RESUMO
Cryptosporidiosis has been reported as an important cause of neonatal diarrhea and mortality in cattle, sheep, and other ruminants, but its impact on alpaca health has not been studied thoroughly. In this study, we have determined the prevalence and evaluated the role of cryptosporidiosis as a risk factor for diarrhea occurrence in newborn alpacas. During the calving season (January-March) of 2006, stool specimens (N = 1312) were collected from 24 herds of newborn alpacas in Puno and Cuzco, departments that account for the largest populations of alpacas in Peru. All the specimens were microscopically screened for Cryptosporidium spp. using the acid-fast technique. The association between Cryptosporidium detection and diarrhea was analyzed using χ2 test and generalized lineal model. Cryptosporidium species were determined by PCR-RFLP analysis of the small subunit rRNA gene. Cryptosporidium oocysts were detected in 159 of 1312 (12.4%) newborn alpacas. Results of the analyses demonstrated that crypstosporidiosis was significantly associated with diarrhea (PR = 3.84; CI95% 2.54-5.81; p < 0.0001). Only Cryptosporidium parvum was detected in the 153 Cryptosporidium-infected animals. Thus, there is an association of C. parvum infection with diarrhea in neonatal alpacas.
Assuntos
Camelídeos Americanos/parasitologia , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/isolamento & purificação , Diarreia/veterinária , Animais , Animais Recém-Nascidos , Cryptosporidium parvum/classificação , Cryptosporidium parvum/citologia , Cryptosporidium parvum/genética , Diarreia/epidemiologia , Diarreia/parasitologia , Fezes/parasitologia , Oocistos/citologia , Peru/epidemiologia , Prevalência , Subunidades Ribossômicas Menores/genética , Fatores de RiscoRESUMO
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
Cryptosporidium can infect a wide variety of vertebrate animals, including mammals, birds, amphibians, reptiles, and fish. There are few molecular characterizations of Cryptosporidium isolated from water buffalo. Thus, the present study investigated the occurrence and molecular characterization of Cryptosporidium spp. in water buffalos by nested-PCR. Non-diarrheic feces were obtained from 122 water buffalo calves. All samples were tested by nested-PCR based on the 18S rRNA gene, after which positive samples were analyzed by RFLP and genetic sequencing. Sixteen fecal (13.1%) samples were positive, and RFLP showed that fifteen presented patterns consistent with C. ryanae and one with C. parvum. Sequencing of the gp60 gene from the C. parvum positive sample indicated the subtype IIaA20G1R1. This is the first identification of the IIaA20G1R1 subtype in water buffalos.
Assuntos
Búfalos , Doenças dos Bovinos/epidemiologia , Criptosporidiose/epidemiologia , Cryptosporidium parvum/isolamento & purificação , Animais , Bovinos , Cryptosporidium , Cryptosporidium parvum/classificação , Fezes/parasitologiaRESUMO
Fecal specimens from 432 pre-weaned calves younger than 35 days were collected over a 2-year period (2010-2012) from 74 dairy cattle farms in the central area of Colombia. These samples were microscopically examined for the presence of Cryptosporidium oocysts, and positive specimens were selected for molecular examination. Microscopy revealed that 115 calves (26.6%) from 44 farms (59.5%) tested positive. Oocyst shedding was recorded in calves aged 3-day-old onwards, although the infection rate peaked at 8-14 days (40.7%). Infection rates were higher in diarrheic (52.2%) than in non-diarrheic calves (19.9%) (p < 0.0001, χ2), and infected calves had up to seven times more probability of having diarrhea than non-infected calves. Cryptosporidium species and subtypes were successfully identified in 73 samples from 32 farms. Restriction and sequence analyses of the SSU rRNA gene revealed C. parvum in all but two isolates identified as Cryptosporidium bovis. Sequence analyses of the 60-KDa glycoprotein (gp60) gene revealed eight subtypes within the IIa family. An unusual subtype (IIaA18G5R1) was the most prevalent and widely distributed (more than 66% specimens and 68% farms) while the subtype most frequently reported in cattle worldwide (IIaA15G2R1) was found in less than 13% of specimens and 16% farms. The remaining subtypes (IIaA16G2R1, IIaA17G4R1, IIaA20G5R1, IIaA19G6R1, IIaA20G6R1, and IIaA20G7R1) were restricted to 1-3 farms. This is the first large-sample size study of Cryptosporidium species and subtypes in Colombia and demonstrates the genetic uniqueness of this protozoan in cattle farms in this geographical area.
Assuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/epidemiologia , Cryptosporidium parvum/isolamento & purificação , Diarreia/veterinária , Oocistos/genética , Animais , Bovinos , Colômbia/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/classificação , Cryptosporidium parvum/genética , Indústria de Laticínios , Diarreia/parasitologia , Fazendas , Fezes/parasitologia , Oocistos/classificação , Oocistos/isolamento & purificação , PrevalênciaRESUMO
We describe two patients with HIV/AIDS who presented pulmonary and intestinal infection caused by Cryptosporidium parvum, with a fatal outcome. The lack of available description of changes in clinical signs and radiographic characteristics of this disease when it is located in the extra-intestinal region causes low prevalence of early diagnosis and a subsequent lack of treatment.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/isolamento & purificação , Enteropatias Parasitárias/parasitologia , Pneumopatias Parasitárias/parasitologia , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Adulto , Coinfecção , Criptosporidiose/diagnóstico , Cryptosporidium parvum/classificação , Evolução Fatal , Fezes/parasitologia , Feminino , Humanos , Enteropatias Parasitárias/diagnóstico , Pneumopatias Parasitárias/diagnóstico , Pessoa de Meia-IdadeRESUMO
We evaluated the presence of DNA of Giardia, Toxoplasma, and Cryptosporidium by PCR, and of Giardia and Cryptosporidium genera by immunofluorescence antibody test (IFAT), in water samples, before, during, and after plant treatment for drinkable water. We applied this method in 38 samples of 10 l of water taken from each of the water treatment steps and in 8 samples taken at home (only for Toxoplasma PCR) in Quindio region in Colombia. There were 8 positive samples for Cryptosporidium parvum (21 %), 4 for Cryptosporidium hominis (10.5 %), 27 for Toxoplasma gondii (58.6 %), 2 for Giardia duodenalis assemblage A (5.2 %), and 5 for G. duodenalis assemblage B (13.1 %). By IFAT, 23 % were positive for Giardia and 21 % for Cryptosporidium. An almost perfect agreement was found between IFAT and combined results of PCR, by Kappa composite proportion analysis. PCR positive samples were significantly more frequent in untreated raw water for C. parvum (p = 0.02). High mean of fecal coliforms, high pH values, and low mean of chlorine residuals were strongly correlated with PCR positivity for G. duodenalis assemblage B. High pH value was correlated with PCR positivity for C. parvum. Phylogenetic analysis of DNA sequences was possible, showing water and human clinical sequences for Toxoplasma within the same phylogenetic group for B1 repeated sequence. PCR assay is complementary to IFAT assay for monitoring of protozoa in raw and drinkable water, enabling species identification and to look for phylogenetic analysis in protozoa from human and environmental sources.
Assuntos
Cryptosporidium parvum/isolamento & purificação , Água Potável/parasitologia , Giardia lamblia/isolamento & purificação , Toxoplasma/isolamento & purificação , Purificação da Água , Animais , Sequência de Bases , Colômbia , Criptosporidiose/parasitologia , Cryptosporidium parvum/classificação , Cryptosporidium parvum/genética , DNA de Protozoário/genética , Fezes/parasitologia , Técnica Direta de Fluorescência para Anticorpo/métodos , Giardia lamblia/classificação , Giardia lamblia/genética , Giardíase/parasitologia , Humanos , Filogenia , Reação em Cadeia da Polimerase/métodos , Proteínas de Protozoários/genética , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasmose/parasitologiaRESUMO
The presence of Cryptosporidium spp. in a cattle herd registered with an outbreak of diarrhea was investigated and the the molecular subtyping of Cryptosporidium parvum was characterized. Fecal samples from 85 Nellore beef cattle (Bos indicus) were collected and examined with Ziehl-Neelsen modified staining method. Fifty-four cattle (63.52%) had Cryptosporidium spp. oocysts in their feces. Fragments of genes encoding the 18S ribosomal RNA subunit and a 60-kDa glycoprotein (gp60) were amplified by nested PCR accomplished in the 11 most heavily parasitized samples, and the amplicons were sequenced. Eight of the 11 analyzed samples were positive for 18S rRNA sequences and identified monospecific infections with C. parvum. Seven samples were positive for gp60 and identified subtypes IIaA15G2R1 (6/11) and IIaA14G2R1 (1/11). This report is the first for C. parvum subtype IIaA14G2R1 in beef cattle in Brazil.
Assuntos
Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , Diarreia/veterinária , Animais , Brasil/epidemiologia , Bovinos , Cryptosporidium parvum/classificação , Cryptosporidium parvum/isolamento & purificação , DNA de Protozoário/genética , Diarreia/epidemiologia , Diarreia/parasitologia , Fezes/parasitologia , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 18S/genética , Análise de Sequência de DNA/veterináriaRESUMO
To further understand the composition of population of parasite in a single host, we analyzed the GP60 gene of Cryptosporidium parvum amplified from DNA of a randomly selected isolate found in the feces of a diarrheic calf from a dairy farm in Central Chile. Direct sequencing of the amplicon yield the IIaA17G4R1 C. parvum subtype. The same amplicon was cloned in Escherichia coli (22 clones) and sequenced, yielding three different GP60 subtypes, IIaA17G4R1 (16/22), IIaA16G4R1 (1/22), and IIaA15G4R1 (1/22), and four sequences with nucleotide substitutions in the serine repeats, which subtype would be otherwise IIaA17G4R1. It is thus possible to determine allelic polymorphism using Sanger sequencing with an additional step of bacterial cloning. The results also indicate the necessity to further characterize parasite populations in a single host to better understand the dynamics of Cryptosporidium epidemiology.
Assuntos
Animais Recém-Nascidos , Doenças dos Bovinos/parasitologia , Cryptosporidium parvum/classificação , Diarreia/veterinária , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/epidemiologia , Chile/epidemiologia , Criptosporidiose/parasitologia , DNA de Protozoário/genética , Diarreia/epidemiologia , Diarreia/parasitologia , Fezes/parasitologia , Polimorfismo GenéticoRESUMO
Cryptosporidium parvum from 73 dairy calves less than two months old from Buenos Aires province (Argentina) were molecularly characterized using sequence analysis of the GP60 gene. Seventy-five sequences were obtained, and seven different subtypes were identified, all belonging to the IIa subtype family. The most common subtypes were IIaA20G1R1 (27/75), IIaA22G1R1 (16/75), and IIaA18G1R1 (13/75). Subtypes IIaA21G1R1, IIaA23G1R1, IIaA16G1R1 and IIaA19G1R1 were found sporadically. Two samples contained mixed infections with IIaA21G1R1 and IIaA22G1R1. A significant association was found between subtypes and geographic location, whereas there was no relation between subtypes and presence of diarrhea. Three of the subtypes found in this study (IIaA16G1R1, IIaA18G1R1, and IIaA19G1R1) were previously identified in humans. These findings suggest that cattle could play an important role in the transmission of cryptosporidiosis to humans in Buenos Aires province.
Assuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , Diarreia/veterinária , Zoonoses/parasitologia , Animais , Animais Lactentes , Argentina , Sequência de Bases , Bovinos , Distribuição de Qui-Quadrado , Cryptosporidium parvum/classificação , DNA de Protozoário/química , DNA de Protozoário/genética , Diarreia/parasitologia , Fezes/parasitologia , Feminino , Humanos , Dados de Sequência Molecular , Contagem de Ovos de Parasitas/veterinária , Reação em Cadeia da Polimerase/veterinária , Análise de Sequência de DNARESUMO
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.