Babesiosis caused by Babesia vogeli in dogs from Uberlândia State of Minas Gerais, Brazil.

Babesia is tick-transmitted protozoan parasites that infect mammalian hosts and have a major impact on farm and pet health-associated costs worldwide. This study aimed to test the prevalence of Babesia spp. infection in a small cohort of dogs at a veterinary hospital and to perform molecular characterization of the Babesia species causing the infection. For the PCR assay, 5 mL of blood was collected by venipuncture of the cephalic or radial veins in 300 dogs of different ages, sex, and breeds, which were presented to the veterinary hospital of the Federal University of Uberlândia between March 2015 and April 2016. In addition, a drop of blood was collected from the marginal blood vessels of the ear of dogs included in this study. Ninety-two (30.67%) were positive for Babesia spp., as determined by microscopic observation of the blood smear, revealing the presence of intra-erythrocyte merozoites. For molecular characterization by PCR, 17 samples were chosen from dogs who were tested positive for Babesia spp. by blood smears. Among them, B. vogeli was found to infect all 17 dogs, as determined by 99-100% sequence identity (closest GenBank match KT246307) using primers PIRO A/PIRO B. Our results indicate that the species observed in these dogs was B. vogeli.

Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil.

Species of Hepatozoon are parasites frequently recorded in snakes. The species identification of this genus was based mostly on the gametocyte morphology and morphometric calculations. For more reliable results, molecular characterization, an initial step for the correct identification of Hepatozoon species, has been used. This study aimed to determine the prevalence and identification of Hepatozoon species in captive snakes from Brazil. To that end, morphological, morphometric, and molecular data were obtained. A total of 157 snakes; 128 venomous (Crotalus durissus) and 29 non-venomous (Epicrates crassus and Boa constrictor) were screened for Hepatozoon blood parasites. Using light microscopy, 20 (12.78%) snakes were found positive for Hepatozoon spp., of which 6/29 (20.7%) were non-venomous and 14/128 (10.9%) were venomous; all with low parasitemia. Polymerase chain reaction (PCR), performed with the primers HepF300/Hep900, confirmed all 20 (100%) samples positive for hemogregarines. Species of Hepatozoon were identified from eight sequenced samples. Two previously described species, Hepatozoon cuestensis and Hepatozoon musa, were identified. The present study is the first to report H. musa within the snake hosts E. crassus and C. durrisus. In addition, a potentially new Hepatozoon species from B. constrictor was identified.

Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in Brazilian captive birds.

A total of 85 fecal samples from captive birds collected from October 2013 to September 2014 in Uberlândia and Belo Horizonte in the state of Minas Gerais (Brazil) were evaluated for the presence of Enterocytozoon bieneusi, Cryptosporidium, and Giardia by PCR. Of these, three birds were found positive for E. bieneusi (3.5%), two for Cryptosporidium (2.3%), and one for Giardia (1.2%). Two genotypes of E. bieneusi were detected by nucleotide sequence analysis of the ITS region, genotypes D and Peru 6 in a swan goose and in two rock pigeons, respectively. For Cryptosporidium and Giardia, nucleotide sequence analysis of the SSU rRNA identified Cryptosporidium baileyi and Duck genotype in a swan goose and a mandarin duck, respectively, and Giardia duodenalis assemblage A in a toco toucon. Our results demonstrate that human-pathogenic E. bieneusi genotypes D and Peru6 and G. duodenalis assemblage A are present in captive birds in Brazil, corroborating their potential role as a source of human infection and environmental contamination.

Label-free detection and enumeration of Giardia cysts in agitated suspensions using in situ microscopy.

Laboratory procedures performed in water treatment studies frequently require the characterization of (oo)cyst suspensions. Standard methods commonly used are laborious, expensive and time-consuming, besides requiring well-trained personnel to prepare samples with fluorescent staining and perform analysis under fluorescence microscopy. In this study, an easy cost-effective in situ microscope was assessed to acquire images of Giardia cysts directly from agitated suspensions without using any chemical labels or sample preparation steps. An image analysis algorithm analyzes the acquired images, and automatically enumerates and provides morphological information of cysts within 10 min. The proposed system was evaluated at different cyst concentrations, achieving a limit of detection of ~30 cysts/mL. The proposed system overcomes cost, time and labor demands by standard methods and has the potential to be an alternative technique for the characterization of Giardia cyst suspensions in resource-limited facilities, since it is independent of experts and free of consumables.

Blastocystis subtype distribution in domestic and captive wild bird species from Brazil using next generation amplicon sequencing.

Blastocystis is a food and water borne intestinal parasite commonly identified in humans and many other animals worldwide. Of the nine potentially zoonotic subtypes of Blastocystis, seven have been reported in bird species. However molecular studies of Blastocystis subtype diversity in birds are limited. In this study, fecal samples from 109 domestic and captive wild birds from Minas Gerais, Brazil were tested for the presence of Blastocystis subtypes using PCR and next generation amplicon sequencing of a fragment of the small subunit ribosomal RNA (SSU rRNA) gene. Birds from 11 orders and 38 species from both local markets and bird conservation facilities were sampled. Blastocystis was present in 14.7% of samples, and eight subtypes, six previously reported (ST5, ST6, ST7, ST10, ST14, ST24) and two novel subtypes (named ST27 and ST28), were identified. The most commonly identified subtypes were ST7 and ST6 identified in 10 (62.5%) and 6 (37.5%) of 16 Blastocystis positive samples. At least one of the three zoonotic subtypes identified (ST5, ST6, and ST7) was found in 81.3% of Blastocystis positive samples. Infection with multiple Blastocystis subtypes was common and identified in 62.5% of positive samples. This study is the first to use next generation amplicon sequencing to characterize Blastocystis subtype diversity in birds. The findings presented here confirm that birds may serve as reservoirs of zoonotic subtypes of Blastocystis and that the role of birds in transmission of Blastocystis to humans requires further study.

Molecular characterization of Cryptosporidium spp. in poultry from Brazil.

Cryptosporidiosis is an important zoonotic disease caused by Cryptosporidium. Infections in birds are mainly caused by C. meleagridis, C. baileyi, and C. galli. C. meleagridis is the third most common cause of cryptosporidiosis in humans and the only Cryptosporidium species known to infect both birds and mammals. One hundred and fifty-five fecal specimens from different poultry species (chicken, turkey, ostrich, helmeted guinea fowl, quail, pheasant, and emu) were collected at local markets in the state of Minas Gerais, Brazil. Twenty-three (14.8%) birds (20 chickens, 2 quails, and 1 turkey) were found Cryptosporidium-positive. This constitutes the first report of Cryptosporidium in turkeys from Brazil. Nucleotide sequence analysis identified C. meleagridis in chickens (15), a turkey (1), and a quail (1), C. baileyi in chickens (4) and a quail (1), and a mixed infection C. meleagridis/C. baileyi in a chicken (1). This is the first report of C. meleagridis in turkeys and quails from Brazil. Using the gp60 gene, three subtype families were identified, IIIa, IIIb and IIIg. Within subtype family IIIg, four subtypes were identified in chickens, two novel (IIIgA25G3R1 and IIIgA21G3R1) and two previously reported (IIIgA22G3R1 and IIIgA24G2R1). Within subtype family IIIb two subtypes were identified, IIIbA24G1R1 in a chicken and IIIbA23G1R1 in a quail. A novel subtype in the family IIIa was identified (IIIaA22G3R1) in a turkey. The finding of C. meleagridis subtypes previously identified in humans (IIIgA22G3R1, IIIbA24G1R1 and IIIbA23G1R1) indicates that they can be potentially zoonotic. Further subtyping studies that clarify genetic diversity of C. meleagridis are required to better understand host specificity, source of infection, and transmission dynamics of C. meleagridis.

Widespread presence of human-pathogenic Enterocytozoon bieneusi genotypes in chickens.

A total of 151 fecal specimens from chickens were randomly collected from local markets in Uberlândia and Belo Horizonte in the state of Minas Gerais, Brazil, to evaluate the presence of Enterocytozoon bieneusi by polymerase chain reaction (PCR). Enterocytozoon bieneusi was identified in 24 fecal samples (15.9%). This represents the first report of E. bieneusi in chickens in Brazil. All PCR-positive specimens were sequenced and 4 genotypes were identified, Peru 6, Peru 11, Type IV, and D. All four genotypes have previously been reported as human pathogens and are potentially zoonotic. Our results demonstrate that human-pathogenic E. bieneusi genotypes are present in chickens in Brazil, corroborating their potential role as a source of human infection and environmental contamination.

Prevalence and risk factors for intestinal protozoa infection in elderly residents at Long Term Residency Institutions in Southeastern Brazil.

This study determined the prevalence of intestinal protozoa in Long Term Residency Institutions for the Elderly (ILPI) in elders, nurses and food handlers, identifying the risk factors associated with the infections. Stool samples taken from the elderly (n = 293), nurses (63) and food handlers (19) were studied. Questionnaires were used with questions related to sociodemographic variables, health, behavior and health characteristics. Stool samples were examined using the techniques of Faust and Ziehl Neelsen, and the prevalence of G. duodenalis, Cryptosporidium spp., E. histolytica/dispar in the elderly was 4.0%, 1.0% and 0.3% respectively. Nurses and food handlers showed 4.8% and 5.2% positivity only for G. duodenalis, respectively. The origin of the individuals and contact with domestic animals has been associated with infection by G. duodenalis in the elderly, and contact with domestic animals was considered a risk factor for infection. The last stool examinations were related to Cryptosporidium spp.. None of the variables were associated with E. histolytica/dispar. The frequency of hand washing was significantly associated with G. duodenalis among nurses. The frequency of positive samples of G. duodenalis, Cryptosporidium spp., E. histolytica/dispar showed that ILPIs environments are conducive to this occurring due to contact between the elderly, nurses and food handlers, which are often poorly trained in hygiene procedures and food handling.