Frequency, spatial distribution, and genetic diversity of Blastocystis among referred individuals to a clinical laboratory: First report of subtype 9 in Brazil.

The enteric protist Blastocystis has a worldwide distribution, however its prevalence in the human population is still underestimated, especially in developing countries where proper diagnosis is not performed in the routine of clinical laboratories. In this study, we aimed to assess the frequency, genetic diversity, and spatial distribution of Blastocystis isolates detected in fecal samples referred to a clinical laboratory for routine examination in inner São Paulo State, Brazil. A total of 348 leftover stool samples available for disposal from female and male individuals with age ranging from 3 months to 88 years were analyzed by both microscopic examination and PCR/sequencing of the SSU rRNA gene. The overall frequency of Blastocystis sp. was 31% (108/348), including 20.1% (70/348) and 31% (108/348) by microscopic examination and PCR/sequencing, respectively. Significant association was found only between Blastocystis infection and age, since the highest rate of positive samples was detected among 5-9 years old individuals (p < 0.0001). In addition, spatial distribution revealed a wide distribution of the positive samples, however they were densely concentrated in more populated areas. Seven subtypes were identified, namely ST1 (40.7%), ST2 (9.2%), ST3 (45.3%), ST4 (0.9%), ST6 (1.8%), ST7 (0.9%) and ST9 (0.9%). The intra-subtype analysis revealed a total of 25 different alleles previously reported. Here, the findings lead us to highlight the following aspects: (1) the identification of a ST9 isolate is a relevant finding since it is considered a very rare subtype in human infections as well as this is the first report in Brazil; (2) the high frequency of Blastocystis in fecal samples submitted for examination in a clinical laboratory points to the need to consider its search in routine parasitological examinations, (3) the spatial distribution of Blastocystis infection was not homogeneous but concentrated in more populated areas where the access for population to diagnostic services in healthcare is likely to be easier and, (4) the genetic variability of Blastocystis isolates suggests exposure of inhabitants living in inner municipalities to different sources of contamination involving anthroponotic and zoonotic transmission pathways.

Molecular identification of hookworm species infecting free-roaming and owned dogs from an urban area in inner São Paulo State, Brazil.

Dogs are the most popular pet animals worldwide, but on the other hand, they are main hosts of pathogens potentially transmissible to humans. The aim of this study was to assess the occurrence of intestinal parasites in free- roaming and owned dogs in an urban area in southeastern Brazil and to identify the hookworm species infecting them. Faecal samples (80 from free-roaming and 53 from owned dogs) were examined for intestinal parasites using concentration methods. DNA extracted from hookworm microscopy-positive samples were tested by PCR targeting the ITS1-5.8S-ITS2 region and the amplicons retrieved were sequenced. Intestinal parasites were detected in 43.60% (58/133) of the dogs and hookworm infection was found at the highest prevalence rate (38.30%), followed by Toxocara canis (10.50%), Trichuris vulpis (2.25%), Giardia spp. (0.75%) and Cystoisospora spp. (0.75%). Out of the 51 samples positive for hookworm eggs, 26 (50.90%) were successfully amplified and sequenced. Single infections with Ancylostoma caninum and Ancylostoma braziliense were recorded in 18 (69.20%) and two (7.70%) isolates, respectively, and mixed infections were found in the remaining six samples (23.10%). Both species were found infecting free-roaming and owned animals, but A. caninum was more common. These findings highlight the public health relevance of dogs as reservoirs of zoonotic parasites, with emphasis on hookworm species commonly implicated in cutaneous larva migrans (CLM) in poor and deprived areas.

Prevalence and genetic characterization of Dientamoeba fragilis in asymptomatic children attending daycare centers.

In order to provide additional data on the prevalence and genetic diversity of Dientamoeba fragilis in human populations, we conducted a study in children from low-income communities in Sao Paulo State, Brazil. Fecal samples from daycare center attendees up to 6 years old (n=156) and staff members (n=18) were submitted to PCR and sequencing of D. fragilis as well as to microscopic examination for the presence of other intestinal parasites. All children assessed were asymptomatic and 10.3% (16/156) were positive for D. fragilis. No worker was found to be positive. An association between Dientamoeba and coinfection with other intestinal parasites was observed. Concerning the genetic diversity, 14 and only two isolates were genotype 1 and genotype 2, respectively. Our findings outline interesting aspects: (1) asymptomatic children as carriers of Dientamoeba in communities in which environmental conditions ensure parasite transmission and, (2) association between Dientamoeba infection in young children and coinfection with other enteric parasites, reinforcing its transmission via the fecal-oral route.

Genetic analysis of Giardia duodenalis isolates from children of low-income families living in an economically successful region in Southeastern Brazil.

Giardia duodenalis is one of the most important and widespread gastrointestinal parasites in the world. Despite its relevance as a causative agent of diarrhea, asymptomatic giardiasis occurs frequently, especially in low resources settings in which children are exposed to many risk factors. Based on microscopic examination and the polymerase chain reaction (PCR) amplification and sequencing of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes, we assessed G. duodenalis occurrence and genetic diversity in isolates of children attending a daycare center and living in low income families, in an economically successful region. Considering both, microscopic examination and PCR/sequencing methods, the overall prevalence of Giardia infection was 51.4%, with the highest frequency in children aged 1-4 years old (p<0.05). Genotyping of 50 isolates revealed that the assemblage A was found in 60% of the samples (30/50), followed by the assemblage B in 38% (19/50) and 2% of mixed-assemblage infections (1/50). At the sub-assemblage level, isolates genotyped as A were AII and among isolates B, BIII and BIV were identified. Both assemblages A and B were detected in children of all age groups, however assemblage A was more prevalent. The detection of anthroponotic assemblages and sub-assemblages (AII, BIII and BIV) reinforces human-to-human transmission, mainly in children of all age groups when they have not yet received toilet training, making them more vulnerable to infection.

Diversity of Blastocystis subtypes in wild mammals from a zoo and two conservation units in southeastern Brazil.

The enteric protist Blastocystis is one of the most commonly parasite reported in humans and a variety of animal hosts worldwide. Regarding genetic diversity, at least 17 subtypes (STs) have been identified in mammals and birds, with eight of them (ST1-8) infecting both humans and animals. Recently, isolates from wild mammalian species have been genetically characterized, however data is still scarce, mainly in Latin America. Here, we aimed to verify the occurrence and genetic diversity of Blastocystis in captive wild mammals kept in one zoo and in two units of protection and conservation in southeastern Brazil. A total of 78 fecal samples (14 pooled and 64 individual samples) were recovered from 102 wild mammals of 35 species included in the following orders: Primates, Carnivora, Artiodactyla, Pilosa, Rodentia and Marsupialia. Zoo and units staff were invited to participated but only 16 fecal samples could be screened. Based on the sequence analyses of SSUrDNA gene, out of 29 PCR products from animal samples, 51.7% (15/29) were successfully sequenced and five Blastocystis subtypes were identified as follows: ST1 (2/15; 13.3%), ST2 (2/15; 13.3%), ST3 (4/15; 26.6%), ST5 (2/15; 13.3%) and ST8 (5/14; 33.3%). Only four isolates from humans were sequenced and identified as ST1 (2 isolates), ST2 and ST3. It was observed that Blastocystis infecting non-human primates belong to ST1 and ST2 and mainly to ST3 and ST8, artiodactyls ST5, carnivores ST1 and ST5 and rodents ST1. In addition, this present study reports some interesting findings: (1) 63% (12/19) of Blastocystis isolates from animals and employees belonged to the potentially zoonotic subtypes ST1-ST3; (2) most of these isolates displayed high identity with publicly available DNA sequences from non-human primates and humans, including primate handlers; (3) Blastocystis ST5 was found infecting the northern tiger cat, a native South American felid and one of the species facing a high risk of extinction in Brazil.

Blastocystis genetic diversity among children of low-income daycare center in Southeastern Brazil.

Blastocystis, an unicellular anaerobic eukaryote, is known to be a very common intestinal parasite found in humans and animals fecal samples worldwide. Currently, there is an increasing interest to yield insights into its prevalence and diversity in human populations living in poor and deprived areas. In this study, we describe the prevalence and genetic variability of Blastocystis isolates obtained from daycare center attendees aged 0 to 6years and staff, as well as some children family members and their dogs in a low-income community in São Paulo State, Brazil. A total of 181 stool samples (123 from daycare children, 14 from workers, 44 from household members and 20 from dogs) were submitted to DNA extraction, tested by polymerase chain reaction (PCR) targeting the SSUrDNA gene and the amplicons retrieved were sequenced. The prevalence of Blastocystis was 40.7% (50/123) in children, 28.6% (4/14) in workers and 50% (22/44) in household members. No dog was found positive. Of the 76 PCR products generated, 57 were successfully sequenced. Four subtypes were identified and the most common were ST1 (54.4%) and ST3 (33.3%), followed by ST2 (7.0%) and ST7 (5.3%). The intra-subtype analysis revealed a total of 10 different alleles previously reported. No statistically significant correlation was observed between subtypes and sociodemographic variables analyzed. Here, the following findings must be highlighted: (1) predominance of subtypes 1 and 3, a pattern that has been observed in many populations worldwide; (2) absence of ST4, a common subtype in Europe but rarely detected in South America's human populations and, (3) human infection with ST7, a subtype primarily found in birds but occasionally seen in human infections, raising the possibility of zoonotic transmission.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins.

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Multilocus sequence typing of Dientamoeba fragilis identified a major clone with widespread geographical distribution.

The flagellated protozoan Dientamoeba fragilis is often detected in humans with gastrointestinal symptoms, but it is also commonly found in healthy subjects. As for other intestinal protozoa, the hypothesis that genetically dissimilar parasite isolates differ in their ability to cause symptoms has also been raised for D. fragilis. To date, only two D. fragilis genotypes (1 and 2) have been described, of which genotype 1 largely predominates worldwide. However, very few markers are available for genotyping studies and therefore the extent of genetic variation among isolates remains largely unknown. Here, we performed metagenomics experiments on two D. fragilis-positive stool samples, and identified a number of candidate markers based on sequence similarity to the phylogenetically related species Trichomonas vaginalis. Markers corresponding to structural genes and to genes encoding for proteases were selected for this study, and PCR experiments confirmed their belonging to the D. fragilis genome; two previously described markers (small subunit ribosomal DNA and large subunit of RNA polymerase II) were also included. Using this panel of markers, 111 isolates of human origin were genotyped, all of which, except one, belonged to genotype 1. These isolates had been collected at different times from symptomatic and asymptomatic persons of different age groups in Italy, Denmark, Brazil and Australia. By sequencing approximately 160kb from 500 PCR products, a very low level of polymorphism was observed across all the investigated loci, suggesting the existence of a major clone of D. fragilis with a widespread geographical distribution.

Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil.

BACKGROUND: Several species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tietê in the State of São Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services. METHODS: Multiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp. RESULTS: By molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites. CONCLUSIONS: This study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis.

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains.

In the present study, we have analyzed by sodium docecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting and Concanavalin A blotting (Con A blotting) proteins of membrane fractions and soluble fractions obtained from Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10), as compared to the reference strain Portland 1. Both Brazilian strains showed a complex and homogeneous electrophoretic pattern of proteins, but some differences could be observed. Several glycoproteins were detected, particularly the proteins of 81, 72, 59 kDa and the protein of 62 kDa in the membrane proteins and cytosol, respectively. Many antigenic components were revealed by anti-Giardia rabbit IgG antibodies in the immunoblotting analysis. Among these components, the membrane protein of 32 kDa and the cytosol protein of 30 kDa could be related to giardin, as previously demonstrated.

Detection of anti-Giardia lamblia serum antibody among children of day care centers.

OBJECTIVES: To detect anti-Giardia lamblia serum antibodies in healthy children attending public day care centers and to assess serological tests as tools for estimating the prevalence of G. lamblia in endemic areas. METHODS: Three separate stool specimens and filter paper blood samples were collected from 147 children ranging from 0 to 6 years old. Each stool sample was processed using spontaneous sedimentation and zinc sulfate flotation methods. Blood samples were tested by indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) for Giardia IgG. RESULTS AND CONCLUSIONS: Of 147 individuals tested, 93 (63.3%) showed Giardia cysts in their feces. Using IIF and ELISA, serum antibodies were detected in 93 (63.3%) and 100 (68%) samples, respectively. Sensitivity of IIF and ELISA was 82% and 72%, respectively. However, ELISA revealed to be less specific (39%) than IIF (70%). IIF also showed a higher concordance with microscopic examination than ELISA.

In vitro antigiardial activity of the cysteine protease inhibitor E-64.

The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors.

Effect of Bifidobacterium animalis on mice infected with Strongyloides venezuelensis.

The administration of viable Bifidobacterium animalis was tested to induce resistance against Strongyloides venezuelensis infection in mice. Effects on parasite burden, worm length, egg output, and intestinal mucosal histology were evaluated. The oral administration of B. animalis, strain 04450B, starting 14 days before the inoculation of nematode larvae significantly decreased the worm burden and egg output. In probiotic treated animals, the percent reduction of adult worms in the intestine was of 33% and the reduction of egg production was of 21%, compared with those of the control group. The duodenum villous height and villous/crypt ratio were significantly higher in probiotic-treated mice, indicating that this group could be experiencing less intestinal damage. The present findings revealed that the administration of B. animalis for the amelioration of host response to nematode infections is biologically plausible and could have some potential for impacting public health. Meanwhile, further study is needed to delineate the nature and identity of the factor(s) involved in these beneficial effects.

Molecular typing of Giardia duodenalis isolates from nonhuman primates housed IN a Brazilian zoo.

Giardia infections in captive nonhuman primates (NHP) housed at a Brazilian zoo were investigated in order to address their zoonotic potential. Fresh fecal samples were collected from the floors of 22 enclosures where 47 primates of 18 different species were housed. The diagnosis of intestinal parasites after concentration by sedimentation and flotation methods revealed the following parasites and their frequencies: Giardia (18%); Entamoeba spp. (18%); Endolimax nana (4.5%); Iodamoeba spp. (4.5%); Oxyurid (4.5%) and Strongylid (4.5%). Genomic DNA extracted from all samples was processed by PCR methods in order to amplify fragments of gdh and tpi genes of Giardia. Amplicons were obtained from samples of Ateles belzebuth, Alouatta caraya, Alouatta fusca and Alouatta seniculus. Clear sequences were only obtained for the isolates from Ateles belzebuth (BA1), Alouatta fusca (BA2) and Alouatta caraya (BA3). According to the phenetic analyses of these sequences, all were classified as assemblage A. For the tpi gene, all three isolates were grouped into sub-assemblage AII (BA1, BA2 and BA3) whereas for the gdh gene, only BA3 was sub-assemblage AII, and the BA1 and BA2 were sub-assemblage AI. Considering the zoonotic potential of the assemblage A, and that the animals of the present study show no clinical signs of infection, the data obtained here stresses that regular coproparasitological surveys are necessary to implement preventive measures and safeguard the health of the captive animals, of their caretakers and of people visiting the zoological gardens.

Characterisation of protease activity in extracellular products secreted by Giardia duodenalis trophozoites treated with propolis.

Results from our laboratory revealed propolis activity on Giardia trophozoites proliferation. Since therapeutic agents can inhibit the activity of proteases related to relevant biologic and physiologic processes of parasites, this study was undertaken to characterise the proteolytic activity of excretory/secretory products (ESP) of trophozoites treated with propolis. ESP was obtained from culture supernatants of trophozoites exposed to 250 and 500 µg mL(-1) of propolis. ESP were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the protein profiles and the protease activity was assayed in gelatin-containing gels. Synthetic inhibitors were used to characterise the protease classes. Treated and non-treated ESP showed a similar protein and hydrolysis pattern. A simple pattern of protein composed by five evident bands of approximately 167, 132, 79, 61 and 51 kDa was found, and the zymograms comprised hydrolysis zones distributed from >170 to 23 kDa. No inhibition was seen on protease activity of propolis-treated trophozoites, whose hydrolysis pattern was similar to control. One may conclude that both ESP degraded gelatin and the activity was predominantly due to cysteine proteases. Although propolis had no effect on the proteolytic activity, further studies could identify the active constituents responsible for propolis antigiardial activity and their mechanisms of action.

Proteolytic activity of excretory/secretory products of Cochliomyia hominivorax larvae (Diptera: Calliphoridae) / Atividade proteolítica de produtos de excreção/secreção de Cochliomyia hominivorax (Diptera: Calliphoridae)

The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30µg, 40µg and 50µg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.(AU)

Os perfis protéicos e a atividade proteolítica dos produtos de excreção/secreção (PE/S) das larvas de primeiro (L1), segundo (L2) e terceiro (L3) estágios de Cochliomyia hominivorax foram estudados em laboratório. Os perfis protéicos foram obtidos por eletroforese em géis de poliacrilamida (SDS-PAGE). Os PE/S de cada fase larval (L1, L2 e L3), tratados com inibidores de proteases, contendo 30µg, 40µg e 50µg de proteína, foram aplicados em géis de poliacrilamida a 10%. A atividade proteolítica dos PE/S na sua forma nativa, foi analisada em géis co-polimerizados com gelatina e por testes colorimétricos usando a azocaseína como substrato, com a caracterização das proteases feita por meio de inibidores sintéticos. Diferentes perfis protéicos foram observados para os instares larvais, com L1 apresentando o perfil mais complexo. Apesar disso, foram observadas várias bandas protéicas comuns a todos os estágios larvais. Os PE/S de todos os instares mostraram atividade proteolítica sobre a gelatina, evidenciada por zonas de proteólise, com predominância de massas moleculares aparentes mais altas em L1, enquanto que para L2 e L3 as zonas de proteólise puderam ser observadas também em regiões de menores massas. Os testes com inibidores de proteases usando a gelatina como substrato mostraram que os PE/S de L1, L2 e L3 eram compostos principalmente de serina-proteases. Adicionalmente, inibição foi observada nos PE/S de L2 tratada previamente com EDTA, um inibidor de metalo-proteases. Os ensaios com a zocaseína revelaram um aumento gradual da atividade proteolítica sobre este substrato com o progresso do desenvolvimento larval, com a mais forte inibição sendo observada após o tratamento com 3,4 dicloroisocumarina (DCI) para os PE/S de L1, L2 e L3. Estes resultados sugerem que as larvas de C. hominivorax produzem diferentes proteases, fato que pode estar relacionado a processos vitais para a sobrevivência do parasita, tais como a penetração nos tecidos dos hospedeiros e nutrição durante os estágios larvais.(AU)

In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64 / Atividade in vitro do inibidor de cisteína-proteases E-64 sobre trofozoítos de Giardia

The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors.

As cisteína-proteases estão entre os alvos mais promissores para o desenvolvimento de novos agentes terapêuticos, visto que participam de eventos fundamentais do ciclo de vida de muitos microorganismos, inclusive Giardia. Como a atividade das proteases pode ser controlada por inibidores específicos, essas substâncias têm sido avaliadas quanto ao potencial antiparasitário. Diante disso, o presente estudo teve por objetivo avaliar o efeito in vitro do inibidor de cisteína-proteases E-64 sobre o crescimento, a aderência e a viabilidade de trofozoítos de cepa de Giardia isolada em Botucatu. Nos ensaios de crescimento e aderência, o número de trofozoítos foi estimado microscopicamente em hemocitômetro, enquanto que a viabilidade celular foi avaliada pelo método do MTT. No presente estudo, embora o metronidazol tenha se apresentado bastante efetivo, o E-64 mostrou ser capaz de inibir o crescimento, a aderência e a viabilidade em taxas superiores a 50%, especialmente nos cultivos expostos à concentração de 100 µM. A despeito de preliminares, esses resultados demonstram que o inibidor E-64 pode interferir em processos primordiais para a sobrevivência do parasita, além do que, abrem novas perspectivas para investigações futuras a fim de se avaliar o real potencial giardicida dos inibidores de proteases.

MOLECULAR TYPING OF Giardia duodenalis ISOLATES FROM NONHUMAN PRIMATES HOUSED IN A BRAZILIAN ZOO / Genotipagem de isolados de Giardia duodenalis de primatas não humanos mantidos em zoológico do Brasil

Giardia infections in captive nonhuman primates (NHP) housed at a Brazilian zoo were investigated in order to address their zoonotic potential. Fresh fecal samples were collected from the floors of 22 enclosures where 47 primates of 18 different species were housed. The diagnosis of intestinal parasites after concentration by sedimentation and flotation methods revealed the following parasites and their frequencies: Giardia (18%); Entamoeba spp. (18%); Endolimax nana (4.5%); Iodamoeba spp. (4.5%); Oxyurid (4.5%) and Strongylid (4.5%). Genomic DNA extracted from all samples was processed by PCR methods in order to amplify fragments of gdh and tpi genes of Giardia. Amplicons were obtained from samples of Ateles belzebuth, Alouatta caraya, Alouatta fusca and Alouatta seniculus. Clear sequences were only obtained for the isolates from Ateles belzebuth (BA1), Alouatta fusca (BA2) and Alouatta caraya (BA3). According to the phenetic analyses of these sequences, all were classified as assemblage A. For the tpi gene, all three isolates were grouped into sub-assemblage AII (BA1, BA2 and BA3) whereas for the gdh gene, only BA3 was sub-assemblage AII, and the BA1 and BA2 were sub-assemblage AI. Considering the zoonotic potential of the assemblage A, and that the animals of the present study show no clinical signs of infection, the data obtained here stresses that regular coproparasitological surveys are necessary to implement preventive measures and safeguard the health of the captive animals, of their caretakers and of people visiting the zoological gardens.

A pesquisa de infecções por Giardia e a caracterização genotípica deste protozoário foi realizada em primatas não humanos (PNH) mantidos em Zoológico a fim de avaliar o seu potencial zoonótico. As amostras dos animais consistiram de fezes colhidas do piso de 22 baias onde eram mantidos 47 primatas de 18 diferentes espécies. Exames coproparasitológicos foram realizados pelos métodos de concentração por sedimentação e centrífugo-flutuação e revelaram a presença dos seguintes parasitas e suas respectivas frequências: Giardia (18%); Entamoeba spp. (18%); Endolimax nana (4.5%); Iodamoeba spp. (4.5%); oxiurídeos (4.5%) e estrongilídeos (4.5%). O DNA extraído de todas as amostras fecais foi submetido à técnica de PCR para a amplificação dos genes gdh e tpi de Giardia, porém, só foram obtidos amplicons das quatro amostras positivas provenientes de Ateles belzebuth, Alouatta caraya, Alouatta fusca and Alouatta seniculus. O seqüenciamento dos fragmentos amplificados foi possível apenas para as amostras oriundas de Ateles belzebuth (BA1), Alouatta fusca (BA2) e Alouatta caraya (BA3), cuja análise fenética de ambos os genes revelou pertencerem ao genótipo A. As análises das sequências de tpi revelaram que todas as amostras pertencem ao subgenótipo AII. No que se refere ao gene gdh as análises revelaram uma amostra pertencente ao subgenótipo AII (BA3) e duas ao subgenótipo A1 (BA1 e BA2). Considerando o potencial zoonótico do genótipo A e o fato de que os animais não apresentavam sintomas de infecção, os dados do presente trabalho salientam a importância de se realizar, periodicamente, exames coproparasitológicos dos animais de zoológico, para implementação de medidas preventivas para resguardar a saúde dos animais em cativeiro, a de seus tratadores e dos visitantes de parques zoológicos.

EFFECT OF Bifidobacterium animalis ON MICE INFECTED WITH Strongyloides venezuelensis / Efeito da administração de Bifidobacterium animalis sobre a infecção por Stongyloides venezuelensis em camundongos

The administration of viable Bifidobacterium animalis was tested to induce resistance against Strongyloides venezuelensis infection in mice. Effects on parasite burden, worm length, egg output, and intestinal mucosal histology were evaluated. The oral administration of B. animalis, strain 04450B, starting 14 days before the inoculation of nematode larvae significantly decreased the worm burden and egg output. In probiotic treated animals, the percent reduction of adult worms in the intestine was of 33% and the reduction of egg production was of 21%, compared with those of the control group. The duodenum villous height and villous/crypt ratio were significantly higher in probiotic-treated mice, indicating that this group could be experiencing less intestinal damage. The present findings revealed that the administration of B. animalis for the amelioration of host response to nematode infections is biologically plausible and could have some potential for impacting public health. Meanwhile, further study is needed to delineate the nature and identity of the factor(s) involved in these beneficial effects.

Os efeitos da administração de Bifidobacterium animalis viáveis sobre a infecção por Strongyloides venezuelensis foram avaliados em camundongos experimentalmente infectados. Os parâmetros analisados incluíram a carga parasitária, o comprimento dos vermes, a quantidade de ovos eliminados e a histologia da mucosa intestinal. A administração oral da cepa 04450B de B. animalis, iniciada 14 dias antes da inoculação de larvas do nematódeo, foi acompanhada de uma redução significativa do número de vermes que se estabeleceu no intestino e do número de ovos eliminados nas fezes. Nos animais tratados com o probiótico, o percentual de redução de vermes adultos no intestino foi de 33% e da produção de ovos foi de 21%, em comparação com os do grupo controle. O comprimento das vilosidades do duodeno e a relação vilus/cripta foram significativamente maiores nos animais tratados, indicando que nestes animais as lesões intestinais foram mais leves. Os resultados do presente trabalho revelaram que a administração de B. animalis com o propósito de modular a resposta do hospedeiro contra infecções por nematódeos é uma possibilidade biologicamente plausível com impacto potencial em saúde pública. No entanto, são ainda necessários mais estudos para esclarecer os mecanismos de ação destes microrganismos e identificar os fatores envolvidos na produção dos efeitos benéficos.

Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis.

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.