Development of Metarhizium anisopliae and Beauveria bassiana formulations for control of malaria mosquito larvae

<p>Abstract</p> <p>Background</p> <p>The entomopathogenic fungi <it>Metarhizium anisopliae </it>and <it>Beauveria bassiana </it>have demonstrated effectiveness against anopheline larvae in the laboratory. However, utilising these fungi for the control of anopheline larvae under field conditions, relies on development of effective means of application as well as reducing their sensitivity to UV radiation, high temperatures and the inevitable contact with water. This study was conducted to develop formulations that facilitate the application of <it>Metarhizium anisopliae </it>and <it>Beauveria bassiana </it>spores for the control of anopheline larvae, and also improve their persistence under field conditions.</p> <p>Methods</p> <p>Laboratory bioassays were conducted to test the ability of aqueous (0.1% Tween 80), dry (organic and inorganic) and oil (mineral and synthetic) formulations to facilitate the spread of fungal spores over the water surface and improve the efficacy of formulated spores against anopheline larvae as well as improve spore survival after application. Field bioassays were then carried out to test the efficacy of the most promising formulation under field conditions in western Kenya.</p> <p>Results</p> <p>When formulated in a synthetic oil (ShellSol T), fungal spores of both <it>Metarhizium anisopliae </it>and <it>Beauveria bassiana </it>were easy to mix and apply to the water surface. This formulation was more effective against anopheline larvae than 0.1% Tween 80, dry powders or mineral oil formulations. ShellSol T also improved the persistence of fungal spores after application to the water. Under field conditions in Kenya, the percentage pupation of <it>An. gambiae </it>was significantly reduced by 39 - 50% by the ShellSol T-formulated <it>Metarhizium anisopliae </it>and <it>Beauveria bassiana </it>spores as compared to the effects of the application of unformulated spores.</p> <p>Conclusions</p> <p>ShellSol T is an effective carrier for fungal spores when targeting anopheline larvae under both laboratory and field conditions. Entomopathogenic fungi formulated with a suitable carrier are a promising tool for control of larval populations of malaria mosquitoes. Additional studies are required to identify the best delivery method (where, when and how) to make use of the entomopathogenic potential of these fungi against anopheline larvae.</p

Automatic Extraction of Nuclei Centroids of Mouse Embryonic Cells from Fluorescence Microscopy Images

Accurate identification of cell nuclei and their tracking using three dimensional (3D) microscopic images is a demanding task in many biological studies. Manual identification of nuclei centroids from images is an error-prone task, sometimes impossible to accomplish due to low contrast and the presence of noise. Nonetheless, only a few methods are available for 3D bioimaging applications, which sharply contrast with 2D analysis, where many methods already exist. In addition, most methods essentially adopt segmentation for which a reliable solution is still unknown, especially for 3D bio-images having juxtaposed cells. In this work, we propose a new method that can directly extract nuclei centroids from fluorescence microscopy images. This method involves three steps: (i) Pre-processing, (ii) Local enhancement, and (iii) Centroid extraction. The first step includes two variations: first variation (Variant-1) uses the whole 3D pre-processed image, whereas the second one (Variant-2) modifies the preprocessed image to the candidate regions or the candidate hybrid image for further processing. At the second step, a multiscale cube filtering is employed in order to locally enhance the pre-processed image. Centroid extraction in the third step consists of three stages. In Stage-1, we compute a local characteristic ratio at every voxel and extract local maxima regions as candidate centroids using a ratio threshold. Stage-2 processing removes spurious centroids from Stage-1 results by analyzing shapes of intensity profiles from the enhanced image. An iterative procedure based on the nearest neighborhood principle is then proposed to combine if there are fragmented nuclei. Both qualitative and quantitative analyses on a set of 100 images of 3D mouse embryo are performed. Investigations reveal a promising achievement of the technique presented in terms of average sensitivity and precision (i.e., 88.04% and 91.30% for Variant-1; 86.19% and 95.00% for Variant-2), when compared with an existing method (86.06% and 90.11%), originally developed for analyzing C. elegans images

Patient attitudes towards medical students at Damascus University teaching hospitals

Background: The cooperation of patients and their consent to involve medical students in their care is vital to clinical education, but large numbers of students and lack of experience as well as loss of privacy may evoke negative attitudes of patients, which may sometimes adversely affect the clinical teaching environment. This study aimed to explore the attitudes of patients towards medical students at Damascus University hospitals, and to explore the determinants of those attitudes thus discussing possible implications applicable to clinical teaching. Methods: This cross-sectional study was conducted at three teaching hospitals affiliated to the Faculty of Medicine at Damascus University. Four hundred patients were interviewed between March and April 2011 by a trained sociologist using a structured questionnaire. Results: Of the patients interviewed, 67.8 % approved the presence of medical students during the medical consultation and 58.2 % of them felt comfortable with the presence of students, especially among patients with better socio-economic characteristics. 81.5 % of the patients agreed to be examined by students in the presence of the supervisor, while 40.2 % gave agreement even in the absence of the supervisor. Privacy was the most important factor in the patients ’ reticence towards examination by the students, whilst the relative safety and comfort if a supervisor was available determined patients ’ agreement

Metarhizium brunneum Blastospore Pathogenesis in Aedes aegypti Larvae: Attack on Several Fronts Accelerates Mortality

Aedes aegypti is the vector of a wide range of diseases (e.g. yellow fever, dengue, Chikungunya and Zika) which impact on over half the world's population. Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have been found to be highly efficacious in killing mosquito larvae but only now are the underlying mechanisms for pathogenesis being elucidated. Recently it was shown that conidia of M. anisopliae caused stress induced mortality in Ae. aegypti larvae, a different mode of pathogenicity to that normally seen in terrestrial hosts. Blastospores constitute a different form of inoculum produced by this fungus when cultured in liquid media and although blastospores are generally considered to be more virulent than conidia no evidence has been presented to explain why. In our study, using a range of biochemical, molecular and microscopy methods, the infection process of Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores was investigated. It appears that the blastospores, unlike conidia, readily adhere to and penetrate mosquito larval cuticle. The blastospores are readily ingested by the larvae but unlike the conidia are able infect the insect through the gut and rapidly invade the haemocoel. The fact that pathogenicity related genes were upregulated in blastospores exposed to larvae prior to invasion, suggests the fungus was detecting host derived cues. Similarly, immune and defence genes were upregulated in the host prior to infection suggesting mosquitoes were also able to detect pathogen-derived cues. The hydrophilic blastospores produce copious mucilage, which probably facilitates adhesion to the host but do not appear to depend on production of Pr1, a cuticle degrading subtilisin protease, for penetration since protease inhibitors did not significantly alter blastospore virulence. The fact the blastospores have multiple routes of entry (cuticle and gut) may explain why this form of the inoculum killed Ae. aegypti larvae in a relatively short time (12-24hrs), significantly quicker than when larvae were exposed to conidia. This study shows that selecting the appropriate form of inoculum is important for efficacious control of disease vectors such as Ae. aegypti

The role of cow urine in the oviposition site preference of culicine and Anopheles mosquitoes

<p>Abstract</p> <p>Background</p> <p>Chemical and behavioural ecology of mosquitoes plays an important role in the development of chemical cue based vector control. To date, studies available have focused on evaluating mosquito attractants and repellents of synthetic and human origins. This study, however, was aimed at seasonal evaluation of the efficiency of cow urine in producing oviposition cues to <it>Anopheles gambiae </it>s.l. and <it>Culex quinquefasciatus </it>in both laboratory and field conditions.</p> <p>Methods</p> <p>Oviposition response evaluation in laboratory conditions was carried out in mosquito rearing cages. The oviposition substrates were located in parallel or in diagonal positions inside the cage. Urine evaluation against gravid females of <it>An. arabiensis </it>and <it>Cx. quinquefasciatus </it>was carried out at Day 1, Day 3 and Day 7. Five millilitres (mls) of cow urine was added to oviposition substrate while de-chlorinated water was used as a control. In field experiments, 500 mls of cow urine was added in artificial habitats with 2500 mls of de-chlorinated water and 2 kgs of soil. The experiment was monitored for thirty consecutive days, eggs were collected daily from the habitats at 7.00 hrs. Data analysis was performed using parametric and non-parametric tests for treatments and controls while attraction of the oviposition substrate in each species was presented using Oviposition Activity Index (OAI).</p> <p>Results</p> <p>The OAI was positive with ageing of cattle urine in culicine species in both laboratory and field experiments. The OAI for anopheline species was positive with fresh urine. The OAI during the rainy season was positive for all species tested while in the dry season the OAI for culicine spp and <it>Anopheles gambiae </it>s.l., changed with time from positive to negative values.</p> <p>Based on linear model analysis, seasons and treatments had a significant effect on the number of eggs laid in habitats, even though the number of days had no effect.</p> <p>Conclusion</p> <p>Oviposition substrates treated with cow urine in both laboratory and field conditions have shown that cow urine left to age from 1-7 days has an influence on oviposition behavioural response in mosquitoes. The analysis of microbial colonies for decaying urine should be investigated along with its associated by-products.</p

Can Insects Develop Resistance to Insect Pathogenic Fungi?

This paper presents new, important information on the microevolution of insect resistance to the insect pathogenic fungus Beauveria bassiana which will have far-reaching implications for the development of insect pathogenic fungi as biological control agents. We placed successive generations of a melanic population of the Greater wax moth, Galleria mellonella, under constant selective pressure from the insect pathogenic fungus, Beauveria bassiana. Enhanced fungal resistance was observed and larvae from the 25th generation were studied in detail to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. There are 3 novel, core findings from the study:1.Antifungal resistance in these insects is pathogen species-specific, and probably arises through trans-generational immune priming. The resistance was less obvious in earlier generations, suggesting subtle cumulative changes that are only fully apparent in the 25th generation. 2.The insect’s fecundity is already pushed close to minimum by its melanic phenotype. Therefore, the additional drain on resources required to boost antifungal defence still more, comes not from further compromising life history traits but via a re-allocation of the insect’s immune defences. Specifically during B. bassiana infection, systemic (fat body and hemocoel) responses, particularly the expression of antimicrobial peptides, are damped down in favour of a tailored repertoire of enhanced responses in the integument (cuticle and epidermis) – the foremost and most important barrier to natural fungal infection. 3.A previously-overlooked range of putative stress-management factors are activated during the specific response of selected insects to B. bassiana. This too occurs primarily in the integument, and contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host’s own immune responses during the battle between host and pathogen.No other study to date has examined so many genes in this context. Indeed, we show that the epidermis has a great capacity to express defense and stress-management genes as well as the fat body (which is the main tissue producing antimicrobial peptides and has been the traditional focus of attention). We therefore propose a “be specific / fight locally / de-stress” model to explain resource allocation and defence priorities for insects selected for superior resistance to insect-pathogenic fungi. However, we also show that these insects are less fecund and probably at no evolutionary advantage in the wild, implying that the risk is small of biological control agents failing in the field

Bovine cryptosporidiosis: impact, host-parasite interaction and control strategies

International audienceAbstractGastrointestinal disease caused by the apicomplexan parasite Cryptosporidium parvum is one of the most important diseases of young ruminant livestock, particularly neonatal calves. Infected animals may suffer from profuse watery diarrhoea, dehydration and in severe cases death can occur. At present, effective therapeutic and preventative measures are not available and a better understanding of the host–pathogen interactions is required. Cryptosporidium parvum is also an important zoonotic pathogen causing severe disease in people, with young children being particularly vulnerable. Our knowledge of the immune responses induced by Cryptosporidium parasites in clinically relevant hosts is very limited. This review discusses the impact of bovine cryptosporidiosis and describes how a thorough understanding of the host–pathogen interactions may help to identify novel prevention and control strategies

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified