RESUMO
Non-human primates (NHPs) are the group that most share infectious agents with humans due to their close taxonomic relationship. The southern brown howler monkeys (Alouatta guariba clamitans) are endemic primates from Brazil and Argentina's Atlantic Forest. This study aimed to investigate the presence of intestinal parasites in free-living (FL) and captive (CA) southern brown howler monkeys. Thirty-nine stool samples were collected in two areas in southern Brazil, 15 FL and 24 CA. Stool sediments obtained by centrifugal sedimentation technique were used for microscopic analysis and direct immunofluorescence assay and evaluated by molecular analysis through amplification and sequencing of TPI fragments. Intestinal parasites Giardia duodenalis, Cryptosporidium spp., and Trypanoxyuris minutus were detected at coproparasitological analysis. This is the first report of the presence of Cryptosporidium spp. in free-living howlers. The molecular characterization of G. duodenalis isolates indicated assemblage B for the first time found in free-living A. guariba clamitans. The high prevalence of G. duodenalis transmission in CA howler monkeys can be explained by direct contact with humans and frequent soil contact. The presence of a potentially zoonotic assemblage in these animals indicates that the process of fragmentation and cohabitation with humans and livestock affects the wildlife, thus indicating a need for eco-health measures.
Assuntos
Alouatta , Giardia lamblia , Giardíase , Doenças dos Macacos , Animais , Alouatta/parasitologia , Brasil/epidemiologia , Doenças dos Macacos/parasitologia , Doenças dos Macacos/epidemiologia , Giardíase/veterinária , Giardíase/parasitologia , Giardíase/epidemiologia , Giardia lamblia/isolamento & purificação , Giardia lamblia/genética , Giardia lamblia/classificação , Fezes/parasitologia , Animais de Zoológico/parasitologia , Cryptosporidium/isolamento & purificação , Cryptosporidium/classificação , Cryptosporidium/genética , Prevalência , Masculino , Animais Selvagens/parasitologia , Feminino , Criptosporidiose/parasitologia , Criptosporidiose/epidemiologiaRESUMO
BACKGROUND: Microscopy and enzyme-linked immunosorbent assay (ELISA) are routinely used for Cryptosporidium diagnosis, without differentiating the parasite species. METHODS: Children's feces were analyzed by modiï¬ed Ziehl-Neelsen (mZN) and ELISA for Cryptosporidium diagnosis and by polymerase chain reaction-restriction fragment length polymorphism for species identification. RESULTS: Cryptosporidium frequency was 2.6%. The sensitivity and specificity of ELISA were 85.7% and 99.7%, respectively, with excellent concordance with mZN (kappa=0.854). Parasite species were characterized as Cryptosporidium hominis (78.3%), Cryptosporidium felis (17.4%), and Cryptosporidium parvum (4.3%). CONCLUSIONS: Coproantigen ELISA is as efficient as mZN for Cryptosporidium diagnosis. Cryptosporidium genotyping suggests anthroponotic and zoonotic transmission to children.
Assuntos
Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , Criança , Criptosporidiose/diagnóstico , Criptosporidiose/parasitologia , Cryptosporidium/classificação , Cryptosporidium/isolamento & purificação , Cryptosporidium parvum/isolamento & purificação , Fezes/parasitologia , Humanos , Polimorfismo de Fragmento de RestriçãoRESUMO
Cryptosporidium felis is the major etiologic agent of cryptosporidiosis in felines and has been reported in numerous human cryptosporidiosis cases. Sequence analysis of the 60-kDa glycoprotein (gp60) gene has been developed for subtyping C. felis recently. In this study, 66 C. felis isolates from the United States, Jamaica, Peru, Portugal, Slovakia, Nigeria, Ethiopia, Kenya, China, India and Australia were subtyped using the newly established tool. Forty-four specimens yielded gp60 sequences, generating 23 subtypes clustered in 4 subtype families (XIXa, XIXc, XIXd and XIXe) with high bootstrap support in a phylogenetic analysis of sequence data. Among them, XIXa showed high genetic diversity at the nucleotide level, with the formation of 18 subtypes from both cats and humans with different geographic distribution. In contrast, all 11 XIXd isolates derived from humans from various countries had identical sequences. Results of this study improve our understanding of the genetic diversity, host specificity and transmission dynamics of C. felis.
Assuntos
Criptosporidiose/transmissão , Cryptosporidium/classificação , Variação Genética , Proteínas de Protozoários/genética , Análise de Sequência de DNA/métodos , Zoonoses/parasitologia , Animais , Austrália , Gatos , Bovinos , China , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Especificidade de Hospedeiro , Humanos , Índia , Jamaica , Quênia , Macaca mulatta , Nigéria , Peru , Filogenia , Filogeografia , Portugal , Eslovênia , Estados Unidos , Zoonoses/transmissãoRESUMO
Cryptosporidium spp. are apicomplexan protozoa associated with chronic diarrhea in AIDS and other immunocompromised patients, and one of the commonest causes of childhood diarrhea and malnutrition, particularly in low-income settings. In Colombia, there are few molecular epidemiological studies on Cryptosporidium spp.; thereby, the transmission dynamics of this parasite in the country is poorly known. This study evaluated the diversity of Cryptosporidium at species, subtype family, and subtype level in children attending various day-care centers in Medellin, Colombia. Two hundred and ninety stool samples from children < 5 years of age were collected from April to November of 2015. All samples were processed by PCR and sequence analysis of the ssu RNA gene and the gp60 gene. An infection rate of 2.4% was observed, with only two Cryptosporidium species identified: C. hominis (6/7) and C. meleagridis (1/7). Cryptosporidium hominis isolates belonged to the subtypes IbA10G2, IaA13R6 and IaA13R7; IIIbA26G1R1 C. meleagridis subtype was also detected. There is a C. hominis predominance in the children evaluated, suggesting an important role of the anthroponotic transmission cycle in the day-care centers analyzed. Further investigation is required to determine infection sources and susceptible hosts in order to define appropriate management of cryptosporidiosis.
Assuntos
Cuidado da Criança/estatística & dados numéricos , Criptosporidiose/epidemiologia , Criptosporidiose/transmissão , Cryptosporidium/isolamento & purificação , Adolescente , Animais , Criança , Pré-Escolar , Colômbia/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium/classificação , DNA de Protozoário/genética , Diarreia/parasitologia , Fezes/parasitologia , Feminino , Genótipo , Humanos , Higiene , Masculino , Reação em Cadeia da Polimerase , Pobreza , Sulfotransferases/genéticaRESUMO
Cryptosporidium spp. is a pathogenic protozoan present in the gastrointestinal tract of several hosts. This protozoan was originally classified as within the Coccidia Class and has recently been reclassified to gregarine based on studies that observed the evolutionary phases from the process of excision and sequencing of the 18S rRNA gene. Molecular biology techniques have become diagnostic tools and have also been used to understand the epidemiology of Cryptosporidium spp., since several species of this genus are very similar morphologically and morphometrically. Molecular techniques have been used in the identification of parasites, at the species and subtypes levels and to study disease transmission. The laboratory diagnosis of human cryptosporidiosis can be made by parasite detection methods, such as optical microscopy, antigens or genetic material detection, as well as serum antibodies raised to Cryptosporidium spp. Molecular methods were developed and allowed, not only an extensive revision of the taxonomy, but also an improvement in the laboratory diagnosis. In Brazil, there are few reports of Cryptosporidium spp. outbreaks in humans and all of them took place in nurseries. A few epidemiological studies developed in Brazil have used molecular methods for the detection of Cryptosporidium spp., as well as genotyping studies of their species and subtypes. The use of real-time PCR, together with microscopy and immunochromatography techniques, would result in a more precise diagnosis of cryptosporidiosis. The analysis of genotypes, subtypes and clonality of Cryptosporidium could be useful to understand and define the prognosis and severity of infections.
Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , DNA de Protozoário/genética , Fezes/parasitologia , Animais , Brasil/epidemiologia , Criptosporidiose/diagnóstico , Criptosporidiose/epidemiologia , Cryptosporidium/classificação , DNA de Protozoário/análise , Genótipo , Humanos , RNA Ribossômico/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
Cryptosporidiosis of calves is caused by the enteroprotozoan Cryptosporidium spp. The disease results in intense diarrhea of calves associated with substantial economic losses in dairy farming worldwide. The aim of this study was to determine calf, herd, and within-herd Cryptosporidium prevalence and identify Cryptosporidium species and subtypes in calves with diarrhea in intensive dairy herds in central Argentina. A total of 1073 fecal samples were collected from 54 randomly selected dairy herds. Cryptosporidium-oocysts were isolated and concentrated from fecal samples using formol-ether and detected by light microscopy with the modified Ziehl-Neelsen technique. Overall prevalence of oocyst-excreting calves was found to be 25.5% (274/1073) (95% C.I. 22.9; 28.1%). Of the herds studied, 89% (48/54) included at least one infected calf, whereas within-herd prevalence ranged from the absence of infection to 57% (20/35). A highly significant association was found between the presence of diarrhea and C. parvum infection (χ2 = 55.89, p < 0.001). For species determination, genomic DNA isolated from oocyst-positive fecal samples was subjected to PCR-RFLP of the 18S rRNA gene resulting exclusively in Cryptosporidium parvum identification. C. parvum isolates of calves displaying diarrhea and high rate of excretion of oocysts were subtyped by PCR amplification and direct sequencing of the 60 kDa glycoprotein (GP60) gene. Altogether five GP60 subtypes, designated IIaA18G1R1, IIaA20G1R1, IIaA21G1R1, IIaA22G1R1, and IIaA24G1R1 were identified. Interestingly, IIaA18G1R1 and IIaA20G1R1 were predominant in calves with diarrhea and high infection intensity. Notably, IIaA24G1R1 represents a novel, previously unrecognized C. parvum subtype. The subtype IIaA18G1R1, frequently found in this study, is strongly implicated in zoonotic transmission. These results suggest that calves might be an important source for human cryptosporidiosis in Argentina.
Assuntos
Doenças dos Bovinos/epidemiologia , Criptosporidiose/epidemiologia , Cryptosporidium parvum/classificação , Cryptosporidium/classificação , Diarreia/veterinária , Animais , Argentina/epidemiologia , Bovinos , Doenças dos Bovinos/parasitologia , Criptosporidiose/parasitologia , Criptosporidiose/transmissão , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Cryptosporidium parvum/genética , Cryptosporidium parvum/isolamento & purificação , Diarreia/epidemiologia , Diarreia/parasitologia , Fezes/parasitologia , Feminino , Glicoproteínas/genética , Humanos , Oocistos , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Prevalência , ZoonosesRESUMO
Cryptosporidiosis is an emergent zoonotic disease caused by the globally distributed protozoa Cryptosporidium spp. Although several Cryptosporidium studies related to humans and many animal species have been published, there are still limited studies on the epidemiology of Cryptosporidium infection in bats. The aim of this study was to determine the occurrence of Cryptosporidium spp. and to perform the molecular characterization of Cryptosporidium species and genotypes in fecal samples from bats in an urban area of the municipality of Araçatuba, state of São Paulo, Brazil. Nested PCR targeting the 18S rRNA, actin, and HSP-70 genes was performed to screen 141 fecal samples from bats and detected Cryptosporidium spp. in 16.3% (23/141) of the samples. Bidirectional sequencing identified three novel Cryptosporidium bat genotypes (XVI, XVII, and XVIII) and a new genotype (18SH) genetically similar to Cryptosporidium avium in six species of bats. This is the first report on the occurrence and molecular characterization of Cryptosporidium spp. in Brazilian bats. Zoonotic Cryptosporidium species were not found in fecal samples from bats living in an urban area in the municipality of Araçatuba, state of São Paulo, Brazil.
Assuntos
Quirópteros/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/classificação , Cryptosporidium/genética , Actinas/genética , Animais , Brasil/epidemiologia , Cryptosporidium/isolamento & purificação , Fezes/parasitologia , Genótipo , Proteínas de Choque Térmico HSP70/genética , Humanos , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , Zoonoses/parasitologiaRESUMO
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 26 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.(AU)
O estudo foi realizado num total de 25 propriedades localizadas nos assentamentos São José I e Salvador, situados nos municípios de Brejo Alegre e Birigui, no estado de São Paulo. Um registro de variáveis foi elaborado, incluindo dados como sexo, raça e idade dos animais. Foram colhidas 231 amostras de fezes de bovinos de um a seis meses de idade, sendo 128 fêmeas e 103 machos, 131 mestiços e 100 da raça Holandesa. Entre os 231 bovinos examinados, 17 (7,36%) foram positivos para Cryptosporidium spp. tanto pela coloração negativa de verde malaquita como pela nested-PCR. Das 17 amostras positivas, 14 apresentaram amplificação pela eletroforese em gel de agarose suficiente para fazer o sequenciamento de DNA. Essas sequências foram detectadas similaridade genética entre 99% e 100% com as seguintes espécies. Uma sequência foi semelhante com C. parvum (referência: AB513880.1), uma com C. bovis (MF074602.1), duas com C. ryanae (KT922233.1), uma com C. felis (KM977642.1) e nove foram semelhantes com C. andersoni (MF350628). O estudo caracteriza a presença do Cryptosporidium spp. em bovinos oriundos de propriedades produtoras de leite na região Noroeste do estado de São Paulo, sendo o C. andersoni a espécie mais prevalente nesses animais, principalmente em uma faixa etária diferente das descritas por diversos autores. A presença de C. parvum indica que os bezerros da região estudada devem ser considerados como uma fonte potencial de oocistos de espécies zoonóticas. Identificamos com ineditismo o C. felis em bovinos na América, o que corrobora outros estudos realizados na Polônia e Espanha e evidencia a presença de espécies de Cryptosporidium em fezes em hospedeiros não naturais.(AU)
Assuntos
Animais , Recém-Nascido , Bovinos , Cryptosporidium/classificação , Cryptosporidium/isolamento & purificação , Criptosporidiose/classificação , Coccidiose/veterinária , Reação em Cadeia da Polimerase/veterinária , População RuralRESUMO
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 26 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.
O estudo foi realizado num total de 25 propriedades localizadas nos assentamentos São José I e Salvador, situados nos municípios de Brejo Alegre e Birigui, no estado de São Paulo. Um registro de variáveis foi elaborado, incluindo dados como sexo, raça e idade dos animais. Foram colhidas 231 amostras de fezes de bovinos de um a seis meses de idade, sendo 128 fêmeas e 103 machos, 131 mestiços e 100 da raça Holandesa. Entre os 231 bovinos examinados, 17 (7,36%) foram positivos para Cryptosporidium spp. tanto pela coloração negativa de verde malaquita como pela nested-PCR. Das 17 amostras positivas, 14 apresentaram amplificação pela eletroforese em gel de agarose suficiente para fazer o sequenciamento de DNA. Essas sequências foram detectadas similaridade genética entre 99% e 100% com as seguintes espécies. Uma sequência foi semelhante com C. parvum (referência: AB513880.1), uma com C. bovis (MF074602.1), duas com C. ryanae (KT922233.1), uma com C. felis (KM977642.1) e nove foram semelhantes com C. andersoni (MF350628). O estudo caracteriza a presença do Cryptosporidium spp. em bovinos oriundos de propriedades produtoras de leite na região Noroeste do estado de São Paulo, sendo o C. andersoni a espécie mais prevalente nesses animais, principalmente em uma faixa etária diferente das descritas por diversos autores. A presença de C. parvum indica que os bezerros da região estudada devem ser considerados como uma fonte potencial de oocistos de espécies zoonóticas. Identificamos com ineditismo o C. felis em bovinos na América, o que corrobora outros estudos realizados na Polônia e Espanha e evidencia a presença de espécies de Cryptosporidium em fezes em hospedeiros não naturais.
Assuntos
Animais , Recém-Nascido , Bovinos , Coccidiose/veterinária , Criptosporidiose/classificação , Cryptosporidium/classificação , Cryptosporidium/isolamento & purificação , População Rural , Reação em Cadeia da Polimerase/veterináriaRESUMO
Cats and dogs are hosts of a large number of gastrointestinal parasites and can shed helminth eggs and protozoan oocysts in their feces. The close relationship between companion animals and humans intensifies human exposure to zoonosis caused by parasites. In this study, 177 fecal samples were collected: 128 from dogs and 49 from cats of both sexes and varied ages. One or more intestinal parasites were observed in 56.2% (72/128) of the dog fecal samples and in 53.0% (26/49) of the cat fecal samples. Parasitic monoinfection was present in 70.8% (51/72) of dog samples and in 46.1% (12/26) of cat samples, whereas multi-infection was observed in 29.2% (21/72) and 53.8% (14/26) of dog and cat samples, respectively. The detection frequency of Cryptosporidium spp. was 22.6% (40/177) using Ziehl-Neelsen staining. DNA was extracted from all samples and the Cryptosporidium small subunit ribosomal ribonucleic acid (SSU rRNA) gene was amplified from 5.6% (10/177) of the fecal samples using nested polymerase chain reaction (PCR). Amplification was achieved in 4.6% (6/128) of the dog samples and in 8.2% (4/49) of the cat samples. DNA sequencing of the nested PCR positive samples identified Cryptosporidium canis in 66.6% (4/6) and Cryptosporidium parvum in 33.3% (2/6) of the dog samples and Cryptosporidium felis in 75% (3/4) and Cryptosporidium parvum in 25% (1/4) in the cat samples. The present study thus demonstrated significant levels of gastrointestinal parasite infection in companion animals and highlighted the presence of zoonosis agents.