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Giardia duodenalis is a flagellate protozoan that multiplies in the small intestine of a wide variety of hosts, animals and humans. It has a worldwide distribution, however it is considered a neglected disease by the World Health Organization (WHO). Nowadays, rabbits are being chosen as pets, especially by children. There are already reports of the occurrence of G. duodenalis in rabbits from other countries, but research has not been carried out in Brazil yet. Thus, the objective of our work was to verify the occurrence and molecularly characterize G. duodenalis that affect pet rabbits, through the polymerase chain reaction (PCR) in the northwest region of the state of São Paulo, Brazil. Fecal samples from 100 rabbits were collected, which later underwent a process of DNA extraction and amplification by nested-PCR (nPCR), using the SSU rRNA gene, and ß-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) to determine the assemblage. A questionnaire was answered by the owners with information about gender, age, deworming, diarrhea, water source, food, place of residence and contact with other animals. From those samples, 40 were positive for G. duodenalis. Good quality of the SSU rRNA gene by nPCR were obtained from two samples. For the first time, we report the occurrence of G. duodenalis assemblage A on pet rabbits in Brazil.

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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.

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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.

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The Leishmania infantum (synonym, Leishmania chagasi) causes life-threatening infection, namely canine leishmaniosis (CanL), which is a chronic zoonosis prevalent in various countries and spread by the bite of the infected Lutzomyia female sandfly in South America. The objective of the study was to assess the effectiveness of a polymer matrix collar containing made up of 10% imidacloprid and 4.5% flumethrin for the prevention of canine leishmaniosis from the hyperendemic region falling under Araçatuba municipality (Brazil). The research included a total of 146 dogs chosen from 75 households. Test were initiated via physical examination; weighing and biological sample collection (blood, popliteal lymph node and conjunctival swab) of these dogs were done in March 2018 (Day 0; GA, control = 69, GB, treated = 77) to initiate laboratory tests. Post-inclusion, the animals were monitored on the 120th, 240th, 360th and 480th days, respectively. The usage of collars continued between 0 and 480 days before being substituted in second (D240) and fourth (D480) follow-up visits. On the whole, 25 dogs in GA (36.2%) and three in GB (3.9%) were found positive for L. infantum infection in a minimum of one diagnostic test used in the research. Therefore, the average collar effectiveness for protection from L. infantum infection was 89.2% (p < .01). In the last follow-up, the average incidence density rate for GA was 30.7%, whereas for GB, it was 2.9%. The imidacloprid/flumethrin collars evaluated in the research were found to be safe and extremely efficient for the prevention of L. infantum infection through Lutzomyia species among the large population of dogs in highly prone endemic regions. This is a dependable and efficient technique aimed at reducing the occurrence and propagation of this illness among the population of canines, which would eventually reduce the human-health-related hazards. In Brazil, Lutzomyia spp. is a leading vector of the infection; thus, the collar can be used to limit infection in dogs and humans.

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The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples.

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Abstract The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples.

Resumo O objetivo deste trabalho foi validar um protocolo de nested PCR em tempo real em um tubo (nPCR-TR-1T) seguida de sequenciamento genético para detectar e caracterizar as espécies e genótipos de Cryptosporidium em aves. Um total de 443 amostras de DNA genômico, extraído de amostras fecais de aves, foi analisado pela nPCR-TR-1T e pela nested PCR convencional. Pela nPCR-TR-1T, foi observada positividade para Cryptosporidium spp. de 20,3% (90/443), em contraste com a nested PCR convencional, que apresentou positividade de 8,1% (36/443). O teste de sensibilidade analítica mostrou que a nPCR-TR-1T detecta aproximadamente 0,5 oocisto (2 esporozoítos) por reação. A avaliação da especificidade analítica não revelou amplificação de microrganismos que comumente apresentam amplificação inespecífica com primers utilizados para o diagnóstico de Cryptosporidium spp. O cálculo da repetibilidade evidenciou o mesmo resultado em 27 de 30 amostras (90%). Em relação à reprodutibilidade da nPCR-TR-1T, foi observado o mesmo resultado em 80% (24/30) das amostras examinadas. Foi possível realizar o sequenciamento em todas as 90 amostras amplificadas pela nPCR-TR-1T, com identificação de C. baileyi, C. galli, C. meleagridis, C. proventriculi e Cryptosporidium genótipo I em aves. O sequenciamento dos fragmentos amplificados pela nested PCR convencional foi possível em 10/36 (27,8%) das amostras positivas.

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This study aimed to perform the detection and molecular characterization of Giardia spp. in Psittaciformes from the Southern and Southeastern regions of Brazil. Fecal samples were obtained from 359 adult exotic captive Psittaciformes belonging to 13 genera, randomly selected from 33 aviaries located in the Southern and Southeastern regions of Brazil during a bird exhibition at the 2015 Ornithological Championship of the Ornithological Federation of Brazil (FOB). Nested polymerase chain reaction targeting the small subunit rRNA gene identified Giardia spp. in 93/359 (25.9%) fecal samples and 25/33 (75.8%) aviaries. Genetic sequencing identified G. psittaci in 12 birds from six genera. Zoonotic Giardia species was not detected in fecal samples from Psittaciformes.

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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.

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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.

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This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

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