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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.

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RESUMO

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.

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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.

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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.

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