Cryptosporidium spp. in dogs - prevalence and genotype distribution

Background: Cryptosporidium spp. is a zoonotic protozoan parasite that affects the gastrointestinal tract of humans and animals. The disease can cause acute and chronic diarrhoea and even death in both humans and animals. In this study, it was aimed to determine the prevalence and genotype distribution of Cryptosporidiosis in shelter dogs in Diyarbakir province located in the Southeastern Anatolia Region of Turkey. Materials, Methods & Results: The animal material of the study consisted of 100 dogs of different breeds and sexes. Faecal samples were collected from the rectum with disposable latex gloves and placed in individual sample containers. All of the samples were examined for Cryptosporidium spp. by Kinyoun Acid Fast and Nested PCR methods. In the Kinyoun Acid Fast staining method, firstly, smear preparations were prepared from fresh faecal samples, fixed in pure methanol for 1 min and allowed to dry. The slides were kept in Kinyoun Carbol-Fuxin for 5 min, dipped in 50% ethyl alcohol, shaken, washed in tap water, kept in 1% sulphuric acid for 2 min and washed in tap water. The slides were kept in methylene blue for 1 min, washed in tap water and allowed to dry. After drying, immersion oil was dripped and examined under a microscope at 100 magnification. DNA extraction was performed from all samples using GeneMATRIX Stool DNA Purification Kit according to the manufacturer's protocol. After Nested PCR analysis was performed. In the PCR step, primers 5'-TTCTAGAGCTAATACATGCG-3' and 5'- CCCATTTCCTTCCTTCGAAACAGGA-3' were used to amplify the 1325 bp gene region. In the nested PCR step, primers 5'- GGAAGGGTTGTATTTATTTATTAGATAAAG-3' and 5'-AAGGAGTAAGGAACAACCTCCA-3' were used to amplify the 826-864 bp gene region. As a result of both methods, a prevalence of 3% was determined. The infection rate was higher in males (3.57%) than females (2.27%) and in younger than 1 year (5.56%) than in older than 1 year (1.56%). The DNA sequences obtained from the sequence analysis of 3 positive PCR samples were analysed in BioEdit software. A phylogenetic tree was constructed with the data set created by using the 18s rRNA gene sequences obtained from the NCBI genbank database and the DNA sequences obtained as a result of the study, and it was shown which Cryptosporidium species the study samples were related to. Today, many Cryptosporidium species have been identified and most of these species have host adaptation. Although C. canis is the most common species in dogs, C. muris, C. meleagridis, and C. parvum have also been detected. Among these species, C. parvum is recognized as a zoonotic species infecting a wide range of mammals. In this study, DNA sequencing of nested PCR positive samples revealed that 3 samples were zoonotic C. parvum. Discussion: This suggests that dogs may be a reservoir for zoonotic transmission of Cryptosporidium. Consequently, it is recommended that people should be informed about the potential for transmission of this protozoan to humans and animals and that control programmes should be implemented, including the prevention of free entry of stray dogs into public places and homes.

Cryptosporidium diagnosis in different groups of children and characterization of parasite species.

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 modified 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.

Cryptosporidiosis outbreak in Amazonia, French Guiana, 2018.

BACKGROUND: Cryptosporidiosis outbreaks in South America are poorly documented. In March 2018, 51 cases of cryptosporidiosis were reported in Maripasoula, a village located in a remote forest area along the border between Surinam and French Guiana. METHOD: To identify the origin of the epidemic, we performed epidemiological, microbiological, and environmental investigations. Only the cases involving diarrhoea and Cryptosporidium-positive stool were considered as bona fide, while cases involving diarrhoea and close contact with a confirmed case were classified as "possible". RESULTS: We identified 16 confirmed cases and 35 possible ones. Confirmed cases comprised nine children (median age of 18 months, range: 6-21), one immunocompromised adult and six soldiers. One child required a hospitalisation for rehydration. All 16 Cryptosporidium stools were PCR positive, and sequencing of the gp60 gene confirmed only one Cryptosporidium hominis subtype IbA10G2. Tap water consumption was the only common risk factor identified. Contamination of the water network with Cryptosporidium parvum subtype IIdA19G2 was found. CONCLUSION: Water quality is a major public health issue in Amazonian French Guiana, especially for population at risk (children, people with comorbidity, travelers). For them, alternative water supply or treatment should be implemented.

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.

Cryptosporidium parvum as a risk factor of diarrhea occurrence in neonatal alpacas in Peru.

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.

Prevalence of Cryptosporidium parvum in dairy calves and GP60 subtyping of diarrheic calves in central Argentina.

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.

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.

Cryptosporidium parvum in captive primates of Parque Municipal Danilo Galafassi, Paraná, Brazil / Cryptosporidium parvum em primatas cativos do Parque Municipal Danilo Galafassi, Paraná, Brasil

A wide variety of terrestrial and aquatic animal species have been identified as hosts of species and genotypes of Cryptosporidium spp., which are important pathogens, however, little is known about their distribution in wild populations. Recent studies associating parasitological findings and molecular techniques have provided a new insight into host specificity and its potential transmission to humans.The objective of this study was to investigate the presence of Cryptosporidium spp. in feces of Callithrixsp. and Ateles paniscus, identify the species, and evaluate their phylogenetic relationships with other representatives of the genus. Four samples of feces were collected from an enclosure where three Callithrix jacchus and one Callithrix penicillate live; in addition, five samples were collected from an enclosure of an Ateles paniscus from Parque Municipal Danilo Galafassi, located in the city of Cascavel-PR. These samples were sent to the UFPR Biotechnology Laboratory, where the modified Ziehl-Neelsen staining technique was performed on microscope slides with fecal smear. Positive samples were submitted to DNA purification, extraction, PCR, and sequencing of the nuclear SSU rRNA region. Phylogenetic analysis based on Maximum Parcimony and Bayesian Inference were performed. Fifty percent (2: 4) of the feces samples from the enclosure of the Callithrix spp. and 60 % (3: 5) of samples from the Ateles paniscus enclosure were positive for Cryptosporidium spp. The phylogenetic analysis showed that the parasite found in both species of primates was recovered nested with others genotypes of C. parvum, and the genotype found in Callithrix spp. has high similarity with that one founded in several domestic animals. This is the first report of C. parvum in A. paniscus. Because it is an important zoonosis which does not have treatment, preventive measures must be adopted to avoid the spread of the disease.(AU)

Uma grande variedade de espécies animais terrestres e aquáticas tem sido identificada como hospedeiros de espécies e genótipos de Cryptosporidium spp., que são importantes agentes patogênicos, mas pouco se conhece sobre a sua distribuição nas populações silvestres. Estudos recentes associando achados parasitológicos e técnicas moleculares têm proporcionado uma nova visão em relação à especificidade do hospedeiro e seu potencial de transmissão para o homem. O objetivo desse estudo foi pesquisar a presença de Cryptosporidium spp. em fezes de Callithrix spp. e Ateles paniscus, identificar a espécie e avaliar o seu relacionamento filogenético com outros representantes do gênero. Foram coletadas quatro amostras de fezes de um recinto onde convivem três Callithrix jacchus e um Callithrix penicillata e cinco amostras de um recinto onde vive um Ateles paniscus do Parque Municipal Danilo Galafassi localizado na cidade de Cascavel-PR. As amostras foram enviadas ao Laboratório de Biotecnologia da UFPR onde foi realizada a técnica de coloração de Ziehl-Neelsen modificado em lâminas de esfregaço de fezes. As amostras positivas foram submetidas à purificação, extração de DNA, PCR, e sequenciamento da região nuclear SSU rRNA. Foram realizadas análises filogenéticas baseadas em Máxima Parcimônia e Inferência Bayesiana. Cinquenta por cento (2:4) das amostras de fezes do recinto dos Callithrix...(AU)

Multilocus fragment analysis of Cryptosporidium parvum from pre-weaned calves in Colombia.

The intra-species genetic diversity of Cryptosporidium parvum in dairy cattle farms in the central area of Colombia was investigated using a multilocus fragment typing approach with nine variable-number tandem-repeat (VNTR) loci and the gp60 gene. Genomic DNA of 70 C. parvum isolates from pre-weaned calves in 32 farms was analysed. Most markers showed two (ML1, MSB, CP47, and MSC6-7) or three alleles (5B12, Cgd2_3850, and Cgd6_5400), although they exhibited a major allele accounting for more than 69% of specimens, which explains their low discriminatory index. The TP14 microsatellite was monomorphic while a total of six alleles were found at the ML2 microsatellite. The two novel allelic variants (219bp, 245bp) exhibited by more than 36% of specimens at the latter locus were a remarkable finding. The 10-markers typing tool provided a Hunter-Gaston discriminatory value of 0.940 (95% CI, 0.918 - 0.961) and differentiated 22 multilocus subtypes (MLTs). Nevertheless, the combination of the three most informative markers (ML2, gp60, and Cgd2_3850) differentiated 68% of MLTs and hardly impaired the discriminatory index. The fact that many MLTs (13/22) were distinctive for individual farms provides evidence for the endemic nature of the infection and the major role played by transmission within farms. The eBURST algorithm suggested a low degree of genetic divergence. All but three MLTs were clustered in a clonal complex with a star-like topology typical of clonal expansion, however linkage analysis did not find evidence of linkage disequilibrium. Bayesian analysis also identified a genetic structure with K = 3 being the best estimation of ancestral clusters, although a large proportion of isolates (35%) could not be allocated to a single population, which indicates their mixed origin. The results confirm the genetic distinctiveness of C. parvum in cattle farms in this geographical area. This is the first multilocus analysis on the intra-specific variability of Cryptosporidium from calves in South America.

A highly sensitive method for detecting Cryptosporidium parvum oocysts recovered from source and finished water using RT-PCR directed to Cryspovirus RNA.

Sensitive detection of Cryptosporidium oocysts is important because the protozoan can cause clinical infection in humans at extremely low numbers. In the present study, 1.5 × 102, 1.0 × 103, or 1.0 × 104C. parvum oocysts were spiked into 10 l of source or finished water in triplicate followed by recovery using Envirochek HV sampling capsules. One subsample of the recovered oocysts was analyzed by commercial immunofluorescence assay (IFA), while a second subsample was subjected to DNA-RNA extraction, followed by RT-PCR using primers directed to the gene encoding Cryspovirus capsid. IFA analysis of Envirochek filter eluates of finished water detected oocysts at all 3 C. parvum oocyst doses, but only at the 1.0 × 103 and 1.0 × 104 doses in source water. Cryspovirus RT-PCR appeared to offer greater sensitivity than IFA because C. parvum oocysts were detected using this molecular technique in both source and finished water concentrates at all 3 spiking levels. A linear relationship was observed between log oocysts spiking dose and the relative intensity of the Cryspovirus RT-PCR signal for finished water, but not for source water. These data indicate that Cryspovirus RT-PCR is a sensitive method for detecting C. parvum oocysts in source and finished water.

Cryptosporidium parvum in captive primates of Parque Municipal Danilo Galafassi, Paraná, Brazil / Cryptosporidium parvum em primatas cativos do Parque Municipal Danilo Galafassi, Paraná, Brasil

A wide variety of terrestrial and aquatic animal species have been identified as hosts of species and genotypes of Cryptosporidium spp., which are important pathogens, however, little is known about their distribution in wild populations. Recent studies associating parasitological findings and molecular techniques have provided a new insight into host specificity and its potential transmission to humans.The objective of this study was to investigate the presence of Cryptosporidium spp. in feces of Callithrixsp. and Ateles paniscus, identify the species, and evaluate their phylogenetic relationships with other representatives of the genus. Four samples of feces were collected from an enclosure where three Callithrix jacchus and one Callithrix penicillate live; in addition, five samples were collected from an enclosure of an Ateles paniscus from Parque Municipal Danilo Galafassi, located in the city of Cascavel-PR. These samples were sent to the UFPR Biotechnology Laboratory, where the modified Ziehl-Neelsen staining technique was performed on microscope slides with fecal smear. Positive samples were submitted to DNA purification, extraction, PCR, and sequencing of the nuclear SSU rRNA region. Phylogenetic analysis based on Maximum Parcimony and Bayesian Inference were performed. Fifty percent (2: 4) of the feces samples from the enclosure of the Callithrix spp. and 60 % (3: 5) of samples from the Ateles paniscus enclosure were positive for Cryptosporidium spp. The phylogenetic analysis showed that the parasite found in both species of primates was recovered nested with others genotypes of C. parvum, and the genotype found in Callithrix spp. has high similarity with that one founded in several domestic animals. This is the first report of C. parvum in A. paniscus. Because it is an important zoonosis which does not have treatment, preventive measures must be adopted to avoid the spread of the disease.

Uma grande variedade de espécies animais terrestres e aquáticas tem sido identificada como hospedeiros de espécies e genótipos de Cryptosporidium spp., que são importantes agentes patogênicos, mas pouco se conhece sobre a sua distribuição nas populações silvestres. Estudos recentes associando achados parasitológicos e técnicas moleculares têm proporcionado uma nova visão em relação à especificidade do hospedeiro e seu potencial de transmissão para o homem. O objetivo desse estudo foi pesquisar a presença de Cryptosporidium spp. em fezes de Callithrix spp. e Ateles paniscus, identificar a espécie e avaliar o seu relacionamento filogenético com outros representantes do gênero. Foram coletadas quatro amostras de fezes de um recinto onde convivem três Callithrix jacchus e um Callithrix penicillata e cinco amostras de um recinto onde vive um Ateles paniscus do Parque Municipal Danilo Galafassi localizado na cidade de Cascavel-PR. As amostras foram enviadas ao Laboratório de Biotecnologia da UFPR onde foi realizada a técnica de coloração de Ziehl-Neelsen modificado em lâminas de esfregaço de fezes. As amostras positivas foram submetidas à purificação, extração de DNA, PCR, e sequenciamento da região nuclear SSU rRNA. Foram realizadas análises filogenéticas baseadas em Máxima Parcimônia e Inferência Bayesiana. Cinquenta por cento (2:4) das amostras de fezes do recinto dos Callithrix...

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.

First identification of Cryptosporidium parvum subtype IIaA20G1R1 in water buffalos (Bubalus bubalis).

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.

Genetic uniqueness of Cryptosporidium parvum from dairy calves in Colombia.

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.

Does the use of tubular digesters to treat livestock waste lower the risk of infection from Cryptosporidium parvum and Giardia lamblia?

Worldwide, high incidences of cryptosporidiosis and giardiasis are attributed to livestock waste. Quantitative microbial risk assessment can be used to estimate the risk of livestock related infections from Cryptosporidium parvum and Giardia lamblia. The objective of this paper was to assess the occupational and public health risks associated with management of raw and anaerobically digested livestock waste in two rural communities in Costa Rica based on fomite, soil and crop contamination and livestock waste management exposure pathways. Risks related to cattle waste were greater than swine waste due to cattle shedding more (oo)cysts. Cryptosporidium parvum also posed a greater risk than Giardia lamblia in all exposure pathways due to livestock shedding high loads of Cryptosporidium parvum oocysts and oocysts' lower inactivation rates during anaerobic digestion compared with Giardia lamblia cysts. The risk of infection from exposure to contaminated soil and crops was significantly lower for a community using tubular anaerobic digesters to treat livestock waste compared to a community where the untreated waste was applied to soil. The results indicate that treatment of livestock waste in small-scale tubular anaerobic digesters has the potential to significantly decrease the risk of infection below the World Health Organization's acceptable individual annual risk of infection (10-4).

Detection of Zoonotic Enteropathogens in Children and Domestic Animals in a Semirural Community in Ecuador.

UNLABELLED: Animals are important reservoirs of zoonotic enteropathogens, and transmission to humans occurs more frequently in low- and middle-income countries (LMICs), where small-scale livestock production is common. In this study, we investigated the presence of zoonotic enteropathogens in stool samples from 64 asymptomatic children and 203 domestic animals of 62 households in a semirural community in Ecuador between June and August 2014. Multilocus sequence typing (MLST) was used to assess zoonotic transmission of Campylobacter jejuni and atypical enteropathogenic Escherichia coli (aEPEC), which were the most prevalent bacterial pathogens in children and domestic animals (30.7% and 10.5%, respectively). Four sequence types (STs) of C. jejuni and four STs of aEPEC were identical between children and domestic animals. The apparent sources of human infection were chickens, dogs, guinea pigs, and rabbits for C. jejuni and pigs, dogs, and chickens for aEPEC. Other pathogens detected in children and domestic animals were Giardia lamblia (13.1%), Cryptosporidium parvum (1.1%), and Shiga toxin-producing E. coli (STEC) (2.6%). Salmonella enterica was detected in 5 dogs and Yersinia enterocolitica was identified in 1 pig. Even though we identified 7 enteric pathogens in children, we encountered evidence of active transmission between domestic animals and humans only for C. jejuni and aEPEC. We also found evidence that C. jejuni strains from chickens were more likely to be transmitted to humans than those coming from other domestic animals. Our findings demonstrate the complex nature of enteropathogen transmission between domestic animals and humans and stress the need for further studies. IMPORTANCE: We found evidence that Campylobacter jejuni, Giardia, and aEPEC organisms were the most common zoonotic enteropathogens in children and domestic animals in a region close to Quito, the capital of Ecuador. Genetic analysis of the isolates suggests transmission of some genotypes of C. jejuni and aEPEC from domestic animals to humans in this region. We also found that the genotypes associated with C. jejuni from chickens were present more often in children than were those from other domestic animals. The potential environmental factors associated with transmission of these pathogens to humans then are discussed.

Cryptosporidium parvum Among Coprolites from La Cueva de los Muertos Chiquitos (600-800 CE), Rio Zape Valley, Durango, Mexico.

: In the present study, 90 coprolites from La Cueva de los Muertos Chiquitos (CMC) were subjected to enzyme-linked immunosorbent assay (ELISA) tests for 3 diarrhea-inducing protozoan parasites, Entamoeba histolytica , Giardia duodenalis , and Cryptosporidium parvum , to determine whether these parasites were present among the people who utilized this cave 1,200-1,400 yr ago. These people, the Loma San Gabriel, developed as a culture out of the Archaic Los Caracoles population and lived throughout much of present-day Durango and Zacatecas in Mexico. The Loma San Gabriel persisted through a mixed subsistence strategy of hunting-gathering and agricultural production. The results of ELISA testing were negative for both E. histolytica and G. duodenalis across all coprolites. A total of 66/90 (∼73% prevalence) coprolites tested positive or likely positive for C. parvum . The high prevalence of C. parvum among CMC coprolites contributes to our growing knowledge of the pathoecology among the Loma San Gabriel who utilized CMC. Herein, we report the successful recovery of C. parvum coproantigens from prehistoric coprolites. The recovery of these coproantigens demonstrates the existence of C. parvum in Mesoamerica before European contact in the 1400s.

INTESTINAL AND PULMONARY INFECTION BY Cryptosporidium parvum IN TWO PATIENTS WITH HIV/AIDS.

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.

Detection by PCR of pathogenic protozoa in raw and drinkable water samples in Colombia.

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.

First genetic identification of Cryptosporidium parvum subtype IIaA14G2R1in beef cattle in Brazil.

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.