5^th International Conference on Parasitology & Microbiology July 12-13, 2018 Paris, France

Elsa Rush EYANG ASSENGONE is a PhD student at the Ecole Doctorale Regionale d’Afrique Centrale (EDR). She is a first year PhD student. She is doing her work at the department of Parasitology of the International Centre of Medical Research (CIRMF) at Franceville in Gabon

Loa loa is a filarial worm restricted to West and Central Africa. Numerous imported cases are reported, including encephalitis cases, which are problematic in that treatment impedes control of other filaria in areas where this filaria is co-endemic with other filaria. Furthermore, the lack of a suitable animal model induces a paucity of material for study of the molecular and immunological bases underlying the relationship with its human host. Therefore, a recombinant DNA technology approach is required. But this approach needs a suitable method for extraction of nucleic acid. In this view, microfilaria was split into triplicate patches of 1, 10, 100, 1000 and 1500 to evaluate six DNA extraction methods, namely, phenol-chloroform (A), Qiagen Kit (B), salting-out (C), methanol (D), CTAB (E) and Tris-EDTA (F). The DNA extracts were analysed spectrophotometrically at 260,230,280 and 320nm using the Nanoview and Qubit methods. Determination of the concentration, yield and quality of the extracted DNA showed that concentrations varied from 0.5ng/ml to 26.30± 3ng/ml. The highest yield achieved using method B (5.77 ± 1.41ng/ml) and the lowest with method C (0.032ng). The quality of DNA was assessed by the A260/A280 (0.9 to 1. 9) and the A260/A230 ratio (0 to 18.5). The time required, hazardous exposure, simplicity of execution and costs were also compared. Taking together, the results obtained suggest that the use of the Qiagen and phenol-chloroform may be suitable for cloning, whereas the Tris-EDTA may be suitable for field use

Aisan asalipisheh has completed her Master at the age of 36 years Zanjan Azad University in Iran She is the Microbiologist and Hygienist and microbiology lab manager at Unilever company, and befor that used to work as anicrobiolgist in Mahram company (Sauce and dressing) and also microbiology lab manager at Gelatin Halal company

Introduction & Aim: Pseudomonas aeruginosa is the major cause of nosocomial infections and is considered as an opportunistic pathogen. Several reports indicate that the organism can also cause infections in healthy hosts. Further, evidence has suggested that there are no major differences in virulence between clinical and environmental isolates. Consuming raw vegetables that have been contaminated by this organism could have serious implications on human health. The aim of this study was to identifiy Pseudomonas aeruginosa virulence factor genes (Exotoxin A, Exoenzyme S, Elastase and Alginate genes) which isolated from salads. Materials & Methods: In this cross-sectional study, 200 salad samples were collected from restaurants located in Qazvin during 6 months. After identification of isolates by biochemical tests, the antibiotic susceptibility test (Kirby-Baur method) was done according to CLSI advice against 13 antibiotics. After DNA extraction from isolates, PCR with specific primers were done for detection of Exotoxin A, Exoenzyme S, Elastase and Alginate genes. Results: Pseudomonas aeruginosa was isolated from 12.5% of samples. Cefotaxime with 95% and imipenem with 21/6% showed the highest and lowest resistance against isolates respectively. Frequency of Exotoxin A, Exoenzyme S, Elastase and Alginate genes were 44%, 16%, 44% and 44%, respectively. Conclusion: Our results showed that the frequency of Pseudomonas aeruginosa in salads was high. This study confirms the rapidity and sensitivity of PCR analysis in identifying isolates which contribute in early monitoring, accurate analysis and control of microbial risks in food products

Afnan Alqurashi has completed her Bachelor of Science degree of Laboratory Medicine from Umm Al-Qura University, KSA and the Master’s of health science in Laboratory Science from Qunipiac University, USA. Recently, she is a PhD student in University of Salford, UK. The main reserch interst is in molecular biology and parasitiology

Toll-like receptors (TLRs) play an important role in innate immunity that recognise pathogen-associated molecular patterns (PAMPs). In general, TLR receptor polymorphism has been found to be associated with resistance and susceptibility to infectious disease. In this project, we closely studied the TLR9 polymorphism in relation to sustbility of diseases in cattle. TLR9 responds to CpG DNA which is commonly found in bacteria, viruses and parasites and that induces proinflammatory cytokines. TLR9 polymorphism has been linked to many infectious diseases like malaria, tuberculosis, etc. The aim of this study is to investigate the association of nucleotide polymorphism in TLR9 using Next Generation Sequencing (NGS) from FTA-cards and susceptibility to parasitic diseases in natural populations of cattle. The FTA-cards contain blood samples from cattle that were collected in previous study. We have extracted the DNA from FTA-card under diffrent conditions. According to our data, Quigen kit was the best kit to extraxct the DNA from FTA-card. Subsequently, we genotyped the TLR9 gene from the FTA-card. In conclusion, we have generated an optimum condition for DNA extraction and purification from FTA-card for down-stream application like Sagner sequensing and NGS. In the future study, we will generate primer library for indentification of SNP’s with the TLR9 gene by using the NGS technique

Muyassar Tarabulsi is currently a 3rd year PhD student in Salford University School of Environment and Life Sciences. She is a holder of MBBS degree in Medicine of Vector Biology from the University of Salford, UK

Toxoplasma gondii is an obligate intracellular organism that has worldwide distribution and is highly prevalent among animals and humans. Although usually asymptomatic, it can cause serious conditions in immunocompromised individuals (pneumonia, ocular toxoplasmosis and abortions). Many studies have been investigated to find T. gondii among cancer patients. However, none have addressed the significance of the infection with this ubiquitous parasite among this group. We aimed to investigate the relationship between T. gondii infection and lung cancer. In a study done at Salford University, 72 lung samples from patients with lung cancer were investigated for T. gondii infection by nested PCR and immunohistochemistry (IHC) and it documented striking 100% prevalence. We recruited 10 lung samples via bronchoalveolar lavage from healthy individuals to be used as a control group for the lung cancer patients study. Samples were tested with nested PCR targeting five T. gondii specific markers (B1, SAG1, SAG2 3′, SAG2 5′ and SAG3) and only one sample was positive for T. gondii infection with all five markers and in IHC. Statistical analysis revealed an extremely significant difference between the lung cancer patients and the non-cancerous control group (P<0.0001). We aim to explore further lung cancer samples as well as control samples to investigate the association between T. gondii infection and lung cancer by confocal microscopy, RT-PCR and q-PCR

José Eduardo Aguayo Flores has completed his Medical studies at the National Polytechnic Institute and did a research stay at the Conde de Valenciana Foundation Ophthalmology Institute. He is a Medical and Optometrist Researcher in the Biochemistry Laboratory in the area of Immunology, a Researcher in the Ministry of Health of Mexico and carries out research in the Ophthalmologist area. He has contributed multiple investigations that resulted in several articles published in renowned journals

Entamoeba histolytica (Eh), produce amebic colitis and can migrate to the liver producing amebic liver abscess (ALA). Neutrophils are the first and main line of the host defense during the invasion of Eh and have a close contact with it. The trophozoites can adhere to the host cells through different lectins such as Gal/GalNac, 220 kDa and 112 kDa adhesin among others. Recently it has been reported that Eh induces the NETs formation. NETs are composed mainly of DNA, histones and myeloperoxidase (MPO). The present study was to evaluate the possible effect of carbohydrates in the NETS formation after the interaction of Eh trophozoites with mouse neutrophils in presence or absence of N-acetyl-D-galactosamine, N-acetyl-glucosamine and D-mannose at different times and concentrations. For detect NETs formation we used a specific polyclonal antibody against MPO, and DNA was detected by Sytox green stain. DNA was quantified by Picogreen assay. The amoeba viability was determined by trypan blue exclusion dye. We observed the formation of NETS during the interaction of Eh and neutrophils in the presence of N-acetyl-D-galactosamine and D-acetyl-glucosamine. No differences in NETs release were observed in presence or absence carbohydrates. We evaluated the DNA concentration in presence of the carbohydrates we can observe an important increase of DNA concentration. The viability of Eh trophozoites diminished importantly in presence of N-acetyl-D-galactosamine with the other two carbohydrates the decrement was lesser. We can conclude that carbohydrates allow that the neutrophils increase the release and formation of NETs and consequently damage to Eh

Maricela Gómez Salvador is a Medical student, studying 12th semester of Medicine in Escuela Superior de Medicina del Instituto Politecnico Nacional, with 4 years in the program Beca de Estímulo Institucional de Formación de Investigadores (BEIFI). She currently holds a rotating internship in the Hospital General Tacuba ISSSTE, having collaborations in the Centro Medico Nacional Siglo XXI, with publication of article in the national magazine of the IMSS

Ascorbic acid removes hypochlorous acid and other free radicals, golden hamster (Mesocricetus auratus) were treated with ascorbic acid and were inoculated with Entamoeba histolytica trophozoites. In this article we will review the MPO role in the amebic liver abscess (ALA) evolution in hamsters treated with ascorbic acid. Two animal groups were formed: (1) Animals that received an intrahepatic inoculation of 1X106 E. histolytica trophrozoites and were sacrificed at 3, 6, 12 hours post-inoculation (Hepatic lesion group); (2) Animals with oral administration of 800 mg/kg of Ascorbic acid every 24 hours during the previous three days before the inoculation of the 1X106 E. histolytica trophrozoites (hepatic lesion+ascorbic acid treatment group). The animals of every group were sacrificed, the size and characteristics of the hepatic lesions were determined, and serum samples were taken, as well as samples from hepatic tissue, this was performed to quantify gene expression and the MPO activity in the hepatic tissue. Ascorbic acid treatment in the ALA susceptibility model in hamsters, induce different changes in the ALA percentage, as well as gene expression and MPO activity, probably due to HOCl removal and free radicals present in the amebic hepatic lesion. Although an increase in MPO gene expression was observed, it did not surpass the basal control level, indicating that the ALA has a significant influence in the MPO gene expression. This could indicate that ascorbic acid is a free radical collector that protects hamsters from liver damage by Entamoeba histolytica and that it does not affect the MOP

Ascorbic acid removes hypochlorous acid and other free radicals, golden hamster (Mesocricetus auratus) were treated with ascorbic acid and were inoculated with Entamoeba histolytica trophozoites. In this article we will review the MPO role in the amebic liver abscess (ALA) evolution in hamsters treated with ascorbic acid. Two animal groups were formed: (1) Animals that received an intrahepatic inoculation of 1X106 E. histolytica trophrozoites and were sacrificed at 3, 6, 12 hours post-inoculation (Hepatic lesion group); (2) Animals with oral administration of 800 mg/kg of Ascorbic acid every 24 hours during the previous three days before the inoculation of the 1X106 E. histolytica trophrozoites (hepatic lesion+ascorbic acid treatment group). The animals of every group were sacrificed, the size and characteristics of the hepatic lesions were determined, and serum samples were taken, as well as samples from hepatic tissue, this was performed to quantify gene expression and the MPO activity in the hepatic tissue. Ascorbic acid treatment in the ALA susceptibility model in hamsters, induce different changes in the ALA percentage, as well as gene expression and MPO activity, probably due to HOCl removal and free radicals present in the amebic hepatic lesion. Although an increase in MPO gene expression was observed, it did not surpass the basal control level, indicating that the ALA has a significant influence in the MPO gene expression. This could indicate that ascorbic acid is a free radical collector that protects hamsters from liver damage by Entamoeba histolytica and that it does not affect the MOP

Maricela Gómez Salvador is a Medical student, studying 12th semester of Medicine in Escuela Superior de Medicina del Instituto Politecnico Nacional, with 4 years in the program Beca de Estímulo Institucional de Formación de Investigadores (BEIFI). She currently holds a rotating internship in the Hospital General Tacuba ISSSTE, having collaborations in the Centro Medico Nacional Siglo XXI, with publication of article in the national magazine of the IMSS

Joshua Angelo H Mandanas is a High School Valedictorian in 2009 and obtained his Degree of Bachelor of Science in Medical Technology in 2014 (Magna Cum Laude) in University of Perpetual Help Laguna. He is also a Registered Medical Technologist. He then finished his Master’s in Public Health Major in Tropical Medicine last year (2017) in University of the Philippines Manila. Currently, he is one of the Philippines’ youngest national Lecturers of Parasitology and Immunology. He is also a Post-graduate student in the College of Medicine of University of the Philippines Manila taking up certificate in Biochemistry.

The study aims to identify the metabolites of the ethanolic leaf extract of Tabernaemontana pandacaqui Poir by phytochemical screening and to determine the in vitro effect of ethanolic leaf extract of Tabernaemontana pandacaqui Poir on adult Caenorhabditis elegans. 2 kilos of Tabernaemontana pandacaqui Poir young leaves were collected by manual picking. Authentification of the leaves was also done. The leaves were air dried for 5 days and subjected to overnight incubation in oven. The selected leaves were then soaked in 2600 mL of solvent (70% ethanol) with dilution ratio of 1:6. The mixture was allowed to stand in room temperature overnight and was filtered for phytochemical analysis. Caenorhabditis elegans N2 strain were grown in Nematode Growth Medium Agar. Adult worms in the final culture were particularly used in the research. The ethanolic extract of Tabernaemontana pandacaqui Poir was positive for carbohydrates, flavonoids and tannins, with tannins as a proven antihelminthic. Lower survival rates were seen following longer exposure (24 hours vs. 48 hours) at concentrations 250 ug/mL and 1000 ug/mL. There is statistically significant difference between length of exposure at 24 hrs and baseline (p<0.001), 48 hrs and baseline (p<0.000) and no statistically significant difference between 24 hrs and 48 hrs. The efficacy of the ethanolic extract of Tabernaemontana pandacaqui Poir can be shown at lengths of exposure at 24 hrs and 48 hrs. Ethanolic extract of Tabernaemontana pandacaqui Poir can have a potential antihelminthic efficacy against in vitro culture of adult Caenorhabditis elegans at lengths of exposure of 24 and 48 hours. This pioneer study can be used as a basis for potential antihelminthic drug development against nematodes. We used a natural extract which has lower side effects and is effective.

Radia Belkhelfa Slimani has completed her PhD in Biochemstry and Immunology from the university of Sciences and Technology, Algiers, Algeria. She is working in the field of development of novel therapeutics and vaccines on leishmaniasis at the Laboratory of Cellular and Molecular Biology in the faculty of Biology at the University of Sciences and Technology in Algiers, Algeria. She is a lecturer in pharmacology and immunology and participate in different project on leishmaniasis. She has published 3 papers in reputed journals and counts several participations in world and national conferences in parasitology and immunology.

Immunization with killed Leishmania promastigotes was considered as safe but ‎gave variable levels of protection. ‎ The aim of this study was to identify an appropriate leishmania vaccine ‎adjuvant based on high Th1 cytokine production. ‎We evaluated the adjuvant properties of caffeic acid (CA), a polyphenol, in cutaneous leishmaniasis. We thus performed 9 weeks infection of BALB/c mice with L. major promastigotes, after a pre-treatment with autovlaved Leishmana major (ALM) alone or in association with CA or Freund’s adjuvant (FA). Our results showed that L. major infection induced neutrophils and macrophages influx correlated with elevated IL-17 and MCP-1 levels, and reduced iNOS/Arg1 ratio and IFN- in footpad lesion. In parallel, ALM-CA, or ALM pre-treatment increased protection as defined by smaller lesion size, correlated to the restoration of iNOS/Arg1 ratio and IFN- level and to reduced IL-17 and MCP-1 levels compared to ALM-F vaccinated groups. Additionally, the effect of ALM-CA was evaluated on bone marrow derived cells from susceptible BALB/c mice. 24h after L. major infection, macrophages and dendritic cells exhibited higher iNOS/Arg1 ratio in the culture supernatant after ALM-CA stimulation. This was correlated to enhanced dendritic cells and macrophages maturation as assessed by MHC class II and CD40 expression. In conclusion, our results indicated that CA, when combined to ALM synergizes with L. major antigens for actively priming innate cells, and inducing early optimal Th1 response, leading to iNOS-dependant leishmanicidal activity of phagocytes. Thus, the use of polyphenols, as immuno-modulator molecules, opens up ‎the way for new therapeutic strategies. ‎ Key word: ALM, Dendritic cells, MHC class II, , Caffeic acid, IFN-phagocytes