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Microsporidia are enteric intracellular pathogens infecting animals and humans. It causes life-threatening diarrhoea in humans. Microsporidia species have been isolated from several domestic and wild animals, however, data on microsporidia infection in animals in Nigeria is still very scanty. This study was conducted to clarify and improve our knowledge of the possibility of the zoonotic origin of Enterocytozoon species in the environment. Two hundred and fifty (250) animal faecal samples from various farm animals were examined for their frequency of microsporidia spores using Weber’s chromotrope stain and Indirect Fluorescence Antibody Test (IFAT). Enterocytozoon bienuesi were detected in 6 (24.0%) turkeys, 2 (8.0 %) goats, 3(12.0 %) cattle, and 4(16.0%) rams. No reactivity was detected with other species of microsporidia by IFAT. This study confirms the presence of E. bienuesi in the animals examined and shows that animals can be a significant source of zoonotic transmission in the environment.

L. feeleii is one of the most frequent Legionella species isolated from natural pools of the central region of Spain. This study aimed to evaluate its ecology and to identify this Legionella species as a respiratory pathogen. A PCR assay for detecting the L. feeleii mip gene was developed to identify it in clinical and environmental samples. Culture and PCR were performed in environmental samples from four drinking water treatment plants (DWTPs). Free L. feeleii was only detected in raw water samples (3.4%), while L. feeleii as an Acanthamoeba endosymbiont was found in 30.7% of raw water, 11.5% of decanter biofilm, and 32% of finished water samples. Therefore, Acanthamoeba spp. plays an essential role in the multiplication, persistence, and spread of Legionella species in the environment. The first case of Legionnaires' disease caused by L. feeleii in Spain is described in this study. The case was diagnosed in an older woman through PCR and sequencing from urine and sputum samples. A respiratory infection could be linked with health care procedures, and the patient presented several risk factors (age, insulin-dependent diabetes, and heart disease). The detection of non-L. pneumophila, such as L. feeleii, is a factor that must be considered when establishing or reviewing measures for the control and prevention of legionellosis. This work was supported by Instituto de Salud Carlos III (PI12/02725) (PI17/01670) (Co-funded by European Regional Development Fund/European Social Fund “A way to make Europe”/“Investing in your future”); and Fundación Universitaria San Pablo CEU (USPCEU-PC07/2013 and USP-PC07/2014). LV was supported by Universidad San Pablo CEU/Spain (FPI Grants). TG was supported by CAPES/Brazil (BEX 9132/13-9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

This work reports the application of photocatalytic disinfection to the inactivation of Acanthamoeba trophozoites, a free-living pathogenic amoeba. Two types of photocatalytic reactors configurations have been used: i) a slurry reactor using suspended titanium dioxide (TiO2); and, ii) a fixed-bed reactor using immobilized TiO2 onto glass Raschig rings. The effect of the chemical composition of water has been analysed, comparing the efficiency of the process in deionized water (DW) and synthetic wastewater treatment plant effluent (SWTPE). The inactivation of Acanthamoeba spp. has been compared to that of Escherichia coli bacteria, being also analysed the concomitant inactivation of both microorganisms. Our results show that 99% of inactivation of E. coli and Acanthamoeba spp. can be achieved using photocatalysis in both reactor configurations, but interestingly, the kinetics of inactivation of both microorganisms together differs from that found with them separately. Particularly, E. coli seems to be more resistant to the inactivation in the presence of Acanthamoeba spp. which has been justified by the screen effect caused by the bigger size of Acanthamoeba spp. This observation is more pronounced in DW as the composition of the SWTPE prevent the microorganisms from suffering osmotic and/or mechanical stress and protect cellular structures to the attack of reactive oxygen species (ROS). On the other hand, the difference between the inactivation rate of E. coli and Acanthamoeba, points out the importance of the different inactivation mechanisms, suggesting that the entry of small TiO2 particles into the cytoplasm of the Acanthamoeba cells provokes the attack of inner structures and as a consequence a faster inactivation. This mechanism is not possible when the catalyst is immobilized leading to a higher cell resistance to inactivation and consequently lower efficiency of the disinfection process.

The present study analyzes at the national level, the presence of circulating Legionella in the artificial aquatic systems of different facilities of all of them state-owned centers throughout Spain for 12 months. 1754 water samples from various state-owned centers were collected from January to December 2014. Samples were collected from the cooling towers and evaporative condensers (CTC), and water distribution networks such as domestic hot water (DHW), cold water for human consumption (CW), sprinkler irrigation systems (SIS), fire sprinkler systems (FSS), and water from decorative fountains (DF). All these facilities are considered, according to current regulations, as potential amplifying systems for bacteria and possible sources of infection by the generation of droplets and aerosols. The isolation and counting of Legionella in water samples was carried out using microbiological culture following the international normative UNE-EN-ISO 11,731:2007 (ISO 11,731:1998) and UNE-EN ISO 8199:2008 (ISO 8199:2005).The quantification of Legionella colonization, the annual distribution, and the geographical distribution of the Legionella isolates recovered in the water were analyzed. Besides, molecular techniques were used for the characterization of the Legionella non-pneumophila isolates. Legionella was recovered from 15.79% of the analyzed water samples. High colonization was more frequently detected in water samples from CTC, DHW, CW, and DF. Regarding the geographic distribution, positive samples of Legionella were obtained in 14 of the 18 Spanish locations analyzed. Legionella non-pneumophila was the most prevalent and was isolated from water samples from 13 different geographical locations (72%). Legionella anisa and Legionella jordanis were the most frequently non-pneumophila species isolated. Legionella donaldsonii was isolated for the first time in the water distribution networks in Spain. Legionella pneumophila sg 2-14 was detected in 13 locations and Legionella pneumophila sg 1 in 11 locations. Therefore, our study concludes that the presence of Legionella pneumophila and Legionella non-pneumophila species in these systems can be a potential threat to public health and should be examined thoroughly with complementary techniques, such as molecular techniques as a screen for routine diagnosis.

open access article Eukaryotic parasites represent a serious human health threat requiring health professionals with parasitology skills to counteract this threat. However, recent surveys highlight an erosion of teaching of parasitology in medical and veterinary schools, despite reports of increasing instances of food and water borne parasitic infections. To address this we developed a web-based resource, DMU e-Parasitology®, to facilitate the teaching and learning of parasitology, comprising four sections: theoretical; virtual laboratory; virtual microscopy; virtual clinical case studies. Testing the package was performed using a questionnaire given to ninety-five Pharmacy students in 2017/18 to assess effectiveness of the package as a teaching and learning tool. 89.5% of students reported appropriate acquisition of knowledge of the pathology, prevention and treatment of some parasitic diseases. 82.1% also welcomed the clinical specialism of the package as it helped them to acquire basic diagnostic skills, through learning infective features/morphology of the parasites.

Free-living amoebae (FLA) are ubiquitous and many isolates have been shown to be infected with amoeba-resisting bacteria, as the example of Acanthamoeba and Legionella interaction. Due to the high environmental prevalence of Acanthamoeba. in the Castilian Plateau (Spain), the aims of this work were to investigate the occurrence of Acanthamoeba and other FLA in water from several sampling points from four Drinking Water Treatment Plants (DWTP) and to investigate the presence of Legionella spp. and other amoeba-resisting bacteria in biofilms in raw and finished water, taking into account that no legislation exists for this protozoa control. Acanthamoeba was detected at different sampling points, and sand filters seemed to contribute to amoebic enrichment. After ozonation, a temporary decrease in viable amoebae was observed. The genotypes detected were T3, T4, and T5, revealing the first report of genotype T5 in waters from this region. Moreover, Balamuthia mandrillaris, Vermamoeba vermiformis and Paravahlkampfia sp. were detected. Regarding Legionella, PCR detection in raw and finished water was higher than by agar culture, but even higher after Acanthamoeba co-culture. Also, Legionella's presence was higher in raw water than in finished water. The decrease of free Legionella observed from raw (27.5%, by PCR) to finished water (3.4% by PCR) contrasted with the increase of Legionella-infected FLA from raw (30.7%) to finished water (52%). At biofilm, free Legionella was not detected, and the percentage of infected FLA was low (3.8%). Legionella species identified in these samples were L. drozanskii, L. donaldsonii and L. feeleii. Additionally, Acanthamoeba co-culture led to the isolation of Pseudomonas aeruginosa, P. stutzeri, P. fluorecens, Achromobacter xylosoxidans and Stenotrophomonas maltophilia. The highly disseminated presence of Acanthamoeba and the detection of amoeba-resisting bacteria inside amoebae highlight the importance of developing methods for controlling FLA in order to limit human pathogenic amoeba-resisting bacteria survival to the water purification processes.

The teaching of medical parasitology is facing important challenges including the need to reverse the current downward trend in the teaching status of this science reported in developed countries, despite increasing food and water parasitic outbreaks in these countries. Moreover, the teaching of this science should be adapted to the rapidly increasing biomedical and technological achievements in our societies, so we can meet future students’ interests and expectations as well as being able to supply future work placement needs. Thus, parasitologists from different European Universities [De Montfort University, DMU, UK; and the Spanish University of San Pablo CEU (USP-CEU) and Miguel Hernández de Elche], are developing a complete on-line package for teaching and learning medical parasitology, named DMU e-Parasitology (http://parasitology.dmu.ac.uk). This novel package includes a virtual laboratory and microscope with a complete library of digitised 2D slides of parasites in clinical samples. Recently, we have been successful in using 3D super-resolution microscopy (3D Cell Explorer; Nanolive), to incorporate 3D microscopic images (multiple-viewpoint-holographic images, 96 z-stacks) of important protozoan (e.g. http://parasitology.dmu.ac.uk /learn/3D_Parasitology/Acanthamoeba_cyst_1.htm) and fungi human parasites fixed on slides, of. In contrast to images created from pre-stained clinical samples, in which structures of the parasites were indistinguishable from the background, although insight of the morphological structure of the infective forms of the parasites could be seen in the 3D z-stack images in each fixed culture samples provided. However, we believe that such images will have little applicability as a potential diagnostic tool, requiring further development. We have also created an e-learning unit on parasite cell culture (http://parasitology.dmu.ac.uk /learn/lab/parasite_cell_cultures/story_flash.html), which show all the practices and procedures to work in a parasite culture unit in conjunction with detailed information and videos of parasitologists/technicians working in real conditions with parasite cultures. In order to validate this unit, we will use a blended learning approach with final year BSc Biomedical Science students and MSc Advanced Biomedical Science at DMU that voluntarily enrol to receive formative training in these topics. This training consists of two sessions, which will be delivered in the first week of April 2019, when these volunteer students have completed the DMU e-Parasitology’s Parasite Cell Unit. The first session, mostly theoretical, will provide an overall description of how to work in a parasite culture unit in conjunction with explanatory mini-videos, in which students will be able to observe different parasites in culture conditions and specific 2D (clinical samples) and 3D (fixed culture samples) slides. Thus, students will be able to observe the morphological structures of the infectious forms of these pathogens in three formats: as culture (live and fixed) and in a human tissue sample. In the second session students will use a class II biological safety cabinet to manage human cells and perform routinely tasks such as grow, culture and count these cells. This paper will provide an overall description of these novel resources for teaching/learning parasite culture and their effectiveness for teaching these important laboratory skills to future healthcare professionals.

Higher Education Students (HES) may find the lectures as something far away from their everyday life. Professors feel a big gap between themselves and their pupils. Furthermore, some students can find that subjects are not connected in between them and make close boxes of knowledge. To show them the great net of knowledge they can build, we have redesigned an old strategy, the use of blogs in education, to break that gap and increase student interest in the lectures. After the introduction of the Bologna Process in the European Higher Education Area, annual subjects have almost disappeared in behalf of shorter ones. This reduction might help the students pass their courses, but it brings up the problem of compartments in knowledge. Because of this, our project has been focused on the students of the Pharmacy Faculty which have 4 subjects associated to infectious diseases (ID) from 3rd to 6th semester (Microbiology, Parasitology, Immunology, and Biological Analysis) of a curriculum of 5 years (10 semesters). Technological gap is a reality in between students and teachers because the internet and technology are used in a different way. HES have grown in a world where social media is a part of them, sharing their daily life with pictures, videos or comments. We have therefore thought to use this enthusiasm to create a blog to share and associate the knowledge they acquire in our classes, with news they find related to ID. The blog is edited by teachers, but written by students named the “Infectious Gazzete”. Students have looked for information related to ID in different media. Once they have found attractive news, students have to practice their critical thinking and write a small blog entry containing a review of the subject using their lectures notes or books. By means of this activity, students can link the knowledge acquired in the lectures to the latest news in the field of ID. Before the entry is published in the blog, teachers play an editor role where the entry is sent back to students for improvement. Finally, the text is uploaded to the blog. As the blog entries are published in an open blog, their peers are able to read and discuss the news. Because this activity is carried out during 4 semesters, students are also able to have a global and up to date view of every aspect related to ID. Furthermore, students from other Universities (The Montfort University, UK) have joined our initiative contributing to the blog. A survey was carried to analyze students’ opinion on this initiative. Ninety-five students answer the survey with a mean age of 19,11 (± 2,11) years old. After statistically analyzing the results, the medians that favorably scored for the activity were for the statements: In general, I liked this activity. The realization of this activity is interesting and I consider that this project favors my Personal development. None negative impression was observed for any question. Overall our project has improved students’ interest in the different subjects that could be used to interconnect the different topics addressed in the lectures. Moreover, the use of the blog as an open platform, make students feel the responsibility of being well informed and read about a topic before open publishing. The exposure of students to this experience creates a platform where we introduce our subjects into an open discussion in students’ families and friends, increasing the attention and interest of infectious diseases in their everyday life.

Molecular characterization of Cryptosporidium spp. and Enterocytozoon bieneusi has improved our understanding of the transmission of both organisms in humans. In this study, to infer possible infection sources, Cryptosporidium spp. and E. bieneusi in fecal specimens from 90 HIV-infected patients attending antiretroviral clinics in Lagos, Nigeria were detected and genotyped by PCR and DNA sequencing. Cryptosporidium spp. and E. bieneusi were identified in four and five patients, respectively, including the occurrence of subtype IeA11T3G3 of Cryptosporidium hominis in two patients, subtype IIcA5G3k of Cryptosporidium parvum in one patient, and Type IV of E. bieneusi in four patients. Among the remaining positive patients, one had mixed infection of Cryptosporidium meleagridis and C. hominis and one had mixed E. bieneusi genotypes. These data highlight a possible difference in major transmission routes (anthroponotic vs. zoonotic) between Cryptosporidium spp. and E. bieneusi in HIV+ patients in the study area.

An international innovative teaching group from different EU Universities (De Montfort University, Leicester, UK; University of San Pablo CEU, Madrid, Spain; University of Miguel Hernandez, Elche, Spain) and biomedical scientists registered by the Health and Care Professions Council (HCPC, UK) are developing a complete e-learning package in medical parasitology for undergraduate and postgraduate students that study Health Sciences. This package, named DMU e-Parasitology, is accessible through the DMU website (http://parasitology.dmu.ac.uk) and will present different modules including a virtual laboratory module for the study of traditional and novel biomedical laboratory techniques and equipment for detecting, identifying and studying human pathogens, specifically parasites. These techniques could also be potentially used to study other pathogens such as bacteria or viruses. The virtual biomedical laboratory is under development, but is available in the DMU website here: http://parasitology.dmu.ac.uk/learn/laboratory.htm. To develop this new module of the DMU e-Parasitology, we are using Storyline 360 software and the scaffolding and methods used to build the theoretical module (Peña-Fernández et al., 2017) [1]. To facilitate the navigation, study and comprehension of the final user, we have divided the virtual laboratory into a series of sub-sections that include different units; the sub-sections so far are: microscopes (with units such as the electron microscope); molecular biology (e.g. polymerase chain reaction and gel electrophoresis); biological safety cabinets and cell/parasite culture; biochemical and immunological techniques (e.g. magnetic immunoseparation); histology (e.g. microtome) and staining techniques (e.g. Kinyoun staining). The virtual laboratory units are highly interactive and present short videos of academics and/or technicians working in real conditions with the different laboratory equipment such as a thermocycler, a microtome, or a biological safety cabinet, as well as performing a specific technique such as a staining to determine pathogens. Therefore, the user of this virtual environment will receive a complete and “real” experience of the work in a biomedical laboratory. The DMU e-Parasitology package, and specifically its virtual laboratory environment, could help technicians and students across the world to learn how to work in a biomedical laboratory as well as to perform techniques to identify and diagnose human pathogens such as microsporidia or Plasmodium spp. Thus, the virtual resource is supported by a virtual library that includes a real collection of clinical slides that will provide the user with the functionality of a light and/or an immunofluorescence microscope. In conclusion, the virtual laboratory may serve as a high quality and reliable on-line environment for the learning of techniques and equipment. These resources can be used to improve the learning of undergraduate and postgraduate students of human health sciences as well as to develop CPD training. Moreover, the virtual laboratory module may impact in the teaching of laboratory techniques and skills in developing countries due to their limited resources. This communication will explore the design and development of the virtual laboratory environment that will be publicly accessible by the end of 2018.

The genus Legionella comprises more than 60 species, and about half are associated with infection. Legionella pneumophila is the most commonly associated with these infections and by far the most studied, but L. non-pneumophila species, such as L. feeleii, L. anisa, etc., may also present clinical importance. Free-living amoebae are their preferred environmental host, where these bacteria not only survive but also succeed in multiplying, and this relationship can lead to an increase in bacterial virulence. The goal of this study was to evaluate the alterations of Legionella pathogenicity due to its interaction with Acanthamoeba. For this, the expression of protein effectors SdhA, LegK2, and SidK were evaluated in L. pneumophila and L. feeleii, before and after infecting Acanthamoeba. Additionally, the host response was evaluated by measuring the production of IL-6, IL-8, and IFN-γ in infected macrophages. Regarding the virulence factors, an increase in SdhA expression was observed after these bacteria infected Acanthamoeba, with a higher increase in the macrophage cultures infected with L. feeleii. Also, an increase in the expression of LegK2 was observed after infecting Acanthamoeba, but it was more intense in the cultures infected with L. pneumophila. With regard to SidK, it was increased in L. feeleii after infecting Acanthamoeba, however the same effect was not observed for L. pneumophila. In cytokine production, the effect on IL-6 and IL-8 was similar for both cytokines, increasing their concentration, but higher production was observed in the cultures infected with L. feeleii, even though it demonstrated slightly lower production with the inoculum obtained from Acanthamoeba. Concerning IFN-γ, induction was observed in both species but higher in the infection by L. pneumophila. Nevertheless, it is not known if this induction is enough to promote an efficient immune response against either L. pneumophila or L. feeleii. Altogether, these alterations seem to increase L. feeleii virulence after infecting Acanthamoeba. However, this increase does not seem to turn L. feeleii as virulent as L. pneumophila. More studies are necessary to understand the aspects influenced in these bacteria by their interaction with Acanthamoeba and, thus, identify targets to be used in future therapeutic approaches.

This work reports the application of photocatalytic disinfection to the inactivation of Acanthamoeba trophozoites, a free-living pathogenic amoeba. Two types of photocatalytic reactors configurations have been used: i) a slurry reactor using suspended titanium dioxide (TiO

The release of biological hazards during biological incidents, bioterrorism or outbreaks of infection has shown to represent a significant challenge for healthcare professionals as it can involve significant numbers of patients and represent a global public health threat. Healthcare educators should provide students with the necessary skills in medical preparedness and response to these incidents to protect the public. However, this is not standardised in the medical curriculum. An innovative teaching group at De Montfort University (DMU, UK) in collaboration with first responders that worked during the 2014-2016 Ebola outbreak in a Public Health England (PHE) mobile laboratory in Sierra Leone, have developed different, novel teaching training sessions to provide health science students with the necessary skills to respond to these events. We have tested the training sessions with students from three different undergraduate (BSc Biomedical and Medical Science) and postgraduate (MSc Advanced Biomedical Science) human health programmes at DMU. In general, these sessions were shown to be successful in providing students with basic skills to respond to minor biological incidents (Peña-Fernández et al., 2017), although we do not know if these could be adopted to develop standardized curricula across any human health degree in the European Union (EU). Therefore, the purpose of this work was twofold: a) to assess the effectiveness of the specialised training session that covers the medical response to protect public health with medical students; and b) to determine the effectiveness of the training session, initially developed in England, in a non-English EU university. To meet these objectives, we have simplified this specialised training and delivered it to final year students of the Medicine degree at the University of San Pablo CEU (USP-CEU, Spain) during an Eramus+ mobility grant for academics in April 2017. The two hour training provided consisted of developing a complete intervention programme to deal with an outbreak of Crimean-Congo haemorrhagic fever (CCFH) virus following the steps of evidence-based public health. CCFH is a haemorrhagic fever virus causing devastating disease symptoms that result in intense and prolonged suffering in humans and has become an increasing global health concern. This paper will describe the teaching resources used and a comprehensive analysis of students’ feedback to this training. Briefly: the specific questionnaire used has shown high levels of engagement and satisfaction [100% (31.2% agreed; 68.8% strongly agreed)] with the USP-CEU medical students. Despite its short duration, this training would be successful in providing medical students with the necessary skills to respond to a biological event. Thus, 100% (31.2% agreed; 68.8% strongly agreed) of these students reported that they learnt how to establish some public health interventions to protect humans in the aftermath of an outbreak of infection. Moreover, all USP-CEU responders have described that they gained appropriate knowledge of public health prevention and preparedness against these events (37.5% agreed; 62.5 strongly agreed). Finally, the Erasmus+ mobility grant for academics has been shown to be a relevant tool and resource to strengthen curricula development and validation in higher education.

BACKGROUND: The use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation. METHODS: Viability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry. RESULTS: Trypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane. CONCLUSIONS: The present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out. This project was supported by Grant PI12/ 02725 from Instituto de Salud Carlos III (FISS) and FEDER by grant USPCEU-PC07/2013 of the Fundación Universitaria San Pablo CEU and by grant CEU-Banco de Santander. TSG was supported by Research Program EADS Construcciones Aeronáuticas S.A. - Brasil I and by CAPES/Brazil (Grant BEX 9132/13-9). Sí

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link Recent outbreaks threatening public health involving contaminated water or food with different microorganisms such as Escherichia coli or Cyclospora spp. in Europe as well as the 2014-16 Ebola crisis in West Africa have highlighted the relevance of teaching parasitology and infectious diseases to future biomedical scientists. The BSc Biomedical Science (BMS) programme at De Montfort University (DMU, Leicester, UK) is accredited by the Institute of Biomedical Sciences (IBMS). However, the dedicated time to study these topics was very little, limited to two 15 credit modules focused on general and basic medical microbiology. A series of important modifications have been undertaken since 2016 to ensure that our graduates receive comprehensive knowledge in parasitology/infectious diseases following the subject-specific threshold standards described by the Quality Assurance Agency (QAA, 2015). New lectures in these topics (virus, fungi, helminths, infectious diseases) were incorporated in our Basic Microbiology level 4 module after expanding this to 30 credits in the academic course 2016/17. Highly specialised training related with emerging parasites (e.g. Cyclospora, microsporidia) and haemorrhagic fever virus (e.g. Crimean-Congo Haemorrhagic Fever virus) was introduced in our level 6 Medical Microbiology module (15 credits), based on a successful novel teaching experience created by first responders to the Ebola outbreak in West Africa. Validated feedback questionnaires to assess the training were collected as well as final module level feedback (MLF) for each module through Blackboard. Students have highlighted high levels of engagement in both modules, specifically in the training provided in Medical Microbiology (n=121). Thus, some students have highlighted in the MLF that the case studies (e.g. related with free-living amoebas and Cyclospora spp.) “improved my understanding” and were “engaging”. The specific questionnaire has highlighted that 77.5% of final year students enjoyed the novel workshop created to respond to an outbreak of infection affecting the UK following evidence-based public health methodology. However, the module that has shown more improvement in the students’ satisfaction was Basic Microbiology (n=196), which showed a significant increase from 20% in the 2015/16 academic year to 64% in the current academic session. The MLF of Basic Microbiology has also reported the following results: 95% (41% definitely agree, 54% agree) of students have highlighted that this first year module provided them with opportunities to apply what they learnt; and 94% considered that the feedback provided was clear and fair. Despite these results should be considered as preliminary, we consider that the initial changes undertaken in the BMS programme at DMU to date could improve students’ understanding of emerging diseases, specifically about how to study and prevent emerging infectious diseases to protect public health. Future proposed improvements include the development of a complete e-learning package for students and academics for teaching these topics in collaboration with Universities from Spain and practising Biomedical Scientists from the National Health Service. These resources will be accessible throughout 2017/18.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link The study of parasitology has become essential to build future health care professionals with skills to respond to public health threats such as the recent outbreaks due to Cryptosporidium spp. or Giardia in the United Kingdom (UK). To facilitate the teaching of parasitology, which negligible across the different undergraduate and taught masters degrees at De Montfort University (DMU, Leicester, UK), a group from different EU Universities [DMU and the Spanish universities: University of San Pablo CEU (USP-CEU) and University Miguel Hernández], clinicians and practising Biomedical Scientists from the UK National Health Service are developing an on-line package for teaching and learning parasitology named DMU e-Parasitology. This package will have three sections or modules: a theoretical module with mini e-learning units to study major human parasites such as Leishmania spp. or malaria; a virtual laboratory module with units to enhance the learning and study of parasitic diseases such as culture of parasites, staining and use of light microscope to identify these organisms or molecular techniques for the identification of parasites to species level; and a virtual microscope with a real slide collection of clinical samples of parasites. The e-Parasitology resource is being created for undergraduate/postgraduate human health science students, with corresponding degrees of difficulty. Units will include a tool to assess the learning process of the students, in form of a quiz, activity and/or exam, and several formative activities throughout each mini-module. The development of this teaching resource will cover a gap in the traditional teaching and learning methods that are currently used and provided in the participating Universities. The DMU e-Parasitology will aid to our undergraduate students to gain a significant knowledge in parasitology by promoting self-learning. A unit related with Toxocara, a helmintiasis with prevalence rates that can reach as high as 40% or more in different parts of the world, was firstly developed to use as a model for the development of the DMU e-Parasitology. Three undergraduate students that studied parasitology during the first term in 2016/17 [n=27; 6 European Credit Transfer and Accumulation System credits (ECTS); 3rd year module] from the bilingual Pharmacy and Biotechnology degree at USP-CEU were voluntarily recruited to provide comprehensive feedback for this model unit at the beginning of the second term. This unit was tested with these students because of their comprehensive knowledge of parasitology. Students described it as interactive and presenting the appropriate content and resources to study the parasitic disease addressed (toxocariasis). Limitations were the poor navigability in the formative exercise section and the excessive information provided in some slides that could hinder their understanding. The team has addressed these limitations and is using this unit as a model to build the DMU e-Parasitology, which will be accessible through the DMU website (http://parasitology.dmu.ac.uk) in 2018. We consider that this teaching and learning resource will overcome barriers of time, space, equipment and resources; and may help students and scientists around the world in the diagnostic of different parasitic diseases that impact human health.

The free-living amoebae, Acanthamoeba, can act as opportunistic parasites on a wide range of vertebrates and are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices, and their ubiquitous distribution. Subgenus classification based on morphology is being replaced by a classification based on the sequences of the 18S rRNA gene with a total of 18 different genotypes (T1-T18). A new environmental strain of Acanthamoeba isolated from a waste water treatment plant is presented in this study as a candidate for the description of the novel genotype T19 after phylogenetic analysis.

A year-long longitudinal study was undertaken to evaluate the presence of Cryptosporidium spp. in drinking water treatment plants (DWTPs), wastewater treatment plants (WWTPs) and freshwater bathing beaches (FBBs) from the central area of Spain. Water samples were collected according to USEPA Method 1623, and concentrated by the IDEXX Filta-Max® system. Cryptosporidium species were detected based on PCR-restriction fragment length polymorphism and sequence analyses of the ssuRNA gene. C. hominis and/or C. parvum isolates were subtyped by DNA sequencing of the Gp60 gene. Among 150 samples, 23 (15.3%) were positive by IFAT and 40 (26.7%) by PCR. Cryptosporidium spp. was more frequent in WWTPs (26.2 and 50.8%) and FBBs (12.5 and 17.5%) by IFAT and PCR respectively. Effluent waters from DWTPs were negative for this parasite suggesting that they are suitable for public use. Tertiary treatment in the WWTPs demonstrated a high removal efficiency of Cryptosporidium in the samples evaluated. Cryptosporidium species identified included C. hominis, C. parvum, C. ubiquitum, C. andersoni and C. muris. Subtyping analysis revealed C. hominis IbA10G2 and IeA11G3T3 alleles, which is the first report of the latter in water samples. Cryptosporidium highest frequency was observed in winter and spring. Our data provide information about the occurrence and diversity of Cryptosporidium in water of human use from the central area of Spain.

Microsporidia are ubiquitous fungi with genomes that have undergone a strong reduction to the extreme cases of Encephalitozoon cuniculi and Encephalitozoon intestinalis. Genetic variability within species of the Encephalitozoon genus has been reported, with most of the studies based on the internal transcribed spacer (ITS) of the rDNA. However, in contrast to the picture of E. cuniculi and Encephalitozoon hellem, where different strains have been identified, no genetic variability has yet been observed in E. intestinalis. We have analysed tandem repeats included in putative coding sequences which could be used as polymorphic markers in E. intestinalis. Eight candidate loci (M2, M2A, M3, M5, M7, M7A, M8 and PTP1) were established and 9 E. intestinalis cultured strains from North America, South America and Europe were analysed. M2, M7 and PTP1 nucleotide sequences were identical among the different strains and the GenBank sequence. In contrast, we observed variants in 4 markers (M2A, M3, M7A and M8) which did not correspond to their respective reference sequences. The most noticeable finding was that with the M5 marker two genotypes were defined among the different strains studied, demonstrating genotypic variability of E. intestinalis. Although the diversity described is certainly not high, which can be explained by a lower chance of genetic variability in its minimal genome, we have demonstrated that polymorphisms actually exist in E. intestinalis. Epidemiological studies using this genetic marker should now be conducted to elucidate the genetic variability in E. intestinalis and improve our knowledge of the epidemiology of this microsporidia.

a b s t r a c t Background: Microsporidiosis is a life threatening opportunistic infection of AIDS patients. The infection is usually restricted to specific anatomical areas, but could become systemic depending on the involved species. Genital microsporidiosis in female patients is rare. Objective: To report genital microsporidiosis in female AIDS patients. Methods: Tissues samples from the genital tract (ovary, fallopian tubes and uterus) of eight deceased women who died of wasting syndrome associated to AIDS and disseminated microsporidiosis at the Institute of Tropical Medicine Pedro Kouri were collected between 1997 and 2005. Using an indirect immunohistochemistry assay the microsporidia species involved in those cases were identified. Results: We report several cases of microsporidial infection of the female genital tract. Six out of eight women with the disseminated form of the disease showed the presence of microsporidia in the genital tract. Encephalitozoon cuniculi and Encephalitozoon hellem were identified in the internal lining epithelium of the fallopian tubes and endometrium. Conclusions: Microsporidia species could disseminate to other organs and become systemic in severe immunocompromised cases. To our knowledge this is the greatest number of female genital tract

Environmental contamination with zoonotic microorganisms can have significant health and environmental implications due to their potential for producing large numbers of transmissive stages and their ability to survive in the environment for prolonged periods of time. This is especially relevant in urban environments because of the dramatic human population boom and urbanization. Cryptosporidium spp. is a zoonotic pathogen which can affect animals and humans. Although a major public health concern there is limited information about the occurrence and circulation of Cryptosporidium spp. in urban environments. 27 soil and animal faecal samples from birds, water birds, cats and dogs were collected in public parks and recreation areas in Leicester centre city between January and February 2016. Samples were collected on days with no rain to avoid problems of sample integrity. After appropriate preparation of each sample, detection of Cryptosporidium spp. was performed with the conventional parasitological technique of Kinyoun's acid-fast staining and the immunoassay ImmunoCard STAT!®. None of the 27 samples collected was positive for these emerging pathogens, although we found structures that may be related to Cryptosporidium in one of the faecal smears from a dog. Despite the preliminary data described indicating a limited presence of Cryptosporidium spp. in the public parks and recreational areas monitored, further more detailed knowledge of their circulation in these ecosystems will be crucial to assess the public risk to develop novel strategies to protect humans and increase the awareness of parasite contamination of our environment.

Although a public health concern, there is limited information about the presence of antibiotic-resistant human pathogenic bacteria in the UK environment. Identification of environmental contaminants, including biological hazards, is critical to protect the public health. Klebsiella pneumoniae and Escherichia coli are considered important Gram-negative opportunistic pathogens. Urban animals may play a significant zoonotic role in the dispersion of these pathogens. 46 faecal samples were collected in different public parks in Leicester city in April-May 2016. Samples were collected in days of no rain to avoid problems with the integrity of the sample. Animal species were identified by a veterinarian: 18 avian (5 pigeons, 13 waterfowls), 15 dog, 3 cat, 7 fox and 3 unidentified. Faecal samples were analysed using routine coprological methods of modified Telemann and MIF. Seven Extended-Spectrum -Lactamases genes for Gram-negative bacteria, and bacteria species (K. pneumoniae and E. coli) were determined using specific primers by PCR. DNA was extracted by bead disruption of prepared samples using the Fast-DNA-Spin kit, according to the manufacturer’s instructions. We detected genes for enzymes for those -Lactamases in 11 samples (specifically CTX-M-15), which corresponded with 6 samples form waterfowls, 3 from dogs and 2 from foxes. None of the samples was identified for K. pneumoniae or E. coli. Although the results are preliminary, the data obtained in this study has revealed the presence of antibiotic resistant bacteria in urban areas that are highly frequented by citizens, which may require recovery and restoration in addition to pro-active public health measures to protect public health.

Humans are increasingly being exposed to a different range of biological hazards in the urban media. The identification of these hazards and the implementation of intervention mechanisms to protect the public health are therefore necessary. Microsporidia, a new emerging pathogenic group, forms environmentally resistant spores, and has been related with recent water- and food-borne outbreaks. “Urban” animals can be reservoirs of these parasites playing a role in the environmental contamination of cities. Accidental ingestion of microsporidia spores while playing or spending time in recreational areas may be a significant risk for people. However, there is limited information about the presence of microsporidia in the UK environment. 18 faecal and 9 soil samples were collected in recreational areas in Leicester city between January and February 2016. A veterinarian identified the possible animal species of the stool samples: 8 avian (pigeons and water birds), 6 dogs, 3 cats and 2 unidentified due to diarrhoea. After appropriate preparation of each sample, detection of microsporidia was performed with the conventional parasitological technique of Weber’s Trichrome staining. We observed microsporidia spores in two samples from dogs and none were identified in soil samples. Although our results should be taken as preliminary, they might indicate a possible potential zoonotic role of domestic dogs in the transmission of these pathogens. Dogs are in close contact to human, so further research is critical to assess the public risks of these pathogens in the urban media and the identification of applicable interventions to decontaminate the environment to protect humans.

Legionnaires' disease is a severe form of pneumonia, with worldwide relevance, caused by Legionella spp. Approximately 90% of all cases of legionellosis are caused by Legionella pneumophila, but other species can also be responsible for this infection. These bacteria are transmitted by inhalation of aerosols or aspiration of contaminated water. In Spain, environmental studies have demonstrated the presence of Legionella non-pneumophila species in drinking water treatment plants and water distribution networks. Aware that this evidence indicates a risk factor and the lack of routine assays designed to detect simultaneously diverse Legionella species, we analyzed 210 urine samples from patients presenting clinical manifestations of pneumonia using a semi-nested PCR for partial amplification of the 16S rDNA gene of Legionella and a diagnostic method used in hospitals for Legionella antigen detection. In this study, we detected a total of 15 cases of legionellosis (7.1%) and the first case of Legionnaires' disease caused by L. anisa in Spain. While the conventional method used in hospitals could only detect four cases (1.9%) produced by L. pneumophila serogroup 1, using PCR, the following species were identified: Legionella spp. (10/15), L. pneumophila (4/15) and L. anisa (1/15). These results suggest the need to change hospital diagnostic strategies regarding the identification of Legionella species associated with this disease. Therefore, the detection of Legionella DNA by PCR in urine samples seems to be a suitable alternative method for a sensitive, accurate and rapid diagnosis of Legionella pneumonia, caused by L. pneumophila and also for L. non-pneumophila species.

Several studies have demonstrated that the soil of public parks presents an important source of infection which has a significant impact on public health. Children are the main group affected by accidentally ingestion of contaminated soil. This study was performed in order to identify the presence of zoonotic parasites in dog and cat faecal and soil samples from public parks of Madrid, Spain. Six hundred twenty-five and seventy-nine soil and faecal samples (presumably from dogs and cats) respectively were collected from 67 parks. Intestinal parasites were identified in 27 parks (40.3%), which were contamined with Giardia sp. (19.4%), microsporidia (19.4%), Toxocara spp. (16.4%), Cryptosporidium sp. (6%), Entamoeba histolytica (3%) and Ancylostomidae (3%). Combinations of two or more intestinal parasites were found in 11 parks, and it was common to find Giardia and microsporidia together in samples. Intestinal parasites were detected in 18% (112/625) of soil samples. The most frequent parasite species found in the examined soil samples were Toxocara spp. (16.4%), followed by Giardia sp. (4.5%) and Strongyloides sp. larvae (3%). The zoonotic parasites found in the 79 faecal samples were Giardia sp. (17.7%), Cryptosporidium sp. (9%), E. histolytica (2.5%), Trichuris vulpis (1.3%), Toxascaris leonina (1.3%) and microsporidia spores (28%). Microsporidia characterization by amplification of DNA confirmed 10 samples as positive, eight for E. bieneusi and two for E. hellem by PCR. The role of those parasites in the environment are discussed.

Enterocytozoon bieneusi is a microsporidian parasite that infects many vertebrate animals, including humans. The rDNA internal transcribed spacer (ITS) shows a hypervariable sequence; however, so far no clear information has been inferred about strain evolution in this species. We reviewed all the sequences described and performed a phylogenetic study. Four groups of sequences strongly differentiated from each other were detected, although most of the isolates (94%) corresponded to group I. The highly diverse sequences of this group were analyzed using median-joining networks. The host species (humans, pets, swine, cattle, birds, and wild animals) and the continents of origin of the isolates were considered. Central haplotypes in the network were obtained from very diverse hosts and geographical origins. The results show that although E. bieneusi has a broad host specificity, transmission is not completely free: some strains were able to circulate within a given host species and were only occasionally transmitted to another host. Additionally, while not relevant for swine or cattle hosts, geography seems to be a relevant factor for human infection by E. bieneusi .

Resistance of Nosema ceranae to different exposure conditions has been evaluated by using Sytox green and DAPI (4′,6-diamidino-2-phenylindole) to test spore viability. High thermotolerance at 60 and 35°C and resistance to desiccation were observed. However, a significant decrease in viability after freezing and a rapid degeneration of spores maintained at 4°C were also detected.

The efficacy of the anti-inflammatory drug Bobel-24 against experimental infection by Cryptosporidium parvum was evaluated in neonatal lambs. The animals were treated by oral administration of the drug at 50 or 500 mg/kg of body weight. The prophylactic/therapeutic treatment was started 4 h before inoculation of the lambs with oocysts and was continued for eight consecutive days. The therapeutic treatment was initiated at the onset of diarrhoea, after confirmation of infection, and was continued for six consecutive days. Infection was monitored by daily examination of faecal samples from the first day until 30 days post-inoculation. The criteria considered in evaluating development of the infection and the drug activity were: oocyst shedding, presence of diarrhoea and weight gain at 15 and 30 days post-inoculation. Bobel-24 was effective as a prophylactic/therapeutic treatment at the lowest dose (50 mg/kg of body weight); in the group treated with this dose of drug there was a longer prepatent period, a shorter patent period and a lower intensity of oocyst excretion than in the untreated control group, and the differences were all statistically significant (P

SUMMARYIn humanToxocara canisinfection, an association has been shown between high IgG avidity in the chronic phase and low IgG avidity in recently acquired toxocarosis. The evolution of the antibody response in terms of avidity has been carried out through aT. canisinfection in BALB/c mice. Infection withT. canisembryonated eggs (EE) was carried out with single doses (SD) of 6, 12, 50, 100, 200 or 1000 EE/mouse and with multiple doses (MD) of 200 and 1000 EE. Specific antibodies againstT. canis(IgM+G, IgG, IgG1 and IgM) were detected by ELISA and Western Blot (WB) techniques in the presence and absence of urea. With the ELISA method, an increase in the avidity index (AI) of around 50% was detected from days 40–80 p.i. to the end of the study, with all the doses studied. The WB method showed the presence of high avidity antibodies bound to 100 kDa and 75 kDaT. canisproteins in all the cases when the IgM+G and the IgG1 antibodies were investigated. Antibodies of variable avidity were observed in those sera that recognized the group of low molecular weight proteins, between 37 kDa and 25 kDa.

Microsporidia are intracellular obligate parasites which have recently been found to be related to fungi. They have a unique extrusion apparatus that is able to inject the sporoplasm directly into the target cell without using receptors. Encephalitozoon microsporidia are a source of morbidity and mortality in humans. It has been suggested that microsporidia may modulate the host cell cycle and apoptosis. We report here that caspase-3 cleavage is inhibited at different times of Vero cell infection by Encephalitozoon microsporidia and that the phosphorylation and translocation of p53 to the nucleus, previous steps for the activation of this protein, do not occur after infection of Vero cells. Consequently, the transcriptional function of p53 is impaired during the infection cycle as demonstrated by luciferase reporter assays. Thus, to our knowledge, for the first time it is shown that an intracellular parasite may be able to multiply in the host cell without activating the p53 apoptotic pathway of that cell. However, changes in the expression of Bcl-2 or Bax levels were not observed.

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for onlyEnterocytozoon bieneusi(9.7%), 5 forEncephalitozoon intestinalis(4%), and one forEncephalitozoon hellem(0.8%). Coinfections were detected in eight additional pigeons:E. bieneusiandE. hellemwere detected in six animals (4.8%);E. bieneusiwas associated withE. intestinalisin one case (0.8%); andE. hellemandE. intestinaliscoexisted in one pigeon. No positive samples forEncephalitozoon cuniculiwere detected. The internally transcribed spacer genotype could be completed for oneE. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in anE. hellemSpanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans forE. intestinalisandE. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.

Legionella spp. is the causative agent of Legionnaires' disease and is transmitted through aerosols emanating from man-made water systems. Legionella resistance to water treatments has been related to its association with environmental amoebae such as Acanthamoeba. Due to the high presence of this protozoon in Spain and the high rate of notification of Legionnaires' disease of this country, the aims of this work were to study the coexistence of these bacteria and protozoa in water as well as their interaction. The usefulness of Acanthamoeba co-culture for the isolation of environmental Legionella was also studied. For this purpose, 70 water samples were collected in 2011 from three Drinking Water Treatment Plants, three Wastewater Treatment Plants and five Natural Pools in Spain. Acanthamoeba was found by PCR in 87.1% (61/70) samples and, by culture in 85.7% (60/70) samples. Legionella was detected by PCR in 58.6% (41/70) of water samples, in 5.7% (4/70) by agar culture and 75.7% (53/70) by Acanthamoeba co-culture. From the 54 Acanthamoeba water isolates, Legionella was detected in 43 of them independently of Acanthamoeba's genotype (T3, T4 and T11). Legionella feeleii, Legionella birminghamiensis, Legionella gresilensis/berliardensis, Legionella fairfieldensis, Legionella drozanski and Legionella falloni were identified. In conclusion, our results showed that environmental Acanthamoeba is infected by Legionella to a high percentage, and due to its ubiquity, high resistance and its pathogenic potential per se, new methods for its elimination should be studied. Also, the high effectivity of Acanthamoeba co-culture for Legionella detection has been shown.

Microsporidia are ubiquitous parasites infecting all animal phyla and we present evidence that supports their zoonotic potential. Fecal samples taken from domestic (cats and dogs), farm (pigs, rabbits and ostriches) and wild animals (foxes) from different provinces of Spain were evaluated for microsporidia infection by light microscopy and PCR. After Microsporidia species identification, E. bieneusi genotypes were additionally studied by sequence analysis of the ITS region. Eighty-five samples out of 159 exhibited structures that were compatible with microsporidia spores by Webeŕs stain with 37 of them being confirmed by PCR. Microsporidia species identified included E. bieneusi, E. intestinalis and A. algerae. We report the first diagnosis of E. intestinalis and E. bieneusi in ostriches and A. algerae in pigs. We also provide new information on the molecular characterization of E. bieneusi isolates both in rabbits and ostriches. All of the E. bieneusi genotypes identified belonged to the zoonotic group of genotypes (Group I) including genotypes A (dogs), I (pigs), D (rabbits and foxes) and type IV (ostriches). Our results demonstrate that microsporidia are present in domestic, farm and wild animals in Spain, corroborating their potential role as a source of human infection and environmental contamination.

Objective: We recently demonstrated that the biggest reduction of T cells in septic patients was produced in the γδ T subset. This depletion was directly proportional to the severity of the septic process, and it was associated with mortality. We hypothesized that microsporidia can harness the deficit of γδ T cells in septic patients to proliferate and contribute to the worsening of the sepsis. Methods: In this retrospective study, we analyzed anti-Encephalitozoon cuniculi antibody levels in sera from 46 septic patients, and compared them with a similar control group of healthy subjects. As a secondary objective we aimed to relate anti-E. cuniculi antibody levels with αβ and γδ T cells in these patients. Results: Forty-eight percent of septic patients were positive for IgE anti-E. cuniculi vs 13.0% of healthy subjects (OR: 3.67, CI95% 1.64-8.20, P=0.001). The frequency of αβ and CD56+ γδ T cell subsets decreased in septic patients with positive anti-E. cuniculi IgE antibodies. This decrease was more potent in the CD3+CD56+ γδ T cell subset. The genitourinary focus (urinary tract infections and pyelonephritis) produced a significant higher percentage of anti-E. cuniculi positive cases (11/13 (84.6%), OR=11.0, CI 95% 2.1-58.5, P=0.003). Conclusion: There was a greater level expression of IgE anti-E. cuniculi in septic patients, reaching almost 50% of positive. The presence of IgE anti-E. cuniculi in septic patients was related to a decrease of αβ and γδ T cells in peripheral blood. This decrease was more potent in the CD3+CD56+ γδ T cell subset. Microsporidia may be heavily involved in the physio-pathological evolution of sepsis.