Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda.

AIMS: Develop a species-specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. METHODS AND RESULTS: The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan-genomic analyses to identify unique regions that could be exploited by species-specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. CONCLUSION: Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end-point mPCR.

The Zebrafish Perivitelline Fluid Provides Maternally-Inherited Defensive Immunity.

In the teleost egg, the embryo is immersed in an extraembryonic fluid that fills the space between the embryo and the chorion and partially isolates it from the external environment, called the perivitelline fluid (PVF). The exact composition of the PVF remains unknown in vertebrate animals. The PVF allows the embryo to avoid dehydration, to maintain a safe osmotic balance and provides mechanical protection; however, its potential defensive properties against bacterial pathogens has not been reported. In this work, we determined the global proteomic profile of PVF in zebrafish eggs and embryos, and the maternal or zygotic origin of the identified proteins was studied. In silico analysis of PVF protein composition revealed an enrichment of protein classes associated with non-specific humoral innate immunity. We found lectins, protease inhibitors, transferrin, and glucosidases present from early embryogenesis until hatching. Finally, in vitro and in vivo experiments done with this fluid demonstrated that the PVF possessed a strong agglutinating capacity on bacterial cells and protected the embryos when challenged with the pathogenic bacteria Edwardsiella tarda. Our results suggest that the PVF is a primitive inherited immune extraembryonic system that protects the embryos from external biological threats prior to hatching.

Development of phage delivery by bioencapsulation of artemia nauplii with Edwardsiella tarda phage (ETP-1).

This study proposed that phage-enriched artemia could be a useful tool for transferring phage into the cultured fish (larvae or adult) as a feed, and introduce mode of phage administration and its safety in concern of tissue adaptation for efficient phage therapy in aquatic animals. First, whether Edwardsiella tarda phage (ETP-1) could attach or ingest by the artemia and optimum time period for the ETP-1 enrichment with artemia were investigated. ETP-1 dispersion, abundance and persistency, and zebrafish immune transcriptional responses and histopathology were evaluated after feeding the fish with ETP-1-enriched artemia. Hatched artemia nauplii (36 h) were enriched with 1.90 × 1011 PFUmL-1 of ETP-1, and maintained at 25 °C. The highest enrichment level was obtained after 4 h (3.00 × 109 PFUmL-1), and artemia were alive and active similar to control for 8 h. ETP-1 disseminated dose dependently to all the tissues rapidly (12 h). However, when feeding discontinued, it drastically decreased at day 3 with high abundance and persistency in the spleen (1.02 × 103) followed by the kidney (4.00 × 101) and the gut (1 × 101 PFUmL-1) for highest ETP-1-enriched artemia dose. In contrast, during continuous delivery of ETP-1-enriched artemia, ETP-1 detected in all the tissues (at day 10: gut; 1.90 × 107, kidney; 3.33 × 106, spleen; 5.52 × 105, liver; 6.20 × 104 PFUmL-1mg-1 tissues). Though the phage abundance varied, results indicated that oral fed ETP-1-enriched artemia disperse to the neighboring organs, even the absence of host as phage carrier. Non-significant differences of immune transcriptional and histopathology analysis between ETP-1-enriched artemia fed and controls suggest that no adverse apparent immune stimulation in host occurred, and use of ETP-1 at 1011 PFUmL-1 was safe. With further supportive studies, live artemia-mediated phage delivery method could be used as a promising tool during phage therapy against pathogenic bacteria to control aquatic diseases.

Typhlocolitis by Edwardsiella tarda in a cow

Background: Edwardsiella tarda has been report as etiology of gastroenteritis in both human and veterinary medicine,usually associated with exposure to aquatic environments in immunocompromised individuals. The present report describesa case of typhlocolitis in a cow in the west region of Santa Catarina state, Brazil.Case: After clinically evaluation of animal and proceeding euthanasia and realized the necropsy. Tissue samples were collected, and routinely processed for histological examination. Histopathological lesions were described as mild, moderateand severe. Tissue and swabs samples of small and large intestine were sent to standard microbiological culture processing.At necropsy, cattle presented severe dehydration and emaciation. Eye and vaginal mucosa were severely congested. Theopening of the abdominal cavity revealed a great amount of greenish fluid and a large amount of fecal material, associatedwith diffuse severe peritonitis, evidenced by hyperemia and severe deposition of fibrin in the visceral and parietal peritonealsurface. In the serosa of the cecum, there were two points of rupture, observed in the proximal colon. The mucosa of cecumand colon were severely edematous, hyperemic, and presented diffusely distributed pinpoint round hemorrhages, as wellas fibrinonecrotic material adhered to the surface. Histologically, in the mucosa of cecum and colon, moderate to severediffuse inflammatory infiltrate of neutrophils, lymphocytes and plasma cells associated with multifocal severe necrosiswere observed. Moderate diffuse fibrin deposition was evidenced in the submucosa and muscular, as well as multifocalmoderate necrosis in the muscular layer. In the serous, severe diffuse inflammatory infiltrate of neutrophils associated withfibrin deposition and innumerous coccoid bacterial colonies were observed. The samples subjected to bacterial isolationshowed growth of Edwardsiella tarda. All samples were negative for Salmonella spp. and...(AU)

Typhlocolitis by Edwardsiella tarda in a cow

Background: Edwardsiella tarda has been report as etiology of gastroenteritis in both human and veterinary medicine,usually associated with exposure to aquatic environments in immunocompromised individuals. The present report describesa case of typhlocolitis in a cow in the west region of Santa Catarina state, Brazil.Case: After clinically evaluation of animal and proceeding euthanasia and realized the necropsy. Tissue samples were collected, and routinely processed for histological examination. Histopathological lesions were described as mild, moderateand severe. Tissue and swabs samples of small and large intestine were sent to standard microbiological culture processing.At necropsy, cattle presented severe dehydration and emaciation. Eye and vaginal mucosa were severely congested. Theopening of the abdominal cavity revealed a great amount of greenish fluid and a large amount of fecal material, associatedwith diffuse severe peritonitis, evidenced by hyperemia and severe deposition of fibrin in the visceral and parietal peritonealsurface. In the serosa of the cecum, there were two points of rupture, observed in the proximal colon. The mucosa of cecumand colon were severely edematous, hyperemic, and presented diffusely distributed pinpoint round hemorrhages, as wellas fibrinonecrotic material adhered to the surface. Histologically, in the mucosa of cecum and colon, moderate to severediffuse inflammatory infiltrate of neutrophils, lymphocytes and plasma cells associated with multifocal severe necrosiswere observed. Moderate diffuse fibrin deposition was evidenced in the submucosa and muscular, as well as multifocalmoderate necrosis in the muscular layer. In the serous, severe diffuse inflammatory infiltrate of neutrophils associated withfibrin deposition and innumerous coccoid bacterial colonies were observed. The samples subjected to bacterial isolationshowed growth of Edwardsiella tarda. All samples were negative for Salmonella spp. and...

Discovery of immunoglobulin T in Nile tilapia (Oreochromis niloticus): A potential molecular marker to understand mucosal immunity in this species.

Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebrates, by protecting the organism from a wide variety of pathogens. Nile tilapia (Oreochromis niloticus) is extensively cultivated worldwide, with a strong established market demand. It constitutes one of the model species for the study of fish immunology and its genome is currently fully sequenced. The presence of the immunoglobulin M gene in this species is well documented, as well as its major role in systemic immunity. To date, the IgT gene from O. niloticus has not been identified and, therefore, no information is available on the role of this immunoglobulin isotype in the immune response in tilapia. In the present work, novel secreted and membrane immunoglobulin T isotypes and a fragment of IgM were isolated from tilapia head kidney lymphocytes. Their transcriptional profiles were analyzed by quantitative PCR in larval development and in different tissues of healthy or lipopolysaccharide/Edwardsiella tarda-challenged tilapia adults. The presence of IgT and IgM were detected in early stages of larval development. Additionally, these genes exhibited differential expression profiles in basal conditions and after E. tarda infection in adult tilapia, in accord with the proposed effector functions of these immunoglobulins in the systemic and mucosal compartments. Our results suggest the potential involvement of this new Ig in mucosal immunity in tilapia.

Recombinant Hsp33 and OmpC protein can serve as promising divalent vaccine with protection against Vibrio anguillarum and Edwardsiella tarda in flounder (Paralichthys olivaceus).

Vibrio anguillarum and Edwardsiella tarda are severe aquaculture pathogens shared similar epidemiological characteristics and susceptible to flounder (Paralichthys olivaceus). In our previous studies, recombinant(r) protein heat shock protein 33 (rHsp33) from V. anguillarum and outer membrane protein C (rOmpC) from E. tarda were proved to have protection against V. anguillarum and E. tarda, respectively. In this paper, the cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, and the protection of divalent vaccine candidate (rHsp33 + rOmpC, rHC) against both V. anguillarum and E. tarda were evaluated. RHC, rHsp33, and rOmpC were vaccinated to flounder, respectively, and the percentages of surface immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBLs), serum IgM, specific antibodies against V. anguillarum or E. tarda, specific antibodies against rHsp33, rOmpC or rHC, the expression of immune-related genes and relative percent survival (RPS) against V. anguillarum or E. tarda were measured. The results showed that: RHC could induced the enhancement of sIg + cells and high levels of specific antibodies against both V. anguillarm and E. tarda; Also a significant increase of specific antibodies against rHsp33, rOmpC or rHC, and up-regulation of gene expression of CD3, CD4-1, CD4-2, CD8α, CD8ß and IgM in spleen, head-kidney, and hindgut, RPS of 70 ±â€¯3.45% against V. anguillarum and 60 ±â€¯1.48% against E. tarda, respectively. In addition, rHsp33 induced specific antibodies against E. tarda and rOmpC, and had a RPS of 43.3 ±â€¯3.73% against E. tarda; rOmpC could evoke specific antibodies against V. anguillarum and rHsp33, and had a RPS of 44 ±â€¯1.27% against V. anguillarm; The results demonstrated that there was cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, rHC as a divalent vaccine can induce significant immune response and efficient protection against both E. tarda and V. anguillarum in flounder.

Edwardsiella tarda outbreak affecting fishes and aquatic birds in Brazil.

BACKGROUND: Edwardsiella tarda infections are frequent causes of severe outbreaks in the fish farming industry besides representing possible zoonotic risks. However, naturally occurring outbreaks that affect various species besides fishes are seldom described. AIM: To report an outbreak of acute mortality caused by E. tarda affecting multiple species that inhabited a natural pond in the state of São Paulo, Brazil. MATERIALS AND METHODS: Three adult tilapias, three Mallard ducks and one Snow egret were necropsied and subjected to further microbiological tests. Gross and microscopic lesions were documented. The antibiotic susceptibility and phylogenetic similarities among fish and avian strains were also determined. The E. tarda species was confirmed through MALDI-TOF, partial sodB sequencing and phylogenetic analysis. RESULTS: Macroscopical findings between the three species included intestinal dilatation, mucosal hyperaemia and mucous to liquid contents. Common histopathology findings included acute enteritis, increased number of intraepithelial lymphocytes with bacteria adhered to the intestinal epithelium and lymphoid depletion in the spleen. E. tarda was isolated from several organs from all affected species. The phylogeny employing amplified fragment length polymorphism (AFLP) of eleven strains revealed high similarity (>90%) among the isolates regardless of the affected species or sampled organs. Ten isolates of E. tarda showed susceptibility to all tested antibiotics. CONCLUSIONS: E. tarda was identified as the cause of death of the species examined. Further studies would be necessary to determine the virulence of these strains and the possible risks regarding public health.

Effect of microalgae on intestinal inflammation triggered by soybean meal and bacterial infection in zebrafish.

Soybean meal has been used in many commercial diets for farm fish; despite this component inducing intestinal inflammation. On the other hand, microalgae have increasingly been used as dietary supplements in fish feed. Nevertheless, the vast quantity of microalgae species means that many remain under- or unstudied, thus limiting wide scale commercial application. In this work, we evaluated the effects to zebrafish (Danio rerio) of including Tetraselmis sp (Ts); Phaeodactylum tricornutum (Pt); Chlorella sp (Ch); Nannochloropsis oculata (No); or Nannochloropsis gaditana (Ng) as additives in a soybean meal-based diet on intestinal inflammation and survival after Edwardsiella tarda infection. In larvae fed a soybean meal diet supplemented with Ts, Pt, Ch, or Ng, the quantity of neutrophils present in the intestine drastically decreased as compared to larvae fed only the soybean meal diet. Likewise, Ts or Ch supplements in soybean meal or fishmeal increased zebrafish survival by more than 20% after being challenged. In the case of Ts, the observed effect correlated with an increased number of neutrophils present at the infection site. These results suggest that the inclusion of Ts or Ch in fish diets could allow the use of SBM and at the same time improve performance against pathogen.

Novel IFNγ homologue identified in Nile tilapia (Oreochromis niloticus) links with immune response in gills under different stimuli.

Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. This cytokine plays a very important role in defining Th1 immune response in all vertebrates. In the present study, we identified and isolated for the first time the gene coding for Nile tilapia (Oreochromis niloticus) IFNγ from spleen lymphocytes. The isolated tilapia IFNγ has between 24 and 62% of amino acid identity as compared to reported sequences for other teleost fishes. It has close phylogenetic relationships with IFNγ molecules belonging to the group of Perciforms and presents the typical structural characteristics of gamma interferon molecules. The tissue expression analysis showed that IFNγ is expressed constitutively in head kidney, skin, intestine, muscle and brain. Its expression was not detected in gills by conventional RT-PCR. However, under conditions of stimulation with Poly I:C and LPS, IFNγ expression was up-regulated in gills after 24 h post-stimulation. IFNγ expression was also induced in gills 24 h after Edwardsiella tarda infection suggesting its important role in immunity against intracellular bacteria. The recombinant protein produced in Escherichia coli induced Mx gene transcription in head kidney primary culture cells. These results are the first steps to characterize the role of tilapia IFNγ in the defense against pathogens in tilapia. Furthermore, the isolation of this molecule provides a new tool to characterize the cellular immune response to various stimuli in this organism.

Near-drowning-associated pneumonia with bacteremia caused by coinfection with methicillin-susceptible Staphylococcus aureus and Edwardsiella tarda in a healthy white man: a case report.

BACKGROUND: Edwardsiella tarda is an Enterobacteriaceae found in aquatic environments. Extraintestinal infections caused by Edwardsiella tarda in humans are rare and occur in the presence of some risk factors. As far as we know, this is the first case of near-drowning-associated pneumonia with bacteremia caused by coinfection with methicillin-susceptible Staphylococcus aureus and Edwardsiella tarda in a healthy patient. CASE PRESENTATION: A 27-year-old previously healthy white man had an episode of fresh water drowning after acute alcohol consumption. Edwardsiella tarda and methicillin-sensitive Staphylococcus aureus were isolated in both tracheal aspirate cultures and blood cultures. CONCLUSION: This case shows that Edwardsiella tarda is an important pathogen in near drowning even in healthy individuals, and not only in the presence of risk factors, as previously known.

Mortalidade de tambacus (Colossoma macropomum x Piaractus mesopotamicus) infectados por Edwardsiella tarda / Mortality of tambacus (Colossoma macropomum x Piaractus mesopotamicus) infected by Edwardsiella tarda

O presente trabalho relata um surto de mortalidade de tambacus (Colossoma macropomum x Piaractus mesopotamicus) criados em tanques escavados da Fazenda-Escola da UCDB. Os peixes apresentaram sintomas clínicos de letargia, anorexia, aumento da produção de muco, nado desordenado e comportamento de buscar a superfície da água. Ao exame necroscópico de três peixes foram evidenciadas hemorragias nas nadadeiras e pele, opacidade de córnea, hemoperitôneo, distensão e repleção da vesícula biliar e congestão e hemorragia do tubo digestivo. O exame microbiológico dos materiais coletados das lesões dos tambacus foi positivo para a bactéria Edwardsiella tarda. A análise de qualidade de água indicou grande quantidade de fitoplânctons que proliferaram em função do excesso de matéria orgânica, caracterizando a eutrofização da água. Atribuiu-se a causa da morte dos tambacus à infecção oportunista pela E. tarda, favorecida pelo desequilíbrio devido ao excesso de matéria orgânica em suspensão na água. As mortes cessaram após a correção dos parâmetros da qualidade da água do tanque.

An outbreak of mortality of tambacus (Colossoma macropomum x Piaractus mesopotamicus) cultivated in dug tanks at UCDB was reported. Animals had clinical surface symptoms of lethargy, anorexia, increased mucus production, cluttering and swimming toward the water. Macroscopic examination of three fishes showed hemorrhages of the fins and skin, corneal opacity, hemoperitoneum, gallbladder distension and repletion, congestion and hemorrhages of the digestive tract. Water quality analysis indicated large amounts of phytoplankton that proliferated as a result of the excessive organic matter causing eutrophication of the water. The microbiological examination of tambacus lesions revealed the presence of the bacterium Edwardsiella tarda. The cause of the tambacus death was attributed to opportunistic infection by E. tarda, favored by the imbalance due to the intense organic matter in suspended in the water. Deaths stopped after the correction of the water quality parameters.

Adhesive and invasive capacities of Edwardsiella tarda isolated from South American sea lion.

Edwarsiella tarda is a zoonotic bacterium that can be isolated from humans, animals and the environment. Although E. tarda is primarily considered a fish pathogen, it is the only species of its genus considered to be pathogenic for humans as well. A survey of zoonotic intestinal bacteria in fresh feces from South American sea lions (SASL) Otaria flavescens, reported E. tarda as the most frequently isolated species. In this study, we used HEp-2 cells to establish in vitro the adherence and invasive ability of 17 E. tarda strains isolated from SASL fecal material. All the strains were able to adhere and invade HEp-2 cells with adhesion and invasion percentages ranging from 56 to 100% and 21 to 74%, respectively. Despite the expression of these pathogenic factors, further investigation is needed to determine whether this bacterium could play a role as primary pathogen for this and other species of pinnipeds.

Adhesive and invasive capacities of Edwarsiella tarda isolated from South American sea lion

Edwarsiella tarda is a zoonotic bacterium that can be isolated from humans, animals and the environment. Although E. tarda is primarily considered a fish pathogen, it is the only species of its genus considered to be pathogenic for humans as well. A survey of zoonotic intestinal bacteria in fresh feces from South American sea lions (SASL) Otaria flavescens, reported E. tarda as the most frequently isolated species. In this study, we used HEp-2 cells to establish in vitro the adherence and invasive ability of 17 E. tarda strains isolated from SASL fecal material. All the strains were able to adhere and invade HEp-2 cells with adhesion and invasion percentages ranging from 56 to 100% and 21 to 74%, respectively. Despite the expression of these pathogenic factors, further investigation is needed to determine whether this bacterium could play a role as primary pathogen for this and other species of pinnipeds.

Adhesive and invasive capacities of Edwarsiella tarda isolated from South American sea lion

Edwarsiella tarda is a zoonotic bacterium that can be isolated from humans, animals and the environment. Although E. tarda is primarily considered a fish pathogen, it is the only species of its genus considered to be pathogenic for humans as well. A survey of zoonotic intestinal bacteria in fresh feces from South American sea lions (SASL) Otaria flavescens, reported E. tarda as the most frequently isolated species. In this study, we used HEp-2 cells to establish in vitro the adherence and invasive ability of 17 E. tarda strains isolated from SASL fecal material. All the strains were able to adhere and invade HEp-2 cells with adhesion and invasion percentages ranging from 56 to 100% and 21 to 74%, respectively. Despite the expression of these pathogenic factors, further investigation is needed to determine whether this bacterium could play a role as primary pathogen for this and other species of pinnipeds.

Novel brain lesions caused by Edwardsiella tarda in a red tilapia (Oreochromis spp.).

The histological lesions caused by Edwardsiella tarda in a variety of fish species, including tilapia, have been well characterized. There are apparent differences in the type of inflammatory response manifested by these different species, which may be due to the fish species itself, the phase of infection, or the virulence factors produced by different strains of E. tarda. In catfish, systemic abscesses involving muscles of the flank or caudal peduncle are the most common lesions. By contrast, infection in tilapia and red sea bream is more likely to be associated with granulomatous inflammation. Necrotic meningitis, encephalitis, and vasculitis with fibrinoid necrosis of the blood vessels walls, as well as the formation of a plaque-like structure in the brain, are described in the current study. The presence of E. tarda was confirmed by microbiological isolation and a positive nested polymerase chain reaction in paraffin wax-embedded tilapia tissues.

Isolation and characterizaton of Edwardsiella tarda from pacu Myleus micans / Isolamento e caracterização de Edwardsiella tarda em pacu Myleus micans

Investigaram-se as causas da mortalidade de peixes ocorrida em janeiro de 2005 na bacia do Rio São Francisco, Brasil. Edwardsiella tarda foi isolada dos rins de pacu Myleus micans. O isolado, denominado Et-LIS, caracterizado por bastonetes Gram negativos móveis, foi identificado por testes bioquímicos e confirmado pelo kit comercial Bactray. A susceptibilidade a 10 drogas das 12 testadas foi determinada pelo método de difusão de discos, enquanto as características de virulência foram avaliadas mediante inoculação experimental em Cyprinus carpio e em Oreochromis spp. Ambas as espécies desafiadas apresentaram sinais compatíveis com infecção por E. tarda. As tilápias (Oreochromis spp.) morreram 48h após a inoculação, enquanto as carpas (Cyprinus carpio) sobreviveram por 72h. Este é o primeiro relato da ocorrência de E. tarda em pacu.(AU)

Isolation and characterizaton of Edwardsiella tarda from pacu Myleus micans / Isolamento e caracterização de Edwardsiella tarda em pacu Myleus micans

Investigaram-se as causas da mortalidade de peixes ocorrida em janeiro de 2005 na bacia do Rio São Francisco, Brasil. Edwardsiella tarda foi isolada dos rins de pacu Myleus micans. O isolado, denominado Et-LIS, caracterizado por bastonetes Gram negativos móveis, foi identificado por testes bioquímicos e confirmado pelo kit comercial Bactray. A susceptibilidade a 10 drogas das 12 testadas foi determinada pelo método de difusão de discos, enquanto as características de virulência foram avaliadas mediante inoculação experimental em Cyprinus carpio e em Oreochromis spp. Ambas as espécies desafiadas apresentaram sinais compatíveis com infecção por E. tarda. As tilápias (Oreochromis spp.) morreram 48h após a inoculação, enquanto as carpas (Cyprinus carpio) sobreviveram por 72h. Este é o primeiro relato da ocorrência de E. tarda em pacu.

Empiema pleural causado por Edwardsiella tarda / Pleural empyema caused by Edwardsiella tarda

Os autores descrevem o primeiro caso registrado de empiema pleural causado por Edwardsiella tarda, uma bactéria Gram negativa, e fazem uma revisão da literatura das infecções causadas por este patógeno