamics Are the Major Driver of Microbial Diversity and Composition in Intensive Freshwater Aquaculture.

Aquaculture facilities such as fishponds are one of the most anthropogenically impacted freshwater ecosystems. The high fish biomass reared in aquaculture is associated with an intensive input into the water of fish-feed and fish excrements. This nutrients load may affect the microbial community in the water, which in turn can impact the fish health. To determine to what extent aquaculture practices and natural seasonal cycles affect the microbial populations, we characterized the microbiome of an inter-connected aquaculture system at monthly resolution, over 3 years. The system comprised two fishponds, where fish are grown, and an operational water reservoir in which fish are not actively stocked. Clear natural seasonal cycles of temperature and inorganic nutrients concentration, as well as recurring cyanobacterial blooms during summer, were observed in both the fishponds and the reservoir. The structure of the aquatic bacterial communities in the system, characterized using 16S rRNA sequencing, was explained primarily by the natural seasonality, whereas aquaculture-related parameters had only a minor explanatory power. However, the cyanobacterial blooms were characterized by different cyanobacterial clades dominating at each fishpond, possibly in response to distinct nitrogen and phosphate ratios. In turn, nutrient ratios may have been affected by the magnitude of fish feed input. Taken together, our results show that, even in strongly anthropogenically impacted aquatic ecosystems, the structure of bacterial communities is mainly driven by the natural seasonality, with more subtle effects of aquaculture-related factors.

Temporal Resistome and Microbial Community Dynamics in an Intensive Aquaculture Facility with Prophylactic Antimicrobial Treatment.

Excessive use of antimicrobials in aquaculture is concerning, given possible environmental ramifications and the potential contribution to the spread of antimicrobial resistance (AR). In this study, we explored seasonal abundance of antimicrobial resistance genes and bacterial community composition in the water column of an intensive aquaculture pond stocked with Silver Carp (Hypophthalmichthys molitrix) prophylactically treated with sulfamethoprim (25% sulfadiazine; 5% trimethoprim), relative to an adjacent unstocked reservoir. Bacterial community composition was monitored using high-throughput sequencing of 16S rRNA gene amplicons in eight sampling profiles to determine seasonal dynamics, representing principal stages in the fish fattening cycle. In tandem, qPCR was applied to assess relative abundance of selected antimicrobial resistance genes (sul1, sul2, dfrA1, tetA and blaTEM) and class-1 integrons (int1). Concomitantly, resistomes were extrapolated from shotgun metagenomes in representative profiles. Analyses revealed increased relative abundance of sulfonamide and tetracycline resistance genes in fishpond-03, relative to pre-stocking and reservoir levels, whereas no significant differences were observed for genes encoding resistance to antimicrobials that were not used in the fishpond-03. Seasons strongly dictated bacterial community composition, with high abundance of cyanobacteria in summer and increased relative abundance of Flavobacterium in the winter. Our results indicate that prophylactic use of sulfonamides in intensive aquaculture ponds facilitates resistance suggesting that prophylactic use of these antimicrobials in aquaculture should be restricted.

Evidence of Increased Antibiotic Resistance in Phylogenetically-Diverse Aeromonas Isolates from Semi-Intensive Fish Ponds Treated with Antibiotics.

The genus Aeromonas is ubiquitous in aquatic environments encompassing a broad range of fish and human pathogens. Aeromonas strains are known for their enhanced capacity to acquire and exchange antibiotic resistance genes and therefore, are frequently targeted as indicator bacteria for monitoring antimicrobial resistance in aquatic environments. This study evaluated temporal trends in Aeromonas diversity and antibiotic resistance in two adjacent semi-intensive aquaculture facilities to ascertain the effects of antibiotic treatment on antimicrobial resistance. In the first facility, sulfadiazine-trimethoprim was added prophylactically to fingerling stocks and water column-associated Aeromonas were monitored periodically over an 11-month fish fattening cycle to assess temporal dynamics in taxonomy and antibiotic resistance. In the second facility, Aeromonas were isolated from fish skin ulcers sampled over a 3-year period and from pond water samples to assess associations between pathogenic strains to those in the water column. A total of 1200 Aeromonas isolates were initially screened for sulfadiazine resistance and further screened against five additional antimicrobials. In both facilities, strong correlations were observed between sulfadiazine resistance and trimethoprim and tetracycline resistances, whereas correlations between sulfadiazine resistance and ceftriaxone, gentamicin, and chloramphenicol resistances were low. Multidrug resistant strains as well as sul1, tetA, and intI1 gene-harboring strains were significantly higher in profiles sampled during the fish cycle than those isolated prior to stocking and these genes were extremely abundant in the pathogenic strains. Five phylogenetically distinct Aeromonas clusters were identified using partial rpoD gene sequence analysis. Interestingly, prior to fingerling stocking the diversity of water column strains was high, and representatives from all five clusters were identified, including an A. salmonicida cluster that harbored all characterized fish skin ulcer samples. Subsequent to stocking, diversity was much lower and most water column isolates in both facilities segregated into an A. veronii-associated cluster. This study demonstrated a strong correlation between aquaculture, Aeromonas diversity and antibiotic resistance. It provides strong evidence for linkage between prophylactic and systemic use of antibiotics in aquaculture and the propagation of antibiotic resistance.

Antibody response and resistance of Cyprinus carpio immunized with cyprinid herpes virus 3 (CyHV-3).

CyHV-3 (Cyprinid herpesvirus-3) is a large DNA virus that causes a fatal disease in koi and common carp. Infection with wild type or attenuated virus induces an immune response that renders the fish resistant to further virus challenges. The kinetics and affinity of the antibody response in immune fish depend on the temperature of the water. Virus-inoculated fish produce anti-CyHV-3 antibodies, which gradually decrease during 280 days post infection to a level slightly above that of naïve fish. The protection against the virus is proportional to the titer of anti-virus antibodies in recently inoculated fish. Nevertheless, these immunized fish, even with no-longer detectable antibodies, are resistant to virus infection, probably due to the subsequent rapid response of high affinity anti-virus antibodies. The fact that anti-virus antibodies neutralize in vitro the pathogenic effects of the virus emphasizes the central role probably played by the antibodies in anti-CyHV-3 protection in vivo.

Detection of carp interstitial nephritis and gill necrosis virus in fish droppings.

Carp interstitial nephritis and gill necrosis virus (CNGV) is an unclassified large DNA virus that morphologically resembles members of the Herpesviridae but contains a large (ca. approximately 280-kbp) linear double-stranded DNA. This virus has also been named koi herpesvirus, koi herpes-like virus, and cyprinid herpesvirus 3. CNGV is the cause of a lethal disease that afflicts common carp and koi. By using immunohistochemistry, molecular analysis, and electron microscopy we previously demonstrated that this virus is present mainly in the intestine and kidney of infected fish. Based on these observations, we postulated that viruses and/or viral components may appear in droppings of infected carp. Here we report that (i) by using PCR we demonstrated that fish droppings contain viral DNA, (ii) fish droppings contain viral antigens which are useful for CNGV diagnosis, and (iii) fish droppings contain active virus which can infect cultured common carp brain cells and induce the disease in naïve fish following inoculation. Thus, our findings show that CNGV can be identified by using droppings without taking biopsies or killing fish and that infectious CNGV is present in the stools of sick fish. The possibility that fish droppings preserve viable CNGV during the nonpermissive seasons is discussed.

Protection of cultured Cyprinus carpio against a lethal viral disease by an attenuated virus vaccine.

Massive mortality of koi and common carp--Cyprinus carpio species--has been observed since 1998 in many countries worldwide, resulting in severe economic losses. The cause of the disease is an as yet unclassified large DNA virus, designated carp nephritis gill necrosis virus (CNGV) or koi herpes virus (KHV). Previously, we demonstrated that the wild type CNGV lost its pathogenecity following serial transfer in cell culture, and that clones isolated from the attenuated population can be used as a prophylactic vaccine. Here, we describe the basic conditions required for proper fish immunization so that a protection protocol may be devised. We demonstrated that carps are very sensitive to the pathogenic and the attenuated viruses, and short immersion of fish in water containing the viruses is sufficient for infection. The infection of fish with the pathogenic and the attenuated viruses is temperature-restricted; fish held at the non-permissive temperature, immediately following infection, were not affected by the pathogenic virus, and were not rendered resistant to the disease. Thus, propagation of the virus in the fingerlings is a pre-requisite for immunization. In order to increase the number of random mutations in the genome of the attenuated virus, and thus, reduce the possibility of the attenuated virus reverting to pathogenic, we irradiated it and selected additional clones appropriate for vaccination. The results of our study suggest that a safe and efficient prophylactic vaccine can be developed by selecting an appropriate attenuated virus.

Description of an as yet unclassified DNA virus from diseased Cyprinus carpio species.

Numerous deaths of koi and common carp (Cyprinus carpio) were observed on many farms throughout Israel, resulting in severe financial losses. The lethal viral disease observed is highly contagious and extremely virulent, but morbidity and mortality are restricted to koi and common carp populations. Diseased fish exhibit fatigue and gasping movements in shallow water. Infected fish had interstitial nephritis and gill necrosis as well as petechial hemorrhages in the liver and other symptoms that were not consistent with viral disease, suggesting a secondary infection. Here we report the isolation of carp nephritis and gill necrosis virus (CNGV), which is the etiologic agent of this disease. The virus propagates and induces severe cytopathic effects by 5 days postinfection in fresh koi or carp fin cell cultures (KFC and CFC, respectively), but not in epithelioma papillosum cyprini cells. The virus harvested from KFC cultures induced the same clinical signs, with a mortality of 75 to 95%, upon inoculation into naive koi and common carp. Using PCR, we provide final proof that the isolated virus is indeed the etiologic agent of food and ornamental carp mortalities in fish husbandry. Electron microscopy revealed viral cores with icosahedral morphology of 100 to 110 nm that resembled herpesviruses. Electron micrographs of purified pelleted CNGV sections, together with viral sensitivities to ether and Triton X-100, suggested that it is an enveloped virus. However, the genome of the isolated virus is a double-stranded DNA (dsDNA) molecule of 270 to 290 kbp, which is larger than known herpesviruses. The viral DNA seems highly divergent and bears only small fragments (16 to 45 bp) that are similar to the genomes of several DNA viruses. Nevertheless, amino acid sequences encoded by CNGV DNA fragments bear similarities primarily to members of the Poxviridae and Herpesviridae and to other large dsDNA viruses. We suggest, therefore, that the etiologic agent of this disease may represent an as yet unclassified virus species that is endemic in C. carpio (carp).

Pathogenesis of acute viral disease induced in fish by carp interstitial nephritis and gill necrosis virus.

A lethal disease of koi and common carp (species Cyprinus carpio) has afflicted many fish farms worldwide since 1998, causing severe financial losses. Morbidity and mortality are restricted to common carp and koi and appear in spring and autumn, when water temperatures are 18 to 28 degrees C. We have isolated the virus causing the disease from sick fish, propagated it in koi fin cell culture, and shown that virus from a single clone causes lethal disease in carp and koi upon infection. Intraperitoneal virus injection or bathing the fish in virus-containing water kills 85 to 100% of the fish within 7 to 21 days. This virus is similar to the previously reported koi herpesvirus; however, it has characteristics inconsistent with the herpesvirus family, and thus we have called it carp interstitial nephritis and gill necrosis virus. We examined the pathobiology of this disease in carp by using immunohistochemistry and PCR. We found large amounts of the virus in the kidneys of sick fish and smaller amounts in liver and brain. A rapid increase in the viral load in the kidneys was detected by using both immunofluorescence and semiquantitative PCR. Histological analyses of fish at various times after infection revealed signs of interstitial nephritis as early as 2 days postinfection, which increased in severity up to 10 days postinfection. There was severe gill disease evidenced by loss of villi with accompanying inflammation in the gill rakers. Minimal focal inflammation was noted in livers and brains. This report describes the etiology and pathology of a recently described viral agent in fish.

Efficient vaccine against the virus causing a lethal disease in cultured Cyprinus carpio.

We have isolated a virus, which causes a mortal disease in cultured ornamental Koi and Common carps (Cyprinus carpio) in many countries worldwide. This unclassified virus, which causes nephritis and gill necrosis, and so has been given the name carp nephritis and gill necrosis virus (CNGV), has a morphology resembling the herpes virus, but bears a genomic DNA of ca 250-300 kbp. So far, both others and we have been unable to find CNGV-DNA sequences possessing a significant similarity to known DNA viruses. The virus induces a lethal disease when water temperature ranges between 18 and 25 degrees C (permissive temperature). In this report, we demonstrate that carps, exposed to the virus at 23 degrees C for 3-5 days and then transferred to the non-permissive temperature of 30 degrees C, became resistant to a challenged infection and their sera demonstrated a high level of virus-specific antibodies. We have isolated attenuated non-pathogenic viruses that render virus-vaccinated carps resistant to the disease. Furthermore, vaccinated fish developed high levels of antibodies against the virus. We suggest, therefore, that this attenuated virus could be used as a live vaccine for the eradication of the mortal disease afflicting Common and ornamental carp fisheries in many countries.