Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3

ABSTRACT Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1-3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS-PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1-3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.

An Unusual Case of Extra-Enteric Blastocystosis in the Uterine Cervix.

Extra-enteric infections by Blastocystis spp. have rarely been documented. Here, we report a case of extra-enteric blastocystosis in a patient with minimal cervicitis symptoms. A 47-year-old Hispanic female patient was attended in a primary health centre in Michoacan state, Mexico, for her routine gynaecological medical examination. As only symptom, she referred to a slight vaginal itching. The presence of several vacuolar-stages of Blastocystis spp. were identified by Papanicolaou staining; molecular identification was attempted by culture-PCR sequencing of a region of 18S gene from cervical and faecal samples obtained 2 months after cytological examination, even when patient declared that she tried self-medicating with vaginal ovules. Blastocystis ST1 was identified only in the faecal sample. The presence of Blastocystis spp. in the cervix of a patient with scarce symptomatology, demonstrates the extraordinary flexibility of this microorganism to adapt to new environments and niches.

Plasma cytokine levels and cytokine gene polymorphisms in Mexican patients during the influenza pandemic A(H1N1)pdm09.

BACKGROUND: In Mexico, the initial severe cases of the 2009 influenza pandemic virus A (H1N1) [A(H1N1)pdm09] were detected in early March. The immune mechanisms associated with the severe pneumonia caused by infection with this new virus have not been completely elucidated. Polymorphisms in interleukin genes have previously been associated with susceptibility to infectious diseases due to their influence on cytokine production. OBJECTIVES: The present case-control study was performed to compare several immunologic and genetic parameters of patients and controls during the initial phase of the pandemic. STUDY DESIGN: Sixty-five patients who were hospitalized due to infection with the influenza A(H1N1)pdm09 virus and 46 healthy controls were studied. A hemagglutination inhibition assay (HIA) was performed to measure anti-influenza antibody titers in these subjects. Protein levels of the cytokines interleukin (IL)-4, IL-6, IL-8, IL-10, tumor necrosis factor-α (TNFα), interferon gamma (IFNγ), transforming growth factor beta (TGFß)1 and TGFß2 were quantified in plasma. Single nucleotide polymorphisms in IL6, IL10 and TNFα were also assessed. RESULTS: Influenza patients had lower antibody titers and produced significantly higher levels of IL-6, IL-10 and TNFα than healthy controls. The frequencies of the TNFα -308G, IL-10 -592C and IL-10 -1082A alleles and the IL10 -1082(A/A) genotype were associated with susceptibility to severe disease, while the haplotypes TNFα AG and IL-10 GTA and GCA were associated with protection from severe disease [P=0.016, OR (CI)=0.11 (0.01-0.96); P=0.0187, OR (CI)=0.34 (0.13-0.85); P=0.013, OR (CI)=0.39 (0.18-0.83)]. CONCLUSIONS: This study demonstrates that the influenza A(H1N1)pdm09 patients and healthy controls have different profiles of immune parameters and that there is an association between IL-10 and TNFα polymorphisms and the outcome of this disease.

Chinchilla laniger can be used as an experimental model for Taenia solium taeniasis.

Chinchilla laniger has been reported as an experimental definitive host for Taenia solium; however no information about its suitability and yield of gravid tapeworm proglottids containing viable and infective eggs has been published. In total 55 outbred female chinchillas were infected with 4 cysticerci each; hosts were immunodeppressed with 6 or 8 mg of methyl-prednisolone acetate every 14 days starting the day of infection and their discomfort was followed. Kinetics of coproantigen ELISA or expelled proglottids was used to define the infection status. Efficiency of tapeworm establishment was 21% and of parasite gravidity was 8%; chinchillas showed some degree of suffering along the infection. Viability of eggs obtained from gravid proglottids was tested comparing methods previously published, our results showed 62% viability with propidium iodide, 54% with trypan blue, 34% with neutral red, 30% by oncosphere activation and 7% with bromide 3-(4,5-dimetil-tiazol-2-il)-2,5-difenil-tetrazolio (MTT) reduction; no statistical differences were obtained between most techniques, except activation. Four piglets were infected with 50,000 eggs each, necropsy was performed 3 months later and, after counting the number of cysticerci recovered, the percentage of infection was similar to data obtained with T. solium eggs recovered from humans. Our results demonstrate that the experimental model of T. solium taeniasis in C. laniger is a good alternative for providing eggs and adult tapeworms to be used in different types of experiments; optimization of the model probably depends on the use of inbred hosts and on the reduction of infected animals' suffering.

Immunolocalization of TSOL18 and TSOL45-1A, the successful protective peptides against porcine cysticercosis, in Taenia solium oncospheres.

Taenia solium life cycle includes humans as definitive hosts and pigs as intermediate hosts. One of the measures to stop the life cycle of this parasite is by vaccination of pigs. In experiments performed in pigs with TSOL18 and TSOL45-1A, two recombinant T. solium proteins, 99.5% and 97.0% protection was induced, respectively. The purpose of this paper was to localize these antigens in all stages of the parasite (adult worms, oncospheres and cysticerci) by immunofluorescence, with the use of antibodies against TSOL18 and TSOL45-1A that were obtained from the pigs used in the vaccination experiment. Results show that TSOL18 and TSOL45-1A are expressed on the surface of T. solium oncospheres and not in tapeworms or cysticerci, indicating that they are stage-specific antigens. This, therefore, might explain the high level of protection these antigens induce against pig cysticercosis.

Short report: evaluation of a self-detection tool for tapeworm carriers for use in public health.

The current study was designed to evaluate a tool for the self-identification of tapeworm carriers. Clinical and animal health care practitioners and schoolteachers were trained regarding the life cycle, risk factors, and control measures related to infection with Taenia solium. More than 120 small glass bottles with a few tapeworm segments fixed in formaldehyde and an instructional guide were distributed among all clinical practitioners (physicians and nurses) working in health centers. The guide contained 10 key points on how to ask questions about tapeworm infections. Information on taeniosis and cysticercosis was also provided to the general population via different media. Seven tapeworm carriers were recorded in the official epidemiology surveillance system the year previous to the study, compared with the year after the study, when 41 tapeworm carriers (37 Taenia saginata; 4 Taenia solium) were recorded. Six times more tapeworm carriers were notified after the study. All four persons with Taenia solium were treated, thereby eliminating the parasite and subsequently preventing any new cases of human and swine cysticercosis that might have arisen from them.

Cloning, characterization, and functional expression of Taenia solium calreticulin.

Calreticulin is an endoplasmic reticulum protein involved in the homeostasis of intracellular Ca++ and other physiological processes. A complementary DNA clone containing the complete coding sequence of Taenia solium calreticulin (TsCRT) was isolated and characterized. Recombinant TsCRT was expressed in bacteria as a 50-kDa protein that specifically bound calcium when tested in a radioassay. The deduced amino acid sequence has 47-50% identity with other reported calreticulins. Poor recognition of TsCRT by human and pig sera with confirmed cysticercosis discourages its use for diagnosis of the disease. However, further characterization and localization studies could provide insights into the role of TsCRT in T. solium physiology and host-parasite interactions.

Induction of protection against porcine cysticercosis by vaccination with recombinant oncosphere antigens.

Two recombinant Taenia solium oncosphere antigens, designated TSOL18 and TSOL45-1A, were investigated as vaccines to prevent transmission of the zoonotic disease cysticercosis through pigs. Both antigens were effective in inducing very high levels of protection (up to 100%) in three independent vaccine trials in pigs against experimental challenge infection with T. solium eggs, which were undertaken in Mexico and Cameroon. This is the highest level of protection that has been achieved against T. solium infection in pigs by vaccination with a defined antigen. TSOL18 and TSOL45-1A provide the basis for development of a highly effective practical vaccine that could assist in the control and, potentially, the eradication of human neurocysticercosis.