Distribution, extracellular virulence factors and antibiogram of motile aeromonads in fresh water ornamental fishes and immune response of Cyprinus carpio against Aeromonas hydrophila infection

The thesis deals with the prevalence and distribution of motile aeromonads in selected ornamental fishes. The presence of motile aeromonads in ornamental fishes and associated carriage water is well documented. Though aeromonads are a part of autochthonous flora of natural waters, disease outbreak occurs as a result of environmental stress on the cultured species and virulence of the pathogens. While ornamental aquaculture in many parts of the world is highly organized and practiced scientifically, it is highly unorganized in India. The culture ponds/tanks are often maintained in very poor manner and the fishes are subjected to high degree of stress during transportation from the production facility to retail vendors. The situation is no better at retail outlets, where fishes are maintained in crowded condition without proper aeration or food. All these could result in high prevalence of diseases caused by motile aeromonads. No systematic study has been carried out to understand the prevalence of motile aeromonads in ornamental fishes and carriage water . It also gives an account of the production of extracellular virulence factors and the antibiogram of the different species of motile aeromonads isolated. The growth characteristics and virulence potential of a representative strain of Aeromonas hydrophila is also studied. The nucleotide sequencing of the strain was carried out and sequences deposited in Genbank. Survival and immune response of Cyprinus carpio under different stress conditions and on probiotic treatment with Bacillus NL110, when challenged with A. hydrophila is also dealt within this thesis.

Aeromonas detection and their toxins from drinking water from reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in Sao Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A. allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%). The results revealed that 70% of A. caviare, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern.

Dynamics of Aeromonas species isolated from wastewater treatment system

Aeromonas are widely distributed in the aquatic environment, and are considered to be emerging organisms that can produce a series of virulence factors. The present study was carried out in a sanitary sewage stabilization pond treatment system, located in Lins, State of Sao Paulo, Brazil. Most probable number was applied for estimation of the genus Aeromonas. Colony isolation was carried out on blood agar ampicillin and confirmed by biochemical characterization. Aeromonas species were isolated in 72.4% of influent samples, and in 55.2 and 48.3% of effluent from anaerobic and facultative lagoons, respectively. Thirteen Aeromonas species were isolated, representing most of the recognized species of these organisms. Even though it was possible to observe a tendency of decrease, total elimination of these organisms from the studied system was not achieved. Understanding of the pathogenic organism`s dynamics in wastewater treatment systems with a reuse potential is especially important because of the risk it represents.

Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei

Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hly A/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hly A/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.

Detection of metallo-beta-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei

Aims: To determine the prevalence and expression of metallo-beta-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil. Methods and Results: Eighty-seven Aeromonas isolates belonging to Aeromonas hydrophila (n = 41) and Aer. jandaei (n = 46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97.6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla(IMP), bla(VIM) and bla(SPM-1) were not detected. On the other hand, production of MBL activity was detectable in 87.8% and 10.9% of the cphA-positive Aer. hydrophila and Aer. jandaei isolates respectively. Conclusions: Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity. Significance and Impact of the Study: These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of water-borne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.

Challenge of pacu (Piaractus mesopotamicus) fed diets supplemented with vitamins C and E by Aeromonas hydrophila under different temperature

Pacus Piaractus mesopotamicus alimentados com dietas contendo três níveis de vitaminas C e E (zero, 250 e 500mg/kg de ração) foram desafiados, em diferentes temperaturas, com Aeromonas hydrophila. Os peixes foram mantidos em caixas plásticas de 300L e, com o objetivo de reduzir as reservas vitamínicas, durante os primeiros 60 dias, foram alimentados com dietas isentas das vitaminas C e E. Após esse período, os peixes foram estocados na densidade inicial de 14 peixes/caixa e as dietas-teste foram oferecidas durante 60 dias. Ao final do experimento, todos os peixes foram infectados com 6×10(6) UFC de A. hydrophila/peixe, injetada intraperitonealmente. Não houve interação nível de vitamina C vs. nível de vitamina E quanto à mortalidade. A suplementação com as vitaminas C e E não reduziu a taxa de mortalidade dos peixes desafiados com A. hydrophila. Independentemente da suplementação vitamínica, após o desafio, os peixes menores apresentaram maior taxa de mortalidade que os maiores e o grupo mantido em ambiente com temperatura mais alta apresentou maior taxa de mortalidade após o desafio.

Eficácia do florfenicol e da oxitetraciclina no controle de Aeromonas hydrophila em pacu (Piaractus mesopotamicus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histopathological biomarkers in pacu (Piaractus mesopotamicus) infected with aeromonas hydrophila and treated with antibiotics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enterotoxigenicidade de cepas de Aeromonas sp. isoladas em diferentes pontos do fluxograma de abate bovino

Com o objetivo de verificar a capacidade enterotoxigênica de cepas de Aeromonas sp. isoladas em diferentes produtos e locais no fluxograma de abate bovino, foram testadas 102 cepas (18 da espécie A. hydrophila, 65 da espécie A. caviae e 19 atípicas) ante os testes de inoculação intragástrica em camundongo lactente e em alça intestinal ligada de coelho. Revelaram-se como produtoras de enterotoxinas três (16,7%) cepas da espécie A. hydrophila, originárias das mãos do manipulador antes que ele iniciasse seus trabalhos e da carne desossada pronta para o consumo, e uma (1,5%) da espécie A. caviae, também isolada das mãos. Os resultados são preocupantes pela presença de cepas enterotoxigênicas de bactérias do gênero Aeromonas em indústria de alto nível higiênico-sanitário.

Bactérias do gênero Aeromonas em água de matadouro bovino

Verificou-se a ocorrência de bactérias do gênero Aeromonas em amostras de água (abastecimento/residuária) obtidas em matadouro bovino. Analisaram-se a água utilizada nas dependências internas, a água dos currais, utilizada na dessedentação, pré-higienização e tranqüilização dos animais e a água residuária da lavagem das carcaças. Das 30 amostras representativas de cada tipo, bactérias do gênero Aeromonas foram isoladas em 10 (33,3%) amostras da água dos currais e em 10 (33,3%) amostras da água residuária da lavagem de carcaças. Nenhuma das amostras da água tratada de abastecimento das instalações revelou-se positiva no isolamento. As espécies isoladas foram Aeromonas hydrophila em duas (2,2%) e Aeromonas caviae em 19 (21,1%) amostras. Uma cepa considerada atípica foi isolada da água dos currais. Os resultados evidenciaram que a água dos currais pode ser uma importante fonte de contaminação, principalmente para a pele, e através dela as Aeromonas sp. podem chegar à sala de matança.

Aeromonas spp. Em água de pisciculturas da região da baixada ocidental Maranhense

Among the widely distributed bacterial agents in the aquatic ecosystem, can be outstanding the family Aeromonadaceae detected such in fish as in the water of this system. This study intended to verify the occurrence of Aeromonas spp. in pond water of fishfarms located in Occidental Lowland Region of Maranhao. Twelve properties located in Pinheiro, Palmeirandia and Perimirim cities had been selected. The harvest of the samples occurred in the period from October, 2008 to March, 2009. A total of 48 pond water samples were harvested. Four water samples of each fishfarm. Aeromonas were confirmed in 100% of the samples. The 89 isolate identified had been classified in three species, A. hydrophila (88%), A. caviae (9%), A. veronii sobria (3%). The pond waters of the fishfarms analyzed presented contamination with potentially pathogenic species of aeromonas which represents risk for people's health, especially for the organisms cultivated in these properties.

Hematology of Piaractus mesopotamicus fed diets supplemented with vitamins C and E, challenged by Aeromonas hydrophila

Piaractus mesopotamicus were fed diets supplemented with three vitamin C and/or E levels (zero, 250, and 500 mg vitamin/kg dry ration) and challenged with Aeromonas hydrophila. The fish were fed during the first 60 days with diets without vitamins C and E, in an attempt to reduce vitamin sources. After this period, test diets were offered during 60 days. At the end of the experiment, all fish were intraperitoneally injected with 6 x 10(6) Colony forming units (cfu) of A. hydrophila per fish. It was concluded that for pacu, vitamin C and E are essential for protection of erythrocytes. Vitamin C induces an increase in the number of circulating thrombocytes in a dose-response relation. However, just like vitamin deficiency should be avoided, excess vitamins can also cause damage to fish as observed in the hematocrit and hemoglobin values. Based on the hematological responses obtained, the recommended vitamin C and vitamin E levels for P mesopotamicus juveniles are 500 and 250 mg/kg feed, respectively. (C) 2007 Elsevier B.V. All rights reserved.

Inflammation induced by inactivated Aeromonas hydrophila in Nile tilapia fed diets supplemented with Saccharomyces cerevisiae

The inflammatory response and hernatological parameters among Nile tilapia (Oreochromis niloticus) supplemented with Saccharomyces cerevisiae were evaluated six and 24 h after inoculation with inactivated Aeromonas hydrophila into the swim bladder. Six groups were formed (n = 10 each): G1 was treated with non-supplemented feed+injection with 0.65% saline solution; G2 with non-supplemented feed+ inoculation with A. hydrophila: G3 with feed containing 2% yeast+ injection with saline; G4 with feed containing 2% yeast + inoculation with A. hydrophila: G5 with feed containing 0.3% cell wall + injection with saline: and G6 with feed containing 0.3% cell wall + inoculation with A. hydrophila. In the groups inoculated with bacteria, the responses were more intense (P<0.05) than in those injected with saline. The groups receiving supplement that were inoculated with A. hydrophila accumulated a greater total number of cells at the lesion site (P<0.05) than did the non-supplemented groups, after six and 24 h. The groups receiving cell wall presented greater total accumulation of cells (P<0.005) that did those receiving yeast. The differential count showed that there were significantly greater number of thrombocytes (P< 0.05) and lower number of neutrophils, macrophages and lymphocytes (P<0.05) in the groups that received supplement, after 6 and 24 h, in relation to the non-supplemented groups. The values in the erythrocyte count, hemoglobin concentration and blood measurement indices did not differ statistically. The variation in circulating thrombocyte and leukocyte counts suggests that the inflammatory stimulus caused recruitment from reserve compartments to the blood. The groups that received yeast or yeast cell wall supplements presented increased nonspecific acute inflammatory response, thus suggesting that this has a beneficial effect on the immunological defense system. Published by Elsevier B.V.

Incidence of non-01 Vibrio cholerae and Aeromonas spp. in fresh water in Araraquara, Brazil

The occurrence of Aeromonas spp., Vibrio cholerae, and Plesiomonas shigelloides in fresh water from various sources in Araraquara, State of São Paulo, Brazil was determined. Samples from ten distinct irrigation systems used in vegetable cultivation, from five distinct streams, from two reservoirs, from one artificial lake, and from three distinct springs were analyzed. All isolates were serotyped and tested for hemolysin, cytotoxin, heat-stable (ST) and heat-labile (LT) enterotoxins production; presence of plasmid; autoagglutination and drug resistance. V. cholerae isolates were also tested for cholera enterotoxin (CT) production, and Aeromonas isolates for suicide phenomenon. No P. shigelloides was found. V. cholerae non 01 was found in five irrigation water samples and in three stream samples. Aeromonas sp. were isolated in two samples of irrigation water, in three streams, and in one reservoir. All the V. cholerae and Aeromonas isolates were positive for P-hemolysin production, and all Aeromonas isolates were positive for suicide phenomenon; cytotoxic activities were observed in two Aeromonas strains. Cholera enterotoxin was not found in eight V. cholerae non-01 isolates tested by the Y-1 mouse adrenal cell. All isolates were also negative for the other virulence markers. Ii cholelerae isolates were found to be sensitive to the majority of drugs tested, while Aeromonas strains presented multiple drug resistance..

Prevalence and virulence properties of Vibrio cholerae non-O1, Aeromonas spp. and Plesiomonas shigelloides isolated from Cambe Stream (State of Parana, Brazil)

The incidence of Vibrio cholerae, Aeromonas spp, and Plesiomonas shigelloides was determined in Rater samples from Cambe Stream. The samples were collected from seven different sites. The serogroups, virulence markers and drug resistance profiles were also evaluated. Twelve. Aer. hydrophila, 12 Aer. caviae, eight Aer. sobria, seven Ple. shigelloides and two V. cholerae non-O1 were isolated. They belonged to different serogroups and all produced haemolysis in different assays. Five of the Aeromonas strains and one of V, cholerae non-O1 were positive for enterotoxin activity. Haemagglutination and its inhibition, using erythrocytes of different origins, was variable for Aeromonas spp and V. cholerae, while none of the Plt. shigelloides haemagglutinated in association with any type of erythrocyte. All isolates exhibited multiple drug resistance. These results indicate that the occurrence of V. cholerae non-O1, Aeromonas spp, and Ple. shigelloides, in water used for vegetable irrigation, human recreation and animal consumption, among others, represents a potential risk for humans.