Marco Lalle

Genotyping of Giardia duodenalis From Humans and Dogs From Mexico Using a β-Giardin Nested Polymerase Chain Reaction Assay

Cysts of Giardia duodenalis were collected in Mexico from symptomatic children (n = 9) and from pet dogs (n = 5), and they were directly characterized by nested polymerase chain reaction (PCR) amplification of the β-giardin gene. Eight isolates of human origin established as in vitro cultures and 2 reference strains, representing assemblages A and B of G. duodenalis, were also analyzed. PCR–restriction fragment length polymorphism showed that all isolates belonged to assemblage A. Sequence analyses indicated that the large majority of isolates were of the A1 genotype; interestingly, 2 human isolates displayed the A3 genotype, which has been previously identified in human isolates from Italy. The presence of cysts of the A1 and A3 genotypes in isolates from pet dogs is consistent with their role as reservoirs for human infection, although further studies are needed to confirm the occurrence of zoonotic transmission. Remarkably, cysts of assemblage B have not been found in any of the Mexican isolates studied to date.

Anisakis pegreffi etiological agent of gastric infections in two Italian women.

Two cases of gastric anisakiasis have been documented in two Italian women who had consumed raw anchovies (Engraulis encrasicolus). The first patient was a 49-year-old woman presenting with epigastric pain and bloody vomiting after ingestion of marinated (vinegar) raw anchovies. During the esophagogastroduodenoscopy (EGDS) a white color worm was detected and extracted from cardia by means of biopsy forceps. The second patient was a 59-year-old woman with irritable bowel syndrome and gastritis, who underwent to periodical EGDSs. In the course of the last EGDS, a white color round worm on antrum and a small polyp on the fundus of the stomach were observed. The two nematodes have been identified as L3 larvae of the genus Anisakis by a light microscope, and as Anisakis pegreffi by polymerase chain reaction-restriction fragment length polymorphism. The molecular identification of the etiological agent at the species level allows to identify what Anisakidae species play a zoonotic role and which are the fish host species.

High genetic polymorphism among Giardia duodenalis isolates from Sahrawi children

Human giardiasis, the gastrointestinal infection caused by two genetically different groups (or assemblages) of Giardia duodenalis, is very common worldwide, and its prevalence is higher in developing countries. However, few surveys in these regions have been performed to include a genetic characterization of the parasite, which is necessary to unravel the complex epidemiology of the infection. In this work, we screened 120 faecal samples collected from Sahrawi children in 2003-2005, and found 41 (34.2%) of them to be positive, using immunofluorescent microscopy, for the presence of G. duodenalis cysts. Molecular characterization of the isolates was performed by RFLP and/or sequence analysis of the triose phosphate isomerase (tpi) and the glutamate dehydrogenase (gdh) genes. The results disclosed an unexpectedly high genetic polymorphism among isolates of both assemblages A and B, and a large percentage of the sequences (50% for the tpi gene, and 90% for the gdh gene) from assemblage B isolates characterized by the presence of overlapping nucleotide peaks at specific positions in the chromatograms, which can be attributed to mixed infections or to allelic sequence heterozygosity of single cysts. Notably, this phenomenon was not observed in sequences from assemblage A isolates. These results suggest that the genetic structure is different in isolates of assemblages A and B.

A novel Giardia duodenalis assemblage A subtype in fallow deer

The molecular identification of species and genotypes of Giardia spp. infecting wild mammals represents the most reliable tool to understand the role played by these animals as reservoirs of cysts infectious for human and other animals. Of 139 fecal samples collected from fallow deer (Dama dama L.) hunted in a Natural Reserve of northern Italy, the prevalence of Giardia sp. was 11.5% (16 of 139 animals), and it was higher in fawns than in older animals. Fragments of the β-giardin and triose phosphate isomerase (tpi) genes were successfully polymerase chain reaction amplified and sequenced from 8 isolates. No sequence variation was observed between isolates at the 2 genetic loci. Sequence and phylogenetic analyses identified a Giardia duodenalis subtype that clusters with assemblage A isolates and that shows homologies of 98 and 97% at the β-giardin and tpi loci, respectively, compared with the A1 subtype. Because the G. duodenalis subtype found in fecal samples of fallow deer has never been detected previously, its role as a pathogen for humans and domestic animals is unknown, but, considering its genetic distinctiveness, it is likely to be low.

Anisakidae infection in fish of the Aegean Sea

Nematode worms of the family Anisakidae are the causative agents of infections in humans when fish is consumed raw and of serious allergies up to the death, when fish is consumed raw or cooked by previously sensitized people. From April until November 2009, 462 fish belonging to 26 species, fished in three areas of the Aegean Sea were tested for Anisakidae larvae. Anisakidae larvae were detected in 87 (18.83%) fish of 13 species. These larvae were identified by morphology as the third-stage larvae of the genera Hysterothylacium sp. or Anisakis. Larvae of the genus Anisakis were identified by PCR-RFLP as belonging to A. simplex s.str., A. pegreffii, or as hybrids between A. simplex s.str and A. pegreffii.

Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions

Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium‐infected erythrocytes, the contribution of lipid‐mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol‐rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium‐infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.

International ring trial to detect anti-Trichinella IgG by ELISA on pig sera.

To determine the reproducibility and robustness of an ELISA to detect anti-Trichinella IgG in pig sera which was previously validated at the Community Reference Laboratory for Parasites (CRLP), a ring trial was organized involving European and extra-European reference laboratories for Trichinella. The sensitivity and specificity of the assay determined by the CRLP validation resulted to be 100% and 98.29%, respectively. The assay was reproducible, moreover, based on the receiver-operator characteristic (ROC) curve, the sensitivity and specificity of the assay reached 97.5% and 96.9%, respectively. The analysis of the differences in optical density (OD) between duplicates indicated a high repeatability of the ELISA with about 95% of the differences between -0.16 and 0.17 absorbance units. The accuracy of the test was determined by calculating the area under the ROC curve (AUC). Overall, the ELISA index (I(E)) showed a very high accuracy (AUC=0.9965) and it performed significantly better than the mean of the duplicated ODs (AUC=0.9387). Of the 21 participating laboratories, nine performed the test without any modification of the original protocol, and 14 with some modifications. Of the laboratories that followed the protocol exactly, three produced false-negatives; whereas of the laboratories that modified the protocol, five produced false-negatives (differences between these two groups of laboratories were not significant, p=0.18). When comparing these two groups of laboratories, the AUCs were very similar (0.9988 and 0.9955, respectively). Finally, a normal mixed multiple model effect was used to evaluate if the I(E) obtained was only related to the serum or to other parameters such as the laboratory, dilution of the serum tested and application of the proposed protocol. The variability found in the test results was mainly due to the serum samples. The assay proposed is robust and reproducible and can be used for monitoring the lack of Trichinella infection in domestic pigs.

Involvement of 14-3-3 protein post-translational modifications in Giardia duodenalis encystation

14-3-3s are a family of phosphoserine/phosphothreonine binding proteins directly affecting many protein functions by regulating enzyme activity, intracellular localisation or mediating protein-protein interaction. The single 14-3-3 (g14-3-3) of the flagellated parasite Giardia duodenalis is phosphorylated at residue threonine 214 (T214) and polyglycylated at the extreme C-terminus in a stage-specific manner. To define the role of each post-translational modification, Giardia transgenic lines expressing a N-terminally FLAG-tagged g14-3-3, or the single point mutant T214A, or the E246A and the E247A mutants of the putative polyglycylation sites, were generated in this study. By affinity chromatography and MALDI-MS analysis, Glu246 was identified as the only site of polyglycylation. The absence of a polyglycine chain results in the nuclear localisation of the protein at any parasite life-stage, suggesting a role for polyglycylation in 14-3-3 nucleo/cytoplasm shuttling. Moreover, cyst formation was strongly induced in parasites expressing the E246A mutant and delayed in those harbouring the T214A mutant. Finally, in vitro overlay assays with a GST_T214E mutant indicated that phosphorylation can alter in vitro the binding properties of 14-3-3. The present data suggest that g14-3-3 post-translational modifications act in combination to affect encystation efficiency in Giardia.

Indirect versus direct detection methods of Trichinella spp. infection in wild boar (Sus scrofa).

BackgroundTrichinella spp. infections in wild boar (Sus scrofa), one of the main sources of human trichinellosis, continue to represent a public health problem. The detection of Trichinella spp. larvae in muscles of wild boar by digestion can prevent the occurrence of clinical trichinellosis in humans. However, the analytical sensitivity of digestion in the detection process is dependent on the quantity of tested muscle. Consequently, large quantities of muscle have to be digested to warrant surveillance programs, or more sensitive tests need to be employed. The use of indirect detection methods, such as the ELISA to detect Trichinella spp. infections in wild boar has limitations due to its low specificity. The aim of the study was to implement serological detection of anti-Trichinella spp. antibodies in meat juices from hunted wild boar for the surveillance of Trichinella spp. infections.MethodsTwo tests were used, ELISA for the initial screening test, and a specific and sensitive Western blot (Wb) as a confirmatory test. The circulation of anti-Trichinella IgG was determined in hunted wild boar muscle juice samples in 9 provinces of 5 Italian regions.ResultsFrom 1,462 muscle fluid samples, 315 (21.5%, 95% C.I. 19.51-23.73) were tested positive by ELISA. The 315 ELISA-positive muscle fluid samples were further tested by Wb and 32 (10.1%, 95% C.I. 7.29-13.99) of these were positive with a final seroprevalence of 2.2% (95% C.I 1.55-3.07; 32/1,462). Trichinella britovi larvae were detected by artificial digestion in muscle tissues of one (0.07%, 95%C.I. 0.01-0.39) out of the 1,462 hunted wild boars. No Trichinella spp. larvae were detected in Wb-negative wild boar. From 2006 to 2012, a prevalence of 0.017% was detected by muscle digestion in wild boar hunted in the whole Italian territory.ConclusionsThe combined use of both serological methods had a sensitivity 31.4 times higher than that of the digestion (32/1,462 versus 1/1,462), suggesting their potential use for the surveillance of the Trichinella spp. infection in wild boar populations.

The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX.

The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions.