Next-generation sequencing reveals wide genetic diversity of Blastocystis subtypes in chickens including potentially zoonotic subtypes.

Blastocystis sp. is an intestinal protist parasite commonly found in the feces of humans and animals worldwide. Blastocystis exhibits extensive genetic diversity and has been identified in humans and a variety of animals including other mammals and birds. Blastocystis subtypes do not exhibit strict host specificity which raises the possibility of zoonotic transmission through either direct contact or fecal contamination of food or water. However, reports detailing the subtypes and prevalence of Blastocystis in avian species are limited. Therefore, this study investigated the presence of Blastocystis in chickens by molecular characterization of the small subunit rRNA (SSU rRNA) gene. Fecal samples from 130 chickens were collected from local markets in Uberlândia and Belo Horizonte in the state of Minas Gerais, Brazil. To detect and identify subtypes of Blastocystis, a next-generation amplicon sequencing protocol was used. Forty-four of the 130 (33.8%) chickens examined were positive for Blastocystis. Blastocystis subtypes ST6 (23/130; 17.7%), ST7 (43/130; 33.1%), ST10 (1/130; 0.8%), ST14 (5/130; 3.8%), ST25 (1/130; 0.8%), and a novel subtype (ST29) (2/130; 1.5%) were observed. A nanopore sequencing strategy was used to obtain the near full-length SSU rRNA gene nucleotide sequence and validate novel subtype ST29. Mixed infections containing multiple subtypes were common and identified in 63.6% of Blastocystis-positive chickens. All positive samples contained one or both potentially zoonotic subtypes ST6 and ST7. The prevalence of Blastocystis in chickens was high, and molecular characterization mostly identified subtypes previously found in humans. Thus, chickens may be a source of human infection and environmental contamination.

Multilocus genotyping of Giardia duodenalis in Brazilian children.

BACKGROUND: Giardia duodenalis is a parasite of several mammalian species, including humans, distributed worldwide. This research aimed to identify the molecular assemblages/sub-assemblages of G. duodenalis and to determine the intra-assemblage genetic variation of the different genes of assemblages A and B in pre-school children in the cities of Araguari and Uberlândia, Minas Gerais, Brazil. METHODS: The molecular characterization followed ß-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) protocols. RESULTS: Of 226 stool samples, G. duodenalis cysts were found in 45 (19.9%). The tpi gene was amplified in 34 samples: 16 assemblage A, 14 B and four mixed samples A/B. The gdh gene was amplified in 32 samples, including 14 A, 16 B and two A/B. For the bg gene, 19 samples were sequenced: nine assemblage A, five B, three E, and two mixed, A/E and B/E. Animal-specific assemblage E were identified by bg, but were not confirmed for other genes. Twelve samples were characterized by full agreement of the three genes. Two new multilocus genotyping (MLGs) for assemblage A and two new MLGs for assemblage B were also described. CONCLUSIONS: These findings substantiate the importance of using more than one gene protocol since the sensitivity and genetic variability changes with the locus used.Access numbers: The GenBank access numbers for the nucleotide sequences reported in this article are: JQ794877-JQ794890, JX033113-JX033118.

Effects of metronidazole analogues on Giardia lamblia: experimental infection and cell organization.

The chemotherapeutic agents used for the treatment of giardiasis are often associated with adverse side effects and are refractory cases, due to the development of resistant parasites. Therefore the search for new drugs is required. We have previously reported the giardicidal effects of metronidazole (MTZ) and its analogues (MTZ-Ms, MTZ-Br, MTZ-N(3), and MTZ-I) on the trophozoites of Giardia lamblia. Now we evaluated the activity of some giardicidal MTZ analogues in experimental infections in gerbils and its effects on the morphology and ultrastructural organization of Giardia. The giardicidal activity in experimental infections showed ED(50) values significantly lower for MTZ-I and MTZ-Br when compared to MTZ. Transmission electron microscopy was employed to approach the mechanism(s) of action of MTZ analogues upon the protozoan. MTZ analogues were more active than MTZ in changing significantly the morphology and ultrastructure of the parasite. The analogues affected parasite cell vesicle trafficking, autophagy, and triggered differentiation into cysts. These results coupled with the excellent giardicidal activity and lower toxicity demonstrate that these nitroimidazole derivates may be important therapeutic alternatives for combating giardiasis. In addition, our results suggest a therapeutic advantage in obtaining synthetic metronidazole analogues for screening of activities against other infectious agents.

Epidemiological, parasitological and molecular aspects of Giardia duodenalis infection in children attending public daycare centers in southeastern Brazil.

The purpose of this study was to determine the prevalence, associated risk factors and genotype of Giardia duodenalis infection in children attending public daycare centers in the city of Araguari, state of Minas Gerais, Brazil. Fecal samples were collected from 245 children aged 0-5 years, and questionnaires were asked about sociodemographic and hygiene-related characteristics. At the daycare centers where children tested positive, fecal samples were collected from the staff handling food, and from family members and domestic animals. Positive samples were analyzed at the dehydrogenase glutamate (gdh) locus to determine the genotype. The prevalence of G. duodenalis was 51.8%, and drinking unfiltered and unboiled water (OR 2.12, CI 1.26-3.69, p<0.001) and washing hands only with water (OR 2.14, CI 1.19-4.04, p<0.001) were related risk factors. No association was found between test-positive children and their family members, domestic animals and food handlers. An analysis of the sequences of 30 samples revealed that they all belonged to genotype B.

Giardia duodenalis: pathological alterations in gerbils, Meriones unguiculatus, infected with different dosages of trophozoites.

To examine the infection kinetics and development of alterations in the small intestine of gerbils (Meriones unguiculatus), 72 gerbils were divided into six groups (A to F), with A serving as control and the others inoculated with increasing doses of trophozoites from Giardia duodenalis human isolate. The infection kinetics and the development of histopathological alterations were monitored by optical scanning electron microscopy (SEM). A 12-day prepatent period was observed, with intermittent elimination up to day 35 after inoculation. Statistically significant differences were found between the mean number of trophozoites recovered, per group, on the days of sacrifice, and a positive correlation between the inoculum dosage and the number of trophozoites recovered. Morphometrically, the villus:crypt ratio showed a drop in all the groups when compared with the control group. SEM revealed an increase in mucus production in the inoculated animals and the presence of trophozoite clusters at the top and base of the villi. The dosage of trophozoite inoculum does not interfere in the ability for infection to occur or in the development of histopathological alterations generated by intestinal colonization.

Hematological and coagulation profiles in dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866).

Hematological and coagulation profiles were studied in crossbred dogs experimentally infected with Angiostrongylus vasorum. Two groups of five dogs were experimentally inoculated with 50 and 100 third stage infective larvae (L(3)) of A. vasorum per kilogram of body weight. A third group of five uninfected animals was used as control. One sample of 10 ml of blood was collected from each animal on the 10, 20, 30, and 45 days after inoculation (dai) and at 30-day intervals thereafter for the remainder of the 210-day experimental period. The blood sample was used for the complete hemogram and platelet count, as well as measurements of prothrombin time, partial thromboplastin time and factors V and VIII. Anemia was observed in infected dogs, 6 weeks after the infection. The eosinophils presented peaks in four periods after infection. Thrombocytopenia became accentuated on the 72 dai. Decreased prothrombin time activity and increased partial thromboplastin time were observed at the 6 and 9 weeks after infection and decreased of factors VIII and V activities occurred from 4 to 6 weeks after infection. It may be conclude that infection by A. vasorum in dogs may cause a discrete anemia during the acute phase which is probably regenerative. In addition, important hemostatic alterations due to the infection suggest a chronic intravascular consumption coagulopathy.