Occurrence and molecular characterization of Giardia duodenalis in child population from Colombia.

Giardia duodenalis is one of the most prevalent human intestinal parasite, with children living in developing countries being particularly at risk of infection. The occurrence and molecular diversity of G. duodenalis was investigated in stools specimens from 307 individuals aged one to nineteen years in Colombia. Samples were collected in three educational establishments (n: 163) and two hospital laboratories (n: 144) from urban and rural areas. Feces were concentrated using a biphasic sedimentation method and wet mounts of the sediment were examined by light microscopy. G. duodenalis assemblages and sub-assemblages were determined on positive samples by PCR of the triose phosphate isomerase (tpi), ß-giardin (bg) and small-subunit (ssu) rRNA genes. G. duodenalis infection was detected by microscopy in 23 individuals (7.5%). The protozoan was more prevalent among specimens collected in educational establishments (11.6%) than in those obtained from hospital laboratories (2.8%). Infection was most common in individuals from urban areas and children aged 1-5 years. No significant association between diarrhea and infection could be demonstrated. Twenty Giardia-positive samples were successfully allocated to assemblage B (n: 11), sub-assemblage AII (n: 7), and assemblage A (n: 2). Results indicate the potential for transmission of G. duodenalis infection in children attending educational establishments and individuals from urban areas, where transmission seems to be primarily anthroponotic.

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.

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.

Multilocus typing and population structure of Cryptosporidium from children in Zaragoza, Spain.

A multilocus typing approach with eight variable-number tandem-repeat (VNTR) loci and the GP60 gene was used to analyze the inter- and intra-species variation of 44 Cryptosporidium isolates from pediatric patients in Zaragoza city (NE, Spain). Restriction and sequence analyses of the SSU rRNA gene revealed that Cryptosporidium transmission is mostly anthroponotic in this area, with the predominance of Cryptosporidium hominis (n: 41) over Cryptosporidium parvum (n: 3). GP60 subtyping showed limited genetic diversity and four subtypes were identified, including IbA10G2 (n: 35), IaA24R3 (n: 6), IIaA15G1R1 (n: 1) and IIaA15G2R1 (n: 2). Five out of eight VNTR loci showed a discriminatory power higher than the GP60 gene, although each locus had a predominant allele exhibited by more than 50% of isolates. All but four alleles were associated to either C. hominis or C. parvum and linked alleles at different loci were found. Multilocus typing substantially increased the discriminatory power (Hunter-Gaston index: 0.807, 95% CI, 0.683-0.926) and revealed that genetic diversity is much higher than that reported by GP60 sequencing, since 17 multilocus subtypes (MLTs) were identified. Nearly half of the specimens were allocated to a single major MLT. However, no more than three specimens were allocated to each of the remaining MLTs. Both phylogenetic and population analyses revealed a population clustering of C. hominis according to the GP60 subtype, which indicates the robustness of this marker to differentiate genetic subpopulations. Subpopulations had an overall clonal genetic structure, although traces of genetic flow between them were also observed.

Validation of fragment analysis by capillary electrophoresis to resolve mixed infections by Cryptosporidium parvum subpopulations.

The potential of capillary electrophoresis (CE)-based DNA fragment analysis to identify mixed infections by Cryptosporidium parvum subpopulations was validated using high-resolution slab-gel electrophoresis. A selection of genomic DNA samples from C. parvum isolates with CE electropherogram profiles indicative of two concurrent alleles at one or more of six mini and microsatellite loci (MSB, MS5, ML1, ML2, TP14, 5B12) were analysed. These loci were PCR-amplified and products separated on precast Spreadex EL600 slab gels. ML1 PCR products differing by as little as 3 bp in length were visible after Spreadex gel electrophoresis and fragments were clearly separated for all but the ML2 and 5B12 loci, which generated stutter bands. No stuttering was seen for the remaining markers, having three or more nucleotide motifs in the repeat region. For each sample, the two bands of interest were excised separately, DNA extracted and re-amplified by PCR. Sequencing of these PCR products revealed the expected sequences for both alleles at most samples, except for the longest ML2 and 5B12 alleles which generated indeterminate sequences. Two novel MS5 alleles were successfully sequenced after PCR re-amplification. These findings demonstrate the utility of high-resolution Spreadex gels for analysing the polymorphism of satellite markers of Cryptosporidium isolates and support the validity of CE as a reliable and sensitive tool for detecting mixed Cryptosporidium subpopulations in a single-host infection.

Host association of Cryptosporidium parvum populations infecting domestic ruminants in Spain.

A stock of 148 Cryptosporidium parvum DNA extracts from lambs and goat kids selected from a previous study examining the occurrence of Cryptosporidium species and GP60 subtypes in diarrheic lambs and goat kids in northeastern Spain was further characterized by a multilocus fragment typing approach with six mini- and microsatellite loci. Various degrees of polymorphism were seen at all but the MS5 locus, although all markers exhibited two major alleles accounting for more than 75% of isolates. A total of 56 multilocus subtypes (MLTs) from lambs (48 MLTs) and goat kids (11 MLTs) were identified. Individual isolates with mixed MLTs were detected on more than 25% of the farms, but most MLTs (33) were distinctive for individual farms, revealing the endemicity of cryptosporidial infections on sheep and goat farms. Comparison with a previous study in calves in northern Spain using the same six-locus subtyping scheme showed the presence of host-associated alleles, differences in the identity of major alleles, and very little overlap in MLTs between C. parvum isolates from lambs and those from calves (1 MLT) or isolates from lambs and those from goat kids (3 MLTs). The Hunter-Gaston index of the multilocus technique was 0.976 (95% confidence interval [CI], 0.970 to 0.982), which supports its high discriminatory power for strain typing and epidemiological tracking. Population analyses revealed the presence of two host-associated subpopulations showing epidemic clonality among the C. parvum isolates infecting calves and lambs/goat kids, respectively, although evidence of genetic flow between the two subpopulations was also detected.

Multilocus fragment typing and genetic structure of Cryptosporidium parvum Isolates from diarrheic preweaned calves in Spain.

A collection of 140 Cryptosporidium parvum isolates previously analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and sequence analyses of the small-subunit (SSU) rRNA and 60-kDa glycoprotein (GP60) genes was further characterized by multilocus fragment typing of six minisatellite (MSB and MS5) and microsatellite (ML1, ML2, TP14, and 5B12) loci. Isolates were collected from diarrheic preweaned calves originating from 61 dairy cattle farms in northern Spain. A capillary electrophoresis-based tool combining three different fluorescent tags was used to analyze all six satellites in one capillary. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis of a selection of isolates for every allele. Size discrepancies at all but the 5B12 locus were found for those isolates that were typed by both techniques, although identical size differences were reported for every allele within each locus. A total of eight alleles were seen at the ML2 marker, which contributed the most to the discriminatory power of the multilocus approach. Multilocus fragment typing clearly improved the discriminatory power of GP60 sequencing, since a total of 59 multilocus subtypes were identified based on the combination of alleles at the six satellite loci, in contrast to the 7 GP60 subtypes previously reported. The majority of farms (38) displayed a unique multilocus subtype, and individual isolates with mixed multilocus subtypes were seen at 22 farms. Bayesian structure analysis based on combined data for both satellite and GP60 loci suggested the presence of two major clusters among the C. parvum isolates from cattle farms in this geographical area.

La criptosporidiosis en la region andina de Colombia: seroprevalencia y reconocimiento de antígenos / Cryptosporidiosis in Andean Region of Colombia: seroprevalence and antigen recognition

El objetivo de este estudio fue investigar, por primera vez, la seroprevalencia de la criptosporidiosis en habitantes urbanos y rurales de distintos departamentos de la región andina de Colombia. Se estudió también el reconocimiento de antígenos de Cryptosporidium parvum por los sueros. Entre junio de 1996 y octubre de 1998 se recogieron 1 778 muestras séricas de personas seleccionadas mediante un muestreo "de conveniencia". La detección de anticuerpos (IgM, IgA e IgG) anti-C. parvum se realizó mediante la técnica de ELISA y el reconocimiento de antígenos, por una técnica de inmunoelectrotransferencia. Se halló una prevalencia de 83,3%, mientras que los porcentajes de anticuerpos fueron 72,2% de IgM; 27,7% de IgA y 27,6% de IgG. Los porcentajes de seropositividad más altos se obtuvieron en mujeres, menores de 30 años de edad y personas de procedencia rural. Se observó que la seroprevalencia de IgM disminuye a medida que aumenta la edad, mientras que las de IgG e IgA aumentan al par que la edad. Estos tres isotipos reconocieron con mayor frecuencia los antígenos de 51 a 69 kDa, que pueden considerarse inmunodominantes. Se destacó la inmunorreactividad de IgM e IgA a fracciones proteínicas de 12 a 14 kDa y de 42 a 48 kDa, respectivamente, lo que podría indicar exposición al parásito. Estos resultados parecen indicar que la criptosporidiosis es endémica en esa región de Colombia, por lo que es posible atribuir muchos casos de síndrome diarreico a C. parvum.

The objective of this study was to investigate, for the first time, the seroprevalence of cryptosporidiosis among urban and rural inhabitants in several departments of the Andean region of Colombia. The antigen recognition of Cryptosporidium parvum was also studied with sera. Between June 1996 and October 1998 1 778 serum samples were collected from people selected through convenience sampling. The detection of anti-C. parvum antibodies (IgM, IgA, and IgG) was carried out with enzyme-linked immunosorbent assay, and antigen recognition was done with immunoblotting. A prevalence of 83.3% was found, and the antibody percentages were 72.2% for IgM, 27.7% for IgA, and 27.6% for IgG. Higher seropositivity percentages were found among women, persons less than 30 years old, and residents of rural areas. IgM seroprevalence decreased with age, while IgG and IgA seroprevalences increased with age. These three immunoglobulin isotypes most frequently recognized the antigens from 51 to 69 kDa, which can be considered immunodominant. Of note was the immunoreactivity of IgM and IgA to protein fractions from 12 to 14 kDa and from 42 to 48 kDa, respectively, which could indicate exposure to the parasite. These results indicate that cryptosporidiosis is endemic in the Andean region of Colombia, and that it is possible to attribute many cases of diarrheal syndrome to C. parvum.