Detecção de genótipos de Giardia duodenalis zoonóticos e específicos de ruminantes domésticos em lagartos selvagens

Giardia duodenalis is a zoonotic parasite that infects the gut of a wide range of vertebrates, including numerous wildlife species. However, little is known about this protozoan parasite in reptiles. Fecal samples from 31 wild lizards were collected in Galicia (northwest Spain) and screened for the presence of Giardia by PCR amplification and sequencing of the ITS1-5.8S-ITS2 region in the ribosomal unit. This allowed detection of the parasite in 5 samples (16.1%), and enabled identification of G. duodenalis assemblage A2 in two samples of Iberian rock lizard (Iberolacerta monticola), G. duodenalis assemblage B in other two samples of I. monticola, and G. duodenalis assemblage E in one sample of Bocage’s wall lizard (Podarcis bocagei). The results obtained after PCR amplification and sequencing of the SSU-rDNA gene confirmed the presence of G. duodenalis assemblage A in two samples of I. monticola. This is the first report of G. duodenalis in free-living lizards, although further studies are needed to distinguish between actual infection and mechanical dissemination of cysts. The detection of zoonotic and livestock-specific assemblages of G. duodenalis demonstrates the wide environmental contamination by this parasite, possibly due to human activitiesGiardia duodenalis é um parasito zoonótico que infecta o intestino delgado de uma ampla gama de vertebrados, sendo detectado em numerosas espécies selvagens. No entanto, pouco se conhece sobre a presença deste parasito protozoário em répteis. Para estudar a presença de Giardia, foram obtidas amostras fecais provenientes de 31 lagartos e coletadas em diferentes localizações de Galicia (Noroeste da Espanha). Mediante a aplicação da técnica de PCR e posterior sequenciamento da região ITS1-5.8S-ITS2 da unidade ribossômica, detectou-se Giardia em 5 amostras (16,1%), identificando-se o genótipo A2 de G. duodenalis em 2 amostras de lagartos da montanha (Iberolacerta monticola), G. duodenalis genótipo B em outras 2 amostras de I. monticola e G. duodenalis genótipo E em outra amostra de lagarto de Bocage (Podarcis bocagei). Os resultados obtidos, após amplificação e sequenciamento de um fragmento do gene SSU-rDNA, confirmam a presença de G. duodenalis genótipo A em 2 amostras de I. monticola. Esta é a primeira vez que se descreve G. duodenalis em lagartos selvagens, embora sejam necessários outros estudos complementares para confirmar se estes animais sofrem uma infecção real ou se apenas atuam como disseminadores mecânicos da contaminação ambiental. Além disso, a detecção de genótipos zoonóticos e específicos de ruminantes domésticos demonstra a contaminação do ambiente selvagem por G. duodenalis, possivelmente devido à atividade humanaThis study was funded by the Spanish Ministry of Education and Science (grant no. CGL-2007-60656) and by the Department of Culture, Education and University of the Autonomous Government of Galicia (grant no. GPC2014-069)S

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

Molecular characterization of Babesia and Theileria species in ticks collected in the outskirt of Monte Romano, Lazio Region, Central Italy

In 2012-2013, an investigation was carried out in the Viterbo province, Lazio region, on ticks and tick-borne Apicomplexan protozoa of the Babesia and Theileria genera. This followed the reporting of high density of ticks by soldiers operating in a military shooting range, and the signaling by owners and local veterinary authorities of several cases of babesiosis among cattle. A total of 422 ticks were collected from 35 heads, whereas 96 ticks were collected by dragging. Ticks were identified as Rhipicephalus (Boophilus) annulatus Say (n=373), Rhipicephalus bursa Canestrini & Fanzago (n=63), Rhipicephalus sanguineus/turanicus (n=32), Hyalomma marginatum Koch (n=49) and Dermacentor marginatus Sulzer, 1776 (n=1). A randomly selected sample of ticks (235 from animals and 36 by dragging) was analyzed using molecular methods to detect species of Babesia and Theileria. In total, 11 ticks collected from animals (4.7%) and two ticks (5.5%) collected by dragging were positive. Sequencing of PCR products of the small subunit ribosomal RNA gene revealed Babesia caballi (n=2), Babesia bigemina (n=3), Theileria sergenti/buffeli/orientalis (n=7) and Theileria equi (n=1). None of the detected species has been associated with human infection

Detection of tick-borne pathogens in ticks collected in the suburban area of Monte Romano, Lazio Region, Central Italy

Background. A study on tick species characterization and tick borne pathogens detection was performed within a survey conducted during 2012 and 2013 in the Viterbo province (Lazio Region, Central Italy). Seven sites were selected for the study investigation, including two farms and a military zone. Methods. A total of 255 ticks, Rhipicephalus (Boophilus) annulatus (n =215), Rhipicephalus bursa (n = 28), and Hyalomma marginatum (n = 12) were screened individually by molecular methods for the tick borne bacterial agents: Borrelia burgdorferi sensu lato group, Bartonella spp., Coxiella burnetii, Ehrlichia spp., Francisella spp., and Rickettsia spp. Results and Conclusion. Overall, 182 ticks (71%) were infected with one pathogen but co-infections were also found. Tick borne pathogens identified were C. burnetii, B. burgdorferi s.l.., Bartonella spp., Rickettsia spp., Francisella spp., and Ehrlichia spp. In R. bursa and H. marginatum, the presence of B. burgdorferi s.l. was positively correlated with that of C. burnetii, Rickettsia spp., and Bartonella spp. and their coinfection probabilities were 29.8%, 22.7% and 11.7%, respectively. The Probability of coinfection for Francisella spp. and Rickettsia spp. and for Francisella spp. and Bartonella spp. was 14.9% and 17.9%, respectively. In R. (Boophilus) annulatus, the probability of coinfection between C. burnetii and B. burgdorferi s.l. was 11.3%, while those between C. burnetii and Bartonella and between B. burgdorferi s.l. and Bartonella were 0.8%. Further studies are needed in order to assess the risk associated with these tick-borne pathogens, somewhat unusual in Central Italy

Selected clones from the library of <i>Giardia duodenalis</i> Ad-28 strain and their genome localizations on both assemblages B (GS) and A (WB).

*<p>Orthologous gene identified on the basis of the similarity of the encoded peptide.</p

Sensitivity of 4E1-HP and 5C1-P21 PCR assays.

<p>Panel A: sensitivity test on serial dilutions of reference DNAs (WB for assemblage A and Ad28 for assemblage B). Left images show results obtained with assemblage A; right images show results obtained with assemblage B. The amount of DNA template, reported as the number of cysts equivalent, is shown at the top of the images. Panel B: results of assays tested on different proportions of the same reference DNAs. The following A to B ratios were tested: 6∶4 (1); 7.5∶2.5 (2,); 9∶1 (3); 1∶9 (4); 2.5∶7.5 (5); and 4∶6 (6). N indicates the negative control.</p

PCR assays at six independent genetic loci.

<p>Locus names are shown at the upper left corner of each panel. Capital letters in the upper lane indicate the reference DNA in each sample: A for assemblage A, B for assemblage B. Neg indicates the negative control (no DNA). Lower cases in the lower lane indicate the specific primer pair used for each reaction: a for the assemblage A specific pair; b for the assemblage B specific pair; a+b for the two primer pairs combined. Sizes of the assemblage-specific bands are indicated on the right. Size markers are indicated on the left.</p

Primer design for the 4E1-HP assay.

<p>Alignment of the sequence of the 4E1 clone (assemblage B) with the homologous sequence of HP 13988 (assemblage A). Dots correspond to identical nucleotides. The primers designed for assemblage A amplification are underlined with single lines, whereas the primers designed for assemblage B amplification are underlined with double lines.</p

List of primers for the assemblage-specific PCRs and sizes of the amplicons.

<p>List of primers for the assemblage-specific PCRs and sizes of the amplicons.</p