Mycobacteriosis caused by Mycobacterium marinum in reared mullets: first evidence from Sardinia (Italy)

Mycobacterium marinum is a slow‐growing non‐tuberculous mycobacterium, and it is considered the most common aetiologic agent of mycobacteriosis in wild and cultured fish. The diagnosis is principally made by histology when positive Ziehl–Neelsen stain granulomas are detected. The aim of this study was to investigate the occurrence of mycobacteriosis in extensively cultured Mugilidae of two lagoons (Cabras and San Teodoro) from Sardinia by the use of histology, microbiology, PCR and DNA sequencing. Nine of 106 mullets examined were affected by mycobacteriosis, and the spleen was the most affected organ. The histology detected higher rate (100%) of infection in spleen than the culture and PCR (75% and 62.5%, respectively). The sequencing of hsp65 gene identified M. marinum as the primary cause of mycobacteriosis in the mullets examined. Mullets affected by mycobacteriosis were mainly fished in the San Teodoro lagoon characterized by critical environmental conditions. Histology remains the most common method in detecting fish affected by mycobacteriosis, and PCR‐based methods are essential for species identification. Our finding are worthy of attention because mycobacteriosis caused by M. marinum in reared mullets was evidenced for the first time in Sardinia, suggesting that this disease may be underestimated also in other cultured fish species.     

Diseases of captive yellow seahorse Hippocampus kuda Bleeker,pot‐bellied seahorse Hippocampus abdominalis Lesson and weedy seadragon Phyllopteryx taeniolatus (Lacépède)

Seahorses, pipefish and seadragons are fish of the Family Syngnathidae. From 1998 to 2010, 172 syngnathid cases from the Toronto Zoo were submitted for post‐mortem diagnostics and retrospectively examined. Among the submitted species were yellow seahorses Hippocampus kuda Bleeker (n = 133), pot‐bellied seahorses Hippocampus abdominalis Lesson (n = 35) and weedy seadragons Phyllopteryx taeniolatus (Lacépède; n = 4). The three most common causes of morbidity and mortality in this population were bacterial dermatitis, bilaterally symmetrical myopathy and mycobacteriosis, accounting for 24%, 17% and 15% of cases, respectively. Inflammatory processes were the most common diagnoses, present in 117 cases. Seven neoplasms were diagnosed, environmental aetiologies were identified in 46 cases, and two congenital defects were identified.     

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.     

Ocular localization of mycobacterial lesions in tank‐reared juvenile cobia,Rachycentron canadum

Severe clinical mycobacteriosis with consistent ocular lesion localization was diagnosed in a population of 800 juvenile tank‐reared Cobia (Rachycentron canadum) which experienced a sudden increase in mortality approximately 5 months after arriving into Trinidad and Tobago from Florida, USA. Moderate daily mortality (15–20 animals per day) persisted for just over 1 month. Moribund fish displayed circling behaviour and had an open‐mouth gape upon death. Fish consistently presented with bilateral exophthalmia, corneal cloudiness and hyphema. Non‐branching acid‐fast rods were detected in aqueous humour touch preparations. Histological analysis revealed severe bilateral intra‐ocular granulomatous responses in all specimens. Mycobacterium sp. was identified using a real‐time PCR assay detecting the RNA polymerase β‐subunit (rpoB) gene in different tissue samples. Specimens did not present with characteristic granulomatous responses usually seen in viscera. To the best of our knowledge, this represents only the third documentation of piscine mycobacterial infection presenting with only localized ocular lesions, and the second documented case of mycobacteriosis in cobia. It is, however, the first documentation of an ocular presentation of mycobacteriosis in a marine species and is the first documentation of such a presentation in cobia.     

Comparison of the Intestinal Bacterial Flora in Healthy and Intestinal‐diseased Seahorses Hippocampus trimaculatus,Hippocampus erectus,and Hippocampus spinosissimus

This investigation examined the intestinal microbial flora among healthy and intestinal‐diseased seahorses Hippocampus trimaculatus, Hippocampus erectus, and Hippocampus spinosissimus by utilizing polymerase chain reaction‐denaturing gradient gel electrophoresis (DGGE) and densitometric analysis. Results demonstrated that 16 disparate DGGE bands belong to six major bacterial groups, which were Vibrionace, Enterobacteriacea, Rhodobacteraceae, Sphingomonadaceae, Flavobacteriaceae, and Alcaligenaceae. It was found that Vibrionaceae was the dominant population among the healthy and intestinal‐diseased seahorses. Vibrio ponticus strain XJ3 and Vibrio neptunius strain WT82, especially V. ponticus strain XJ3 of high abundance, were identified for the first time in seahorses and concluded to be intestinal disease pathogens because of their co‐existence in three intestinal‐diseased seahorse species and other reported fish or oyster. In comparison, uncultured bacterium clone Alcaligenaceae, Vibrio sp., uncultured bacterium clone Rhodobacteraceae, Serratia nematodiphila strain, and Serratia marcescens strain comprised the basic intestinal bacterial flora of all healthy seahorses. This study is the first to report the presence of S. nematodiphila in seahorses.     

Effects of sodium butyrate stimulation on immune‐related mRNA‐miRNA network in intestine of grass carp

Sodium butyrate is one of the most popular feed additives in animal husbandry. In recent years, sodium butyrate has been increasingly used as supplement in aquaculture. The present study is to investigate the intestinal mRNA and microRNA response to diet with sodium butyrate in grass carp (Ctenopharyngodon idella), an important aquaculture species in China. mRNA and microRNA profiles of intestine of grass carp fed with diet contained 0, 1.0, 2.5, 5.0, 7.5 and 10.0 g/kg sodium butyrate were obtained by RNA‐seq using Illumina Hiseq 2,500 platform. The feeding trial was performed using 18 individuals of 1‐year‐old grass carp (n = 3 for each group) and lasted for 40 days in tanks in laboratory. A total of 349,860,852 sequence reads were generated from six intestinal libraries. Functional analysis of differentially expressed genes showed that genes participated in immune pathways tend to be activated by sodium butyrate supplementation. A total of 700 microRNAs were obtained, including 275 conserved microRNAs and 425 novel microRNAs which are potentially involved in regulating 14,300 genes. Spearman's correlation analysis identified 18 pairs of microRNA‐mRNA associated with immune pathways (p < .01 and R<?0.5). The potential genes targeted by microRNAs include CXCL12, AKT1S1, Cab39 and MHCII which are important genes associated with intestinal immune pathways. To our knowledge, this is the first integrated profiling of both mRNA and microRNA in intestine with supplementation of sodium butyrate in grass carp. The present results suggest that sodium butyrate affects intestinal immune system by regulating microRNA‐mRNA interaction in fish.     

Labeo rohita and Argulus siamensis infection: Host size,local inflammatory reaction and immunity modulate ectoparasite load on fish

Parasite species often show a heterogeneous, highly dispersed pattern of infestation within hosts. Varieties of factors including morphological, physiological, immunological and nutritional characteristics affect the infestation level of a specific parasite in homogenous pray. Limited attempts, however, have been made to explore such underlying drivers of infestation pattern. Here, three stages of Labeo rohita (fingerling, juvenile and pre‐adult) were challenged with ectoparasite, Argulus siamensis in same aquaria. The parasite load on individuals was determined at 5‐day interval for 1 month. The load was found to be highest in pre‐adult stage followed by juveniles and fingerlings. On day 20 post infection, the load of parasite on pre‐adult fish was high along with detectable skin damages. Skin tissues were collected for immune gene expression analysis and histopathology. Histological studies showed increased melanization in the dermis and mild inflammatory cellular reactions in pre‐adult fish whereas, massive subcutaneous myositis with engorged blood vessels were observed in fingerlings. The expression levels of various inflammation and innate immune‐related genes viz., interleukin (IL)‐8, IL‐10, IL‐11, IL‐15, natural killer enhancing factor, toll‐like receptor 4, apolipoprotein A–I and immunoglobulin Z were significantly high in skin samples of infected fingerlings as compared to other two growth stages or controls of each stage. On the other hand, the expression of immunoglobulin M was down‐regulated in all infected samples as compared to their respective controls. The results thus depict that local immuno‐inflammatory response plays a significant role in determining susceptibility of host in intra‐specific group, and has important implications for ecology and aquaculture.     

Cloning and expression analysis of innate immune genes from red sea bream to assess different susceptibility to megalocytivirus infection

As suggested by the Office International des Epizooties (OIE), fishes belonging to the genus Oplegnathus are more sensitive to megalocytivirus infection than other fish species including red sea bream (Pagrus major). To assess the roles of the innate immune response to these different susceptibilities, we cloned the genes encoding inflammatory factors including IL‐8 and COX‐2, and the antiviral factor like Mx from red sea bream for the first time and performed phylogenetic and structural analysis. Analysed expression levels of IL‐1β, IL‐8 and COX‐2 and the antiviral factor like Mx genes performed with in vivo challenge experiment showed no difference in inflammatory gene expression or respiratory burst activity between red sea bream and rock bream (Oplegnathus fasciatus). However, the Mx gene expression levels in red sea bream were markedly higher than those in rock bream, suggesting the importance of type I interferon (IFN)‐induced proteins, particularly Mx, during megalocytivirus infection, rather than inflammation‐related genes. The in vitro challenge experiments using embryonic primary cultures derived from both fish species showed no difference in cytopathic effects (CPE), viral replication profiles, and inflammatory and Mx gene expression pattern between the two fish species.     

Characterization of Mycobacterium salmoniphilum as causal agent of mycobacteriosis in Atlantic salmon,Salmo salar L., from a freshwater recirculation system

Thirty Atlantic salmon, Salmo salar L., with low corporal condition relative to other fish present in the culture system, were sampled from a freshwater recirculation pisciculture located in Chile. The most characteristic signs and lesions were cachexia and presence of multiple greyish‐white granulomas within internal organs. The external and internal lesions, along with the microscopic, histologic and biochemical findings, were consistent with mycobacteriosis. The identification of Mycobacterium salmoniphilum as the causal agent of the lesions was possible through the use of molecular analyses. This study represents the first report of Mycobacterium salmoniphilum in a freshwater recirculation system and the first case of fish mycobacteriosis described in Chile.     

Availability of culture filtrate protein-10 (CFP-10) secreted from Mycobacterium pseudoshottsii for mycobacteriosis diagnosis in ginbuna crucian carp Carrasius auratus langsdorfii

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed-type hypersensitivity (DTH) response against culture filtrate protein-10 (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT-6 and CFP-10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT-6 and CFP-10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon-γ1-1-encoding gene (IFN-γ1-1). In contrast, IFN-γ1-1 expression accompanied by DTH response was observed only in the CFP-10-DNA plasmid-injected fish. Furthermore, fish that had been prophylactically injected with CFP-10-DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP-10 may facilitate diagnosis of mycobacteriosis.     

Evaluation of some intestinal cytokines genes expression and serum innate immune parameters in common carp (Cyprinus carpio) fed dietary loquat (Eriobotrya japonica) leaf extract

The aim of this study was to illustrate the effects of dietary loquat leaf extract (LLE) on the expression of some intestinal cytokines as well as serum innate immune parameters in common carp (Cyprinus carpio) fingerlings. The fish were fed with experimental diets supplemented with 0 (control), 0.25% (0.25LLE), 0.5% (0.5LLE) and 1% (1LLE) LLE over a 7‐week period. At the end of the trial, the serum lysozyme (Lys) activity, ACH50, total Ig as well as the expression of IL1B, IL8, TNF‐alpha, LYZ and TGF‐β in intestine were evaluated. The results showed that administration of 0.5% or 1% LLE significantly increased serum total Ig. However, in case of serum Lys activity significant elevation was observed just in fish fed 0.5% LLE. Also, supplementation of diet with LLE significantly increased ACH50 compared to the control group, regardless of inclusion levels. Gene expression studies revealed upregulation of TNF‐alpha, IL1B, IL8 and LYZ in intestine of fish fed LLE. However, the effects varied dependent on LLE levels and the tested immune related gene. Also, in case of TGF‐β significant downregulation was observed just in 1% LLE treatment. In conclusion, dietary LLE supplementation significantly upregulated immune related genes in intestine and improves innate immune responses. Altogether, LLE can be recommended as fish immunostimulant in early stage of carp culture.     

Detection and identification of aquatic mycobacteria in formalin-fixed, paraffin-embedded fish tissues

The isolation of mycobacteria from field samples is problematic, and isolation of the bacterium is sometimes not even attempted. The detection of mycobacteria through traditional histology using formalin‐fixed, paraffin‐embedded (FFPE) tissues is neither sensitive nor specific. However, detection of mycobacterial DNA from FFPE specimens, suspected of being infected with mammalian mycobacteriosis, is a routine clinical procedure. In the present study, a polymerase chain reaction (PCR)‐based method was used to detect and identify mycobacteria in FFPE specimens sampled from fish suspected of being infected with fish mycobacteriosis. A total of 45 fish tissue samples, comprising of 12 tissue samples obtained from experimentally infected fish and the remainder from fish naturally infected with mycobacteria, were analysed using a PCR protocol which amplifies a fragment of the mycobacterial 65 kDa heat‐shock protein (hsp65) gene. PCR‐restriction enzyme analysis and/or sequencing were employed to further analyse the PCR amplicons. The PCR results were compared with those obtained by histology and culture. Mycobacterial DNA was detected in 34 of the 45 samples examined, of which 16 samples (47%) showed granulomatous reactions on histological examination. Using histology as the gold standard, no false‐negative PCR results were obtained. Also, considering the presence or absence of granulomas as a diagnostic criterion, the sensitivity and specificity of PCR in 42 of the FFPE tissues were 16/16 (100%) and 8/26 (～30.8%), respectively. Corresponding microbiological cultures were available for 15 cases, of which 13 were pure Mycobacterium cultures. Of these, 13 were PCR positive (100% sensitivity and 50% specificity). The PCR‐based methods used here proved sensitive, specific and rapid for the detection of mycobacteria in routinely processed paraffin wax‐embedded and formalin‐fixed histological samples, and the results of the study suggest that this method has potential use in retrospective epidemiological studies.     

Identification and characterization of the pathogen associated with skin ulcer syndrome in lined seahorse,Hippocampus erectus

The lined seahorse, Hippocampus erectus, is an Atlantic species, which mainly inhabits shallow sea beds or coral reefs. Seahorse has become very popular in China due to its wide use in traditional Chinese medicine (TCM). However, the aquaculture of the lined seahorse has been threatened by a variety of diseases, especially bacterial infections. Skin ulcer syndrome becomes more and more common in the culture of seahorses, leading to skin ulcer, liver erosion and minority tail rotting. These diseased seahorses die in only 72 hr, causing huge economic losses in aquaculture. Pathogen HMT‐1 was isolated from the lined seahorses with skin ulcer syndrome, which was identified as Vibrio harveyi. The median lethal dose (LD₅₀) of HMT‐1 was 2.88 × 10⁸ cfu/ml. The pathogenic mechanism of HMT‐1 was preliminarily studied, and the pathological changes of the diseased lined seahorses were also investigated. Moreover, the activities of ACP, complement 3, SOD and LZM at different time post injection were measured by commercial kits. Our findings can provide reliable reference for diagnosis and treatment in the future.     

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.