Antibody Based Diagnostic for Detection Of Vibrios and their Biological Control using Antagonistic Bacteria in Macrobrachium Rosenbergii Larval Rearing Systems

The main objective of the work undertaken here was to develop an appropriate microbial technology to protect the larvae of M.rosenbergii in hatchery from vibriosis. This technology precisely is consisted of a rapid detection system of vibrios and effective antagonistic probiotics for the management of vibrios. The present work was undertaken with the realizations that to stabilize the production process of commercial hatcheries an appropriate, comprehensive and fool proof technology is required primarily for the rapid detection of Vibrio and subsequently for its management. Nine species of Vibrio have been found to be associated with larvae of M. rosenbergii in hatchery. Haemolytic assay of the Vibrio and Aeromonas on prawn blood agar showed that all isolates of V. alginolyticus and Aeromonas sp., from moribund, necrotized larve were haemolytic and the isolates of V.cholerae, V.splendidus II, V.proteolyticus and V.fluvialis from the larvae obtained from apparently healthy larval rearing systems were non-haemolytic. Hydrolytic enzymes such as lipase, chitinase and gelatinase were widespread amongst the Vibrio and Aeromonas isolates. Dominance of V.alginolyticus among the isolates from necrotic larvae and the failure in isolating them from rearing water strongly suggest that they infect larvae and multiply in the larval body and cause mortality in the hatchery. The observation suggested that the isolate V. alginolyticus was a pathogen to the larvae of M.rosenbergii. To sum up, through this work, nine species of Vibrio and genus Aeromonas associated with M.rosenbergii larval rearing systems could be isolated and segregated based on the haemolytic activity and the antibodies (PA bs) for use in diagnosis or epidemiological studies could be produced, based on a virulent culture of V.alginolyticus. This could possibly replace the conventional biochemical tests for identification. As prophylaxis to vibriosis, four isolates of Micrococcus spp. and an isolate of Pseudomonas sp. could be obtained which could possibly be used as antagonistic probiotics in the larval rearing system of M.rosenbergii.

Salmonella cross contamination in retail chicken outlets and the efficacy of spice extracts on salmonella enteritidis growth inhibhition on various surfaces

The source of samonella cross contamination in 15 retail chicken outlets in aresidual area in coimbatore city ,sourthern India was studied. Chopping boards and the butchers hands were predominant followed by knives and the weighing balance tray. Serotyping of the salmonella strains revealed that all strains were salmonella enteritis, except one which was found to be salmonella cerro.The anti bacterial activity of commonly used spices were evaluated.

Caso sospechoso de envenenamiento por araña reclusa (loxosceles) y revisión de la literatura

El Loxoscelismo en una enfermedad causada por la picadura de una araña del género Loxosceles, que se puede manifestar con compromiso cutáneo y/o sistémico e incluso llevar a la muerte. La distribución geográfica de la araña es mundial, con mayor incidencia en países como Perú, Chile y Brasil, y en algunos lugares de Norte América. En Colombia, se ha identificado este tipo de arácnido, sin embargo no se ha reportado ningún caso, se cree que probablemente exista subregistro. El cuadro clínico puede ir desde pacientes asintomáticos hasta dermonecrosis severa, con gran riesgo de secuelas e incapacidad funcional. Cuando hay compromiso sistémico, cursa con falla renal, hemólisis, coagulación intravascular diseminada y alto riesgo de muerte. El diagnóstico es clínico y epidemiológico, pues no hay pruebas serológicas confirmatorias disponibles. A pesar de que se han descrito múltiples opciones terapéuticas, no hay un consenso que permita hacer recomendaciones, por la escasa evidencia científica que existe al respecto. Este artículo presenta el caso de un varón de 11 años, con antecedente de picadura por animal desconocido, con evolución clínica sugestiva de loxoscelismo cutáneo y sistémico, que recibió manejo con suero antiarácnido, oxígeno hiperbárico, inhibidor de polimorfonucleares y reconstrucción quirúrgica. Se revisa la literatura y la situación de la enfermedad en Colombia, con el fin de llamar la atención sobre una enfermedad poco conocida y favorecer la sospecha diagnóstica y el enfoque adecuado.

Hemi-maxilectomia no tratamento de fibroma oral em cão : caso clínico

A cavidade oral é o quarto local mais afetado por neoplasias em cães e gatos, representando 3% em gatos e 6% em cães, podendo ser benignas ou malignas e de origem dental ou extradental. O objetivo do trabalho foi relatar o tratamento cirúrgico por hemi-maxilectomia parcial num cão de 10 anos de idade, da raça pastor alemão, atendido no Hospital Veterinário diagnosticado histopatologicamente por fibroma oral. Durante o exame físico foi observado um aumento de volume do tipo tumoral, no palato duro, estendendo-se desde o canino esquerdo até o primeiro molar, com tecido necrosado. Foram realizados hemograma, bioquímica sérica, urinálise, radiografia do tórax e biopsia. O tratamento cirúrgico instituído foi eficaz, pois após um ano não se observou qualquer sinal de recidiva tumoral.

Estudo da presença de Mycobacterium avium subespécie paratuberculosis numa exploração leiteira no Litoral Centro de Portugal Continental

A Paratuberculose, conhecida também como Doença de Johne, é uma afeção infecciosa que provoca uma enterite granulomatosa crónica causada pelo agente Mycobacterium avium subespécie paratuberculosis (Map). É uma doença de carácter contagioso, de distribuição mundial, atingindo os mamíferos, particularmente os pequenos e grandes ruminantes, equinos, suínos, búfalos, coelhos, etc. Tem um grande impacto económico, nomeadamente na redução da produção leiteira, na redução dos teores de proteína no leite, na susceptibilidade a outras doenças, no refugo de animais e no aumento dos custos na sanidade. Pensa-se que também terá impacto a nível de Saúde Pública uma vez que o Map pode estar associado á doença de Crohn em humanos em que o leite cru, leite em pó ou leite pasteurizado podem ser os veículos de transmissão, porém não existem estudos suficientes para sustentar este acontecimento. A técnica de diagnóstico “Gold Standard” é a cultura microbiológica de fezes, no entanto é um procedimento muito demorado podendo levar até 4 meses para se observarem as colónias bacterianas, uma vez que o seu crescimento é muito lento. Deste modo, existem outros testes, tais como o PCR e ELISA, com elevada especificidade para o agente que fornecem resultados mais rápidos e que permitem minimizar os falsos-positivos, apesar da sua reduzida sensibilidade (por volta dos 50%). Este estudo é baseado na observação das instalações da exploração e consequente comparação com parâmetros analisados e documentados em literatura. Para além disso, são utilizadas informações fornecidas pelo Software da exploração com o intuito de estudar a incidência da Paratuberculose, ao longo dos anos, de modo a comprovar se as medidas de maneio e de higiene adotadas pela exploração afetada contribuem ou não de alguma maneira para o controlo/erradicaçãoda doença de Johne.

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity

Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO center dot) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage. (c) 2007 Elsevier B.V. All rights reserved.

Modelling acidosis and the cell cycle in multicellular tumour spheroids

A partial differential equation model is developed to understand the effect that nutrient and acidosis have on the distribution of proliferating and quiescent cells and dead cell material (necrotic and apopotic) within a multicellular tumour spheroid. The rates of cell quiescence and necrosis depend upon the local nutrient and acid concentrations and quiescent cells are assumed to consume less nutrient and produce less acid than proliferating cells. Analysis of the differences in nutrient consumption and acid production by quiescent and proliferating cells shows low nutrient levels do not necessarily lead to increased acid concentration via anaerobic metabolism. Rather, it is the balance between proliferating and quiescent cells within the tumour which is important; decreased nutrient levels lead to more quiescent cells, which produce less acid than proliferating cells. We examine this effect via a sensitivity analysis which also includes a quantification of the effect that nutrient and acid concentrations have on the rates of cell quiescence and necrosis.

Mutation of toxB and a truncated version of the efa-1 gene in Escherichia coli O157 : H7 influences the expression and secretion of locus of enterocyte effacement-encoded proteins but not intestinal colonization in calves or sheep

Enterohemorrhagic Escherichia coli (EHEC) strains comprise a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in humans and animals. Non-O157:H7 EHEC strains contain the gene efa-1 (referred to in previous publications as efa1), which influences adherence to cultured epithelial cells. An almost identical gene in enteropathogenic E. coli (lifA) mediates the inhibition of lymphocyte proliferation and proinflammatory cytokine synthesis. We have shown previously that significantly lower numbers of EHEC 05 and 0111 efa-1 mutants are shed in feces following experimental infection in calves and that these mutants exhibit reduced adherence to intestinal epithelia compared with isogenic wild-type strains. E. coli O157:H7 strains lack efa-1 but encode a homolog on the pO157 plasmid (toxB/l7095) and contain a truncated version of the efa-1 gene (efa-1'/z4332 in O island 122 of the EDL933 chromosome). Here we report that E. coli O157:H7 toxB and efa-1' single and double mutants exhibit reduced adherence to cultured epithelial cells and show reduced expression and secretion of proteins encoded by the locus of enterocyte effacement (LEE), which plays a key role in the host-cell interactions of EHEC. The activity of LEE1, LEE4, and LEE5 promoters was not significantly altered in E. coli O157:H7 strains harboring toxB or efa-1' mutations, indicating that the effect on the expression of LEE-encoded secreted proteins occurs at a posttranscriptional level. Despite affecting type III secretion, mutation of toxB and efa-1' did not significantly affect the course of fecal shedding of E. coli O157:H7 following experimental inoculation of 10- to 14-day-old calves or 6-week-old sheep. Mutation of tir caused a significant reduction in fecal shedding of E. coli O157:H7 in calves, indicating that the formation of AE lesions is important for colonization of the bovine intestine.

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.

Regulation of cardiac myocyte cell death.

Cardiac myocyte death, whether through necrotic or apoptotic mechanisms, is a contributing factor to many cardiac pathologies. Although necrosis and apoptosis are the widely accepted forms of cell death, they may utilize the same cell death machinery. The environment within the cell probably dictates the final outcome, producing a spectrum of response between the two extremes. This review examines the probable mechanisms involved in myocyte death. Caspases, the generally accepted executioners of apoptosis, are significant in executing cardiac myocyte death, but other proteases (e.g., calpains, cathepsins) also promote cell death, and these are discussed. The two principal cell death pathways (death receptor- and mitochondrial-mediated) are described in relation to the emerging structural information for the principal proteins, and they are discussed relative to current understanding of myocyte cell death mechanisms. Whereas the mitochondrial pathway is probably a significant factor in myocyte death in both acute and chronic phases of myocardial diseases, the death receptor pathway may prove significant in the longer term. The Bcl-2 family of proteins are key regulators of the mitochondrial death pathway. These proteins are described and their possible functions are discussed. The commitment to cell death is also influenced by protein kinase cascades that are activated in the cell. Whereas certain pathways are cytoprotective (e.g., phosphatidylinositol 3'-kinase), the roles of other kinases are less clear. Since myocyte death is implicated in a number of cardiac pathologies, attenuation of the death pathways may prove important in ameliorating such disease states, and possible therapeutic strategies are explored.

‘Symptomless’ infection by Botrytis cinerea

The study was carried out to clarify the nature of symptomless infection by Botrytis cinerea and to what extent it differs from aggressive necrotic infection in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless plants were produced by dry spore inoculation in plants growing in controlled environmental conditions or in glasshouses. Plating out of surface-disinfected and non-surface-disinfected samples of inoculated, apparently healthy, plants on selective medium revealed that the fungus was spreading from the initial inoculation site to newly developing plant organs both internally and externally. Similar findings were obtained in microscope experiments in which host plants were inoculated with GFP labelled B. cinerea and symptomless spreading was monitored under confocal laser scanning microscope. Spore germination on leaf surface was followed by development of sub-cuticular vesicles and plant cell damage in the infected epidermal cell and a few nearby cells. Sparsely branched long hyphae arose from the vesicles and spread on the leaf surface; spread was mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells. In the late symptomless phase, mycelium arising from single vesicles formed several mycelial networks on leaves. Experiments were carried out to compare the extent of gene expression in symptomless and necrotic infections, using RT-qPCR. Expression of selected genes was quantified in tissue samples based on the amount of mRNA of the respective genes found. In both host species, the mRNA concentration of signalling genes bcg1, bmp1 and calcineurin, and the pathogenicity genes bcsod1 and bcpg1 were similar to or slightly greater in symptomless samples than in necrotic samples. The mRNA of the signalling gene bac and pathogenicity genes bcbot1 and bcnep1, were not detected or detected in lower abundance than in necrosis. In lettuce, the leaves developing distant from the site of inoculation showed similar results to A. thaliana, but in healthy leaves close to the site of inoculation mRNA concentrations of bac and bcnep1 were similar to necrotic samples. Thus, in both host species, the fungus grew along with the plant and moved to newly growing plant parts without producing symptoms; during this growth some pathogenicity genes were less expressed than in necrotic infection.

Microbial culture and checkerboard DNA-DNA hybridization assessment of bacteria in root canals of primary teeth pre- and post-endodontic therapy with a calcium hydroxide/chlorhexidine paste

Aim. To investigate the root canal microbiota of primary teeth with apical periodontitis and the in vivo antimicrobial effects of a calcium hydroxide/chlorhexidine paste used as root canal dressing. Design. Baseline samples were collected from 30 root canals of primary teeth with apical periodontitis. Then, the root canals were filled with a calcium hydroxide paste containing 1% chlorhexidine for 14 days and the second bacteriologic samples were taken prior to root canal filling. Samples were submitted to microbiologic culture procedure to detect root canal bacteria and processed for checkerboard DNA-DNA hybridization. Results. Baseline microbial culture revealed high prevalence and cfu number of anaerobic, black-pigmented bacteroides, Streptococcus, and aerobic microorganisms. Following root canal dressing, the overall number of cfu was dramatically diminished compared to initial contamination (P < 0.05), although prevalence did not change (P > 0.05). Of 35 probes used for checkerboard DNA-DNA hybridization, 31 (88.57%) were present at baseline, and following root canal dressing, the number of positive probes reduced to 13 (37.14%). Similarly, the number of bacterial cells diminished folowing application of calcium hydroxide/chlorhexidine root canal dressing (P = 0.006). Conclusion. Apical periodontitis is caused by a polymicrobial infection, and a calcium hydroxide/chlorhexidine paste is effective in reducing the number of bacteria inside root canals when applied as a root canal dressing.

Structural changes in the epithelium of the small intestine and immune cell infiltration of enteric ganglia following acute mucosal damage and local inflammation

An acute enteritis is commonly followed by intestinal neuromuscular dysfunction, including prolonged hyperexcitability of enteric neurons. Such motility disorders are associated with maintained increases in immune cells adjacent to enteric ganglia and in the mucosa. However, whether the commonly used animal model, trinitrobenzene sulphonate (TNBS)-induced enteritis, causes histological and immune cell changes similar to human enteric neuropathies is not clear. We have made a detailed study of the mucosal damage and repair and immune cell invasion following intralumenal administration of TNBS. Intestines from untreated, sham-operated and TNBS-treated animals were examined at 3 h to 56 days. At 3 h, the mucosal surface was completely ablated, by 6 h an epithelial covering was substantially restored and by 1 day there was full re-epithelialisation. The lumenal epithelium developed from a squamous cell covering to a fully differentiated columnar epithelium with mature villi at about 7 days. Prominent phagocytic activity of enterocytes occurred at 1-7 days. A surge of eosinophils and T lymphocytes associated with the enteric nerve ganglia occurred at 3 h to 3 days. However, elevated immune cell numbers occurred in the lamina propria of the mucosa until 56 days, when eosinophils were still three times normal. We conclude that the disruption of the mucosal surface that causes TNBS-induced ileitis is brief, a little more than 6 h, and causes a transient immune cell surge adjacent to enteric ganglia. This is much briefer than the enteric neuropathy that ensues. Ongoing mucosal inflammatory reaction may contribute to the persistence of enteric neuropathy.

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1 beta (IL-1 beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alphalbeta (CINC-2 alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process. Copyright (c) 2007 John Wiley & Sons, Ltd.

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: Biological effects benchmarking relatively to jararhagin, a P-IIISVMP

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding. (C) 2007 Elsevier Ltd. All rights reserved.