The Impact of Physical Effort on the Gut Microbiota of Long-Distance Fliers.

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.

Cryptosporidium proventriculi in Captive Cockatiels (Nymphicus hollandicus).

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.

Giardiasis in children and dogs, and the first report of assemblage E in dogs from northeastern Brazil.

Diagnosis is crucial for controlling giardiasis. We determined the prevalence and genetically characterize isolates of Giardia duodenalis of children and dogs from rural communities in northeastern Brazil. G. duodenalis cysts were concentrated by centrifugal flotation/sedimentation. Molecular characterization was carried out using the loci ssu-rRNA, bg, tpi, and gdh. By parasitological techniques, Giardia spp. infection was detected in 72/192 children (37.5%; 95% CI: 30.6%-44.7%) and 24/139 dogs (17.3%; 95% CI: 11.4%-24.6%). By molecular analysis, infection was detected in 60/141 children (42.5%; 95% CI: 34.3%-51.2%) and 26/92 dogs (28.3%; 95% CI: 19.4%-38.6%). The total prevalence of giardiasis was 54.9% in children (106/193; 95% CI: 47.1%-61.6%) and 32.9% in dogs (47/143; 95% CI: 25.2%-41.2%). Zoonotic assemblages A and B of G. duodenalis were detected in children, and assemblage E of G. duodenalis was detected in one child and two dogs. Parallel use of parasitological and molecular techniques proved to be a more effective strategy for detecting giardiasis in children and dogs from endemic areas.

Canine visceral leishmaniasis and Rhipicephalus sanguineus: evaluation and comparison of classical techniques.

The aim of this study was evaluating the association and correlation between the diagnostics tests used for Leishmania spp. detection in dogs and ticks. We evaluated 99 dogs and 990 Rhipicephalus sanguineus. In dogs, we used bone marrow aspirates and lymph node fine-needle aspiration biopsy (FNAB) for direct parasitological examinations and real time-polymerase chain reaction (RT-PCR) and collected blood samples for enzyme-linked immunosorbent assays (ELISA). In ticks, two laboratory techniques [immunohistochemistry to lipophosphoglycan (IHC) and RT-PCR] were performed in the intestine, ovaries and salivary glands. With respect to the measurement of diagnostic performance in dogs, lymph node RT-PCR proved to be the best test followed by ELISA and bone marrow RT-PCR. In ticks, intestine IHC were considered as a gold standard for diagnosis of leishmaniasis with intestinal RT-PCR being the best diagnostic test. To arrive at the correlation between laboratory techniques for dogs and their ticks, we evaluated the diagnostic test used for dogs with tests performed in R. sanguineus, which used lymph node FNAB as the gold standard. The intestine IHC technique showed strongest association. We demonstrated that the best tissue for Leishmania spp. detection in dogs was the lymph node and the intestine in case of ticks. As for laboratory techniques, the isolated analysis of each species presented a strong agreement between immunohistochemistry and RT-PCR when compared to its gold standard. In addition, we concluded that the immunohistochemistry of ticks' intestines was a better technique for diagnosing Leishmania spp. in R. sanguineus, thereby showing almost perfect correlation with the lymph node FNAB.

Cryptosporidium parvum in brown brocket (Mazama gouazoubira) from Brazil: First report of the subtype IIaA16G3R1 in cervids.

This research had as objective to evaluate the occurrence and to characterize genetically the infections by Cryptosporidium in Mazama gouazoubira. By a non-invasive harvest methodology using trained sniffer dogs to locate fecal samples of cervids, 642 fecal samples were obtained from six Brazilian localities. The cervids species responsible for the excretion of each fecal sample were identified by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), using the mitochondrial cytochrome b target gene (cyst b) and the restriction enzymes Sspl, AflIII and BstN. From this identification, 437 fecal samples of M. gouazoubira were selected for research of Cryptosporidium spp. performed through negative staining with malachite green and polymerase chain reaction (nPCR), with the subunit of 18S rRNA gene, followed by sequencing the amplified products. In the samples that were diagnosed the presence of parasite species with zoonotic potential, genotyping was also performed using nPCR with the subunit of GP60 gene. Statistical analysis consisted of the Fisher exact test to verify the association of the presence of the enteroparasite in relation to the presence of cattle in each locality, and the McNemar tests and Kappa correlation coefficient used to compare the results obtained between the two diagnostic techniques. In the fecal samples of M. gouazoubira the occurrences of Cryptosporidium were diagnosed in 1.6% (7/437) and 1.1% (5/437), respectively, through nPCR and microscopy. Cryptosporidium. parvum was diagnosed in 100% (7/7) of the samples submitted to sequencing (18S gene). The IIaA16G3R1 subtype was diagnosed in five of the C. parvum samples submitted to genotyping (GP60 gene). This is the first world report of C. parvum in M. gouazoubira and subtype IIaA16G3R1 in cervids.

Enteric parasitic infections in children and dogs in resource-poor communities in northeastern Brazil: Identifying priority prevention and control areas.

The aim of this study was to determine the prevalence and risk factors of the main enteric parasitic infections that affect children and dogs in the municipality of Ilhéus, Bahia, Brazil; and to identify the geopolitical areas that should receive priority interventions to combat them. Between March and November 2016, fecal samples of 143 dogs and 193 children aged 1 month to 5 years were collected in 40 rural and semirural communities using a systematic sampling approach, stratified by district. Samples were collected by legal guardians of the children and / or dog owners. Eggs, larvae, cysts and oocysts of parasites were concentrated by centrifugal-flotation and centrifugal-sedimentation, and acid-resistant staining was used to visualize parasites. One hundred and thirty-two children (68.4%), 111 dogs (77.6%) and 199 (73.7%) dog fecal samples collected from streets were parasitized. Giardiasis, cryptosporidiosis, amoeba infections and hookworm were the most frequent infections in all studied populations, in addition to trichuriasis in dogs and ascaridiasis in children. A predominance of Giardia and hookworms was observed in children and dogs, respectively. The coastal districts of Aritaguá, Olivença and the main district had a higher parasitic diversity and overlapping of important potential zoonotic infections. Age over one year (p<0.001), adjusted OR = 3.65; 95% CI = 1.86-7.16) and income below the minimum monthly salary (p = 0.02, adjusted OR = 2.78, 95% CI = 1.17-6.59) were the main factors associated with intestinal parasitic infections in children and dogs, respectively. The coastal districts of Aritaguá and Olivença and the main district should be prioritized through enteric disease control programs, and the factors associated with infections must be considered in the design of health interventions in these districts. The integration between affirmative income actions and investments to improve the health infrastructure of these communities may work more effectively than current preventive measures to combat enteric parasites.

Deprivation of dietary fiber enhances susceptibility of mice to cryptosporidiosis.

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.

TF-Test Quantified: a new technique for diagnosis of Schistosoma mansoni eggs.

OBJECTIVES: Laboratory diagnosis of Schistosoma mansoni eggs is routinely performed by conventional quantitative techniques through the parasitological examination of human faeces. 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 faeces. 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 faecal debris. The next stage of this study is the field validation of the TFT-Quant technique with the use of human faecal 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.

OBJECTIFS: Le diagnostic en laboratoire des œufs de Schistosoma mansoni est systématiquement effectué à l'aide de techniques quantitatives conventionnelles faisant appel à l'examen parasitologique de selles humaines. Cependant, la sensibilité diagnostique de ce type d'examen varie de faible à modérée. Nous avons voulu développer une nouvelle technique parasitologique appelée TF-Test Quantified (TFT-Quant), pour la détection quantitative des œufs de S. mansoni dans les selles humaines. MÉTHODES: Quatre étapes de l'étude ont été réalisées sur 43 souris infectées par des cercaires de S. mansoni. Ces expériences ont permis de définir un protocole opérationnel pour TFT-Quant et de comparer cette nouvelle technique à la technique classique Helm-Teste. RÉSULTATS: Les résultats montrent une bonne efficacité diagnostique de TFT-Quant, dont la sensibilité et la spécificité ont atteint 100%, ce qui indique une concordance kappa (k) «presque parfaite¼. CONCLUSIONS: Cette nouvelle technique a permis un gain quantitatif dans la détection des œufs de S. mansoni, largement exempts de débris fécaux. La prochaine étape de cette étude est la validation sur le terrain de la technique TFT-Quant avec l'utilisation d'échantillons de selles humaines provenant d'une région endémique pour la schistosomiase mansonique au Brésil (Microregion Jequitinhonha, dans l'Etat de Minas Gerais). Parallèlement à cette validation, des algorithmes informatiques de calcul seront développés pour permettre le diagnostic quantitatif automatisé des œufs de S. mansoni.

Prevalence of Ehrlichia canis (Rickettsiales: Ehrlichieae) DNA in Tissues From Rhipicephalus sanguineus (Acari: Ixodidae) Ticks in Areas Endemic for Canine Monocytic Ehrlichiosis in Brazil.

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.

First description of Giardia duodenalis in buffalo calves (Bubalus bubalis) in southwest region of São Paulo State, Brazil.

We performed molecular characterization of Giardia duodenalis in buffalo calves from the Southwest region of São Paulo State, Brazil. A total of 183 fecal samples of Murrah breed buffaloes up to six months of age were collected. We examined these samples by the polymerase chain reaction (PCR) targeting the small-subunit ribosomal RNA gene and positive samples were characterized using additional PCR assays targeting a portion of the beta-giardin, the glutamate dehydrogenase and the triose-phosphate isomerase genes. Based on the SSU rRNA nPCR, the presence of G. duodenalis was confirmed in 12 (6.56%) of fecal samples, of these, five, four and three samples were positive for the tpi, bg and gdh genes, respectively. Assemblage identification by sequencing was successful in 6 of 12 samples and sequence analysis showed 100% genetic similarity with G. duodenalis assemblage E. This observation represents the first detection of G. duodenalis assemblage E in buffaloes calves in Brazil.

First description of Cryptosporidium parvum in carrier pigeons (Columba livia).

The carrier pigeon and the domestic pigeon are different breeds of the species Columba livia. Carrier pigeons are used for recreational activities such as bird contests and exhibitions. Due to the close contact with humans, these birds may potentially represent a public health risk, since they can host and disseminate zoonotic parasites, such as those belonging to the genus Cryptosporidium (phylum Apicomplexa). The purpose of this work was the detection by microscopic and molecular techniques of Cryptosporidium spp. oocysts in fecal samples of carrier pigeons, and subsequently to sequence the 18S ribosomal RNA marker of positive samples to identify the species. A total of 100 fecal samples were collected individually in two pigeon breeding facilities from Formiga and Araçatuba, cities located in Minas Gerais state and São Paulo state, Brazil, respectively. The age of the birds ranged from one to 12 years; 56 were females and 44 males. Fecal smears were stained with negative malachite green, whereas the molecular characterization was based on the sequence of a ∼800bp fragment of the 18S rRNA gene. Microscopic examination of fecal smears revealed 4% (4/100) oocyst positivity. On the other hand, 7% (7/100) of positivity were found using nested PCR. Three samples were 99% to 100% similar to Cryptosporidium parvum 18S rDNA type A (Genbank AH006572) and the other three samples had 99% to 100% similarity to C. parvum 18S rDNA type B (Genbank AF308600). To our knowledge, this is the first report of C. parvum oocysts in carrier pigeons.

First description of Cryptosporidium hominis GP60 genotype IkA20G1 and Cryptosporidium parvum GP60 genotypes IIaA18G3R1 and IIaA15G2R1 in foals in Brazil.

The present study focuses on Cryptosporidium infections of foals in Brazil. A total of 92 animals of different breeds from 11 farms in the vicinity of Araçatuba in the state of São Paulo, were examined. According to PCR targeting the 18S rRNA gene, Cryptosporidium sp. DNA was detected in 21.7% (20/92) of foals. Good quality 18S rRNA, actin, HSP70 and gp60 genes nPCR amplicons were obtained from five fecal samples. PCR amplification and sequencing of a fragment of the GP60 sporozoite surface glycoprotein gene revealed C. parvum genotypes IIaA18G3R1, IIaA15G2R1. Interestingly, we also detected in two foals a GP60 genotype related to the human parasite C. hominis.

Cryptosporidium baileyi in wild captive psittacines in Brazil.

Cryptosporidiosis in birds manifests as an acute or chronic disease of the respiratory or digestive tracts. The objective of our study was to perform the molecular characterization of Cryptosporidium spp. in wild psittacines kept in captivity at the Araçatuba Municipal Zoo, São Paulo, Brazil. A total of 47 fecal samples were collected from Amazona aestiva, Psittacara leucophthalma, and Ara ararauna. All samples were collected at the time the birds defecated. DNA extraction was performed using the ZR Fecal DNA MiniPrep™ kit (Zymo Research). Screening for Cryptosporidium spp. was accomplished using nested PCR targeting the 18S RNA gene and sequencing of amplified fragments. Positivity for Cryptosporidium spp. (10.64%; 5/47) was found in A. ararauna (4) and P. leucophthalma (1) samples. The amplified fragments were sequenced and showed 100% genetic similarity with Cryptosporidium baileyi.

Validation of a new technique to detect Cryptosporidium spp. oocysts in bovine feces.

Due to its important zoonotic potential, cryptosporidiosis arouses strong interest in the scientific community, because, it was initially considered a rare and opportunistic disease. The parasitological diagnosis of the causative agent of this disease, the protozoan Cryptosporidium spp., requires the use of specific techniques of concentration and permanent staining, which are laborious and costly, and are difficult to use in routine laboratory tests. In view of the above, we conducted the feasibility, development, evaluation and intralaboratory validation of a new parasitological technique for analysis in optical microscopy of Cryptosporidium spp. oocysts, called TF-Test Coccidia, using fecal samples from calves from the city of Araçatuba, São Paulo. To confirm the aforementioned parasite and prove the diagnostic efficiency of the new technique, we used two established methodologies in the scientific literature: parasite concentration by centrifugal sedimentation and negative staining with malachite green (CSN-Malachite) and Nested-PCR. We observed good effectiveness of the TF-Test Coccidia technique, being statistically equivalent to CSN-Malachite. Thus, we verified the effectiveness of the TF-Test Coccidia parasitological technique for the detection of Cryptosporidium spp. oocysts and observed good concentration and morphology of the parasite, with a low amount of debris in the fecal smear.

Identification of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus actively infesting dogs.

Sand flies are recognized as the major vector of canine visceral leishmaniasis. However, in some areas of Brazil where sand flies do not occur, this disease is found in humans and dogs. There has been speculation that ticks might play a role in transmission of canine visceral leishmaniasis and the DNA of Leishmania spp. has been reported in whole ticks. We investigated the presence of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus ticks collected from tick-infested dogs in two cities of Brazil. We used 66 dogs that tested positive and 33 that tested negative for Leishmania spp. according to direct cytological examination assays. Ten ticks were collected from each dog and dissected to collect the intestines, ovaries, and salivary glands for immunohistochemistry (IHC) and diagnostic real-time polymerase chain reaction (RT-PCR). IHC results showed Leishmania spp. in 98, 14, and 8 % of the intestines, ovaries, and salivary glands, respectively. Real-time PCR showed that 89, 41, and 33 % of the tick intestine, ovary, and salivary glands, respectively, were positive for Leishmania spp. The verification of promastigotes of Leishmania spp. by two independent techniques in ticks collected from these urban region dogs showed that there is need for clarification of the role of ticks in the transmission of canine visceral leishmaniasis in Brazil.