Transmission of Mycobacterium chelonae and Mycobacterium marinum in laboratory zebrafish through live feeds

The zebrafish (Danio rerio) is a popular vertebrate model organism used in a wide range of research fields. Mycobacteriosis, caused by Mycobacterium species, is particularly concerning because it is a common disease associated with chronic infections in these fish. Infections are also a source of uncontrolled experimental variance that may influence research results. Live feeds for zebrafish are common and include paramecia (Paramecium caudatum), brine shrimp (Artemia franciscana) and rotifers (Branchionus spp.). Although nutritionally beneficial, live feeds may pose a biosecurity risk. In this study, we investigate transmission of Mycobacterium chelonae and Mycobacterium marinum through these three live feeds. We show that all three live feeds ingest both M. marinum and M. chelonae and can transmit mycobacterial infections to zebrafish. This observation emphasizes the need for live feeds to be included in the consideration of potential biosecurity risks. This study is of importance to other beyond the zebrafish community, including those of additional aquatic models and those using live feeds for other types of aquaculture.     

Mycobacteria in aquarium fish: results of a 3‐year survey indicate caution required in handling pet‐shop fish

Fish are commonly infected with non‐tuberculous mycobacteria (NTM), which should be regarded as potential pathogens when handling aquarium fish and equipment. This study examined 107 aquarium fish from pet shops. Cultivation of the fish samples using different selective media was conducted for identification of NTM. Isolates were identified using the GenoType Mycobacterium common mycobacteria and additional species assays, sequencing of the 16S rRNA and rpoB genes, and real‐time PCR assay for identification of Mycobacterium (M.) marinum. Among the investigated fish, 79.4% (85/107) were positive for mycobacteria, with 8.2% (7 of 85) having two mycobacterial species present. Among the positive fish, the common pathogens M. marinum, Mycobacterium fortuitum (M. fortuitum group) and Mycobacterium chelonae were identified in approx. 90% of fish and other NTM species in 10%, including Mycobacterium peregrinum/septicum, Mycobacterium gordonae, Mycobacterium arupense, Mycobacterium kansasii, Mycobacterium ulcerans and Mycobacterium setense. The well‐known human pathogen M. marinum was present in 10.6% of the positive fish (9 of 85). The species of mycobacteria identified in the study are not only recognized as aquarium fish pathogens, but can also cause pathology in humans. Microbiological and clinical communities should therefore be sensitized to the role of NTM in infections associated with exposure to aquarium fish.     

Evidence of fish and human pathogens associated with doctor fish (Garra rufa,Heckel, 1843) used for cosmetic treatment

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G. rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A. hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M. goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well‐known agents of zoonosis. Indeed, M. goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M. goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.     

Heat‐inactivated Mycobacterium bovis protects zebrafish against mycobacteriosis

Control of mycobacterial infection constitutes a priority for human and animal health worldwide. However, effective vaccines are needed for the control of human and animal tuberculosis (TB). Adult zebrafish have become a useful model for studying the pathophysiology of mycobacterial infection and for the development of novel interventions for TB control and prevention. Recently, parenteral and oral immunization with the heat‐inactivated Mycobacterium bovis vaccine (M. bovis IV) protected wild boar against TB. The objectives of this study were to provide additional support for the role of M. bovis IV in TB control using the zebrafish model and to conduct the first trial with this vaccine for the control of fish mycobacteriosis. The results showed that M. bovis IV protected zebrafish against mycobacteriosis caused by low and high infection doses of Mycobacterium marinum and provided evidence suggesting that the protective mechanism elicited by M. bovis IV in zebrafish as in other species is based on the activation of the innate immune response through the C3 pathway, with a role for the regulatory protein Akr2 in this process. These results encourage the use of M. bovis IV for TB control in different species.     

Mytilus species under rope culture in Scotland: implications for management

Mussel (Mytilus spp.) production is one of the most economically important aspects of global aquaculture and, in Scotland, production has increased from 262 t in 1986 to 4,219 t in 2006. Until recently, mussel production in Scotland was considered to be based exclusively on the blue mussel Mytilus edulis, the native species. In Europe, production is known to consist of M. edulis and M. galloprovincialis, while a third less profitable species M. trossulus occurs in the Baltic Sea, where it is unexploited. In Scotland, the sympatric occurrence of M. edulis, M. trossulus, M. galloprovincialis and their hybrids in cultivation in Loch Etive has recently been reported, with significant losses in production attributed to the presence of M. trossulus. Samples of mussels were taken at three depths from 10 rope-farm sites in Loch Etive. The distribution of the Mytilus species and their hybrids in Loch Etive was investigated using the Me 15/16 nuclear DNA locus for species and hybrid identification. All three species and their hybrids were detected and the data were in agreement with the Hardy–Weinberg model suggesting panmixia. Frequencies of M. galloprovincialis and its hybrids were very low. Overall, M. trossulus (37%) was found to be more common than M. edulis (30%) and 23% of the sampled mussels were M. trossulus × M. edulis hybrids. Species distribution did not correlate with year of settlement nor with salinities taken at the time of sampling. There were significant differences in species distribution due to site location and depth, with M. edulis being more frequent at 8 m depth and M. trossulus more common at shallower depths. These differences might be exploitable in management strategies for continuing production, for example to decrease the prevalence of M. trossulus at shellfish farms in favour of the more desirable M. edulis.     

Investigation of co‐infections with pathogens associated with gill disease in Atlantic salmon during an amoebic gill disease outbreak

Gill diseases are a complex and multifactorial challenge for marine farmed Atlantic salmon. Co‐infections with putative pathogens are common on farms; however, there is a lack of knowledge in relation to the potential effect co‐infections may have on pathology. The objective of this study was to determine the prevalence and potential effects of Neoparamoeba perurans, Desmozoon lepeophtherii, Candidatus Branchiomonas cysticola, Tenacibaculum maritimum and salmon gill poxvirus (SGPV) during a longitudinal study on a marine Atlantic salmon farm. Real‐time PCR was used to determine the presence and sequential infection patterns of these pathogens on gill samples collected from stocking until harvest. A number of multilevel models were used to determine the effect of these putative pathogens on gill health (measured as gill histopathology score), while adjusting for the effect of water temperature and time since the last freshwater treatment. Results indicate that between 12 and 16 weeks post‐seawater transfer (wpst), colonization of the gills by all pathogens had commenced and by week 16 of marine production each of the pathogens had been detected. D. lepeophtherii and Candidatus B. cysticola were by far the most prevalent of the potential pathogens detected during this study. Detections of T. maritimum were found to be significantly correlated with temperature showing distinct seasonality. Salmon gill poxvirus was found to be highly sporadic and detected in the first sampling point, suggesting a carryover from the freshwater stage of production. Finally, the model results indicated no clear effect between any of the pathogens. Additionally, the models showed that the only variable which had a consistent effect on the histology score was N. perurans.     

Experimental challenge studies in Vietnamese catfish,Pangasianodon hypophthalmus (Sauvage), exposed to Edwardsiella ictaluri and Aeromonas hydrophila

The two main diseases in the pangasius catfish industry are bacillary necrosis of Pangasianodon (BNP) and motile aeromonas septicaemia (MAS), where the aetiological agents have been identified as Edwardsiella ictaluri and Aeromonas hydrophila, respectively. In this study, apparently healthy Pangasianodon hypophthalmus were exposed to E. ictaluri, A. hydrophila or both bacterial species by intraperitoneal injection or immersion. There were 20 fish per treatment group, and the bacterial isolates used for the study were recovered from natural infections of BNP or MAS in farmed Vietnamese P. hypophthalmus. The results of the experimental infections mimicked the natural disease outbreaks reported from these pathogens in P. hypophthalmus. Furthermore, it was clearly demonstrated that E. ictaluri was only recovered from the fish exposed to the bacterium and not recovered from the animals receiving A. hydrophila.     

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 28 days at 10³ 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.     

Comparison of taste and odour characteristics of three mass‐produced aquaculture clams in China

Differences in taste and odour between three kinds of clam with the highest aquacultural production in China were investigated. Meretrix petechialis, Mactra veneriformis and Ruditapes philippinarum (fresh and dried product) were analysed firstly by electronic tongue and electronic nose. Fresh and dried clams could be easily distinguished, and there was little difference between uncooked fresh clams, while greater difference occurred between R. philippinarum and other fresh clams after cooking. The total free amino acid (FAA) content of uncooked clams increased after cooking but decreased in dried clam; the highest proportion was of sweet taste FAAs. Ala, Glu, Arg, 5′‐adenosine monophosphate and 5′‐inosine monophosphate were the most important active taste compounds. The umami intensity order was found to be fresh R. philippinarum, followed by M. petechialis and M. veneriformis, and finally dried R. philippinarum. The greatest number of volatile compounds was found in dried clam, while M. petechialis and M. veneriformis had the most compounds in common.     

Biosecurity in Shrimp Farming: Pathogen Exclusion through Use of SPF Stock and Routine Surveillance

Biosecurity, as it is being applied to shrimp aquaculture, may be defined as the practice of exclusion of specific pathogens from cultured aquatic stocks in brood stock facilities, hatcheries, and farms, or from entire regions or countries for the purpose of disease prevention. To make a biosecurity program a functional concept in shrimp aquaculture, the relevant risks should be identified and the appropriate biosecurity measures put into practice to mitigate those risks. Examples of biosecurity measures put into place for this purpose may include such basics as site selection when the intent is to locate a new shrimp culture facility in an area where certain diseases are not enzootic. Standard facilitylfarm operating procedures can be adapted to minimize the risks of disease introduction and spread within a facility through such concepts as pretreatment of all source water, and reduced or “zero” water exchange. Stocking shrimp culture facilities with domesticated shrimp stocks that are free of specific diseases (“Specific Pathogen Free” or SPF) and or with stocks resistant to specific disease agents (SPR) is perhaps the most important single component of a biosecurity program. The example set by the development of domesticated SPF stocks of Litopenaeus vannamei has helped to make biosecure shrimp culture feasible. The development of these and other SPF stocks, and the diagnostic methods to develop and monitor them for specific diseases and disease causing agents, have been milestones in the development of the international shrimp farming industry in recent years, and it has contributed to the species rivaliig Penaeus monodon as the dominant farmed shrimp species. The regular monitoring (surveillance) of shrimp stocks in biosecure culture facilities is a necessary component of a biosecurity plan, as is having in place a contingency plan for disease containment and eradication should a breach occur in the physical and managerial components of a biosecure facility and a targeted disease occur.     

Rederivation of a mutant line (prop 1) of zebrafish Danio rerio infected with Pseudoloma neurophilia using in vitro fertilization with eggs from pathogen-free wild-type (AB) females and sperm from prop 1 males

Along with the growing number of laboratories that work with zebrafish (Danio rerio), it is necessary to have animals with good sanitary quality. Specific pathogens can interfere with the experimental results and in the life quality of the animals. Pseudoloma neurophilia is a parasite with high potential for interference in behavioural, morphology, toxicological and genetic research, and is very common in zebrafish facilities. With that, we implemented a protocol for the pathogen elimination in a genetically modified lineage (prop 1) using eggs from specific pathogen-free (SPF) wild-type fish (AB line) for in vitro fertilization, along with water recirculation equipment disinfection, appropriate PCR screening and back crossing protocols. This resulted in SPF prop 1 heterozygotes, which allowed us to move forward with subsequent crossings to develop homozygote prop 1 mutants for our research. Hence, this demonstrates a useful strategy for an individual research laboratory to rederive a specific mutant free line that is not available from other SPF laboratories.     

A new cultivation‐independent tool for fast and reliable detection of Mycobacterium marinum

The Mycobacterium marinum group (MMG) is a class of mycobacteria that includes M. marinum, the cause of chronic systemic infections in fish. This species occasionally causes granulomatous skin lesions in humans. Other members of MMG are mycolactone‐producing mycobacteria (MPM; M. ulcerans, M. shottsii and M. pseudoshottsii). The cultivation‐independent approach presented in this study brings a fast and reliable alternative to classically used cultivation methods. The developed triplex erp/IS2404 qPCR assay is based on a primary species‐specific erp detection, which allows enumeration of MMG in analysed samples, and secondary IS2404 detection is suitable for the differentiation of M. marinum from MPM. The detection of M. marinum in clinical specimens and in artificially contaminated tissue samples has proven its applicability for diagnostic purposes.     

Saprolegnia molecular phylogeny among farmed teleosts in Nova Scotia,Canada

To identify the pathogens causing saprolegniosis among farmed fish in Nova Scotia, 172 infected tissues and 23 water samples were collected from six species of teleosts: Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus), brook trout (Salvelinus fontinalis), striped bass (Morone saxatilis) and rainbow trout (Oncorhynchus mykiss) at nine facilities over a 600 km range. Following laboratory culture, 132 isolates were recovered. Six species of oomycetes were identified from analysis of the internal transcribed spacer (ITS) sequence of the nrDNA: Saprolegnia parasitica, Saprolegnia ferax, Saprolegnia diclina, Saprolegnia aenigmatica, Saprolegnia torulosa, Saprolegnia sp. and Pythiopsis cymosa. Further phylogenetic analyses of the ITS and cytochrome c oxidase subunit 1 (Cox1) regions revealed four strains of Saprolegnia parasitica (named here as S1, S2, S3 and S4), of which S1 and S2 were common (37% and 42% of the isolates), and two strains of S. ferax. Among S. parasitica, S2 and S3 are more closely related to each other than to S1 based on the phylogenetic analyses and predicted RNA secondary structure of the ITS region. Sexual structures with a similar morphology were formed by S1 and S3 in vitro, but were not formed by S2.     

Construction expression and immunogenicity of a novel trivalent outer membrane protein (OmpU-A-II) from three bacterial pathogens in Japanese eels (Anguilla japonica)

Vibrio vulnificus, Edwardsiella anguillarum and Aeromonas hydrophila are three common bacterial pathogens in cultivated eels. To protect farming eels from infection by these pathogens, a trivalent outer membrane protein (OMP) containing partial sequences of OmpU from V. vulnificus, OmpA from E. anguillarum and OmpII from A. hydrophila was expressed and purified; then, the OMP was used as a vaccine to immunize Japanese eels (Anguilla japonica). Whole-blood cell proliferation, antibody titres and complement and lysozyme activities were detected at different days post-immunization (dpi), and the relative per cent survival (RPS) was determined after eels were infected with V. vulnificus, E. anguillarum or A. hydrophila at 28 dpi. The results showed that the OMP significantly stimulates the antibody titres. At 14 days after the challenge (i.e. at 28 dpi), the RPS of OMP against V. vulnificus, E. anguillarum and A. hydrophila was 20%, 70% and 11.1%, respectively. The construction, expression and immunogenicity of a trivalent Omp were reported for the first time, and this study will provide a valuable reference for the development of fish multiplex vaccines.     

Targeted species substitution in giant freshwater prawn trade revealed by genotyping

This study presents the molecular barcoding results of giant freshwater prawns and allied products collected from inland landing centres, markets and stores of Vembanad Lake (Kerala state, India) which is recognized as a natural abode for Macrobrachium rosenbergii. Prawns collected from landing centres of the lake could be easily identified as the above with their large size and morphological characters. There were certain ‘alien’ prawns and prawn products (headless shell on, peeled and deveined) traded in the markets and stores of this region as M. rosenbergii. Genotyping of all these samples using COI and 16S rRNA gene sequences confirmed the speciation of individuals from inland landing centres as M. rosenbergii, ‘alien’ prawns and certain prawn products as M. malcolmsonii. To ensure the same and to detect the presence of any other congeners of genus Macrobrachium inhabiting Vembanad Lake, additional homologous COI and 16S rRNA sequences available in NCBI were acquired and incorporated for molecular analyses. Results generated from NJ tree and genetic distance data confirmed the trade of non‐indigenous Macrobrachium species M. malcolmsonii in Kerala for the first time and its use as a species substituent for M. rosenbergii.     

Dietary supplementation effects of Pediococcus acidilactici as probiotic on growth performance,digestive enzyme activities and immunity response in zebrafish (Danio rerio)

This study was carried out to assess the effects of Pediococcus acidilactici on zebrafish (Danio rerio). Different levels of P. acidilactici including 0, 1 × 10⁶, 2 × 10⁶, 4 × 10⁶ and 8 × 10⁶ colony‐forming unit per g of the diet (cfug^?1) were examined in fish with 120 ± 10 mg weight for 60 days in a completely randomized design. The results showed that the best growth indices were recorded in group 4 × 10⁶ cfug^?1 (p < 0.05). The highest number of total viable count and lactic acid bacteria of intestine were found in group 4 × 10⁶ cfug^?1 (p < 0.05). The maximum activity of digestive enzymes including amylase, lipase, protease and alkaline phosphatase was observed in group 4 × 10⁶ cfug^?1. The highest activity for superoxide dismutase was recorded in group 4 × 10⁶ cfug^?1 while catalase, glutathione peroxidase and glutathione reductase showed the highest activity in group 8 × 10⁶ cfug^?1. The most growth inhibition zone of Aeromonas hydrophylla, Flavobacterium columnare, Vibrio anguillarum and Edwardsiella tarda was found in group 4 × 10⁶ cfug^‐1 (p < 0.05). Therefore, P. acidilactici as a probiotic improved growth and immunity of the zebrafish and could be used by zebrafish farmers.