{"title":"Trichinella-induced immunomodulation: Another tale of helminth success","authors":"F. Bruschi , D.S. Ashour , A.A. Othman","doi":"10.1016/j.fawpar.2022.e00164","DOIUrl":"https://doi.org/10.1016/j.fawpar.2022.e00164","url":null,"abstract":"<div><p><em>Trichinella spiralis</em> is a unique parasite in that both the adults and larvae survive in two different intracellular niches in the same host. The immune response, albeit intense, is highly modulated to ensure the survival of both the host and the parasite. It is skewed to T helper 2 and regulatory arms. Diverse cells from both the innate and adaptive compartments of immunity, including dendritic cells, T regulatory cells, and alternatively activated macrophages are thought to mediate such immunomodulation. The parasite has also an outstanding ability to evade the immune system by several elaborate processes. The molecules derived from the parasites including <em>Trichinella</em>, particularly the components of the excretory–secretory products, are being continually identified and explored for the potential of ameliorating the immunopathology in animal models of diverse inflammatory and autoimmune human diseases. Herein we discuss the various aspects of <em>Trichinella</em>-induced immunomodulation with a special reference to the practical implications of the immune system manipulation in alleviating or possibly curing human diseases.</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S240567662200021X/pdfft?md5=3b275a2f9c12144607480cb01b889a80&pid=1-s2.0-S240567662200021X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91685189","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Miguel Bao , Paolo Cipriani , Lucilla Giulietti , Mohammad Ashraful Alam , Marialetizia Palomba , Simonetta Mattiucci , Arne Levsen
{"title":"Ascaridoid nematodes infecting commercially important marine fish and squid species from Bangladesh waters in the Bay of Bengal","authors":"Miguel Bao , Paolo Cipriani , Lucilla Giulietti , Mohammad Ashraful Alam , Marialetizia Palomba , Simonetta Mattiucci , Arne Levsen","doi":"10.1016/j.fawpar.2022.e00157","DOIUrl":"10.1016/j.fawpar.2022.e00157","url":null,"abstract":"<div><p>Parasitic ascaridoid nematodes occur in a wide range of marine organisms across the globe. Some species of the anisakid family (Ascaridoidea: Anisakidae) can cause gastrointestinal disease in humans (i. e. anisakidosis). Despite their importance as potentially hazardous parasites, the occurrence and infection characteristics of ascaridoids are still poorly known from many host species and geographical areas. This study investigated the diversity and infection levels of ascaridoid parasites in various commercial fish and squid host species off Bangladesh. Fish and squid specimens were visually inspected for nematodes using the UV-press method. Nematodes were assigned to genus level based on morphology and identified by sequence analyses of the entire ITS region and partial 28S rDNA and mtDNA <em>cox2</em> genes. Third-stage larvae (L3) of <em>Anisakis typica</em> occurred at low prevalence (<em>P</em> = 10% and 8%, respectively) in the viscera of <em>Selar crumenophthalmus</em> and <em>Trichiurus lepturus</em>, while <em>Hysterothylacium amoyense</em> occurred in the viscera of <em>Sardinella fimbriata</em> (<em>P</em> = 1%) and the viscera and muscle of <em>Harpadon nehereus</em> (<em>P</em> = 32%) and <em>T. lepturus</em> (<em>P</em> = 76%). <em>Lappetascaris</em> sp. Type A L3 occurred in the mantle of the squid <em>Uroteuthis duvaucelii</em> (<em>P</em> = 11%). <em>Anisakis</em> and <em>Lappetascaris</em> species, and <em>H. amoyense</em> were firstly identified in the Bay of Bengal. The potentially zoonotic <em>A. typica</em> was only found in fish viscera. <em>Hysterothylacium amoyense</em> and <em>Lappetascaris</em> sp., both generally regarded as non-zoonotic, occurred at low prevalence in the muscle or mantle of fish or squid, respectively. Since consumption of raw or lightly processed seafood seems to be rare in Bangladesh, the risk of acquiring anisakidosis from consuming fishery products from off Bangladesh appears to be low. Due to its reddish appearance, the visual presence of <em>H. amoyense</em> larvae in fish flesh may represent a food quality issue.</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/34/dd/main.PMC9065311.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10251856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dante Zarlenga , Peter Thompson , Makedonka Mitreva , Bruce A. Rosa , Eric Hoberg
{"title":"Horizontal gene transfer provides insights into the deep evolutionary history and biology of Trichinella","authors":"Dante Zarlenga , Peter Thompson , Makedonka Mitreva , Bruce A. Rosa , Eric Hoberg","doi":"10.1016/j.fawpar.2022.e00155","DOIUrl":"10.1016/j.fawpar.2022.e00155","url":null,"abstract":"<div><p>Evolution involves temporal changes in the characteristics of a species that are subsequently propagated or rejected through natural selection. In the case of parasites, host switching also plays a prominent role in the evolutionary process. These changes are rooted in genetic variation and gene flow where genes may be deleted, mutated (sequence), duplicated, rearranged and/or translocated and then transmitted through vertical gene transfer. However, the introduction of new genes is not driven only by Mendelian inheritance and mutation but also by the introduction of DNA from outside a lineage in the form of horizontal gene transfer between donor and recipient organisms. Once introduced and integrated into the biology of the recipient, vertical inheritance then perpetuates the newly acquired genetic factor, where further functionality may involve co-option of what has become a pre-existing physiological capacity. Upon sequencing the <em>Trichinella spiralis</em> (Clade I) genome, a cyanate hydratase (cyanase) gene was identified that is common among bacteria, fungi, and plants, but rarely observed among other eukaryotes. The sequence of the <em>Trichinella</em> cyanase gene clusters with those derived from the Kingdom Plantae in contrast to the genes found in some Clade III and IV nematodes that cluster with cyanases of bacterial origin. Phylogenetic analyses suggest that the <em>Trichinella</em> cyanase was acquired during the Devonian period and independently from those of other nematodes. These data may help inform us of the deep evolutionary history and ecological connectivity of early ancestors within the lineage of contemporary <em>Trichinella</em>. Further, in many extant organisms, cyanate detoxification has been largely superseded by energy requirements for metabolism. Thus, deciphering the function of <em>Trichinella</em> cyanase may provide new avenues for treatment and control.</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405676622000129/pdfft?md5=15deba4f5bbda42a49c4ced29c078991&pid=1-s2.0-S2405676622000129-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46463546","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
María Ángeles Gómez-Morales, Simona Cherchi, Alessandra Ludovisi
{"title":"Serological testing for Trichinella infection in animals and man: Current status and opportunities for advancements","authors":"María Ángeles Gómez-Morales, Simona Cherchi, Alessandra Ludovisi","doi":"10.1016/j.fawpar.2022.e00165","DOIUrl":"10.1016/j.fawpar.2022.e00165","url":null,"abstract":"<div><p>Serological tests are widely used for the detection of <em>Trichinella</em> spp. infections in animals and humans. Despite some limitations, (such as low sensitivity in the early period after infection) ELISA and western blot testing have demonstrated good performance when excretory/secretory products from muscle larvae are used as antigens in agreement with the International Commission on Trichinellosis. Over recent decades, considerable progress has been made in the characterization of <em>Trichinella</em>-derived molecules in the hope of improving diagnosis, mainly during the early days post infection. Despite these efforts, validated tests using characterized antigens for early diagnosis are still not available. However, combining currently available sero-diagnostic tools with clinical and epidemiological data provides valuable information on <em>Trichinella</em> infections in humans and animals as shown in this review.</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405676622000221/pdfft?md5=718250a9b4bc6f028e20bc99b7bda5ed&pid=1-s2.0-S2405676622000221-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43703341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Eskild Petersen , Valeria Meroni , Daniel V. Vasconcelos-Santos , Laurent Mandelbrot , Francois Peyron
{"title":"Congenital toxoplasmosis: Should we still care about screening?","authors":"Eskild Petersen , Valeria Meroni , Daniel V. Vasconcelos-Santos , Laurent Mandelbrot , Francois Peyron","doi":"10.1016/j.fawpar.2022.e00162","DOIUrl":"10.1016/j.fawpar.2022.e00162","url":null,"abstract":"<div><p>Prenatal systematic screening for congenital toxoplasmosis has been performed in Austria and France since 1975 and neonatal screening for congenital toxoplasmosis has been part of the New England Newborn screening program since 1986.</p><p>In this narrative review we review the data leading up to the systematic screening programs in Austria and France, highlighting the main finding of the European Union funded research in the 1990s and early 2000s. Different descriptive studies of the effect of pre- or postnatal treatment are discussed. <em>Toxoplasma gondii</em> has different genetic lineages with different pathogenicity in humans. This means that results in areas with a low pathogenic lineage cannot be extrapolated to an area with highly pathogenic lineages. The importance of meat as a source of infection is discussed in the light of an increased prevalence of <em>T.gondii</em> in organic livestock production .</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9249550/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40567126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sanna Malkamäki , Antti Oksanen , Anu Näreaho , Antti Sukura
{"title":"Dispersal of taeniid eggs: Experimental faecal contamination of forest environment followed by DNA detection in wild berries","authors":"Sanna Malkamäki , Antti Oksanen , Anu Näreaho , Antti Sukura","doi":"10.1016/j.fawpar.2022.e00152","DOIUrl":"10.1016/j.fawpar.2022.e00152","url":null,"abstract":"<div><p>To understand Taeniidae epidemiology, the principles of egg-dispersion dynamics under natural conditions must be known. In this study, non-zoonotic <em>Taenia laticollis</em> was used as a model parasite for the family Taeniidae (including <em>Echinococcus</em> spp.). An experiment to investigate dispersion from contaminated faeces to the surroundings was performed both with bilberries (<em>Vaccinium myrtillus</em>) and lingonberries (<em>Vaccinium vitis-idaea</em>), both of which are commercially harvested wild berries in Finland. For this experiment, 30 g of fox faeces was inoculated with 30,000 <em>T. laticollis</em> eggs for the bilberry experiment and 100,000 eggs for the lingonberry experiment. The faecal material was placed in the middle of good berry growth areas in four locations for bilberries and eight locations for lingonberries. After 41–42 days, berries at different distances (0–15 m) from the original contamination spot were collected and delivered to our laboratory. DNA was extracted from washed and sieved material and analysed using <em>T. laticollis</em>-specific semi-quantitative SYBR Green real-time polymerase chain reaction (qPCR). <em>Taenia laticollis</em>-specific DNA was recovered from 67% (8/12) of bilberry samples but not reliably from any of the lingonberry samples 0% (0/24), although the exposure dose was higher for those. The qPCR results suggest that under natural conditions, taeniid egg dispersion from the contamination spot is demonstrated but attachment is berry specific. The surface of bilberries may be more adhesive for taeniid eggs than the waxier and harder pericarp of the lingonberries or there might be a difference in the dispersal mechanism caused by different biotopes.</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405676622000099/pdfft?md5=bef06a8844d817949a6d38b037edbbc3&pid=1-s2.0-S2405676622000099-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44869894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Guy Robinson , Gregorio Pérez-Cordón , Clare Hamilton , Frank Katzer , Lisa Connelly , Claire L. Alexander , Rachel M. Chalmers
{"title":"Validation of a multilocus genotyping scheme for subtyping Cryptosporidium parvum for epidemiological purposes","authors":"Guy Robinson , Gregorio Pérez-Cordón , Clare Hamilton , Frank Katzer , Lisa Connelly , Claire L. Alexander , Rachel M. Chalmers","doi":"10.1016/j.fawpar.2022.e00151","DOIUrl":"10.1016/j.fawpar.2022.e00151","url":null,"abstract":"<div><p>Subtyping <em>Cryptosporidium parvum</em> for outbreak investigations or epidemiological surveillance usually relies on DNA sequence analysis of a gene coding for a 60 KDa glycoprotein (<em>gp60</em>). Although <em>gp60</em> can be useful for allelic discrimination and to help investigate sources and routes of transmission, the presence of common subtypes and recombination during the parasite's sexual life-cycle demand a multilocus-based method for more discriminatory genotyping. While whole genome sequencing would provide the ultimate approach, it is a time consuming and expensive option for faecal parasites such as <em>Cryptosporidium</em> that occur at low density and are difficult to propagate routinely. In this study, we selected and evaluated a panel of previously identified variable-number tandem-repeat (VNTR) markers, to establish a multilocus genotyping scheme based on fragment sizing, appropriate for inter-laboratory surveillance and outbreak investigations. Seven VNTR markers were validated <em>in vitro</em> and demonstrated typeability of 0.85 and discriminatory power of 0.99. The discriminatory power was much greater than the currently used <em>gp60</em> sequencing (0.74), which identified 26 subtypes, compared to 100 different MLVA profiles within the same sample set. The assay was robust, with repeatable results and reproducibility across three laboratories demonstrating the scheme was suitable for inter-laboratory comparison of <em>C. parvum</em> subtypes. As the majority of genotypes (79%) were unique among epidemiologically unrelated samples, there was efficiency to infer linkage, and epidemiological concordance was observed in historical outbreaks. We propose that the multilocus variable number of tandem repeats analysis scheme is suitable to assist outbreak investigations.</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405676622000087/pdfft?md5=78fb0656f2679905f655e9b78aac427a&pid=1-s2.0-S2405676622000087-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44180005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Is Cryptosporidium a hijacker able to drive cancer cell proliferation?","authors":"Gabriela Certad","doi":"10.1016/j.fawpar.2022.e00153","DOIUrl":"10.1016/j.fawpar.2022.e00153","url":null,"abstract":"<div><p>The pathophysiological mechanisms of <em>Cryptosporidium</em> infection are multifactorial and not completely understood. Some advances achieved recently revealed that the infection by <em>Cryptosporidium parvum</em> induces cytoskeleton remodeling and actin reorganization through the implication of several intracellular signals involving, for example, PI3K, Src, Cdc42 and GTPases. It has also been reported that the infection by <em>C. parvum</em> leads to the activation of NF-κβ, known to induce anti-apoptotic mechanisms and to transmit oncogenic signals to epithelial cells. Despite the growing evidence about the hijacking of cellular pathways, potentially being involved in cancer onset, this information has rarely been linked to the tumorigenic potential of the parasite. However, several evidences support an association between <em>Cryptosporidium</em> infection and the development of digestive neoplasia. To explore the dynamics of <em>Cryptosporidium</em> infection, an animal model of cryptosporidiosis using corticoid dexamethasone-treated adult SCID (severe combined immunodeficiency) mice, orally infected with <em>C. parvum</em> or <em>Cryptosporidium muris</em> oocysts was implemented. <em>C. parvum</em>-infected animals developed digestive adenocarcinoma. When mechanisms involved in this neoplastic process were explored, the pivotal role of the Wnt pathway together with the alteration of the cytoskeleton was confirmed. Recently, a microarray assay allowed the detection of cancer-promoting genes and pathways highly up regulated in the group of <em>C. parvum</em> infected animals when compared to non-infected controls. Moreover, different human cases/control studies reported significant higher prevalence of <em>Cryptosporidium</em> infection among patients with recently diagnosed colon cancer before any treatment when compared to the control group (patients without colon neoplasia but with persistent digestive symptoms). These results suggest that <em>Cryptosporidium</em> is a potential oncogenic agent involved in cancer development beyond the usual suspects. If <em>Cryptosporidium</em> is able to hijack signal transduction, then is very likely that this contributes to transformation of its host cell. More research in the field is required in order to identify mechanisms and molecular factors involved in this process and to develop effective treatment interventions.</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405676622000105/pdfft?md5=3b7c32239b58a8c203b11320a9689d8e&pid=1-s2.0-S2405676622000105-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41264241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"A Simple Alcohol-based Method of Oocyst Inactivation for Use in the Development of Detection Assays for Cryptosporidium","authors":"Biniam Hagos, Robert E. Molestina","doi":"10.1016/j.fawpar.2022.e00163","DOIUrl":"10.1016/j.fawpar.2022.e00163","url":null,"abstract":"<div><p><em>Cryptosporidium spp.</em> are obligate, intracellular parasites that cause life-threatening diarrhea among children and immunocompromised adults. Transmission occurs by the fecal-oral route following ingestion of thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia are suitable disinfectants against <em>Cryptosporidium</em> oocysts. Effective concentrations of these chemicals can be toxic and not practical for downstream research use of non-viable oocysts. Oocyst inactivation approaches such as UV light, heat, and treatments with ethanol or methanol are generally more accessible for routine lab use, yet their applicability in <em>Cryptosporidium</em> assay development is limited. The aims of this study were to evaluate methods of inactivation of <em>Cryptosporidium</em> oocysts that can be readily applied in the laboratory and test the utility of whole inactive oocysts in quantitative PCR (qPCR). Experiments were performed on <em>C. parvum</em> oocysts subjected to heat (75 °C/10 min) or treated with increasing concentrations of ethanol and methanol over time. Viability assays based on propidium iodide (PI) staining, <em>in vitro</em> excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations of ≥70% of these chemicals were required to completely inhibit excystation and infection of Hct-8 cells <em>in vitro</em>. Inactivated oocysts stored for up to 30 days at 4 °C retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence. Moreover, non-viable oocysts applied directly in qPCR assays of the COWP gene were useful reference reagents for the identification and quantification of <em>Cryptosporidium</em> in spiked water samples. In summary, we have established a practical approach to inactivate <em>C. parvum</em> oocysts in the laboratory that is suitable for the development of detection or diagnostic assays targeting the parasite.</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f5/25/main.PMC9249555.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40567124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}