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1.
Microbiol Res ; 229: 126325, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31563838

RESUMO

Edwardsiella bacteria cause economic losses to a variety of commercially important fish globally. Human infections are rare and result in a gastroenteritis-like illness. Because these bacteria are evolutionarily related to other Enterobacteriaceae and the host cytoskeleton is a common target of enterics, we hypothesized that Edwardsiella may cause similar phenotypes. Here we use HeLa and Caco-2 infection models to show that microtubules are severed during the late infections. This microtubule alteration phenotype was not dependant on the type III or type VI secretion system (T3SS and T6SS) of the bacteria as ΔT3SS and ΔT6SS mutants of E. piscicida EIB202 and E. tarda ATCC15947 that lacks both also caused microtubule disassembly. Immunolocalization experiments showed the host katanin catalytic subunits A1 and A like 1 proteins at regions of microtubule severing, suggesting their involvement in the microtubule disassembly events. To identify bacterial components involved in this phenotype, we screened a 2,758 transposon library of E. piscicida EIB202 and found that 4 single mutations in the atpFHAGDC operon disrupted microtubule disassembly in HeLa cells. We then constructed three atp deletion mutants; they all could not disassemble host microtubules. This work provides the first clear evidence of host cytoskeletal alterations during Edwardsiella infections.


Assuntos
Edwardsiella/fisiologia , Infecções por Enterobacteriaceae/veterinária , Células Epiteliais/metabolismo , Doenças dos Peixes/metabolismo , Microtúbulos/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CACO-2 , Edwardsiella/genética , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Células Epiteliais/microbiologia , Doenças dos Peixes/microbiologia , Regulação Bacteriana da Expressão Gênica , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Óperon , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo
2.
J Microbiol Biotechnol ; 29(8): 1324-1334, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370117

RESUMO

Fish mycobacteriosis is a common bacterial disease in many species of freshwater and marine fish and has caused severe loss of fish production. Mycobacterium marinum has been the most prevalent pathogen observed in several outbreaks of mycobacteriosis of farmed sturgeons in China. However, the immune responses and pathology of sturgeons in mycobacterial infection are rarely studied. Therefore, we used the Illumina RNA-seq method to analyze the transcriptome profile of Acipenser schrenckii challenged with Mycobacterium marinum. To begin, 168,220 non-redundant contigs were acquired from the infection and control groups, and among these, 33,225 contigs have acquired annotations. A total of 4,043 differently expressed (DE) contigs between the two groups were identified, and among these, 2479 were upregulated and 1564 were down-regulated in the infected fish. A total of 1,340 DE contigs with acquired annotations in KEGG were enriched for 124 pathways including the TNF signaling pathway, and the Toll-like receptor signaling pathway. The roles of DE genes involved in significant pathways and other processes were discussed. The 2,209 DE contigs that have yet to acquire proper annotation may represent candidate genes associated with infection in sturgeons and are expected to serve as immunogenetic resources for further study. To our best knowledge, this is the first transcriptome study on sturgeons under bacterial infection.


Assuntos
Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Peixes/genética , Infecções por Micobactéria não Tuberculosa/genética , Infecções por Micobactéria não Tuberculosa/imunologia , Infecções por Micobactéria não Tuberculosa/veterinária , Transcriptoma , Animais , China , Regulação para Baixo , Doenças dos Peixes/microbiologia , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica , Imunidade , Anotação de Sequência Molecular , Mycobacterium marinum/patogenicidade , Regulação para Cima
3.
Microbiol Res ; 228: 126306, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422233

RESUMO

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mCherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 distinct mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_3474, annotated as fabR and involved in fatty acid metabolism, was screened out and identified as a novel regulator mediating T3SS and T6SS expression. In addition, the fabR mutants displayed critical virulence attenuation in turbot. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be applied for functional genomics studies in various bacteria.


Assuntos
Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Edwardsiella/genética , Regulação Bacteriana da Expressão Gênica/genética , Testes Genéticos/métodos , Genoma Bacteriano/genética , Animais , Mapeamento Cromossômico , Farmacorresistência Bacteriana/genética , Ácidos Graxos/metabolismo , Doenças dos Peixes/microbiologia , Biblioteca Gênica , Genes Reporter/genética , Genômica/métodos , Mutagênese Insercional/métodos , Fatores de Transcrição/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo VI/genética , Virulência , Fatores de Virulência/genética
4.
Curr Microbiol ; 76(10): 1168-1174, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31263923

RESUMO

A Gram-negative, strictly aerobic, motile, rod-shaped bacterium with monopolar flagellum, designated as F51T, was isolated from the skin ulcer of farmed Murray cod sampled from Zhejiang Province, China. Strain F51T grew at 4-37 °C (optimal temperature, 28 °C), pH 5.0-8.5 (optimal pH, 7.5) and NaCl concentration of 0-6.0% (w/v) (optimal concentration, 2.0%). Phylogenetic analysis based on average nucleotide identity (76.2-78.4%) and in silico DNA-DNA hybridization (22.3-23.2%) values revealed that strain F51T forms a distinct lineage in the clade of genus Pseudomonas with less than 98.9% 16S rRNA gene sequence similarity to type strains of the genus and represents a novel species related most closely to Pseudomonas floridensis LMG 30013T. Three housekeeping genes (rpoB, rpoD and gyrB) of strain F51T were analysed to further confirm that the isolate is distinctly delineated from related Pseudomonas species. Chemotaxonomic analysis indicated that the sole respiratory quinone of strain F51T is Q-9; its predominant cellular fatty acids are C16:0, summed feature 3 (iso-C15:0 2-OH and/or C16:1ω7c), summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C10:0 3-OH; and its major polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified glycolipids, three unidentified phospholipids and an unidentified aminophosphoglycolipid. This composition is typical of the chemotaxonomic attributes of Pseudomonas. Based on its phenotypic, chemotaxonomic and phylogenetic features, strain F51T is considered to represent a novel species for which the name Pseudomonas ovata sp. nov. is proposed. The type strain is F51T (= KCTC 62133T = MCCC 1K03458T).


Assuntos
Doenças dos Peixes/microbiologia , Perciformes/microbiologia , Filogenia , Pseudomonas/classificação , Pseudomonas/fisiologia , Animais , Aquicultura , Composição de Bases , China , DNA Bacteriano/genética , Genes Bacterianos/genética , Genoma Bacteriano/genética , Lipídeos/química , Hibridização de Ácido Nucleico , Pseudomonas/química , Pseudomonas/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da Espécie
5.
J Microbiol Biotechnol ; 29(8): 1273-1280, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31337186

RESUMO

Edwardsiella piscicida is the causative agent of edwardsiellosis, which has caused enormous economic losses worldwide. In our previous research, an attenuated live vaccine WED based on the virulent strain E. piscicida EIB202 can effectively protect turbots against edwardsiellosis via intraperitoneal injection, while vaccination by immersion exhibits a weaker effect. During the development of the immersion vaccine, we surprisingly found the counts of ΔpEIB202/ EIB202 colonized on zebrafish were 100 times lower than those of EIB202. However, pEIB202 carries 53 predicted ORFs and has several copies in E. piscicida EIB202, impeding the study of its function. Thus the replication region is located at a 1 980 bp fragment (from 18 837 to 20 816 bp), containing a transcriptional repressor and a replication protein. Moreover, the minimal replication plasmid, named pRep-q77, has low copies in both E. coli and E. piscicida, but is more stable in E. piscicida than in E. coli. This work lays a foundation for further examination of the function of the virulence plasmid pEIB202.


Assuntos
Replicação do DNA , Edwardsiella/genética , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Animais , Proteínas de Bactérias/genética , DNA Bacteriano/isolamento & purificação , Edwardsiella/crescimento & desenvolvimento , Infecções por Enterobacteriaceae , Escherichia coli/genética , Doenças dos Peixes/microbiologia , Brânquias/microbiologia , Análise de Sequência de DNA , Pele/microbiologia , Virulência/genética , Peixe-Zebra/microbiologia
6.
Microbiol Immunol ; 63(9): 379-391, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31310013

RESUMO

The immune system with large number of molecules protects the host against a plethora of continuously evolving microbes. Major histocompatibility complex (MHC) molecules serve as cardinal elements of the adaptive immune system responsible for the activation of the adaptive immunity in the host. The present study reports MHCI molecule in freshwater carp, Catla catla, and its differential expression in immunologically relevant tissues post-infection with Gram-negative and Gram-positive bacteria. The MHCI sequence of C. catla had 502 bp nucleotides encoding putative 146 amino acids. The phylogenetic analysis exhibited its evolutionary conservation within the Cyprinidae family and formed a different clade with the higher vertebrates. Simultaneously, CXCR3 and CXCR4 chemokines were cloned and characterized for their expression in infected tissues. Analysis of immunologically relevant tissues of the infected fish exhibited an increase of MHCI gene expression and the down-regulation of CXCR3 and CXCR4 chemokines, indicating a tricky interaction between the innate and adaptive immune system. It was found that intestine, skin and spleen played a crucial role in the contribution of the defense activity which instigated the self-immunity. These immune activities can provide useful information to understand the interaction of self and non-self- immune system in freshwater fish, Catla catla.


Assuntos
Carpas/genética , Quimiocinas/genética , Clonagem Molecular/métodos , Cyprinidae/genética , Genes MHC Classe I/genética , Receptores CXCR3/genética , Receptores CXCR4/genética , Imunidade Adaptativa/genética , Sequência de Aminoácidos , Animais , Carpas/imunologia , Cyprinidae/imunologia , Regulação para Baixo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Água Doce , Expressão Gênica , Filogenia , Alinhamento de Sequência , Pele/imunologia , Baço/imunologia
7.
J Fish Dis ; 42(8): 1201-1209, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31192462

RESUMO

MALDI-TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on-target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24-hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48-hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI-TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate-dependent.


Assuntos
Bass , Doenças dos Peixes/diagnóstico , Infecções por Bactérias Gram-Negativas/veterinária , Photobacterium/isolamento & purificação , Dourada , Animais , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia
8.
Microb Pathog ; 135: 103612, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31252064

RESUMO

Emergence of antibiotic resistant bacteria has necessitated the drive to explore competent antimicrobial agents or to develop novel formulations to treat infections including Aeromonas hydrophila. The present study investigates the synergistic antibacterial effects of citrus flavonoid rutin and florfenicol (FF) against A. hydrophila in vitro and in vivo. Rutin is extracted and purified from Citrus sinensis peel through preparative HPLC and characterized through TLC, GC-MS and 1H and 13C NMR analyses. Though rutin did not display significant antibacterial activity, it modulated FF activity resulting in four-fold reduction in the MIC value for FF. The anti-biofilm potential of synergistic association of rutin and FF was validated by protein analysis, quantification of exopolysaccharide (EPS) and microscopy studies using sub-MIC doses. Besides antibacterial action, in vivo studies showed that Rutin/FF combination enhanced host immunity by improving blood cell count, anti-protease, and lysozyme activities as well as decreased the oxidative stress and the pathological changes of tilapia Oreochromis niloticus against A. hydrophila infection. No significant DNA damages or clastogenic effects were detected in tilapia challenged with A. hydrophila under Rutin/FF treatment. It is shown that an acute-phase Lipopolysaccharide binding protein (LBP) enhances the innate host defence against bacterial challenge. Semi quantitative RT-PCR and western blot results revealed the significant increase of LBP in the supernatant of tilapia monocytes/macrophages challenged with A. hydrophila upon treatment. The study findings substantiate that the combination of natural molecules with antibiotics may open up possibilities to treat MDR strains.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Doenças dos Peixes/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/veterinária , Rutina/farmacologia , Rutina/uso terapêutico , Tianfenicol/análogos & derivados , Aeromonas hydrophila/crescimento & desenvolvimento , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes/efeitos dos fármacos , Citrus sinensis/química , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Combinação de Medicamentos , Sinergismo Farmacológico , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/patologia , Pesqueiros , Imunidade/efeitos dos fármacos , Imunomodulação , Índia , Testes de Sensibilidade Microbiana , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Rutina/imunologia , Tianfenicol/imunologia , Tianfenicol/farmacologia , Tianfenicol/uso terapêutico , Tilápia/microbiologia , Virulência/efeitos dos fármacos
9.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 759-765, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31222994

RESUMO

As one of the most common pathogens in aquatic animals, Aeromonas hydrophila exhibits a wide range of pathogenicity. Due to factors like unreasonable use of antibiotics and horizontal gene transfer mediated by plasmids, many resistant strains of Aeromonas hydrophila were isolated from ready-to-eat seafood products in retail markets, supermarkets and restaurants. These strains carry many resistance genes. Therefore, it is essential to explore the key control points, and seek for prevention and control strategies so as to effectively alleviate antibiotic resistance. We review here the prevalence of drug resistance of Aeromonas hydrophila in China, and its main infection and resistance mechanisms, and the main means and strategies for reducing and preventing drug resistance. We also address further research directions and focus on drug resistance in Aeromonas hydrophila of the aquatic product.


Assuntos
Aeromonas hydrophila , Antibacterianos , Farmacorresistência Bacteriana , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Aeromonas hydrophila/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , China/epidemiologia , Doenças dos Peixes/epidemiologia , Doenças dos Peixes/microbiologia , Pesqueiros , Peixes , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Pesquisa
10.
Vet Res ; 50(1): 41, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159877

RESUMO

Recently, Vibrio tapetis was isolated for the first time from skin ulcerations in wild-caught common dab (Limanda limanda). To further examine its role in the development of these skin lesions, an in vivo experiment was performed. The significance of the skin barrier and in addition the difference between pigmented and non-pigmented side were investigated. Hence, the skin of common dab was treated in three different ways on both the pigmented and non-pigmented side. On a first "treatment zone", the scales and overlying epidermal tissue were removed whereas in a second zone only the mucus was discarded. The third zone served as a non-treated zone. Thereafter, fish were challenged with V. tapetis. The control group was sham treated. Mortality, clinical signs, severity and size of the developing lesions were recorded. All animals were sacrificed and sampled 21 days post-inoculation. Significantly more fish of the group challenged with V. tapetis died compared to the control group with the highest incidence occurring 4 days post-inoculation. Fish challenged with V. tapetis developed more severe skin ulcerations. In zones where scales and epidermal tissue were removed, the ulcerations were more severe compared to zones where only mucus was eliminated. Ulcerations occurred more frequently, were more severe and larger on the pigmented side. Our data represents prove of V. tapetis as causative agent of ulcerative skin lesions although prior damage of the skin seems to be a major contributing factor. Furthermore, the pigmented side seemed predisposed to the development of skin ulcerations.


Assuntos
Doenças dos Peixes/microbiologia , Linguados , Pigmentação , Dermatopatias Bacterianas/veterinária , Úlcera Cutânea/veterinária , Vibrioses/veterinária , Animais , Dermatopatias Bacterianas/microbiologia , Fenômenos Fisiológicos da Pele , Úlcera Cutânea/microbiologia , Úlcera Cutânea/patologia , Vibrio/crescimento & desenvolvimento , Vibrioses/microbiologia
11.
Microb Pathog ; 132: 261-265, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31078710

RESUMO

Aeromonosis is a fish disease that leads to haemorrhagic septicaemia and high mortality. The detection of early behavioural changes associated to this disease could be helpful in anticipating the initiation of treatment, increasing the probability of success. The influence of this disease on the hypothalamic-pituitary-interrenal (HPI) axis and on the brain expression of heat shock proteins (HSP) is little known. Therefore, the aim of this study was to evaluate the effect of Aeromonas hydrophila infection on individual behaviour and brain expression of genes related to stress (slc6a2, hsp90, hspa12a, hsd20b, hsd11b2, crh) in silver catfish (Rhamdia quelen). Thirty fish were divided into healthy and infected groups. The fish of the infected group were inoculated intramuscularly with 50 µL of bacterial suspension (6.4 × 108 CFU/mL), while control animals received 50 µL of saline. On day five post-infection, animals were submitted to the novel tank test, euthanized, and the brain was collected for molecular analysis. Infected fish swam more in the unknown aquarium and presented an increase in brain expression of genes related to HSP (hspa12a) and the route of cortisol synthesis (crh) when compared to uninfected fish. Therefore, this disease causes hyperlocomotion related to stress.


Assuntos
Aeromonas hydrophila/patogenicidade , Peixes-Gato/microbiologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/fisiopatologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/fisiopatologia , Infecções por Bactérias Gram-Negativas/veterinária , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Animais , Comportamento Animal , Encéfalo/metabolismo , Peixes-Gato/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/genética , Locomoção , Masculino , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética
12.
J Fish Dis ; 42(7): 975-984, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31066066

RESUMO

A previous proteomic study examining the plasma acute-phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5-kDa spot using 2D-PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443-bp nucleotide sequence that was 98.6% similar to type-4 ice-structuring protein LS-12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS-12 following experimental intraperitoneal injection of rainbow trout with either 106 or 108 colony-forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS-12 up to 15 days post-infection in any group. Hepatic LS-12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post-infection in fish injected with 108 CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute-phase response. Under the conditions used, LS-12 is not a positive acute-phase protein in rainbow trout.


Assuntos
Reação de Fase Aguda/veterinária , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Infecções por Flavobacteriaceae/veterinária , Oncorhynchus mykiss/microbiologia , Reação de Fase Aguda/microbiologia , Animais , Proteínas de Peixes/sangue , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/patogenicidade , Reação em Cadeia da Polimerase , Proteômica
13.
J Fish Dis ; 42(7): 1065-1076, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31074078

RESUMO

Co-infection of rainbow trout with infections haematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum is known to occur, and it has been speculated that a combined infection can result in dramatic losses. Both pathogens can persist in fish in an asymptomatic carrier state, but the impact of co-infection has not been well characterized or documented. In this study, it was hypothesized that fish co-infected with F. psychrophilum and IHNV would exhibit greater mortality than fish infected with either pathogen alone. To test this, juvenile rainbow trout were co-infected with low doses of either IHNV or F. psychrophilum, and at 2 days post-initial challenge, they were given a low dose of the reciprocal pathogen. This combined infection caused high mortality (76.2%-100%), while mortality from a single pathogen infection with the same respective dose was low (5%-20%). The onset of mortality was earlier in the co-infected group (3-4 days) when compared with fish infected with F. psychrophilum alone (6 days) or IHNV (5 days), confirming the synergistic interaction between both pathogens. Co-infection led to a significant increase in the number of F. psychrophilum colony-forming units and IHNV plaque-forming units within tissues. This finding confirms that when present together in co-infected fish, both pathogens are more efficiently recovered from tissues. Furthermore, pathogen genes were significantly increased in co-infected groups, which parallel the findings of increased systemic pathogen load. Extensive tissue necrosis and abundant pathogen present intracellularly and extracellularly in haematopoietic tissue. This was pronounced in co-infected fish and likely contributed to the exacerbated clinical signs and higher mortality. This study provides novel insight into host-pathogen interactions related to co-infection by aquatic bacterial and viral pathogens and supports our hypothesis. Such findings confirm that mortality in fish exposed to both pathogens is greatly elevated compared to a single pathogen infection.


Assuntos
Coinfecção/veterinária , Infecções por Flavobacteriaceae/veterinária , Interações Hospedeiro-Patógeno , Oncorhynchus mykiss/microbiologia , Oncorhynchus mykiss/virologia , Infecções por Rhabdoviridae/veterinária , Animais , Infecções Assintomáticas , Coinfecção/mortalidade , Doenças dos Peixes/microbiologia , Doenças dos Peixes/mortalidade , Doenças dos Peixes/virologia , Flavobacterium/genética , Flavobacterium/patogenicidade , Vírus da Necrose Hematopoética Infecciosa/patogenicidade , Células-Tronco
14.
Fish Shellfish Immunol ; 91: 188-193, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31077849

RESUMO

Columnaris disease has long been recognized as a serious problem worldwide which affects both wild and cultured freshwater fish including the commercially important channel catfish (Ictalurus punctatus). The fundamental molecular mechanisms of the host immune response to the causative agent Flavobacterium columnare remain unclear, though gene expression analysis after the bacterial infection has been conducted. Alternative splicing, a post-transcriptional regulation process to modulate gene expression and increase the proteomic diversity, has not yet been studied in channel catfish following infection with F. columnare. In this study, genomic information and RNA-Seq datasets of channel catfish were used to characterize the changes of alternative splicing after the infection. Alternative splicing was shown to be induced by F. columnare infection, with 8.0% increase in alternative splicing event at early infection stage. Intriguingly, genes involved in RNA binding and RNA splicing themselves were significantly enriched in differentially alternatively spliced (DAS) gene sets after infection. This finding was consistent with our previous study in channel catfish following infection with Edwardsiella ictaluri. It was suggested to be a universal mechanism that genes involved in RNA binding and splicing were regulated to undergo differential alternative splicing after stresses in channel catfish. Moreover, many immune genes were observed to be differentially alternatively spliced after infection. Further studies need to be performed to get a deeper view of molecular regulation on alternative splicing after stresses, setting a foundation for developing catfish broodstocks with enhanced disease resistance.


Assuntos
Processamento Alternativo/imunologia , Doenças dos Peixes/imunologia , Infecções por Flavobacteriaceae/veterinária , Ictaluridae , Transcrição Genética/imunologia , Animais , Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/fisiologia , Distribuição Aleatória
15.
Fish Shellfish Immunol ; 91: 1-11, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31085326

RESUMO

The immune mechanism elicited in pufferfish (Takifugu obscurus) against the invasion of Aeromonas hydrophila is still poorly understood. We examined the spleen of pufferfish at the transcriptome and proteome levels by using Illumina-seq and TMT coupled mass spectrometry after 12 h infection by A. hydrophila, respectively. A total of 2,339 genes (1,512 up-regulated and 827 down-regulated) and 537 (237 up-regulated and 300 down-regulated) proteins were identified. GO and KEGG analyses revealed that the responses to stimulus were the main biological processes, intestinal immune network for IgT production and calcium signaling pathway. Fourteen genes (8 up-regulated and 6 down-regulated) and proteins (5 up-regulated and 9 down-regulated) involved immune responses or signal transduction were validated by qRT-PCR and parallel reaction monitoring to confirm the reliability of the transcriptomic and proteomic analyses, respectively. Moreover, qRT-PCR and flow cytometry were used to detect dynamics of the genes in calcium signaling pathway and changes of concentration of cytoplasm Ca2+ in spleen cells within a 72 h challenge. This study provides the findings regarding immune response, especially intestinal immune network for IgT production pathway and calcium signaling pathway at the molecular, protein and cellular in pufferfish after infection by A. hydrophila. These results would provide a new insight and molecular targets into the response to pathogenic infection in pufferfish.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Baço/imunologia , Takifugu/genética , Takifugu/imunologia , Aeromonas hydrophila/fisiologia , Animais , Regulação para Baixo , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Proteoma/genética , Proteoma/imunologia , Transcriptoma , Regulação para Cima
16.
Fish Shellfish Immunol ; 91: 333-342, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31129189

RESUMO

In recent years, microRNAs (miRNAs) have been shown to play important roles in immunity. Analyses of the functions of miRNAs and their targets are useful in understanding the regulation of the immune response. To understand the relationships between miRNAs and their targets during infection, we used zebrafish as an infection model in which to characterize the miRNA and mRNA transcriptomes of zebrafish larvae infected with Vibrio parahaemolyticus. We identified the differentially expressed miRNAs and mRNAs. Overall, 37 known zebrafish miRNAs were differentially expressed in the infection group and 107 predicted target genes of 26 miRNAs were differentially expressed in the mRNA transcriptome. These targets with specific Gene Ontology (GO) terms, such as peripheral nervous system neuron axonogenesis, organophosphate metabolic process, heme binding, protein binding, tetrapyrrole binding, protein dimerization activity, and aromatase activity, which regulate nerve conduction, energy metabolism, hematopoiesis, and protein synthesis. They were also associated with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as phototransduction, tryptophan metabolism, notch signaling, and purine metabolism. Our findings indicate that miRNAs regulate the innate immune response via complex networks, and zebrafish (Danio rerio, dre)-miR-205-3p, dre-miR-141-5p, dre-miR-200a-5p, dre-miR-92a-2-5p, dre-miR-192, and dre-miR-1788 may play important roles in the innate immune response by regulating target genes.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata/genética , MicroRNAs/genética , RNA Mensageiro/genética , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Doenças dos Peixes/microbiologia , Perfilação da Expressão Gênica/veterinária , MicroRNAs/imunologia , RNA Mensageiro/imunologia , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrioses/veterinária , Vibrio parahaemolyticus/fisiologia
17.
Vet Immunol Immunopathol ; 211: 25-34, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084890

RESUMO

Red Mark Syndrome (RMS) is a skin disease reported from farmed rainbow trout. Since the turn of the millennium it has been spreading through Europe. RMS is probably a bacterial disease caused by a Midichloria-like organism (MLO). It is non-lethal and causes little obvious changes in appetite or behavior but results in red hyperaemic skin lesions, which may lead to economic losses due to downgrading. Here we transfer RMS to naïve specific pathogen free (SPF) fish by cohabitation with RMS-affected seeder fish. During disease development we characterize local cellular immune responses and regulations of immunologically relevant genes in skin of the cohabitants by immunohistochemistry and qPCR. Skin samples from SPF controls and cohabitants (areas with and without lesions) were taken at 18, 61, 82 and 97 days post-cohabitation. Gene expression results showed that lesions had a Th1-type profile, but with concurrent high expression levels of all three classes of immunoglobulins (IgD, IgM and IgT). The marked local infiltration of IgD + cells in the skin lesions as well as a highly up-regulated expression of the genes encoding sIgD and mIgD indicate that this immunoglobulin class plays an important role in skin immunity in general and in RMS pathology in particular. The co-occurrence of an apparent B cell dominated immune reaction with a Th1-type profile suggests that the local production of antibodies is independent of the classical Th2 pathway.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Humoral/imunologia , Oncorhynchus mykiss/imunologia , Pele/imunologia , Animais , Doenças dos Peixes/microbiologia , Expressão Gênica , Imunoglobulina D/imunologia , Oncorhynchus mykiss/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Pele/metabolismo
18.
BMC Vet Res ; 15(1): 176, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138199

RESUMO

BACKGROUND: Vibriosis is an important bacterial disease of cultured marine fishes worldwide. However, information on the virulence and antibiotic resistance of Vibrio spp. isolated from fish are scarce. This study investigates the distribution of virulence associated genes and antibiotic resistance patterns of Vibrio spp. isolated from cage-cultured marine fishes in Malaysia. RESULTS: A total of 63 Vibrio spp. isolated from 62 cultured marine fishes in various geographical regions in Peninsular Malaysia were analysed. Forty-two of the isolates (66.7%) were positive for all chiA, luxR and vhpA, the virulence genes produced by pathogenic V. harveyi. A total of 62 Vibrio isolates (98%) had tlh gene of V. parahaemolyticus, while flaC gene of V. anguillarum was detected in 43 of isolates (68%). Other virulence genes, including tdh, trh, hlyA and toxRvc were absent from any of the isolates. Multiple antibiotic resistance (MAR) was exhibited in all strains of Harveyi clade, particularly against ampicillin, penicillin, polypeptides, cephems and streptomycin. The MAR index ranged between 0.06 and 0.56, and 75% of the isolates have MAR index of higher than 0.20. Host species and geographical origin showed no correlation with the presence of virulence genes and the antibiotic resistance patterns of Vibrio spp. CONCLUSIONS: The study indicates that majority of Vibrio spp. isolated from cultured marine fishes possess virulence genes, but were not associated with human pathogen. However, the antibiotics resistance is a real concern and warrants ongoing surveillance. These findings represent an updated knowledge on the risk of Vibrio spp. to human health, and also provides valuable insight on alternative approaches to combat vibriosis in cultured fish.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Doenças dos Peixes/microbiologia , Vibrioses/veterinária , Vibrio/genética , Animais , Aquicultura , Peixes , Malásia , Vibrio/patogenicidade , Virulência/genética
19.
Microb Pathog ; 133: 103548, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31112771

RESUMO

One of the major challenges in Nile tilapia (Oreochromis niloticus L.) farming is the occurrence of bacterial infections, and the Francisella noatunensis subsp. orientalis (FNO) is an important pathogen that has emerged in last decades. Francisellosis outbreaks have been reported in the literature as occurring seasonally when water temperature is below 24 °C. The aim of this study was to quantify the median lethal doses (LD50) of FNO in experimental challenges at 28 °C and 22 °C, and to investigate the impact of temperature changes in whole genome expression using microarray technology. The LD50 for Nile tilapia at 28 °C was ∼105.7, whereas at 22 °C, the LD50 was ∼102.2, showing that the decrease in temperature enhanced disease outcome. Out of 1917 genes screened, a total of 31 and 19 genes were down- and up-regulated at 22 °C, respectively. These genes were grouped by orthology into functional categories of: amino acid, inorganic ion, and carbohydrate transport and metabolism; transcription; and posttranslational modification, protein turnover, and chaperones. Expression of genes related to metabolism, oxidative stress, and thermal shock were regulated by temperature changes, reflecting an ability of FNO to adapt to the environment. Expression of virulence genes usually required for the Francisella genus was not changed between tested temperatures, including that of genes located on the Francisella Pathogenicity Island.


Assuntos
Doenças dos Peixes/microbiologia , Peixes/microbiologia , Francisella/genética , Francisella/metabolismo , Francisella/patogenicidade , Infecções por Bactérias Gram-Negativas/veterinária , Temperatura Ambiente , Transcriptoma , Animais , Ciclídeos/microbiologia , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Dose Letal Mediana , Estresse Oxidativo , Regulação para Cima , Virulência/genética
20.
Microb Pathog ; 133: 103559, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31132417

RESUMO

Aeromonas salmonicida, the oldest known fish pathogen and currently endemic throughout most of the world in both fresh and marine waters, causes severe economic losses to the salmon farming industry. Although there have been many studies on the prevention of furunculosis over the past few decades, it is still prevalent in many fisheries. In this study, a recombinant adenovirus vaccine candidate harboring the highly immunogenic Vapa gene (pAd-easy-cmv-Vapa) was successfully constructed and tested. The immune protection rate and specific antibody levels in the peripheral blood were then determined after immunizing rainbow trout. In addition, relative levels of IgM and IgT in the head kidney and hindgut before and after immunization were measured by quantitative reverse transcription PCR. Western blotting results indicated that the recombinant adenovirus could infect HEK-293 cells and express the A layer protein (encoded by Vapa). Further, survival analysis of fish 28 days after challenge showed that immunization significantly lowered the mortality rate (40%) compared to that in the control group (76.6%) and empty vector group (73.6%). This also led to an increase in specific antibodies in peripheral serum. In addition, levels of IgM and IgT in the head kidney and hindgut were increased to varying degrees. In conclusion, our research provides a candidate vaccine for the prevention of Aeromonas salmonicida A450 infection in rainbow trout and lays the foundation for future research on adaptive immune mechanisms associated with rainbow trout antibodies.


Assuntos
Adenoviridae/genética , Aeromonas salmonicida/imunologia , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Imunização , Vacinas Sintéticas/imunologia , Imunidade Adaptativa , Vacinas contra Adenovirus , Aeromonas salmonicida/genética , Sequência de Aminoácidos , Animais , Anticorpos , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunoglobulina M , Rim/imunologia , Oncorhynchus mykiss , Vacinação , Vacinas Sintéticas/genética
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