Analysis of Kudoa thyrsites (Myxozoa: Myxosporea) spore antigens using monoclonal antibodies

A method employing Percoll gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoa-specific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K. thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K. thyrsites, K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to > 220 kDa, whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses, depending on the Kudoa species. The dominant epitope of the K. thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated, intact, permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting, indicating that these mAbs have potential for use in developing a field-based diagnostic test

Geographic Coincidence of Increased Malaria Transmission Hazard and Vulnerability Occurring at the Periphery of two Tanzanian Villages.

The goal of malaria elimination necessitates an improved understanding of any fine-scale geographic variations in transmission risk so that complementary vector control tools can be integrated into current vector control programmes as supplementary measures that are spatially targeted to maximize impact upon residual transmission. This study examines the distribution of host-seeking malaria vectors at households within two villages in rural Tanzania. Host-seeking mosquitoes were sampled from 72 randomly selected households in two villages on a monthly basis throughout 2008 using CDC light-traps placed beside occupied nets. Spatial autocorrelation in the dataset was examined using the Moran's I statistic and the location of any clusters was identified using the Getis-Ord Gi* statistic. Statistical associations between the household characteristics and clusters of mosquitoes were assessed using a generalized linear model for each species. For both Anopheles gambiae sensu lato and Anopheles funestus, the density of host-seeking females was spatially autocorrelated, or clustered. For both species, houses with low densities were clustered in the semi-urban village centre while houses with high densities were clustered in the periphery of the villages. Clusters of houses with low or high densities of An. gambiae s.l. were influenced by the number of residents in nearby houses. The occurrence of high-density clusters of An. gambiae s.l. was associated with lower elevations while An. funestus was also associated with higher elevations. Distance from the village centre was also positively correlated with the number of household occupants and having houses constructed with open eaves. The results of the current study highlight that complementary vector control tools could be most effectively targeted to the periphery of villages where the households potentially have a higher hazard (mosquito densities) and vulnerability (open eaves and larger households) to malaria infection

A standardized framework for the validation and verification of clinical molecular genetic tests

The validation and verification of laboratory methods and procedures before their use in clinical testing is essential for providing a safe and useful service to clinicians and patients. This paper outlines the principles of validation and verification in the context of clinical human molecular genetic testing. We describe implementation processes, types of tests and their key validation components, and suggest some relevant statistical approaches that can be used by individual laboratories to ensure that tests are conducted to defined standards

Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety

Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animal-derived feeder layers and media containing animal-derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-the-shelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process

Effect of meteorological factors on clinical malaria risk among children: an assessment using village-based meteorological stations and community-based parasitological survey

<p>Abstract</p> <p>Background</p> <p>Temperature, rainfall and humidity have been widely associated with the dynamics of malaria vector population and, therefore, with spread of the disease. However, at the local scale, there is a lack of a systematic quantification of the effect of these factors on malaria transmission. Further, most attempts to quantify this effect are based on proxy meteorological data acquired from satellites or interpolated from a different scale. This has led to controversies about the contribution of climate change to malaria transmission risk among others. Our study addresses the original question of relating meteorological factors measured at the local scale with malaria infection, using data collected at the same time and scale.</p> <p>Methods</p> <p>676 children (6–59 months) were selected randomly from three ecologically different sites (urban and rural). During weekly home visits between December 1, 2003, and November 30, 2004, fieldworkers tested children with fever for clinical malaria. They also collected data on possible confounders monthly. Digital meteorological stations measured ambient temperature, humidity, and rainfall in each site. Logistic regression was used to estimate the risk of clinical malaria given the previous month's meteorological conditions.</p> <p>Results</p> <p>The overall incidence of clinical malaria over the study period was 1.07 episodes per child. Meteorological factors were associated with clinical malaria with mean temperature having the largest effect.</p> <p>Conclusion</p> <p>Temperature was the best predictor for clinical malaria among children under five. A systematic measurement of local temperature through ground stations and integration of such data in the routine health information system could support assessment of malaria transmission risk at the district level for well-targeted control efforts.</p

Incidence and predictors of immune reconstitution inflammatory syndrome in a rural area of Mozambique.

There is limited data on the epidemiology of Immune Reconstitution Inflammatory Syndrome (IRIS) in rural sub-Saharan Africa. A prospective observational cohort study was conducted to assess the incidence, clinical characteristics, outcome and predictors of IRIS in rural Mozambique

Reproductive Phase Locking of Mosquito Populations in Response to Rainfall Frequency

The frequency of moderate to heavy rainfall events is projected to change in response to global warming. Here we show that these hydrologic changes may have a profound effect on mosquito population dynamics and rates of mosquito-borne disease transmission. We develop a simple model, which treats the mosquito reproductive cycle as a phase oscillator that responds to rainfall frequency forcing. This model reproduces observed mosquito population dynamics and indicates that mosquito-borne disease transmission can be sensitive to rainfall frequency. These findings indicate that changes to the hydrologic cycle, in particular the frequency of moderate to heavy rainfall events, could have a profound effect on the transmission rates of some mosquito-borne diseases

Sugar-fermenting yeast as an organic source of carbon dioxide to attract the malaria mosquito Anopheles gambiae s.s.

<p>Abstract</p> <p>Background</p> <p>Carbon dioxide (CO₂) plays an important role in the host-seeking process of opportunistic, zoophilic and anthropophilic mosquito species and is, therefore, commonly added to mosquito sampling tools. The African malaria vector <it>Anopheles gambiae sensu stricto </it>is attracted to human volatiles augmented by CO₂. This study investigated whether CO₂, usually supplied from gas cylinders acquired from commercial industry, could be replaced by CO₂derived from fermenting yeast (yeast-produced CO₂).</p> <p>Methods</p> <p>Trapping experiments were conducted in the laboratory, semi-field and field, with <it>An. gambiae s.s</it>. as the target species. MM-X traps were baited with volatiles produced by mixtures of yeast, sugar and water, prepared in 1.5, 5 or 25 L bottles. Catches were compared with traps baited with industrial CO₂. The additional effect of human odours was also examined. In the laboratory and semi-field facility dual-choice experiments were conducted. The effect of traps baited with yeast-produced CO₂on the number of mosquitoes entering an African house was studied in the MalariaSphere. Carbon dioxide baited traps, placed outside human dwellings, were also tested in an African village setting. The laboratory and semi-field data were analysed by a χ²-test, the field data by GLM. In addition, CO₂concentrations produced by yeast-sugar solutions were measured over time.</p> <p>Results</p> <p>Traps baited with yeast-produced CO₂caught significantly more mosquitoes than unbaited traps (up to 34 h post mixing the ingredients) and also significantly more than traps baited with industrial CO₂, both in the laboratory and semi-field. Adding yeast-produced CO₂to traps baited with human odour significantly increased trap catches. In the MalariaSphere, outdoor traps baited with yeast-produced or industrial CO₂+ human odour reduced house entry of mosquitoes with a human host sleeping under a bed net indoors. <it>Anopheles gambiae s.s</it>. was not caught during the field trials. However, traps baited with yeast-produced CO₂caught similar numbers of <it>Anopheles arabiensis </it>as traps baited with industrial CO₂. Addition of human odour increased trap catches.</p> <p>Conclusions</p> <p>Yeast-produced CO₂can effectively replace industrial CO₂for sampling of <it>An. gambiae s.s</it>.. This will significantly reduce costs and allow sustainable mass-application of odour-baited devices for mosquito sampling in remote areas.</p