Accumulation of oocysts of Cryptosporidium parvum in Biomphalaria glabrata (Pulmonata:Planorbidae) under experimental conditions.

INTRODUCTION: Cryptosporidium oocysts are easily transported to various aquatic environments. The objective of this study was to evaluate B. glabrata mollusks exposed to food containing C. parvum oocysts. METHODS: Six experimental groups were used with B. glabrata either exposed or not to C. parvum oocysts. Microscopic and molecular diagnostics were conducted in water samples and tissues of B. glabrata. RESULTS: By light microscopy, C. parvum oocysts were identified in the water of the exposed groups. C. parvum DNA was not detected in water but was detected in tissue samples. CONCLUSIONS: Further studies should be conducted under natural conditions.

Molecular epidemiology of Blastocystis isolated from animals in the state of Rio de Janeiro, Brazil.

The enteric protist Blastocystis is one of the most frequently reported parasites infecting both humans and many other animal hosts worldwide. A remarkable genetic diversity has been observed in the species, with 17 different subtypes (STs) on a molecular phylogeny based on small subunit RNA genes (SSU rDNA). Nonetheless, information regarding its distribution, diversity and zoonotic potential remains still scarce, especially in groups other than primates. In Brazil, only a few surveys limited to human isolates have so far been conducted on Blastocystis STs. The aim of this study is to determine the occurrence of Blastocystis subtypes in non-human vertebrate and invertebrate animal groups in different areas of the state of Rio de Janeiro, Brazil. A total of 334 stool samples were collected from animals representing 28 different genera. Blastocystis cultivated samples were subtyped using nuclear small subunit ribosomal DNA (SSU rDNA) sequencing. Phylogenetic analyses and BLAST searches revealed six subtypes: ST5 (28.8%), ST2 (21.1%), ST1 and ST8 (19.2%), ST3 (7.7%) and ST4 (3.8%). Our findings indicate a considerable overlap between STs in humans and other animals. This highlights the importance of investigating a range of hosts for Blastocystis to understand the eco-epidemiological aspects of the parasite and its host specificity.

Accumulation of oocysts of Cryptosporidium parvum in Biomphalaria glabrata (Pulmonata: Planorbidae) under experimental conditions

Abstract INTRODUCTION: Cryptosporidium oocysts are easily transported to various aquatic environments. The objective of this study was to evaluate B. glabrata mollusks exposed to food containing C. parvum oocysts. METHODS: Six experimental groups were used with B. glabrata either exposed or not to C. parvum oocysts. Microscopic and molecular diagnostics were conducted in water samples and tissues of B. glabrata. RESULTS: By light microscopy, C. parvum oocysts were identified in the water of the exposed groups. C. parvum DNA was not detected in water but was detected in tissue samples. CONCLUSIONS: Further studies should be conducted under natural conditions.

Infecção natural de pombos (Columba livia) por Cryptosporidium spp / Natural infection of pigeons (Columba livia) with Cryptosporidium spp

O estudo teve como objetivo diagnosticar microscopicamente e molecularmente a presença de oocistos de Cryptosporidium spp. provenientes de amostras fecais de pombos (Columba livia) no município do Rio de Janeiro. As amostras fecais foram coletadas frescas, logo após as aves defecarem e acondicionadas sob refrigeração e encaminhadas para o processamento e pesquisa de oocistos de Cryptosporidium spp. ao laboratório de Protozoologia do Departamento de Parasitologia, da Universidade Federal Rural do Rio de Janeiro. As amostras foram submetidas a técnica de centrifugação e flutuação com solução saturada de açúcar e as lâminas observadas com objetiva de 40X. Nas amostras positivas para a presença de oocistos de Cryptosporidium spp. foi realizada a extração de ácido desoxirribonucleico (DNA) para uso nas reações em cadeia pela polimerase (PCR) e Nested-PCR. Um total de 387 amostras fecais de pombos foram obtidas, no diagnóstico microscópico foi possível a observação de oocistos deste parasita em 81 amostras (20,93%). Destas, 53 amostras amplificaram DNA específico para Cryptosporidium spp. na reação da Nested-PCR. Mundialmente, Cryptosporidium spp. parasitando pombos é assinalado em apenas quatro países: Turquia, China, Irã e Tailândia. O resultado encontrado neste trabalho, em três bairros populosos do município do Rio de Janeiro, revelou ser preocupante, por serem áreas onde há uma quantidade acentuada da população animal e humana, facilitando a dispersão e contaminação ambiental dos oocistos. Este é o primeiro registro no Brasil de parasitismo de Cryptosporidium spp. tendo como hospedeiro o pombo e estes resultados, não devem ser negligenciados.

The aim of this study was to diagnose microscopically and molecularly the presence of Cryptosporidium spp. oocysts from pigeons (Columba livia) faecal samples in Rio de Janeiro city. The fecal samples were collected fresh, after the birds defecated and conditioned under refrigeration and sent to the processing and research of Cryptosporidium spp. oocysts to the protozoology laboratory of the Parasitology Department, Universidade Federal Rural do Rio de Janeiro. The samples were submitted to centrifugation and flotation with saturated sugar solution technique and the slides observed with a 40X objective. In the positive samples for the Cryptosporidium spp. oocysts the extraction of deoxyribonucleic acid (DNA) for polymerase chain reaction (PCR) and NestedPCR was performed. A total of 387 pigeons faecal samples were obtained, in the microscopic diagnosis it was possible to observe oocysts of this parasite in 81 samples (20.93%). Of these, 53 samples amplified DNA specific for Cryptosporidium spp. in the Nested-PCR reaction. Worldwide, Cryptosporidium spp. parasitizing pigeons is reported in only four countries: Turkey, China, Iran and Thailand. The results found in this study, in three populated neighborhoods of Rio de Janeiro city, were worrisome because they are areas where there is a marked quantity of animal and human population, facilitating the dispersion and environmental contamination of oocysts. This is the first record in Brazil of parasitism of Cryptosporidium spp. having as host the pigeon and these results, should not be neglected.

Mussels (Perna perna) as bioindicator of environmental contamination by Cryptosporidium species with zoonotic potential.

Sources of contamination such as animal feces runoff, organic fertilizer application, and the release of partially treated or untreated sewage can lead to the contamination of aquatic environments by Cryptosporidium spp. The quality of mussels as food is closely related to the sanitary conditions of the marine environment where these bivalves are found. Marine mollusks are filter feeders that are able to retain Cryptosporidium oocysts in their tissue, thus functioning as bioindicators. A total of 72 pooled mussel samples of the species Perna perna were collected at two sites (A and B) in the municipality of Mangaratiba, Rio de Janeiro State, Brazil. Sampling involved removal of 30 mussels, from each collection site every month for one year. The 30 mussels from each sampling were then allocated into three groups of 10. Two Cryptosporidium spp. genes (18S and GP60) were targeted for DNA amplification from the samples obtained. After purification, all of the products obtained were sequenced and phylogenetic analyses were performed. Of the 72 samples analyzed using the nested-PCR for the 18S gene target, 29.2% were positive for the presence of Cryptosporidium spp. Of these samples, 52.4% were collected at site A (ie 11/21) and 47.6% at site B (ie 10/21). The 18S genes of all the samples considered positive for Cryptosporidium spp. were sequenced, and the following three species were identified: Cryptosporidium parvum, C. meleagridis, and C. andersoni. Three distinct C. parvum subtypes (IIaA19G2R2; IIaA20G2R2; IIaA20G3R2) were identified using the GP60 gene. More studies to evaluate the zoonotic potential of this species should be performed as both sampling locations contain human and/or animal fecal contaminants.

New Cryptosporidium parvum subtypes of IIa subfamily in dairy calves from Brazil.

Bovine cryptosporidiosis is mainly caused by four distinct species: Cryptosporidium parvum, C. bovis, C. ryanae and C. andersoni. The first, C. parvum, is a major concern in livestock causing economic losses, in addition to public health impact because of its zoonotic characteristics. The present study aimed to determine the occurrence of different species and subtypes of Cryptosporidium using molecular techniques. A total of 143 fecal samples were collected from calves from three dairy farms located in the state of Rio de Janeiro, Brazil. Saturated sugar centrifugal flotation method was used for the microscopic evaluation of the samples. Among these samples, 19.6% (28) were positive by microscopy, and 82.1% (23) of these 28 samples had their diagnosis confirmed by PCR using 18S as gene target. After sequencing, three species of Cryptosporidium were found to infect calves in different age groups. In pre-weaning phase (<2 months), 10% (3/30) of the calves were infected with C. parvum, whereas 14.2% (16/113) of post-weaning calves (≥2 months) were observed to be infected with C. andersoni and 1.8% (2/113) by C. ryanae with the latter diagnosed for the first time in the state of Rio de Janeiro. Those samples identified as C. parvum were further characterized at the GP60 locus, and PCR products were cloned. Eight different subtypes (IIaA20G2R1, IIaA20G2R2, IIaA19G2R1, IIaA19G2R2, IIaA18G1R1, IIaA18G2R2, IIaA16G3R2 and IIaA14G2R2) of C. parvum were identified, all belonging to the IIa family subtype, which is considered of high zoonotic potential. The subtypes mentioned above have not yet been detected in Brazilian cattle, and four of these subtypes (IIaA20G2R2, IIaA19G2R2, IIaA18G2R2 and IIaA14G2R2) had not been diagnosed elsewhere in calves until this study.

Cryptosporidium spp. parasitize exotic birds that are commercialized in markets, commercial aviaries, and pet shops.

The purpose of this study was to genetically characterize and phylogenetically analyze the Cryptosporidium spp. isolated from exotic birds commercialized in popular markets, commercial aviaries, and pet shops located in Rio de Janeiro, Brazil. Fecal samples from individually housed birds were collected and subjected to centrifuge-flotation technique using saturated sugar solution. DNA was isolated from Cryptosporidium positive samples, and 18S subunit rDNA was amplified and processed using nested-polymerase chain reaction (PCR). To identify the protozoan species, the PCR amplicons were used for restriction fragment length polymorphism and sequencing analyses. Of the 103 analyzed fecal samples, seven (6.8%) were positive for Cryptosporidium oocysts. Sequencing and further phylogenetic analyses allowed us to identify the following species: Cryptosporidium parvum in Bengalese finch (Lonchura striata domestica) and avian genotype III in Java sparrow (Padda oryzivora) and cockatiel (Nymphicus hollandicus). The sequences of the Cryptosporidium spp. isolated from canaries (Serinus canarius) were not identifiable within the groups of known species, but they presented a higher genetic similarity with C. parvum. This is the first report in Brazil showing that C. parvum parasitizes Bengalese finches and that avian genotype III parasitizes Java sparrows.

Avaliação clínica e molecular de cães com erliquiose / Clinical and molecular evaluation of dogs with ehrlichiosis

A erliquiose monocítica canina é uma doença cosmopolita causada por Ehrlichia canis e transmitida pelo carrapato Rhipicephalus sanguineus, sendo frequentemente diagnosticada em cães em todo o Brasil. Este trabalho teve por objetivo investigar citológica e molecularmente a infecção por Ehrlichia em 195 cães atendidos no Hospital Veterinário da Universidade Federal de Mato Grosso, analisando os achados clínicos e laboratoriais. Nos 48 cães atendidos com citologia positiva para Ehrlichia sp., foi possível verificar a diversidade de sinais, com predominância estatisticamente significativa de palidez de mucosas (P≤0,05), assim como variados achados hematológicos, ocorrendo tanto anemia, leucopenia e trombocitopenia, quanto normalidade ou aumento dessas células. Ocorreu aumento das proteínas plasmáticas, com hiperglobulinemia, sem, no entanto, haver diferença significativa (P≥0,05), apesar de esse achado ser frequente nessa afecção. Por meio do PCR nested, confirmou-se a infecção por E. canis em cães da cidade de Cuiabá.

The canine monocytic ehrlichiosis is a cosmopolitan disease, caused by Ehrlichia canis, transmitted by ticks Rhipicephalus sanguineus that has been frequently diagnosed in dogs throughout the country. This study aimed to investigate the cytological and molecular Ehrlichia infection in 195 dogs examined at the University Veterinary Hospital of Mato Grosso, by analyzing the clinical and laboratory findings. In 48 dogs with positive cytology for Ehrlichia sp it was possible to detect the diversity of signs, with predominance statistically significant of pallor of mucous membranes (P≤0.05) as well as several hematological findings, occurring anemia, leukopenia and thrombocytopenia, or increased as normal cells. There was increased of plasma proteins, with hyperglobulinemia, however without any significant difference (P≥0.05), although this finding is common in that infection. Through the nested PCR technique it was possible to confirm the infection by E. canis in the city of Cuiaba.

Infecção natural por Cryptosporidium sp. em aves domésticas comercializadas em mercados municipais do Estado do Rio de Janeiro / Natural infection by Cryptosporidium sp. in domestic fowls sold at local markets

O objetivo do trabalho foi comparar a dinâmica, a ocorrência, a morfometria de oocistos e os períodos patentes de Cryptosporidium sp. em aves domésticas, patos (Anas platyrhynchos), pintos (Gallus gallus) e codornas (Coturnix japonica), naturalmente infectadas, provenientes de dois mercados municipais do Estado do Rio de Janeiro, Rio de Janeiro (RJ). Houve diferenças quanto à ocorrência da infecção entre os dois locais e entre pintos e patos, mas não entre codornas. Para a morfometria, foram observadas diferenças estatísticas nas medidas dos diâmetros maior e menor e para o índice morfométrico calculado (P<0,01). Já entre pintos e codornas não houve diferenças (P>0,05). Na comparação do período de eliminação, patos tiveram um maior período com maiores quantidades de oocistos eliminados. Codornas e pintos apresentaram dinâmica de eliminação semelhante e não houve diferença quanto à concentração de oocistos. Pintos foram mais susceptíveis à infecção seguidos por patos e codornas. Pode-se concluir que a infecção natural por Cryptoporidium sp. foi frequente nas aves estudadas. Patos, pintos e codornas podem ser disseminadores do protozoário em mercados municipais do Rio de Janeiro, RJ. Assim, podem constituir risco de infecção.

The objective of the current study was comparing the dynamic and occurrence of Cryptosporidium sp., as well as the morphometry and elimination period of oocysts in naturally infected ducks (Anas platyrhynchos), chickens (Gallus gallus) and Japanese quails (Coturnix japonica) from two local markets of Rio de Janeiro, RJ. There were significant differences considering the occurrence of infection between the two markets, and also between chickens and ducks, but not among Japanese quails. Also, significant statistical differences were observed in morphometry, considering the major and minor diameters of oocysts and the calculated morphometric index (P<0.01); however, there was no difference between chickens and Japanese quails (P>0.05). According to the elimination period, ducks eliminated oocysts for a longer period and in a higher number. Japanese quails and chickens had a similar dynamic of oocysts elimination, no difference in its concentration was observed. Chickens were the most susceptible bird species to Cryptosporidium sp. infection, followed by ducks and Japanese quails. In conclusion, Cryptosporidium sp. natural infection was commonly found in the studied fowls. Ducks, chickens and Japanese quails may disseminate this protozoan in local markets of Rio de Janeiro, representing risk of infection.