Chilean Salmonid Research Articles

Saprolegnia species affecting the salmonid aquaculture in Chile and their associations with fish developmental stage

article The rapid increase in the aquaculture production of salmonids has been followed by a rise in several diseases. In particular, saprolegniosis can account for at least 10% of the annual economic loss in salmonids. In this study, we investigated the main Saprolegnia species involved in saprolegniosis of salmonids in Chile, and their association with specific developmental stages of the host fish. For this purpose, we studied 244 isolates of Saprolegnia- affected Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and king salmon (Oncorhynchus tshawytscha) from the salmon farming regions, using a recently developed identification strategy based on mo- lecular taxonomical operational units. We found that the Saprolegnia species associated with diseased salmon were Saprolegnia australis, Saprolegnia delica, Saprolegnia diclina, Saprolegnia ferax, Saprolegnia parasitica and two new Saprolegnia species observed during this study. In order to determine whether there were any specific species associations with different stages in the fish life cycle, we applied mosaic plots and correspondence anal- yses for categorical data. These analyses showed a strong association of S. parasitica with samples from the adult stage of the fish (χ 2 = 196.29, p b 0.0001), while the species S. australis, S. diclina and Saprolegnia sp. 2 were

Factors affecting the abundance of Caligus rogercresseyi (Boxshall and Bravo) on farmed salmonids in Chile in the period 2006–2007

An epidemiological study was carried out in the period 2006–2007 to identify the factors that affected the abundance of Caligus rogercresseyi in the two susceptible salmonid species reared in Chile, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). The differences recorded between four aquaculture zones were mainly a consequence of the geographical and oceanographic characteristics of each location. The values of abundance were significantly influenced by the environmental variable salinity, temperature and water transparency. In addition, the stocking density, the season of year, level of exposure, depth of water at the farm and cage location, were important variables influencing the abundance of sea lice. No significant differences in sea lice abundance were recorded between rainbow trout and Atlantic salmon.

Deltamethrin resistance in the sea louse Caligus rogercresseyi (Boxhall and Bravo) in Chile: bioassay results and usage data for antiparasitic agents with references to Norwegian conditions

The sea louse Caligus rogercresseyi is a major threat to Chilean salmonid farming. Pyrethroids have been used for anticaligus treatments since 2007, but have shown reduced effect, most likely due to resistance development. Pyrethroid resistance is also a known problem in Lepeophtheirus salmonis in the Northern Hemisphere. This study describes the development of deltamethrin resistance in C.rogercresseyi based on bioassays and usage data for pyrethroids in Chilean aquaculture. These results were compared to bioassays from L.salmonis from Norway and to Norwegian usage data. Available deltamethrin bioassay results from 2007 and 2008, as well as bioassays from Norway, were collected and remodelled. Bioassays were performed on field-collected sea lice in region X in Chile in 2012 and 2013. Bioassays from 2007 were performed prior to the introduction of pyrethroids to the Chilean market. Both the results from 2008 and 2012 showed an increased resistance. Increased pyrethroid resistance was also indicated by the increased use of pyrethroids in Chilean aquaculture compared with the production of salmonids. A similar trend was seen in the Norwegian usage data. The bioassay results from Chile from 2012 and 2013 also indicated a difference in the susceptibility to deltamethrin between male and female caligus.

Introduction, expansion and coexistence of epidemic Flavobacterium psychrophilum lineages in Chilean fish farms

Chile is one of the countries where the development of salmonid farming has been the most successful. The first importation of salmonids in Chile from the northern hemisphere dates back to the late 19th century and the country now ranks as the world second largest producer of farmed salmon. However, the fast increase of infections caused by the bacterium Flavobacterium psychrophilum is a growing concern for this local industry. This pathogen, also recognized as an important problem worldwide, has been first reported in Chile in 1993 and is currently affecting all three cultivated salmonid species: Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch) and rainbow trout (O. mykiss). Here we conducted a MLST (multi-locus sequence typing) analysis of the local genetic diversity of F. psychrophilum to better understand its origin and propagation in the country, and to suggest practices that could contribute to its control in the future. A total of 94 bacterial isolates, collected from the main production zones, were analyzed and compared to those of other origins already available. The data reveal the country-wide distribution of several genotypes closely related to those that are the most prevalent in European and North American fish farms, and overlapping host fish species of the different lineages. This population structure is probably the direct consequence of local fish farming practices that relied until recently on massive import of fish eggs (e.g., 78 million of eggs in 2012) and where mixed-species farms and fish transportation across the country are common.

Caligus rogercresseyi as a potential vector for transmission of Infectious Salmon Anaemia (ISA) virus in Chile

The sea louse Caligus rogercresseyi is the most serious ectoparasite on farmed salmonids in Chile, mainly affecting Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss). In 2007 the highest infestation ever recorded for this sea louse struck the aquaculture industry. In the same year a serious epizootic due to the Infectious Salmon Anaemia (ISA) virus (Isavirus, family Orthomyxoviridae) also occurred.This study shows that C. rogercresseyi is capable of being a mechanical vector for ISA virus. The findings indicate that even after 48hours away from its host C. rogercresseyi can still transmit ISA virus to naïve Atlantic salmon, which was confirmed by sequencing of the virus. Both the amount and prevalence of ISA virus RNA were decreasing the longer C. rogercresseyi was away from its host. This indicates that ISA virus is not replicating in the lice and hence, it is not likely that C. rogercresseyi could be a biological vector.ISA virus positive Caligus sp. nauplius larvae or copepodids were neither detected inside nor outside the Atlantic salmon farming cage experiencing ISA outbreaks. Nor was it possible to detect ISA virus RNA in nauplius larvae or copepodids retained in the lab, even if they were the offspring of ISA virus positive C. rogercresseyi population. This indicates that ISA virus is not vertically transmitted from adult C. rogercresseyi to nauplii. Based on these results it is not very likely that Caligus sp. nauplius larvae or copepodids are vectors for ISA viruses.For the salmonid industry this means that a co-infection with ISA virus and C. rogercresseyi increases the risk of transmission of ISA virus within the site and also to other sites in the same hydrographical area.

Antimicrobial susceptibility and plasmid profiles of Flavobacterium psychrophilum strains isolated in Chile

Flavobacterium psychrophilum is one of the most threatening bacterial infections for freshwater fish species in the world and is of considerable economic significance to aquaculture producers. Up to now, antimicrobial therapies represent the only recourse to control the disease in farmed fish. In this study, a total of 40 F. psychrophilum isolates obtained from Chilean salmonid farms were tested to determine their antimicrobial susceptibility using minimum inhibitory concentration (MIC) determinations and disk diffusion tests, for oxytetracycline (OTC), florfenicol (FLO) and oxolinic acid (OXA) and also to examine the plasmid profile to assist in the characterization of the strains isolated from different salmonid species. In addition, the presence of mutations in the quinolone resistance‐determining region (QRDR) was evaluated. Normalized resistance interpretation (NRI) of MIC data suggested cut-off values for non-susceptible at R>0.203, and 2.35μgmL−1 for OTC, and FLO, respectively. For OXA a slightly larger coefficient of variation in the calculations required a conservative estimate at R≥16μgmL−1. Of the 40 Chilean isolates studied, 90%, 92.5%, and 85% F. psychrophilum were resistant to the three antimicrobials. In the disk diffusion tests, all isolates produced zones of inhibition to OTC30 and FLO30. However, there was a low or no correlation between MIC results and inhibition zone diameter values and valid NRI calculations were not possible to perform. For OXA2 disks, no zones were observed (<7mm). No correlation was found between quinolone resistance and PCR-RFLP analysis; whereas 5 isolates had mutations in the QRDR of gyrA. Thirty-nine of 40 isolates possessed a single plasmid (15 isolates with 3.5kb) or combinations of two plasmids (17 and 7 isolates of sizes 1.5 and 3.5kb and 2.7 and 3.5kb, respectively), but a relationship between plasmid and resistance could not be established. In summary, we demonstrate in this work by MIC assays that high levels of resistance to OTC, FLO and OXA exist among Chilean isolates, which could be associated with the high amounts of antimicrobials applied at the Chilean farms to control outbreaks caused by F. psychrophilum. Disk contents for disk diffusion tests were not suitable for this bacterium. Surveillance in the use of drugs is recommended in cultured freshwater fish to prevent resistance problem.

Use of reverse transcription-real time polymerase chain reaction (real time RT-PCR) assays with Universal Probe Library (UPL) probes for the detection and genotyping of infectious pancreatic necrosis virus strains isolated in Chile

Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal Probe Library (UPL) probes has been developed for the detection and genotyping of Chilean infectious pancreatic necrosis virus (IPNV) isolates from infected cell culture. Partial nucleotide sequences (1175 bp) of the VP2 coding region from a selection of 7 Chilean IPNV isolates showed that they clustered into two main groups strongly correlated with Genogroups 1 and 5 proposed by Blake et al. (2001), corresponding to types West Buxton (WB) and Spajarup (Sp), respectively. Based on the VP2 gene sequences of those 7 Chilean isolates and different reference IPNV strains, 2 sets of candidate primer/UPL probes (# 8 and # 117) were designed and evaluated with a total of 32 field isolates isolated from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Pacific salmon (Oncorhynchus kisutch) farms from 2006 to 2010 in Chile. The UPL probes clearly differentiated the same two major Genogroups that those recognized by sequencing analysis. Among the Chilean isolates examined, 18 yielded amplification with UPL probe # 8, and 14 with probe # 117, respectively corresponding to types Sp and WB, as demonstrated by typing by sequencing. Based on the findings reported below, it has been demonstrated that the combined real time RT-PCR protocol with UPLs approach was efficient in discriminating distinct Genogroups of IPNV cultured in fish cell lines and, therefore, recommended its use for detection and typing of IPN viruses. The study also confirmed the existence of two IPNV type strains in Chilean salmonid aquaculture.

Diversity of Flavobacterium psychrophilum and the potential use of its phages for protection against bacterial cold water disease in salmonids

Flavobacterium psychrophilum causes rainbow trout fry syndrome (RTFS) and cold water disease (CWD) in salmonid aquaculture. We report characterization of F. psychrophilum strains and their bacteriophages isolated in Chilean salmonid aquaculture. Results suggest that under laboratory conditions phages can decrease mortality of salmonids from infection by their F. psychrophilum host strain. Twelve F. psychrophilum isolates were characterized, with DNA restriction patterns showing low diversity between strains despite their being obtained from different salmonid production sites and from different tissues. We isolated 15 bacteriophages able to infect some of the F. psychrophilum isolates and characterized six of them in detail. DNA genome sizes were close to 50 Kbp and corresponded to the Siphoviridae and Podoviridae families. One isolate, 6H, probably contains lipids as an essential virion component, based on its chloroform sensitivity and low buoyant density in CsCl. Each phage isolate rarely infected F. psychrophilum strains other than the strain used for its enrichment and isolation. Some bacteriophages could decrease mortality from intraperitoneal injection of its host strain when added together with the bacteria in a ratio of 10 plaque-forming units per colony-forming unit. While we recognize the artificial laboratory conditions used for these protection assays, this work is the first to demonstrate that phages might be able protect salmonids from RTFS or CWD.

Sequence similarities of the capsid gene of Chilean and European isolates of infectious pancreatic necrosis virus point towards a common origin

The Chilean salmonid industry was developed by importing breeding materials, a practice still in effect due to deficits in the national supply of roe. Importation of breeding materials is often associated with the transmission of pathogens. The objectives of this study were to compare the infectious pancreatic necrosis virus (IPNV) isolates from Chile to those of European origin and to determine the diversity of the Chilean IPNV. The VP2 genes of IPNV from Chilean fish (whose eggs originated from Scotland, Iceland and Norway) were compared to isolates from fish in Norway and Ireland. The results show that the isolates are identical (97-99%) and cluster into one genogroup. Our findings support previous reports of association between the trade-in breeding materials and transmission of pathogens. Furthermore, our results demonstrate the genotypic diversity of Chilean IPNV isolates. These findings have important implications for IPNV disease diagnosis and control in Chile.

Discrimination between farmed and free-living invasive salmonids in Chilean Patagonia using stable isotope analysis

In Chilean Patagonia relatively pristine aquatic environments are being modified by the introduction of exotic salmonids, initially through their deliberate release for sport fishing since the early twentieth century, and more recently via the accidental escape from fish farms. There is therefore a need to reliably distinguish between naturally reproducing and fugitive salmonids associated with the Chilean salmonid farming industry, the second largest in the world. We tested the ability of stable isotope analysis (SIA) and analysis of scale growth profiles to discriminate between farmed and free-living salmonids sampled around the Island of Chiloe. Juvenile Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) from aquaculture facilities were significantly more enriched in δ15N and lipid-corrected δ13C than river-caught individuals. Scale growth slopes during the first year in freshwater were significantly higher in farmed than in wild-caught rainbow trout, indicating faster somatic growth under hatchery conditions. Stable isotopes analysis classified 94% of juvenile Atlantic salmon and rainbow trout to their correct farm or free-living groups. Our results, therefore, can help to elucidate the origin and spread of exotic invasive salmonids in Chile, and address one of the biggest threats to native freshwater fishes in Patagonia and other temperate zones of the Southern Hemisphere.

Problems affecting nitrification in commercial RAS with fixed-bed biofilters for salmonids in Chile

The present case study focused on the problems that affect the nitrification process at three commercial recirculating aquaculture systems (RAS) for salmonids with fixed-bed biofilters operating in Chile, where the main factors were found to be management problems: (1) large variations in daily feeding, which results in unstable nitrogenous compounds (TAN, NO2−, NO3−) concentration; (2) variable daily water exchange, producing unstable culture conditions (variations in pH and temperature); (3) high densities of culture, which results in overall bad culture conditions (high CO2 concentration, high amount of fine solids, high oxygen consumption). When properly managed, the RAS have proven to tolerate up to 15% of daily variation in feeding, as low as 10% of daily “new” water inlet, and densities as high as 60kgfish/m3 without showing any nitrification problems. The results from this study demonstrates that maintaining good water quality is essential to secure an efficient growth of both the target species and the nitrifying bacteria, therefore, the production strategies should consider both the target species and the nitrification process requirements.

Phenotypic, serological and genetic characterization of Flavobacterium psychrophilum strains isolated from salmonids in Chile

Characterization of 20 Flavobacterium psychrophilum strains isolated from farmed Atlantic salmon and rainbow trout in Chile was done using phenotypic, antigenic and genetic techniques. Experimental infections were also performed to assess the virulence of two representative isolates and of the type strain. Biochemical and physiological analyses showed that Chilean F. psychrophilum strains, regardless of the host species, constitute a phenotypically very homogeneous group matching with previous descriptions of this pathogen. However, serological assays indicated the existence of antigenic heterogeneity with four patterns of serological reactions. The first group contained most (14 of 20) of the F. psychrophilum isolates showing cross-reaction with the antisera obtained against Atlantic salmon and rainbow trout isolates. Group 2 corresponded to four other rainbow trout isolates (1658, 1731, 1762 and 29009) that did not agglutinate with anti-1150 serum. Two minor serological groups were identified for the remaining isolates (Groups 3 and 4). Marked homogeneity was also revealed by genetic studies including 16S rRNA alleles, random amplified polymorphic DNA and REP-PCR showing that a major genetic group of F. psychrophilum may be dominant in disease outbreaks in farms. Restriction fragment length polymorphism of PCR analysis showed that gyrase genotypes B-S or B-R were found in Chilean isolates from rainbow trout and Atlantic salmon, whereas genotype A was not found. Virulence assays using Atlantic salmon indicated no relationship between the degree of pathogenicity and the host origin of the F. psychrophilum strains.

First description of serotype O3 in Vibrio anguillarum strains isolated from salmonids in Chile

First description of serotype O3 in <i>Vibrio anguillarum</i> strains isolated from salmonids in Chile

A review of the impacts of salmonid farming on marine coastal ecosystems in the southeast Pacific

AbstractThe production of farmed salmonids in Chile reached 550 000 t in 2004. The industry is considered to be consolidated, but with potential for further expansion to the south into pristine coastal areas. The environmental impacts of the salmonid farming industry in Chile were reviewed in 1996, and evidence at that time did not suggest significant adverse effects. However, after almost ten years of sustained growth, current evidence indicates that significant loss of benthic biodiversity and localized changes in the physico-chemical properties of sediments have occurred in areas with salmonid farms. Furthermore, the presence of these farms significantly increases in pulses the density of dinoflagellates. Data suggest that escaped farmed fish may have an impact on native species, although their survival in the wild appears low. The abundance of omnivorous diving and carrion-feeding marine birds increased from twofold to fivefold in areas with salmon farms compared with control areas without them. It is urgent that an ecosystem approach be implemented to assess all impacts of salmonid farming on coastal ecosystems in southern Chile.

Isolates of Piscirickettsia salmonis from Scotland and Ireland show evidence of clonal diversity.

Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.

The feasibility of using Udonella sp. (Platyhelminthes: Udonellidae) as a biological control for the sea louse Caligus rogercresseyi, Boxshall and Bravo 2000, (Copepoda: Caligidae) in southern Chile

Caligus rogercresseyi is a sea louse of native and farmed fish in southern Chile. Due to the impact that this copepod has on the Chilean salmonid production, it is necessary to search for control mechanisms that can effectively reduce its population. This study focuses on determining the feasibility of using Udonella sp., a common flatworm found on copepod ectoparasites of the Chilean rock cod, Eleginops maclovinus, as a biological control for sea lice. The following aspects were studied: distribution and preference of Udonella sp. regarding both host sex and body location on copepods collected from both the Chilean rock cod and cultured salmonids; the effect of Udonella on both fecundity and survival of the free-living stages; and seasonal variations of population of both Udonella and the copepod. Results indicated that Udonella sp. is frequently found on copepods collected from the Chilean rock cod, but is absent on those from cultured salmonids. The flatworm showed a marked preference for female copepods and the main body locations used by Udonella are the genital complex and egg sacs. The three life stages of Udonella are distributed across the copepod body in different ways since different body locations represent feeding and nursery areas. Udonella has no significant effect either on fecundity or survival of free-living stages. Udonella may maintain its population relatively independent of the Caligus infra-population all year round. Seasonal patterns of variation were not evident for the population parameters of either Caligus or Udonella, probably due to the exceptional environmental conditions that occurred during 1997 that allowed Caligus populations to remain high during fall and winter, instead of decreasing as it has been reported previously. Results suggest that Udonella cannot be used as a biological control for sea lice since it is not found naturally on salmonid species and because the presence of Udonella did not reduce either fecundity or survival of free-living stages of copepods.

Differential infectivity of Caligus flexispina (Copepoda, Caligidae) in three farmed salmonids in Chile

The ectoparasitic copepod, Caligus flexispina, is causing increasing problems in farmed salmonids in southern Chile. Field and experimental approaches were used to assess whether any of the three host species, rainbow trout Oncorhynchus mykiss, atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch, is preferentially colonized. Prevalence, abundance, density and developmental stages attained by the parasite in three host species were compared. Results clearly revealed that rainbow trout is the most susceptible species: under field conditions, C. flexispina is more prevalent and abundant and there is a higher proportion of adult stages. No ovigerous females were found in coho salmon. In experimental infestations, rainbow trout were more heavily colonized by infective copepodids, and these were more likely to reach the adult stage. A mixture of factors inherent to each host-parasite relationship is considered to play a role in these observations because coho salmon is also colonized by copepodids but a low proportion of the parasites reach the adult stage. However, atlantic salmon is less suitable for colonizing larva.

Feasibility of the utilization of Udonella calligorum (plathyhelminthes: udonellidae) in the biological control of caligid copepods parasites on cultured salmonids in Chile

Feasibility of the utilization of Udonella calligorum (plathyhelminthes: udonellidae) in the biological control of caligid copepods parasites on cultured salmonids in Chile

The life cycle of Hysterothylacium aduncum (Nematoda: Anisakidae) in Chilean marine farms

The transmission of the nematode Hysterothylacium aduncum to seawater netpen-cultured salmonids in Chile was studied in order to clarify the life history of this enteroparasite in marine farms and to propose a control strategy. The stomach content analysis of salmonids revealed that trout Oncorhynchus mykiss which are most liable to carry H. aduncum, feed considerably on natural organisms, less so with coho salmon O. kisutch and almost not at all in Atlantic salmon Salmo salar which are not infected. In trout, the most important food item is gammarids followed by stomatopod larvae. Polychaetes and zooplanktonic anomurans are also present. Gammarids from nets and floats of cages seems to be mainly responsible for nematode transmission. The in vitro culture and experimental infection of the nematode demonstrated that the L3 that hatches from the egg easily infects their first intermediate host a calanoid or harpacticoid copepod. In this host the larvae develop to the typical form observed in salmonids and other intermediate or paratenic hosts. From two to four-host life cycles are proposed. A systematic removal of netpen fouling organisms is necessary to eliminate the intermediate hosts and prevent the transmission of the nematode to the salmonids. The prevalence and abundance of the nematode in salmonids are similar to those found in the wild for their natural and definitive host Merluccius australis.

Environment, sustainability and regulation in commercial aquaculture: The case of Chilean salmonid production

Chilean salmon aquaculture has exhibited dynamic growth since its introduction on a commercial scale in the early 1980s (IFOP, 1994). The indicators of growth show no immediate signs of abating, despite lower prices, and Chilean production now ranks second only to Norway in international production. The industry's efficiency and profitability depends primarily on two factors: optimal food utilisation to achieve a maximal rate of growth at a minimum cost and the control of mortality in the farmed fish population. The management of these factors has direct and indirect effects on the fresh water and marine environments of production sites. Many of these effects can be linked to the contamination of aquatic environments. The regulation of the industry to control these effects will determine its sustainability. This paper argues two points. The first point is that responsibility for the monitoring and regulation of the salmon aquaculture industry should lie with the state, especially in terms of environmental quality. The second point is the need to establish the degree to which intensive salmon aquaculture in Chile is sustainable at its current rate of expansion. In both cases, it is stressed that state regulation should be broader and more efficient than at present if the industry is to have a more sustainable future.