Bruno César Miranda Oliveira

A microbiota intestinal é composta por trilhões de microrganismos que exercem funções essenciais no metabolismo energético, na síntese de vitaminas, na modulação do sistema imunológico e na proteção contra patógenos. Em humanos, a interação entre exercício físico e microbiota intestinal tem sido alvo de crescente interesse, uma vez que atletas apresentam perfis microbianos distintos quando comparados a indivíduos sedentários. Diversos estudos demonstraram maior diversidade bacteriana e enriquecimento de vias metabólicas relacionadas à produção de ácidos graxos de cadeia curta em praticantes de atividade física regular, sugerindo benefícios na saúde intestinal e no desempenho físico. Entretanto, a literatura apresenta resultados heterogêneos devido às limitações como a variabilidade dietética, estilo de vida e diferenças genéticas entre os indivíduos avaliados. Modelos animais surgem como ferramentas importantes para reduzir essas variáveis e permitir maior controle experimental. O pombo correio (Columba livia), por exemplo, possui notável resistência física e pode ser submetido a protocolos de voo padronizados, possibilitando a investigação dos efeitos do esforço físico prolongado sobre a microbiota intestinal. Assim, os achados nesse modelo oferecem paralelos relevantes para a medicina humana, auxiliando na compreensão da relação entre exercício, microbiota e saúde.

Flying pigeons (Columbia livia) are extensively studied for their physical endurance and superior sense of orientation. The extreme physical endurance of which these birds are capable creates a unique opportunity to investigate the possible impact of long-distance flying on the taxonomy and metabolic function of the gut microbiota. This project was enabled by access to two groups of pigeons raised by the same breeder in the same conditions, except that one group was trained in long-distance flying and participated in multiple races covering a total distance of over 2600 km over a 9-week period. In contrast, the second group did not fly. The fecal microbiota was analyzed using 16S amplicon sequencing, and the taxonomy and metabolic function were inferred from this sequence data. Based on phylogenetic distance and metabolic function, flying and non-flying pigeons were found to harbor distinct bacterial microbiota. The microbiota taxonomy varied extensively between the birds, whereas the inferred metabolic potential was relatively stable. Age was not a significant determinant of the fecal microbiota profile. In flying birds, the metabolic pathways annotated with biosynthesis were enriched, representing 60% of the 20 metabolic pathways that were most closely associated with flying.

Cockatiels (Nymphicus hollandicus) are among the most commonly sold psittacines pets. The aim of this study was to evaluate the occurrence of Cryptosporidium spp. in domestic N. hollandicus and identify risk factors for this infection. We collected fecal samples from 100 domestic cockatiels in the city of Araçatuba, São Paulo, Brazil. Feces from birds of both genders and older than two months were collected. Owners were asked to complete a questionnaire to identify how they handle and care for their birds. Based on nested PCR targeting the 18S rRNA gene, the prevalence of Cryptosporidium spp. in the cockatiels sampled was 9.00%, 6.00% based on Malachite green staining, 5.00% based on modified Kinyoun straining, and 7.00% when the Malachite green was combined with Kinyoun. Applying multivariate logistic regression to test the association between Cryptosporidium proventriculi positivity and potential predictors showed that gastrointestinal alterations was a significant predictor (p < 0.01). Amplicons from five samples were sequenced successfully and showed 100% similarity with C. proventriculi. In summary, this study demonstrates the occurrence of C. proventriculi in captive cockatiels.

Based on our initial observations showing that mice consuming a probiotic product develop more severe cryptosporidiosis, we investigated the impact of other dietary interventions on the intracellular proliferation of Cryptosporidium parvum and C. tyzzeri in the mouse. Mice were orally infected with oocysts and parasite multiplication measured by quantifying fecal oocyst output. High-throughput sequencing of 16S ribosomal RNA amplicons was used to correlate oocyst output with diet and with the composition of the intestinal microbiota. On average, mice fed a diet without fiber (cellulose, pectin and inulin) developed more severe infections. As expected, a diet without fibers also significantly altered the fecal microbiota. Consistent with these observations, mice fed a prebiotic product sold for human consumption excreted significantly fewer oocysts. The fecal microbiota of mice consuming no plant polysaccharides was characterized by a lower relative abundance of Bacteroidetes bacteria. Since bacterial metabolites play an important role in the physiology of intestinal enterocytes, we hypothesize based on these observations that the impact of diet on parasite proliferation is mediated primarily by the metabolic activity of the anaerobic microbiota, specifically by the effect of certain metabolites on the host. This model is consistent with the metabolic dependence of intracellular stages of the parasite on the host cell. These observations underscore the potential of dietary interventions to alleviate the impact of cryptosporidiosis, particularly in infants at risk of recurrent enteric infections. The infection with Cryptosporidium parasite, a condition known as cryptosporidiosis, is a common cause of infant diarrhea in developing countries. We have previously shown that mice infected with C. parvum, one of the main cause of human cryptosporidiosis, develop a more severe infection if given probiotics. To investigate the mechanism of this effect, we fed mice prebiotics and diet lacking plant fiber. We found that fermentable fiber, whether administered as a prebiotic supplement or as part of the diet, has a protective effect against cryptosporidiosis in mice. We also observed a significant association between the severity of infection and the composition of the gut microbiota. A significant inverse correlation was found between severity of cryptosporidiosis and the ratio between the abundance of bacteria belonging to the phylum Bacteroidetes and the abundance of Firmicutes bacteria. This ratio is frequently viewed as a marker of a healthy microbiota. These results raise the possibility that dietary interventions could be used to alleviate the impact of cryptosporidiosis.

Technologies capable of noninvasively sampling different locations in the gut upstream of the colon enable new insights into the role of organ‐specific microbiota in human health. Herein, an ingestible, biocompatible, battery‐less, 3D‐printed microengineered pill with an integrated osmotic sampler and microfluidic channels for in vivo sampling of the gut lumen and its microbiome upstream of the colon is discussed. The pill's sampling performance is characterized using realistic in vitro models and validated in vivo in pigs and primates. Herein, the results show that the bacterial populations recovered from the pill's microfluidic channels closely resemble the bacterial population demographics of the microenvironment to which the pill is exposed. Herein, it is believed that such lab‐on‐a‐pill devices revolutionize the understanding of the spatial diversity of the gut microbiome and its response to medical conditions and treatments.

The study was conducted on 25 properties of the settlements São José I and Salvador, located in the municipalities of Brejo Alegre and Birigui, in the State of São Paulo, Brazil. A record of variables was elaborated and included data such as gender, breed and age of the animals. A total of 231 stool samples were collected from bovines aged one to six months, 128 being females and 103 males, 131 crossbred and 100 Holstein. Among the 231 samples, 17 (7.36%) were positive for Cryptosporidium spp. both by malachite green negative staining and by nested-PCR. Of the 17 positive samples, 14 were sequenced in agarose gel. These sequences were detected between 99% and 100% of genetic similarity for the following species. One sequence was similar to C. parvum (AB513880.1), one to C. bovis (MF074602.1), two to C. ryanae (KT922233.1), one to C. felis (KM977642.1) and nine were similar for C. andersoni reference MF350628. C. andersoni was found in animals aged 2–6 months, an age group which is different from those described by several authors. The presence of C. parvum indicates that the calves in the studied region should be considered a potential source for zoonotic transmission. For the first time to our knowledge, C. felis was identified in cattle in America.

Objectives Laboratory diagnosis of Schistosoma mansoni eggs is routinely performed by conventional quantitative techniques through the parasitological examination of human feces. However, the diagnostic sensitivity of this type of exam varies from low to moderate. We aimed to develop a new parasitological technique called TF‐Test Quantified (TFT‐Quant), for the quantitative detection of S. mansoni eggs in human feces. Methods Four study stages were performed using 43 mice infected by S. mansoni cercariae. These experiments allowed the definition of an operational protocol for TFT‐Quant and a comparison of this new technique to the conventional Helm‐Teste technique. Results The results show a good diagnostic efficacy of TFT‐Quant, which reached 100% in sensitivity and specificity, indicating an “Almost Perfect” Kappa (k) agreement. Conclusions This new technique provided a quantitative gain in the detection of S. mansoni eggs, largely free of fecal debris. The next stage of this study is the field validation of the TFT‐Quant technique with the use of human fecal samples from an endemic region for mansonic schistosomiasis in Brazil (Microregion Jequitinhonha, State of Minas Gerais). In parallel to this validation, computational algorithms will be developed to allow the automated quantitative diagnosis of S. mansoni eggs.

Canine monocytic ehrlichiosis (CME) is a disease caused by the obligate intracellular bacterium Ehrlichia canis. Tropical lineages of Rhipicephalus sanguineus ticks play an essential role in the transmission of this pathogen. The aim of the present study was to evaluate the prevalence of E. canis DNA in tissue from R. sanguineus ticks in areas endemic for CME in Brazil and quantify levels of E. canis DNA in dissected tissues from these samples. A total of 720 ticks were collected from 72 dogs (36 dogs from the city Araçatuba in São Paulo state and 36 from Campo Grande in the state of Mato Grosso do Sul). Ticks were dissected to collect the guts, ovaries and salivary gland. A quantitative polymerase chain reaction (qPCR) targeting the disulphide bond formation (dsb) protein gene was performed to quantify the level of E. canis infection. The E. canis dsb-qPCR assay was positive for 31.9, 10, and 15.2% of the gut, ovary, and salivary glands, respectively. The average gut, ovary, and salivary gland bacterial load estimated by qPCR was 1.21 × 10³, 2.60 × 10³, and 4.92 × 10³ gene copies/µl, respectively. This is the first report of E. canis DNA in ovaries of R. sanguineus ticks parasitizing dogs in these CME-endemic areas. These observations raise the possibility of E. canis trans-ovarial transmission.

Cryptosporidiosis is one of the leading causes of diarrhea in humans and several vertebrates species. Because surveys of Cryptosporidium genotypes from animals and humans living in the same region are rare, our understanding of the importance of zoonotic transmission in the epidemiology of cryptosporidiosis remains superficial. PCR was used to amplify a portion of the Cryptosporidium 18S small subunit ribosomal RNA gene from fecal DNA from humans and livestock living in Ekiti and Oyo state, Nigeria. PCR-positive samples were further analyzed using PCR targeting the heat-shock protein HSP-70, the actin and the sporozoite glycoprotein gene gp60. A questionnaire was used to collect demographic information. Sixteen of 187 samples collected were Cryptosporidium 18S PCR positive. Of these, 5 samples originating from HIV-positive patients, 5 from otherwise healthy children, 2 from chickens, 3 from goats and 1 from a dog were positive for at least one marker. Sequencing of the 18S rRNA amplicons reveale...