Redox profile of silver catfish challenged with Aeromonas hydrophila and treated with hexane extract of Hesperozygis ringens (Benth.) Epling through immersion bath.

The growing increase in the fish farming sector has favored the establishment of bacterial outbreaks caused by Aeromonas hydrophila in several species. The hexane extract of Hesperozygis ringens (HEHR) (Lamiaceae) leaves increased the survival rate of silver catfish (Rhamdia quelen) experimentally infected by A. hydrophila. However, it is noteworthy that no reports have been found on the possible mechanisms of action of this extract in infected fish. This study aimed to evaluate the effect of the HEHR, administered through single immersion bath, on lipid peroxidation and antioxidant defenses in muscle and liver tissue of silver catfish challenged with A. hydrophila. The results showed that the oxidative status of silver catfish was altered, although oxidative stress was not triggered during the experiment. HEHR at 30 mg/L (HEHR30) was not characterized as a pro-oxidant agent in the presence of infection, unlike florfenicol and HEHR at 15 mg/L treatments in some cases. In short, HEHR30 provided an important increase in hepatic catalase activity, characterizing one of the possible mechanisms involved in the greater survival of fish experimentally infected by A. hydrophila. Additionally, HEHR30 did not induce lipid peroxidation, nor reduced antioxidant defenses of silver catfish infected or not by A. hydrophila.

Essential oil of Aloysia citriodora Paláu and citral: sedative and anesthetic efficacy and safety in Rhamdia quelen and Ctenopharyngodon idella.

OBJECTIVE: To verify the efficacy of citral in inducing sedation and anesthesia in silver catfish (Rhamdia quelen) and grass carp (Ctenopharyngodon idella) and to assess the safety of essential oil (EO) of Aloysia citriodora and citral in inducing and maintaining anesthesia in silver catfish. STUDY DESIGN: Clinical study, randomized, parallel, multi-arm with control group in target species. ANIMALS: A total of 96 juvenile and 72 adult silver catfish and 80 juvenile grass carp were used. METHODS: Silver catfish and grass carp were exposed to different concentrations of citral, 15-675 and 15-600 µL L-1, respectively, during the maximum period of 30 minutes to verify sedation and anesthesia induction and recovery times. In addition, for anesthetic induction, silver catfish were exposed to the EO of A. citriodora and citral at 225 µL L-1 for 3.5 minutes. Then, fish were transferred to an anesthesia maintenance solution at 50 µL L-1 for 10 minutes to assess hematologic and biochemical variables at 60 minutes, 2 and 6 days after treatment. RESULTS: Citral only induced sedation from 15, 25 and 40 µLL-1 in both species. Anesthesia without mortality was induced in silver catfish at 50-600 µL L-1 and grass carp at 75-450 µL L-1. At 675 and 600 µL L-1, mortality was recorded in silver catfish and grass carp, respectively. The EO of A. citriodora and citral were safe in inducing and maintaining anesthesia in silver catfish, with mean corpuscular hemoglobin concentration being the only variable that varied in relation to time and treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Citral was effective in inducing sedation and anesthesia in both species. In addition, A. citriodora EO and citral were safe in inducing and maintaining anesthesia in silver catfish. Both agents are promising substances for the development of new drugs for fish.

The influence of dietary Motore™ supplement on antioxidant status to Aeromonas hydrophila infection in Rhamdia quelen.

This study points to evaluate the effects of pre-treatment with standardized dry extract of Curcuma longa (Motore™) added to the diet (0; 250; 500; and 750 mg/kg) on oxidative stress parameters, longevity, and therapeutic success in Rhamdia quelen experimentally infected with Aeromonas hydrophila (MF 372510). After treatment, the liver and kidney were collected to determine non-enzymatic oxidative parameters such as the formation of thiobarbituric acid reactive substances (TBARS), non-protein thiols (NPSH), and quantification of reactive oxygen species (ROS) levels. Also, two enzymatic antioxidant parameters were evaluated: superoxide dismutase (SOD) and catalase (CAT) activities. The results showed an increase of ROS and TBARS levels, a depletion in NPSH, and a decrease of SOD and CAT activities in infected fish compared to control. The highest Motore™ dose minimized the deleterious effect of A. hydrophila infection improving longevity, oxidative status, and survival rate. The addition of 750 mg Motore™/kg feed is recommended for silver catfish in fish farming. Serious economic losses in Rhamdia quelen culture caused by Aeromonas hydrophila infections can be prevented by the addition of Motore™ to the diet.

Agro food by-products and essential oil constituents curtail virulence and biofilm of Vibrio harveyi.

Vibrio harveyi causes severe loss to the aquaculture industry due to its virulence, which is mediated by Quorum sensing (QS) and biofilm formation. In the current study, we have explored the anti-virulent properties and biofilm disruption ability of luteolin (extracted from coconut shell) and linalool against this important aquaculture pathogen. HPLC analysis of the methanolic extract of coconut shells revealed a single major peak which matched to the standard luteolin which was further elucidated by NMR studies. Further, luteolin and linalool were screened for their ability to inhibit biofilms and various quorum sensing mediated virulence factors of V. harveyi. The Minimum Inhibitory Concentration (MIC) of the two compounds was determined and the sub-inhibitory concentrations of the compounds were able to inhibit biofilm formation. Both the compounds disrupted about 60-70% mature biofilms, which was also visually observed by light microscopy. Both linalool and luteolin exhibited a significant reduction in the production of EPS and alginate in the biofilms matrix of V. harveyi which was confirmed by Scanning Electron Microscopy (SEM). Both compounds inhibited the swarming and swimming motility, the crucial quorum sensing (QS) mediated virulence of V. harveyi. The present study shows the presence of valuable polyphenolic compound like luteolin in coconut shells that are discarded as a waste. From the present study we envisage that luteolin and linalool can serve as potent anti-virulent agents to combat QS mediated infections against aquaculture pathogens.

Aeromonas hydrophila infection in silver catfish causes hyperlocomotion related to stress.

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.

The survival and hepatic and muscle glucose and lactate levels of Rhamdia quelen inoculated with Aeromonas hydrophila and treated with terpinen-4-ol, carvacrol or thymol.

The aim of this study was to evaluate the resistance of Rhamdia quelen (silver catfish) to Aeromonas hydrophila infection after treatment with pure and nanoencapsulated forms either terpinen-4-ol, thymol, or carvacrol and the effects of these treatments on fish metabolic responses. After A. hydrophila inoculation, fish were treated with 30 min daily baths for 6 consecutive days with terpinen-4-ol, thymol, or carvacrol in their pure or nanoencapsulated forms at concentrations of 5, 10, 15 or 25 mg L-1. A positive control group, negative control group and saline group were also included. Survival was evaluated at the end of treatment for six consecutive days. Muscle and liver were collected to determine glucose and lactate levels. The fish treated with the nanoencapsulated form of the compounds had a high survival rate, similar to saline group and negative control groups. The carvacrol, thymol and terpinen-4-ol nanoencapsulated forms improved survival of silver catfish infected with A. hydrophila. Muscle and liver glucose and lactate levels are not indicated as biomarkers because they did not present any correlation between the metabolic state of the fish and the bacterial infection.

Citrobacter freundii infection in silver catfish (Rhamdia quelen): Hematological and histological alterations.

Citrobacter freundii is a fish pathogen known for its ability to cause injury and high mortality. There have been no studies reporting the effect of this bacterium on hematological parameters and internal organ histology in silver catfish (Rhamdia quelen). Therefore, the aim of this study is to evaluate the hematological and histopathological effects of an experimentally induced C. freundii infection in silver catfish. Twenty fish were divided into healthy and infected groups. The fish of the infected group were inoculated intramuscularly with 100 µL of bacterial suspension (6.4 × 108 CFU mL-1), while healthy control animals received 100 µL of sterile saline. On day 18 post-infection, blood and tissues (cephalic kidneys, livers, and spleens) were collected for histological analysis. The infected animals presented high mortality, as well as hematological and histological changes. In relation to hematology, the infected fish presented aregenerative anemia, protein loss, leukopenia with neutropenia, lymphocytosis, and leukoblastosis. Regarding histology, there was liver degeneration, decrease in the amount of renal hematopoietic tissue, and the presence of melanomacrophage centers (MMCs) in the spleen and cephalic kidney of infected fish. In summary, these alterations may contribute to disease pathophysiology, contributing to high mortality of affected fish.

Plant essential oils against bacteria isolated from fish: an in vitro screening and in vivo efficacy of Lippia origanoides / Óleos essenciais de plantas contra bactérias isoladas de peixes: uma triagem in vitro e eficácia in vivo de Lippia origanoides

ABSTRACT: The use of natural products, such as essential oils (EOs), is a potential novel approach to treat fish bacterial infections with a lower risk of developing resistance. There has been a number of studies reporting the activity of EOs as those obtained from the species Achyrocline satureioides, Aniba parviflora, Aniba rosaeodora, Anthemis nobilis, Conobea scoparioides, Cupressus sempervirens, Illicium verum, Lippia origanoides, and Melaleuca alternifolia against bacteria. However, there are few studies investigating the effect of these EOs against fish bacteria. Therefore, the aim of this study was to evaluate the in vitro antibacterial activity of EOs against the following fish bacteria, Aeromonas hydrophila, Citrobacter freundii, and Raoultella ornithinolytica. Additionally, the in vivo antibacterial activity of the EO L. origanoides was evaluated against experimentally induced A. hydrophila infection of silver catfish (Rhamdia quelen). The EO of L. origanoides was chosen as it showed the highest in vitro antibacterial activity, with minimum inhibitory concentrations ranging from 0.2 to 0.8 mg mL-1. This EO also presented a therapeutic success of 58.33%, on a 30 day A. hydrophila infection. Therefore, we suggested that the EO of L. origanoides may be a viable alternative as a treatment for A. hydrophila infection.

RESUMO: O uso de produtos naturais, como óleos essenciais (OEs), é uma nova abordagem potencial para o tratamento de infecções bacterianas em peixes com um baixo risco de desenvolvimento de resistência. Existem estudos reportando a atividade de OEs como aqueles obtidos das espécies Achyrocline satureioides, Aniba parviflora, Aniba rosaeodora, Anthemis nobilis, Conobea scoparioides, Cupressus sempervirens, Illicium verum, Lippia origanoides e Melaleuca alternifolia contra bactérias. Porém, existem poucos estudos investigando o efeito desses OEs supracitados contra bactérias isoladas de peixes. Portanto, o objetivo desse estudo foi avaliar a atividade antibacteriana in vitro desses OEs contra bactérias isoladas de peixes (Aeromonas hydrophila, Citrobacter freundii e Raoultella ornithinolytica). Assim como avaliar a atividade antibacteriana in vivo do OE de L. origanoides contra uma infecção por A. hydrophila experimentalmente induzida em jundiá (Rhamdia quelen). O OE de L. origanoides foi escolhido porque apresentou a melhor atividade antibacteriana in vitro, com concentrações inibitórias mínimas variando de 0,2 a 0,8 mg mL-1. Esse OE apresentou sucesso terapêutico de 58,33% no dia 30 pós-infecção. Portanto, sugerimos que o OE de L. origanoides possa ser uma alternativa viável no tratamento da infecção por A. hydrophila em peixes.

Essential oils of Cunila galioides and Origanum majorana as anesthetics for Rhamdia quelen: efficacy and effects on ventilation and ionoregulation

This study evaluated anesthetic efficacy and possible effects of the essential oils (EOs) of Cunila galioides (EOC) and Origanum majorana (EOO) on ventilatory rate (VR) and ionoregulation in Rhamdia quelen. In the anesthesia assessments, 50, 100, 200 and 300 μL L-1 EOC and 50, 100, 200, 300, 400 and 500 μL L-1 EOO were tested, and time for induction to sedation and anesthesia stages, as well as recovery, were taken. A second trial employed lower concentrations of both EOs, 10, 25, 50 and 100 μL L-1, in order to verify VR and Na+, K+ and Cl- whole body net fluxes. Sedation was achieved with both oils at 100 µL L-1, and anesthesia at ≥ 200 µL L-1. There was no significant difference between control and EO-treated groups regarding VR, but all fish subjected to 100 µL L-1 EOC died within 2 h of exposure. Overall, ionic loss declined in the presence of the EOs. The EOC at 200 - 300 μL L-1 and EOO at 400 - 500 μL L-1 present the potential to promote fast anesthesia in R. quelen.(AU)

No presente estudo foi avaliada a eficácia da anestesia e possíveis efeitos dos óleos essenciais (EOs) de Cunila galioides (EOC) e Origanum majorana (EOO) sobre a taxa ventilatória (VR) e regulação iônica em Rhamdia quelen. Nas avaliações de anestesia, as concentrações de 50, 100, 200 e 300 μL L-1 EOC e 50, 100, 200, 300, 400 e 500 μL L-1 EOO foram testadas, e os tempos para a indução às fases de sedação e de anestesia, assim como recuperação, foram mensurados. Um segundo ensaio empregou concentrações mais baixas de ambos EOs: 10, 25, 50 e 100 μL L-1 a fim de verificar a VR e o fluxo líquido corporal dos íons Na+, K+ and Cl- . A sedação foi alcançada para ambos os óleos em 100 μL L-1, e a anestesia em concentrações ≥ 200 µL L-1. Não houve diferença significativa entre o controle e grupos tratados com EOs em relação a VR, mas todos os peixes submetidos a 100 µL L-1 do EOC morreram dentro de 2 h de exposição. No geral, a perda iônica declinou na presença dos EOs. O EOC em 200 - 300 μL L-1 e o EOO em 400 - 500 μL L-1 apresentam potencial para anestesia rápida em R. quelen.(AU)

Temporal profile of antimicrobial resistance exhibited by strains of Staphylococcus spp. isolated from cases of bovine mastitis for 20 years (1992-2011) / Perfil temporal da resistência antimicrobiana exibido por cepas de Staphylococcus spp. isoladas de casos de mastite bovina durante 20 anos (1992-2011)

Records of in vitro susceptibility tests performed between 1992 and 2011 were retrospectively reviewed in order to evaluate the dynamic profiles of possible changes in antimicrobial resistance of Staphylococcus spp. isolated from milk samples of cows with mastitis during two decades. The results of 2,430 isolates tested by disk diffusion technique for susceptibility to oxacillin, penicillin, ampicillin, cephalexin, norfloxacin, tetracycline, sulfazotrim, gentamicin, and neomycin were analysed. Comparisons were performed between the percentages of resistance to antimicrobials and their classes and also between the decades studied. Additionally, the possible tendency or changes in the behaviour of these pathogens against the major drugs used in the last two decades were evaluated using regression analysis. The highest rates of resistance (P<0.0001) were observed for the beta-lactams (34.3%), with exception of cephalexin (6.9%), and for the tetracyclines (28%). Similar resistance rates (7.6% to 15.7%) were observed among the other drugs. Regression analysis showed a reduction in resistance to penicillin and ampicillin throughout the period, whilst for oxacillin and neomycin a decrease in the resistance was observed during the first decade, followed by an increase. A trend towards decreased resistance was found for sulfazotrim, whereas for the other antimicrobials no decrease was observed. The results indicated no trend towards increased resistance for most antimicrobials tested. Nevertheless, it is necessary to monitor the resistance patterns of these pathogens in order to save these drugs as a therapeutic reserve.

Os registros de resultados de testes de suscetibilidade in vitro realizados no período de 1992 a 2011 foram revisados, retrospectivamente, a fim de avaliar a dinâmica de possíveis mudanças dos perfis de resistência de isolados de Staphylococcus spp., oriundos de amostras de leite de vacas com mastite ao longo do tempo. Os resultados de 2430 isolados testados pela técnica de difusão em disco para oxacilina, penicilina, ampicilina, cefalexina, norfloxacina, tetraciclina, sulfazotrim, gentamicina e neomicina foram analisados. Comparações entre as médias de resistência para os antimicrobianos e suas respectivas classes e em relação ao período analisado foram estabelecidas. O comportamento da resistência para cada antimicrobiano foi avaliado por meio de análise de regressão. As maiores taxas de resistência (P<0,0001) foram observadas para os betalactâmicos (34,3%), com exceção da cefalexina (6,9%), e tetraciclina (28%). Frente aos demais fármacos, os isolados apresentaram médias de resistência semelhantes entre si (de 7,6% a 15,7%). A análise de regressão demonstrou redução da resistência para penicilina e ampicilina ao longo do período analisado. Para a oxacilina e a neomicina, houve um decréscimo da resistência dos micro-organismos testados durante a primeira década; entretanto, houve um aumento no segundo período. Em relação ao sulfazotrim, foi verificada uma tendência à diminuição da resistência dos isolados, ao passo que, para os demais antimicrobianos, nenhum comportamento foi destacado. Os resultados não indicaram tendência de aumento da resistência dos isolados de Staphylococcus spp. para a maioria dos antimicrobianos testados. Contudo, é necessário monitorar os padrões de resistência destes patógenos, a fim de que estes fármacos sejam preservados para garantir uma reserva terapêutica.