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.     

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.     

Further evaluation of the efficacy of emamectin benzoate for treating Pseudocapillaria tomentosa (Dujardin 1843) in zebrafish Danio rerio (Hamilton 1822)

Pseudocapillaria tomentosa is a pathogenic nematode parasite, causing emaciation and severe inflammatory lesions in the intestines in zebrafish Danio rerio (Hamilton 1822). Emamectin benzoate is commercially available analogue of ivermectin used for treating salmon for sea lice, under the brand name SLICE^®, and we have used this for treating zebrafish with the P. tomentosa. Here, SLICE^®, 0.2 per cent active emamectin benzoate, was used for oral treatments at 0.35 mg emamectin benzoate/kg fish/day for 14 days starting at 7 days post‐exposure (dpe). Another experiment entailed initiating treatment during clinical disease (starting at 28 dpe). Early treatment was very effective, but delaying treatment was less so, presumably due to inappetence in clinically affected fish. We evaluated emamectin benzoate delivered in water, using Lice‐Solve? (mectinsol; 1.4% active emamectin benzoate) in two experiments. Application of four 24‐hr treatments, space over 7 days was initiated at 28 dpe at either 0.168 or 0.56 mg emamectin benzoate/L/bath, and both treatments completely eradicated infections. This was 3 or 10 times manufacture's recommended dose, but was not associated with clinical or histological side effects.     

Antibiotic treatment of zebrafish mycobacteriosis: tolerance and efficacy of treatments with tigecycline and clarithromycin

Zebrafish (Danio rerio) are a popular model organism used in a growing number of research fields. Maintaining healthy, disease‐free laboratory fish is important for the integrity of many of these studies. Mycobacteriosis is a chronic bacterial infection caused by several Mycobacterium spp. and is the second most common disease found in laboratory zebrafish. Current mycobacteriosis control measures recommend the removal of infected fish and in severe outbreaks, depopulation. These measures can be effective, but less disruptive measures should be assessed for controlling mycobacteriosis, particularly when valuable and rare lines of fish are affected. Here, the in vivo efficacy of two drug candidates, tigecycline (1 μg g^?1) and clarithromycin (4 μg g^?1), was tested in adult zebrafish experimentally infected with Mycobacterium chelonae. We assessed both short (14 day)‐ and long‐term (30 day) treatments and evaluated fecundity and pathological endpoints. Fecundity and histology results show that zebrafish tolerated antibiotics. Antibiotic treatments did not significantly impact the prevalence of acid‐fast granulomas; however, the severity of infections (acid‐fast granuloma intensity) was significantly decreased following treatments.     

Effects of fish meal replacement by soybean meal with supplementation of functional compound additives on intestinal morphology and microbiome of Japanese seabass (Lateolabrax japonicus)

A 15‐week trial was carried out to estimate the effects of functional compound additives (FCA) on intestinal morphology and microbiome in Japanese seabass, Lateolabrax japonicus, fed diets with soybean meal (SBM) partially replacing fish meal (FM). The formulation of FCA was the mixture of antioxidant, immunopotentiator and Mintrex^® trace elements. Four isonitrogenous (45%) and isolipidic (11%) diets, including FM42 (the control group without FCA), FM35 (15% FM protein substitution level with FCA), FM21 (50% FM protein substitution level with FCA) and FM21‐N (50% FM protein substitution level without FCA) were formulated. Quadruplicate groups of seabass (initial average weight 125.65 ± 0.60 g) were randomly handfed each diet twice daily in seawater floating cage. No significant differences were found in microvillus height (HMV) and muscular thickness (MT) of distal intestine among fish fed FM42, FM35 and FM21, whereas the HMV, MT and fold height (HF) in seabass fed FM42 were significantly higher than those of seabass fed FM21‐N (P < 0.05). Seabass fed FM21‐N had more obvious enteritis parameters such as tissue disruption, wider lamina propria and less mucosal fold than those in seabass fed FM21. High‐throughput sequencing technology of gut flora showed that Proteobacteria was the most dominant bacteria at phylum level among groups. There was no significant difference in relative abundance and composition of gut microflora among groups. In conclusion, supplementation of FCA might partially eliminate SBM‐induced enteritis, but the composition and relative abundance of intestinal microflora were not affected by dietary treatments.     

Revealing the compositions of the intestinal microbiota of three Anguillid eel species using 16S rDNA sequencing

Anguilla japonica, A. marmorata and A. bicolor pacifica are important eel species for aquaculture in Taiwan. In the past, seldom studies focused on the intestinal microbiome of them or the probiotics used in their cultivation. Probiotics are microbes that can improve the health of organisms, and the microbes isolated from the intestines of organisms might be the potential probiotics. Therefore, the aim of this study was to collect the intestinal mucus samples of these three eel species for investigating the autochthonous microbiome using 16S rDNA sequencing. The diversity, abundance of the intestinal microbiome and compositions of all the libraries were identified. Moreover, the composition of intestinal microbiome of eel was suggested that it would be affected by the waters and the characteristics of different eel species. Furthermore, most of the identified predominant genera of the intestinal microbiome of these three eel species, such as Cetobacterium, Clostridium, Shewanella, Acinetobacter and Bacteroides, seemed to have great potential as the probiotics. In the future, more studies shall be conducted to promote the practical application of these candidate probiotics for improving the techniques of eel farming.     

Changes in the intestinal bacterial community during the growth of white shrimp,Litopenaeus vannamei

In this study, we documented the changes in the intestinal bacterial community at four stages in Litopenaeus vannamei: 14 days postlarvae (L14) and 1‐, 2‐ and 3‐month old juveniles (J1, J2, J3), using 454 pyrosequencing techniques. The intestinal bacterial community was dominated by three bacterial phyla, Proteobacteria, Bacteroidetes and Actinobacteria at all stages. However, the relative abundance and bacterial lineages varied at the family level. The intestinal bacterial community of L14 and J1 was similar, with dominant members belonging to the Comamonadaceae of Betaproteobacteria. Conversely, bacterial members affiliated to Flavobacteriaceae of Bacteroidetes were dominant in J2 and Vibrionaceae of Gammaproteobacteria was dominant in J3. The abundance of Microbacteriaceae of Actinobacteria also fluctuated during the four stages. Bacterial members of Flavobacteriaceae and Rhodobacteraceae (Alphaproteobacteria) were present through all growth stages, and likely form the intestinal core microbiome of L. vannamei. However, they varied at the operational taxonomic unit (OTU) level through the growth stages. The intestinal bacterial community of pond‐rearing shrimp included the three main bacterial phyla identified above, and an additional group, Mycoplasmataceae of Mollicutes. Our results demonstrate that the intestinal bacterial community of L. vannamei was highly dynamic during the growth stages. Bacterial members belonging to Commamonadaceae dominated in the earlier growth stage of shrimp, possibly influenced by feeding with Artemia nauplii, but there was a shift to Flavobacteriaceae in the mid and Vibrionaceae in the late growth stages.     

Establishing a zebrafish transgenic line expressing tilapia lysozyme with enhanced antibacterial activity

In recent years, infectious disease caused by Streptococcus agalactiae has increased in aquaculture, threatening the healthy development of tilapia and resulting in substantial economic losses. Cultivating disease‐resistant tilapia by transgenic technology is one strategy that has been developed to address this issue. Here, we used a transposon system to investigate gene expression and tissue lytic activity in transgenic zebrafish expressing the tilapia lysozyme. The transpose vector contained the Tgf2 transposon skeleton, tilapia heat shock protein 70 promoter (Hsp70) and two target genes (tilapia C‐type lysozyme 3 [lysozyme‐C3] and green fluorescent protein [GFP]). The transgenic zebrafish F₀ generation was obtained by microinjecting the donor vector and transposase mRNA into fertilized zebrafish eggs. There was 45% green fluorescence in the zebrafish F₀ generation, which was spread over the entire body based on microscope observation. The F₀ generation was reared to sexual maturity, at which point individuals were mated with wild‐type zebrafish to produce the F₁ generation. Tilapia lysozyme‐C3 expression was detected in the liver of F₁ generation by RT‐PCR and western blotting, but was not detected in the skin, intestine, or muscle. Significantly higher liver bacteriolysis activity was detected in transgenic zebrafish compared with wild‐type zebrafish. Therefore, transgenic zebrafish may have an increased potential for bacterial resistance.     

Morphological characterization of the adherence and invasion of Streptococcus agalactiae to the intestinal mucosa of tilapia Oreochromis sp.: An in vitro model

Streptococcosis in tilapia Oreochromis sp. is possibly the most important bacterial disease for fish production worldwide. In Colombia, streptococcosis is caused by Streptococcus agalactiae (GBS), but in other countries, Streptococcus iniae is also involved. Prevention of streptococcosis is required and must be addressed for economic, social, international trade and public health reasons. This research used an in vitro culture of tilapia intestine to detail the intestinal mucosal response once the pathogen contacts the epithelium. We show that S. agalactiae sheds off its capsule to adhere to the epithelium. The bacterium adheres as a single individuum, in groups or in chains and is able to divide on the apical border of enterocytes. GBS adheres at and invades exclusively through the apical portion of the intestinal folds, using the transepithelial route. Once within the cytoplasm of enterocytes, the bacteria continue to divide. On the basolateral side of the epithelium, the microorganisms leave the cells to reach the propria and travel through the microcirculation. No evidence of an immuno‐inflammatory reaction or goblet cell response in the epithelium or the lamina propria was seen during the process of adherence and invasion of the pathogen.     

Protective efficacy of Nigella sativa seeds and oil against columnaris disease in fishes

Columnaris disease, caused by the bacterium Flavobacterium columnare, is currently the most frequently reported bacterial disease affecting farm‐raised channel catfish in the USA. Common treatments against the disease include the use of medicated feed that has led to emergent antibiotic resistant strains of F. columnare. Nigella sativa (Black cumin) is a medicinal herb commonly used by many cultures as a natural remedy for numerous disorders. Recently, we have discovered the antibacterial activity of N. sativa and its oil extract against F. columnare. In this study, we showed N. sativa oil (NSO) strongly inhibited the growth of all of the strains of F. columnare tested and yielded significantly larger zones of inhibition than those produced by oxytetracyclin. We tested the protective effect against columnaris disease in vivo by incorporating NSO (5%) or N. sativa seeds (NSS) (5%) into fish feeds. Fishes (Ictalurus punctatus and Danio rerio) fed amended diets displayed significantly lower mortality than those fed control diets. Per cent mortalities in control groups ranged from 77% to 44% and from 70% to 18% in zebrafish and channel catfish, respectively. A dose study using different NSS concentrations showed that 5% NSS offered the most protection against columnaris disease in channel catfish.     

Field and laboratory transmission studies of haemic neoplasia in the soft‐shell clam,Mya arenaria,from Atlantic Canada

A two‐year laboratory and field study was initiated in 2001 in response to mass mortalities associated with haemic neoplasia (HN) in 1999 in Prince Edward Island (PEI) soft‐shell clams, Mya arenaria. A laboratory proximity experiment (cohabitation) and an inoculation challenge were conducted with clams and mussels (Mytilus edulis). Three field exposure experiments were also conducted, in which naive clams were held in sediment (in trays) or out of sediment (in mesh bags) at three high HN prevalence sites on PEI. There was a conversion to HN positive in clams in the proximity experiment and in clams injected with whole blood and cell‐free homogenate, but not at statistically significant levels. No mussels or control clams became HN positive. There was a significant conversion to HN positive in as little as 24 and 58 days after transfer with clams held out of sediment and in sediment, respectively. The laboratory and field experiments’ results suggest that HN‐infected clams are spreading the disease through water from infected clams to naïve individuals and via transplantation from affected to unaffected sites. Some environmental conditions (e.g. abnormally high water temperature and hypoxia‐induced sea lettuce [Ulva lacteus] invasion) may make clams susceptible to infections or exacerbate the proliferation of HN.     

First report of Fusarium oxysporum species complex infection in zebrafish culturing system

Fusarium oxysporum species complex (FOSC) is a highly diverse fungus. Recently, F. oxysporum infection was identified from zebrafish (Danio rerio) culturing system in Korea. Initially, a rapid whitish smudge was appeared in the water with the fungal blooming on walls of fish tanks. Microscopic studies were conducted on fungal hyphae, colony pigmentation and chlamydospore formation and the presence of macro‐ and microspores confirmed that the isolated fungus as F. oxysporum. Furthermore, isolated F. oxysporum was confirmed by internal transcribed spacer sequencing which matched (100%) to nine F. oxysporum sequences available in GenBank. Experimental hypodermic injection of F. oxysporum into adult zebrafish showed the development of fungal mycelium and pathogenicity similar to signs observed. Histopathologic results revealed a presence of F. oxysporum hyphae in zebrafish muscle. Fusarium oxysporum growth was increased with sea salt in a concentration‐dependent manner. Antifungal susceptibility results revealed that F. oxysporum is resistant to copper sulphate (up to 200 μg mL^?1) and sensitive to nystatin (up to 40 μg mL^?1). This is the first report of FOSC from zebrafish culture system, suggesting it appears as an emerging pathogen, thus posing a significant risk on zebrafish facilities in the world.     

Effects of Pediococcus pentosaceus supplementation on growth performance,intestinal microflora and disease resistance of white shrimp,Litopenaeus vannamei

Litopenaeus vannamei is economically important shrimp species in worldwide aquaculture. This study was conducted to assess the effect of different levels of probiotic Pediococcus pentosaceus (PP) on growth performance, feed utilization, digestive enzyme activity, intestinal microflora count and body composition of L. vannamei. Four diets containing different concentrations [0 (PP0), 10⁶ (PPI), 10⁷ (PPII) and 10⁸ (PPIII) CFU/g] of PP were formulated. After 8 weeks feeding trial, the experimental shrimps were challenged with Vibrio anguillarum and noted the surveillance. At the end of the feeding trial, the obtained results revealed a significant increase (p < .05) in final body weight, final length, weight gain (WG), survival rate, protease and amylase activities, lactobacillus sp. and Bacillus sp. intestinal count, total haemocyte counts (THC) and lysozyme activity in PPIII group when compared with the other groups. Similarly, WG, amylase activity, Bacillus sp. count, THC and lysozyme activity were significantly enhanced in case of PPII compared to the control group (p < .05). Interestingly, FCR and Vibrio sp. counts were significantly decreased in case of PPIII group when compared to the other groups (p < .05). Also, significant level of surveillance was noted in the challenging test with V. anguillarum. These results suggested that the P. pentosaceus improved the growth performance, digestive enzyme activity, immunity and tolerance against V. anguillarum of L. vannamei.     

A global view of hepatopancreas and intestinal reveals the potential influencing mechanism of aflatoxin B1 on nutrition and metabolism in Litopenaeus vannamei

The present study was to determine the time‐dependent alterations in growth performance, histomorphology, digestive enzymes activities and the genes expression related to target of rapamycin (TOR) signalling pathway involved in hepatopancreas and intestine of Litopenaeus vannamei at 3, 6, 12, 18, 24 and 30 days after Aflatoxin B1 (AFB1) challenge. The results manifested that AFB1 could induce a significant decrease in growth performance and visible variations of the hepatopancreas and intestine structures in shrimp. Meanwhile, the digestive enzymes activities and genes expression level were significantly lower in the experimental group than in the control group and showed a time‐dependent decrease (p < 0.05). The expression levels of tor and s6k were significantly higher in the experimental group than in the control group from days 6 to 24 and showed a tendency to increase first and then decrease as a whole (p < 0.05). Meanwhile, we analysed candidate pathways involved in hepatopancreas and intestine of shrimp in metabolic response to AFB1. The mainly disturbed pathways related to metabolism in hepatopancreas were involved in pyrimidine, glycosylphosphatidylinositol, glycosaminoglycan, vitamin and amino acid biosynthesis and metabolism, while the mainly disturbed metabolic pathways in intestine were glycosaminoglycan, vitamin and amino acid biosynthesis and metabolism.