Survey of canine tick-borne diseases in Lábrea, Brazilian Amazon: ‘accidental findings of Dirofilaria immitis infection / Pesquisa de agentes transmitidos por carrapatos em cães de Lábrea, Amazonas: achados “acidentais” de Dirofilaria immitis

Blood samples were collected from 99 domestic dogs from the urban and rural areas of the Lábrea municipality, state of Amazonas, Brazil. Canine serum samples were tested by immunofluorescence assay against Rickettsia spp., which revealed that only 3.0% (1/33) and 7.6% (5/66) of the dogs from urban and rural areas, respectively, reacted positively to at least one Rickettsia species. DNA was extracted from canine blood and tested by a battery of PCR assays targeting protozoa of the genera Babesia and Hepatozoon, and bacteria of the genera Rickettsia and Ehrlichia and family Anaplasmataceae. All samples were negative in the PCR assays targeting the genera Babesia, Hepatozoon, Ehrlichia and Rickettsia. For Anaplasmataceae, 3% (1/33) and 39.4% (26/66) of the urban and rural dogs, respectively, yielded amplicons that generated DNA sequences 100% identical to the corresponding sequence of Wolbachia endosymbiont of Dirofilaria immitis. Because of these results, all canine DNA samples were further tested in a PCR assay targeting filarial nematodes, which was positive for 18.2% (6/33) and 57.6% (38/66) urban and rural dogs, respectively. Filarial-PCR products generated DNA sequences 100% identical to D. immitis. While tick-borne infections were rare in Lábrea, D. immitis infection rates were among the highest reported in South America.

Amostras de sangue foram coletadas de 99 cães domésticos de áreas urbana e rural do município de Lábrea, estado do Amazonas. Soros caninos foram testados pela técnica de imunofluorescência indireta contra Rickettsia spp., resultando em apenas 3,0% (1/33) e 7,6% (5/66) de cães soropositivos nas áreas urbana e rural, respectivamente. DNA foi extraído do sangue canino e testado por diferentes protocolos da PCR para detecção de protozoários dos gêneros Babesia e Hepatozoon, e bactérias dos gêneros Rickettsia e Ehrlichia e da família Anaplasmataceae. Todas as amostras foram negativas nos protocolos de PCR para os gêneros Babesia, Hepatozoon, Ehrlichia e Rickettsia. Para Anaplasmataceae, 3% (1/33) e 39,4% (26/66) dos cães de áreas urbana e rural, respectivamente, geraram sequências de DNA 100% idênticas ao endosimbionte Wolbachia de Dirofilaria immitis. Posteriormente, as amostras foram testadas pela PCR para nematódeos filarídeos, resultando em 18,2% (6/33) e 57,6% (38/66) de amostras positivas nas áreas urbana e rural, respectivamente. Os produtos geraram sequências de DNA 100% idênticas a D. immitis. Em contraste com várias outras regiões do Brasil, infecções transmitidas por carrapatos foram raras em Lábrea. Por outro lado, as frequências de infecção por D. immitis estiveram entre as mais altas relatadas na América do Sul.

Survey of canine tick-borne diseases in Lábrea, Brazilian Amazon: ‘accidental’ findings of Dirofilaria immitis infection / Pesquisa de agentes transmitidos por carrapatos em cães de Lábrea, Amazonas: achados “acidentais” de Dirofilaria immitis

Blood samples were collected from 99 domestic dogs from the urban and rural areas of the Lábrea municipality, state of Amazonas, Brazil. Canine serum samples were tested by immunofluorescence assay against Rickettsia spp., which revealed that only 3.0% (1/33) and 7.6% (5/66) of the dogs from urban and rural areas, respectively, reacted positively to at least one Rickettsia species. DNA was extracted from canine blood and tested by a battery of PCR assays targeting protozoa of the genera Babesia and Hepatozoon, and bacteria of the genera Rickettsia and Ehrlichia and family Anaplasmataceae. All samples were negative in the PCR assays targeting the genera Babesia, Hepatozoon, Ehrlichia and Rickettsia. For Anaplasmataceae, 3% (1/33) and 39.4% (26/66) of the urban and rural dogs, respectively, yielded amplicons that generated DNA sequences 100% identical to the corresponding sequence of Wolbachia endosymbiont of Dirofilaria immitis. Because of these results, all canine DNA samples were further tested in a PCR assay targeting filarial nematodes, which was positive for 18.2% (6/33) and 57.6% (38/66) urban and rural dogs, respectively. Filarial-PCR products generated DNA sequences 100% identical to D. immitis. While tick-borne infections were rare in Lábrea, D. immitis infection rates were among the highest reported in South America.

Amostras de sangue foram coletadas de 99 cães domésticos de áreas urbana e rural do município de Lábrea, estado do Amazonas. Soros caninos foram testados pela técnica de imunofluorescência indireta contra Rickettsia spp., resultando em apenas 3,0% (1/33) e 7,6% (5/66) de cães soropositivos nas áreas urbana e rural, respectivamente. DNA foi extraído do sangue canino e testado por diferentes protocolos da PCR para detecção de protozoários dos gêneros Babesia e Hepatozoon, e bactérias dos gêneros Rickettsia e Ehrlichia e da família Anaplasmataceae. Todas as amostras foram negativas nos protocolos de PCR para os gêneros Babesia, Hepatozoon, Ehrlichia e Rickettsia. Para Anaplasmataceae, 3% (1/33) e 39,4% (26/66) dos cães de áreas urbana e rural, respectivamente, geraram sequências de DNA 100% idênticas ao endosimbionte Wolbachia de Dirofilaria immitis. Posteriormente, as amostras foram testadas pela PCR para nematódeos filarídeos, resultando em 18,2% (6/33) e 57,6% (38/66) de amostras positivas nas áreas urbana e rural, respectivamente. Os produtos geraram sequências de DNA 100% idênticas a D. immitis. Em contraste com várias outras regiões do Brasil, infecções transmitidas por carrapatos foram raras em Lábrea. Por outro lado, as frequências de infecção por D. immitis estiveram entre as mais altas relatadas na América do Sul.

Vibrio ordalii antimicrobial susceptibility testing--modified culture conditions required and laboratory-specific epidemiological cut-off values.

The present study aimed to determine oxytetracycline (OTC), florfenicol (FLO) and oxolinic acid (OXO) MICs and zone diameters for 24 Chilean Vibrio ordalii isolates using the methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals and the methods for antimicrobial disk susceptibility testing of bacteria isolated from aquatic animals guidelines published by the Clinical Laboratory Standards Institute (CLSI). The results were then used in a normalized resistance interpretation (NRI) analysis to establish tentative laboratory-specific epidemiological cut-off (ECOFF) values. MIC results were similar at the two tested temperatures (22 °C and 18 °C). At 18 °C, the NRI analysis of OTC, FLO and OXO MIC data calculated laboratory-specific ECOFF values and non-wild-type (NWT) rates to be ≤4 mg/l (24%), ≤16 mg/l (4%) and ≤8 mg/l (25%), respectively. Tests performed with all V. ordalii isolates following the officially recommended incubation temperature (22 °C) revealed difficulties in measuring inhibition zone diameters. When disk diffusion tests were performed using Mueller-Hinton agar with 1% NaCl (MHA-1) at 18 °C the inhibition zone diameter distributions showed the formation of WT populations which could be defined using NRI analysis. For OTC the laboratory-specific ECOFF value was ≥38 mm with NWT rate of 16.7%. For FLO and OXO, the laboratory-specific ECOFF values were ≥38 and ≥40, respectively, generating NWT rates of 25 and 46%, respectively. Although the CLSI suggests testing Vibrio spp. on MHA-1 at 22 °C, we found measurements of the 24 isolates were better defined and normally distributed at 18 °C. This is the first study determining the MIC and disk diffusion test of V. ordalii isolated from diseased salmonids, where laboratory-specific ECOFF values could be established. Also resistance to OTC, FLO and OXO among some Chilean isolates was demonstrated.

Development and validation of a method for the simultaneous extraction and separate measurement of oxytetracycline, florfenicol, oxolinic acid and flumequine from marine sediments.

A simple and rapid method for the detection and extraction of oxolinic acid, flumequine, florfenicol and oxytetracycline from marine sediments was developed and validated. The analytes were extracted from the marine sediment using a solution of oxalic acid diluted in methanol with sonication before detection by HPLC using a diode-array detector (florfenicol and oxytetracycline) and fluorescence (oxolinic acid and flumequine). The quantification limits (QL) were 100 ng/g for oxytetracycline and florfenicol and 5 ng/g for oxolinic acid and flumequine. The coefficients of variation of the repeatability and intermediate precision were less than 10% in all of the analytes. The calibration curves were linear between 50 and 500 ng/ml for oxytetracycline and florfenicol and 1 and 20 ng/ml for oxolinic acid and flumequine. The recuperation rate for the analytes was above 86%.

Salmon aquaculture and antimicrobial resistance in the marine environment.

Antimicrobials used in salmon aquaculture pass into the marine environment. This could have negative impacts on marine environmental biodiversity, and on terrestrial animal and human health as a result of selection for bacteria containing antimicrobial resistance genes. We therefore measured the numbers of culturable bacteria and antimicrobial-resistant bacteria in marine sediments in the Calbuco Archipelago, Chile, over 12-month period at a salmon aquaculture site approximately 20 m from a salmon farm and at a control site 8 km distant without observable aquaculture activities. Three antimicrobials extensively used in Chilean salmon aquaculture (oxytetracycline, oxolinic acid, and florfenicol) were studied. Although none of these antimicrobials was detected in sediments from either site, traces of flumequine, a fluoroquinolone antimicrobial also widely used in Chile, were present in sediments from both sites during this period. There were significant increases in bacterial numbers and antimicrobial-resistant fractions to oxytetracycline, oxolinic acid, and florfenicol in sediments from the aquaculture site compared to those from the control site. Interestingly, there were similar numbers of presumably plasmid-mediated resistance genes for oxytetracycline, oxolinic acid and florfenicol in unselected marine bacteria isolated from both aquaculture and control sites. These preliminary findings in one location may suggest that the current use of large amounts of antimicrobials in Chilean aquaculture has the potential to select for antimicrobial-resistant bacteria in marine sediments.

Degradation of the antibiotic oxolinic acid by photocatalysis with TiO2 in suspension.

In the work presented here, a photocatalytic system using titanium Degussa P-25 in suspension was used to evaluate the degradation of 20mg L(-1) of antibiotic oxolinic acid (OA). The effects of catalyst load (0.2-1.5 g L(-1)) and pH (7.5-11) were evaluated and optimized using the surface response methodology and the Pareto diagram. In the range of variables studied, low pH values and 1.0 g L(-1) of TiO(2) favoured the efficiency of the process. Under optimal conditions the evolution of the substrate, chemical oxygen demand, dissolved organic carbon, toxicity and antimicrobial activity on Escherichia coli cultures were evaluated. The results indicate that, under optimal conditions, after 30 min, the TiO(2) photocatalytic system is able to eliminate both the substrate and the antimicrobial activity, and to reduce the toxicity of the solution by 60%. However, at the same time, ∼53% of both initial DOC and COD remain in solution. Thus, the photocatalytical system is able to transform the target compound into more oxidized by-products without antimicrobial activity and with a low toxicity. The study of OA by-products using liquid chromatography coupled with mass spectrometry, as well as the evaluation of OA degradation in acetonitrile media as solvent or in the presence of isopropanol and iodide suggest that the reaction is initiated by the photo-Kolbe reaction. Adsorption isotherm experiments in the dark indicated that under pH 7.5, adsorption corresponded to the Langmuir adsorption model, indicating the dependence of the reaction on an initial adsorption step.

Quantitation and identity confirmation of residues of quinolones in tilapia fillets by LC-ESI-MS-MS QToF.

A method for simultaneous determination of flumequine (FLM), oxolinic acid (OXO), sarafloxacin (SAR), danofloxacin (DAN), enrofloxacin (ENR), and ciprofloxacin (CIP) in tilapia (Orechromis niloticus) fillets, using liquid chromatography-tandem mass spectrometry (LC-ESI-MS-MS QToF) is presented. The quinolones were extracted from the food matrix with a solution of 10% trichloroacetic acid-methanol (80:20 v/v) with ultrasonic assistance. Clean-up of the extract solution was performed by using polymeric solid-phase extraction cartridges. The LC separation was carried out on an octadecyl hybrid silica column (C18, 150 mm x 3 mm, 5 microm). The column temperature was set at 30 degrees C, and gradient elution (0.2 mL min(-1)) was performed using water and acetonitrile, both containing 0.1% of acetic acid, as mobile phase components. The analytes were ionized using electrospray in the positive polarity mode. The following analytical results were obtained: linearity was about 0.99 for all the quinolones; intra and inter-assay precision (RSD%) were lower than 12.7 and 20%, respectively; and recoveries were from 89 to 112%. The quantitation limits were below the maximum residue limits established for the analytes. The method is suitable for the determination of quinolone residues in fish fillets and the QToF technique made it possible to obtain m/z ratios with less than 10 ppm of error for each analyte.

Oxolinic acid photo-oxidation using immobilized TiO(2).

This work studied the photocatalysed oxidation of the antibiotic oxolinic acid (OA) in an annular reactor operated with immobilized TiO(2) on sintered glass cylinders (SGC). Experiments were carried out in 1l solution of OA (18 mg l(-1)) at pH 9 with oxygen bubbling. Irradiation was performed with black light (36 W). The reaction was monitored by COD, TOC and average oxidation state (AOS) calculations. The antibacterial activity of intermediates was followed using the inhibition halo technique on Escherichia coli cultures. The initial antibiotic concentration decreases in one order of magnitude after 60 min irradiation, and was completely eliminated at 100 min reaction. The TOC was reduced in 54% and the AOS reach values around +3 indicating the formation of low molecular weight carboxylic acids. The oxidation reaction fit well with the Langmuir-Hinshelwood kinetic model indicating the dependence of reaction rate with initial adsorption step. The antibacterial activity of the solution decreases with antibiotic removal, demonstrating that intermediates do not present antibiotic activity.

[Residues of tetracycline and quinolones in wild fish living around a salmon aquaculture center in Chile]. / Residuos de tetraciclina y quinolonas en peces silvestres en una zona costera donde se desarrolla la acuicultura del salmón en Chile.

The presence of residues of tetracycline, quinolones and antiparasitic drugs was investigated in wild fish captured around salmon aquaculture pens in Cochamó, Region X, Chile. Residues of both antibiotics were found in the meta [corrected] of two species of wild fish that are consumed by humans, robalo (Elginops maclovinus) and cabrilla (Sebastes capensis) [corrected] These findings suggest that the antibiotic usage in salmon aquaculture in Chile has nvironmental implications that may affect human and animal health. More studies are needed in Chile to determine the relevance of these findings for human and animal health and the environment to regulate this use of antibiotics.

Residuos de tetraciclina y quinolonas en peces silvestres en una zona costera donde se desarrolla la acuicultura del salmón en Chile / Residues of tetracycline and quinolones in wild fish living around a salmon aquaculture center in Chile

La presencia de antibacterianos y antiparasitarios residuales fue investigada en muestras de carne de peces silvestres de consumo humano pescados alrededor de un recinto de acuicultura en Cochamó (41° 29' S; 72° 19'W), X Región, Chile. Esta investigación demostró que peces silvestres, incluyendo róbalo (Elginops maclovinus), cabrilla (Sebastes capensis) y truchas de vida libre (Oncorhynchus mykiss), ingieren alimento artificial para salmón y que la carne de algunos ejemplares de estos peces contienen tetracicilina y quinolona en cantidades detectables. Estos resultados sugieren que el uso de antibacterianos en la acuicultura del salmón, como ha sido demostrado en otros países, tiene efectos ambientales que se proyectan más allá de los recintos de acuicultura. Se indica que dada la relevancia de estos hallazgos para la salud humana y animal, el ambiente requerirá de estudios más amplios y detallados para implementar futuras regulaciones del uso de antibacterianos en acuicultura.

The presence of residues of tetracycline, quinolones and antiparasitic drugs was investigated in wild fish captured around salmon aquaculture pens in Cochamó, Region X, Chile. Residues of both antibiotics were found in the meta of two species of wild fish that are consumed by humans, robalo (Elginops maclovinus) and cabrilla (Sebastes capensis) . These findings suggest that the antibiotic usage in salmon aquaculture in Chile has environmental implications that may affect human and animal health. More studies are needed in Chile to determine the relevance of these findings for human and animal health and the environment to regulate this use of antibiotics.

Structure and medium effects on the photochemical behavior of nonfluorinated quinolone antibiotics.

The photophysical behavior of the quinolone antibiotics, oxolinic (OX), cinoxacin (CNX) and pipemidic (PM) acids was studied as a function of pH and solvent properties. The ground state of these compounds exhibits different protonated forms, which also exist in the first excited states. Theoretical calculations of the Fukui indexes allowed to assigning the different protonation equilibria. The pK values indicate that the acidity of the 3-carboxylic and 4-carbonyl groups increases with the N-atom at position 2 in CNX. It has been found that fluorescence properties are strongly affected by pH, the more fluorescent species is that with protonated carboxylic acid, protonated species at the carbonyl group and the totally deprotonated form present very low fluorescence. The fluorescence behavior also depends on the chemical structure of the quinolone and on the solvent properties. The analysis of the solvent effect on the maximum and the width of the fluorescence band of OX, using the linear solvent-energy relation solvatochromic equation, indicates that the polarizability and hydrogen bond donor ability are the parameters that condition the spectral changes. The hydrogen bond acceptor ability of the solvents also contributes to the spectral shifts of CNX. The compound bearing the piperazinyl group at the position 7, PM only is fluorescent in high protic solvents. These results are discussed in terms of the competition between the intra- and intermolecular hydrogen bonds. The irradiation of OX, CNX and PM using 300 nm UV light led to a very low photodecomposition rate. Under the same conditions the nalidixic acid (NA), a structurally related quinolone, photodecomposes two orders of magnitude faster.

Lipid peroxidation and loss of potassium from red blood cells produced by phototoxic quinolones.

Alterations of the cationic permeability of red blood cell membranes induced by the photosensitiser nalidixic acid were demonstrated by evaluating the potassium loss from intact erythrocytes. The results show that an increase in intracellular potassium efflux, precedes the photohemolysis induced by nalidixic acid. The addition of a nonpermeable osmotic solute, such as sucrose, inhibited photohemolysis but not the potassium loss, indicating a colloid osmotic lysis. Lipid peroxidation induced by nalidixic acid and other photosensitiser quinolones (oxolinic acid and rosoxacin) was time irradiation-dependent. Although rosoxacin was the most photoperoxidative, none of the three quinolones studied produced significant lipid peroxidation. However, of the three quinolones studied, only rosoxacin considerably diminished the percentage of the cholesterol extracted from red blood cell membranes. It is postulated that the increased cation permeability induced by nalidixic and oxolinic acids cannot be attributed to cholesterol oxidation nor to lipid peroxidation; a more probable mechanism is photo-oxidation of amino acid residues of the membrane proteins. However, the lysis induced by rosoxacin is caused by photo-oxidation of cholesterol, not excluding other cellular targets.

Phototoxicity induced by nalidixic and oxolinic acids: decrease in cell survival of chick embryo fibroblasts and Hep-2 cells.

The phototoxic effects of nalidixic and oxolinic acids were evaluated in two types of cultured cells: chick embryo fibroblast and Hep-2 (human laryngo carcinoma cell line). In order to evaluate the phototoxicity induced by nalidixic and oxolinic acids, both cell types were irradiated for 5 min in the presence of each drug. The results showed an inverse relationship between cell survival and the concentration of the drug added to the culture medium. The concentrations of nalidixic and oxolinic acids necessary to induce a phototoxic effect were in the range of therapeutic blood levels. Both chick embryo fibroblasts and Hep-2 cells were more sensitive to the phototoxic effect induced by nalidixic acid than oxolinic acid.

Effects of DNA gyrase inhibitors in a polyamine-auxotrophic strain of Escherichia coli.

The polyamine content of the Escherichia coli polyamine-auxotrophic strain BGA 8 seemed to influence the effects of nalidixic acid, an antibiotic acting on subunit A of DNA gyrase. The growth rate was more affected under conditions of putrescine depletion and the inhibition could be partially relieved if the polycation was added back to the culture. DNA synthesis was likewise more sensitive to nalidixic acid in cultures grown without polyamine. The expression of some proteins characteristic of the heat-shock response, evoked by the antibiotic, showed a different persistence according to the presence or absence of polyamines. Novobiocin, acting on subunit B of gyrase, also promoted a differential effect depending on the polyamine content, but in this case putrescine-supplemented cells were more sensitive. The described findings suggest a role of polyamines in all the reactions carried out by gyrase, perhaps due to the influence of the polycations on the state of DNA aggregation.