Morphology does not allow differentiating the species of the Phlebotomus perniciosus complex: Molecular characterization and investigation of their natural infection by Leishmania infantum in Morocco.

Morphological and DNA-based complemented approaches were applied for characterization of sympatric populations of Phlebotomus longicuspis and Phlebotomus perniciosus in Morocco. Both sand fly species are generally recorded in sympatry in North Africa but on few occasions have been molecularly characterized. The diagnostic confusion of these species has led to errors in their geographical distribution and probably, in the assignment of their role in the transmission of L. infantum. Sand flies were caught inside households in El Borouj, central Morocco, in 2014-2015. For female sand flies, detection of L. infantum natural infection and blood meal identification were carried out. According to morphological identification, Phlebotomus longicuspis s.l. (34.7%) was the second most abundant Phlebotomus species after P. sergenti, followed by atypical Phlebotomus perniciosus (7.1%); 11.6% of the male specimens of P. longicuspis s.l. were identified as P. longicuspis LCx according to the number of coxite setae. The density of Larroussius species was very high (31 Larroussius/light trap/night) in the peripheral neighbourhood of Oulad Bouchair (p = 0.001) where the first case of cutaneous leishmaniasis due to Leishmania infantum was detected in 2017. Phylogenetic trees based on three independent genes highlighted three well-supported clusters within P. perniciosus complex that could be interpreted as corresponding to P. perniciosus, P. longicuspis s.s. and an undescribed species, all coexisting in sympatry. Some females with typical morphology of P. longicuspis were genetically homologous to P. perniciosus. The taxa cannot be differentiated by morphological methods but characterized by a distinctive genetic lineage for which the synapomorphic characters are described. Leishmania infantum was detected in females of all clusters with a low parasite load. Population genetics will help to assess the threat of the geographical spread of L. infantum in Morocco by determining the density, abundance and vector role of the species of the P. perniciosus complex identified correctly.

Acute Anisakiasis: Pharmacological Evaluation of Various Drugs in an Animal Model.

BACKGROUND: The accidental ingestion of the third larval stage of Anisakis can cause acute clinical symptoms, which are relieved via extraction of the larvae. Although this is a highly effective technique, it can only be practiced when the larvae are found in accessible areas of the gastrointestinal tract, and therefore instead the condition has often been treated using various different drugs. AIMS: This study evaluates the effectiveness of gastric acid secretion inhibitors (omeprazole and ranitidine), gastric mucosal protectants (sucralfate) and anthelmintics (mebendazole and flubendazole) in treating anisakiasis in Wistar rats. METHODS: Rats were infected with Anisakis-type I larvae and administered the drugs via a gastric probe. Data were recorded regarding the number of live and dead larvae, their location both within the animal and in its feces, and the presence of gastrointestinal lesions. Additionally, gastric pH was measured and histology performed. RESULTS: While rats in all experimental groups exhibited lesions; those treated with ranitidine and mebendazole showed significantly fewer lesions (50% and 35% of rats exhibited lesions, respectively). Histological examination of the gastric lesions revealed infection-induced changes, but no significant differences were observed between the treated and untreated rats. CONCLUSIONS: Mebendazole was found to be most efficacious in preventing gastrointestinal lesions, followed by ranitidine, which was the most effective antacid of those studied. Both these drugs could thus be considered as part of the conservative management of anisakiasis.

A multi-restriction fragment length polymorphism genotyping approach including the beta-tubulin gene as a new differential nuclear marker for the recognition of the cryptic species Anisakis simplex s.s. and Anisakis pegreffii and their hybridization events.

The detection of Anisakis simplex s.s./A. pegreffii putative hybrids has been a controversial issue in spite of the fact that natural hybridization is an extended process across free living and parasitic organisms. Differential traits of biomedical and ecological importance, such as invasive and allergenic potential have been demonstrated in both cryptic species. Therefore, in this work, we discuss about the potential for hybridization between these anisakid species in sympatric zones, implementing a multi-marker Restriction fragment length polymorphism (RFLP) genotyping approach based on the ribosomal DNA internal transcribed spacer 1 (ITS1), the mitochondrial cytochrome C oxidase 2 (Cox-2) and a new nuclear marker, the highly conserved ß-tubulin gene (ß-TUB). The two cryptic species differed at least in 7 bp in the ß-TUB gene and some larvae with heterozygous genotypes at the 7 diagnostic nucleotide positions were found. Taxonomic, population and genealogical analyses served to support the occurrence of hybridization between both species. Predicted restriction endonucleases enzymes were assayed for Cox-2 and ß-TUB markers. The implemented multi-marker PCR-RFLP allowed us to detect the two pure parental species, F1 hybrids, hybrid backcrossed progeny and individuals with nuclear-mitochondrial discordance, being a useful, simple and reproducible procedure in any laboratory for epidemiological studies.

Molecular diagnosis of Pseudoterranova decipiens s.s in human, France.

BACKGROUND: Anisakis and Pseudoterranova are the main genera involved in human infections caused by nematodes of the Anisakidae family. Species identification is complicated due to the lack of differential morphological characteristics at the larval stage, thus requiring molecular differentiation. Pseudoterranova larvae ingested through raw fish are spontaneously eliminated in most cases, but mechanical removal by means of endoscopy might be required. To date, only very few cases of Pseudoterranova infection have been reported in France. CASE PRESENTATION: A 19-year-old woman from Northeastern France detected, while brushing her teeth, a larva exiting through her mouth. The patient who presented with headache, diarrhea, and abdominal cramps reported having eaten baked cod. The worm was a fourth-stage larva with a size of 22 × 0.9 mm, and molecular biology identified it as Pseudoterranova decipiens sensu stricto (s. s.). In a second P. decipiens infection case, occurring a few months later, a worm exited through the patient's nose after she had eaten raw sea bream. CONCLUSION: These two cases demonstrate that Pseudoterranova infection is not uncommon among French patients. Therefore, molecular techniques should be more widely applied for a better characterization of anisakidosis epidemiology in France.

Molecular epidemiology and risk factors for Anisakis simplex s.l. infection in blue whiting (Micromesistius poutassou) in a confluence zone of the Atlantic and Mediterranean: Differences between A. simplex s.s. and A. pegreffii.

Our study determined parameters of parasitization of Anisakis simplex s.l. in Micromesistius poutassou in a confluence zone of the Atlantic and Mediterranean, with a total prevalence of 82%. Also, in the three seasons analyzed, high prevalence's values were found, reaching 100% in spring; however mean intensity and abundance values were higher in winter. The use of molecular techniques to differentiate between Anisakis genotypes of the larvae characterized allowed obtaining values of 99.7% Anisakis simplex s.l. (50.1% A. simplex s.s., 42.9% A. pegreffii, 7.0% A. simplex s.s. - A. pegreffii hybrids) and 0.3% A. typica. The infections found in the fish were of both single and mixed species, in all the different possible combinations. The presence of A. simplex s.l. in the viscera varied according to genotype and season. Likewise, factors associated with the presence of the parasite in the ventral or dorsal musculature were different, where A. simplex s.s. proportion was double than that of A. pegreffii. The ecology of the two sibling species with regard to their location in fish and the influence of the season were different.

Fishing area and fish size as risk factors of Anisakis infection in sardines (Sardina pilchardus) from Iberian waters, southwestern Europe.

The sardine (Sardina pilchardus) is a fish commonly consumed and appreciated in many countries, although they are more likely to be eaten fresh in western Mediterranean countries such as Spain, Portugal, France or Italy. A molecular epidemiological survey of sardines from 5 fishing areas of the Spanish Mediterranean (Málaga, southern Spain) and Atlantic coasts (southern: Cádiz and Isla Cristina; northern: A Coruña and Ondarroa) was carried out to determine the presence of Anisakis spp. larvae. The highest prevalence of these larvae was observed in fish from A Coruña (28.3%), followed by Ondarroa (5%) and Cádiz (2.5%). No Anisakis larvae were found in fish from Málaga and Isla Cristina. Three Anisakis genotypes were identified: Anisakis simplex sensu stricto, Anisakis pegreffii and a hybrid genotype between these two species. A. pegreffii was the most prevalent species in A Coruña (71% of larvae). Only three Anisakis larvae (9% collected larvae) were located in the musculature of sardines: two were identified as A. pegreffii while the other was a hybrid genotype. Sardine infection was associated with fishing area and fish length/weight (length and weight were strongly correlated; Pearson's correlation 0.82; p<0.001). Risk factor multivariate analysis showed that the risk of infection increases 1.6 times for every additional cm in the length of the sardines from the same fishing area. Comparison of fish of equal length showed that in sardines from A Coruña the risk of parasitization is 11.5 times higher than in those from other fishing areas. Although the risk of infection by Anisakis through consumption of sardines is generally low due to the low epidemiological parameter values (prevalence 10%, mean intensity 1.7 (range 1-5) and mean abundance 0.17), as larger fish are more heavily parasitized, there is an increased risk of infection by Anisakis through consumption of large sardines which are raw or have undergone insufficient treatment (undercooked, smoked, marinated, salted, pickled, freezing,…).

Reconocimiento de los antígenos excretores/secretores de Anisakis tipo I y evolución de la IgE en ratas infectadas experimentalmente / Recognition of excretory/secretory antigens of Anisakis type I and evolution of IgE in experimentally infected rats

INTRODUCCIÓN: Anisakis spp., durante la parasitación, libera antígenos de excreción secreción (ES) que, al ponerse en contacto con el sistema inmunológico del hombre, pueden desencadenar una respuesta de hipersensibilidad mediada por la IgE, provocando diversos síntomas alérgicos. OBJETIVOS: Evaluar la respuesta de la IgE en ratas Wistar tras la infección con larvas L3 del parásito. MÉTODOS: Se ha procedido a la obtención de antígenos ES del parásito y suero anti-Anisakis. Se investigan también en este trabajo ciertos factores que intervienen en la técnica de inmunotransferencia, como la concentración de poliacrilamida empleada en la preparación de los geles, la concentración antigénica utilizada y la temperatura requerida para la desnaturalización de las proteínas. RESULTADOS: Las reacciones inmunológicas (Ag-Ac) observadas mediante esta técnica muestran mayor intensidad con los sueros obtenidos después de la reinfección, los cuales han reconocido proteínas que podrían corresponder al antígeno principal Ani s 1 y a otros polipéptidos de interés en el diagnóstico de la anisakiosis humana. CONCLUSIÓN: En este trabajo, se pone de manifiesto que inmunotransferenciala inmunotransferencia es una técnica útil para detectar anticuerpos de tipo IgE frente a proteínas de Anisakis

INTRODUCTION: Anisakis spp., during parasitism, release excretory-secretory antigens that, in contact with the human immune system, can trigger a hypersensitivity response mediated by IgE, causing various allergic symptoms. OBJECTIVES: To evaluate the IgE response in Wistar rats after infection with L3 larvae of the parasite Anisakis spp. METHODS: Some determining factors involved in the technique have been improved in this work, such as: the concentration of polyacrylamide used in the preparation of the gels, the antigen concentration used, and the temperature required for denaturation of proteins. RESULTS: Immune responses (Ag-Ab) observed by the immunoblotting technique showed a greater intensity with serum obtained after reinfection, which have recognized proteins that may correspond to the major antigen Ani s 1 and other polypeptides of interest in the diagnosis of human anisakiasis. CONCLUSION: This paper concludes that immunoblotting is a useful technique to detect IgE antibodies against Anisakis proteins

[Recognition of excretory/secretory antigens of Anisakis type I and evolution of IgE in experimentally infected rats]. / Reconocimiento de los antígenos excretores/secretores de Anisakis tipo I y evolución de la IgE en ratas infectadas experimentalmente.

INTRODUCTION: Anisakis spp., during parasitism, release excretory-secretory antigens that, in contact with the human immune system, can trigger a hypersensitivity response mediated by IgE, causing various allergic symptoms. OBJECTIVES: To evaluate the IgE response in Wistar rats after infection with L3 larvae of the parasite Anisakis spp. METHODS: Some determining factors involved in the technique have been improved in this work, such as: the concentration of polyacrylamide used in the preparation of the gels, the antigen concentration used, and the temperature required for denaturation of proteins. RESULTS: Immune responses (Ag-Ab) observed by the immunoblotting technique showed a greater intensity with serum obtained after reinfection, which have recognized proteins that may correspond to the major antigen Ani s 1 and other polypeptides of interest in the diagnosis of human anisakiasis. CONCLUSION: This paper concludes that immunoblotting is a useful technique to detect IgE antibodies against Anisakis proteins.