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Molecular characterization of Cryptosporidium parvum and Cryptosporidium hominis GP60 subtypes worldwide/Caracterizacion molecular de los subtipos de la GP60 de Cryptosporidium parvum y Cryptosporidium hominis alrededor del mundo.

INTRODUCTION

Cryptosporidium spp. is a ubiquitous protozoan that infects humans and a large variety of vertebrate animals around the world with significant implications for public health. The impact of this parasite on public health can be found in the high morbidity possible in children and immunocompromised people. In addition, Cryptosporidium spp. causes economic losses due to increases in the rates of morbidity and mortality in animals, and due to negative effects on the development of young animals. The route of infection is fecal-oral, nevertheless, the ingestion of oocysts can occur in several ways such as through person-to-person contact, contact with household pets, farm animals or ingestion of contaminated food, drinking water or water contacted during recreation (1).

Cryptosporidiosis is a significant cause of death in calves, potentially producing economic losses for the farms of some countries, however this has still not been assessed. The most pathogenic species of Cryptosporidium is Cryptosporidium parvum, it is a result of the ability of C. parvum sporozoites to invade the intestinal epithelium, after excystation from the oocyst, producing shortening and destruction of the villi, reducing their absorptive capacity, and leading to negative effects on productive processes in the host, such as growth. C. parvum transmission is characterized by a low infectious dose. Following infection, clinical cases appear between 7 and 30 days after the birth of a calf as acute diarrhea, depression, anorexia, abdominal pain and death as a result of dehydration and cardiovascular failure (2).

Studies of the parasite at the morphological and phenotypic levels are unable to establish taxonomic differences (3); therefore epidemiological studies of the parasite, making use of molecular tools, have been carried out in the past two decades, generating information about the species, its genotypes and its subtypes (4) facilitating an understanding of its epidemiology, taxonomy and evolutionary genetics.

Molecular diagnostic

So long as the morphology of the oocysts does not permit differentiation between the species of Cryptosporidium spp., microscopic identification presents problems for the determination of the species that affects humans or animals and the role of these species in the disease or in its transmission. Furthermore, the majority of infections are subclinical and recognition requires more sensitive methods like the polymerase chain reaction (PCR), the current method of choice for diagnosis of the disease. Thus the identification and evaluation of the prevalence of different species of Cryptosporidium has been achieved using molecular tests (5). For this reason, molecular tools have become the key to identification of species (6) and are recognized as essential for the determination of the taxonomy of Cryptosporidium. These tools underlie the ability to understand the biology, epidemiology and relationship to health, identifying the various species of Cryptosporidium and their populations as genotypes that have not been recognized as distinct species (1). The advances in the techniques of molecular biology have significantly improved the diagnosis of cryptosporidiosis, as well as the genetic characterization of species of Cryptosporidium (7).

INTRODUCCION

Cryptosporidium spp. es un protozoo ubicuo que infecta humanos y una gran variedad de animales vertebrados alrededor del mundo, con una implicacion importante en salud publica. Entre las implicaciones en salud publica se encuentra la alta morbilidad que puede ocasionar este parasito en ninos y personas inmunocomprometidas, que junto con las perdidas economicas generadas por aumento en las tasas de mortalidad, morbilidad y efecto negativo en el crecimiento de animales jovenes. La via de infeccion es fecal-oral, sin embargo, la ingestion de ooquistes puede ocurrir de varias maneras como por ejemplo a traves del contacto de persona a persona, contacto con animales de compania y animales de granja o ingestion de alimento contaminado, agua de bebida y aguas recreacionales (1).

La criptosporidiosis es responsable de una importante fatalidad en terneros, pudiendo producir perdidas economicas en las fincas en varios paises; sin embargo, estas aun no han sido tasadas. La especie de Cryptosporidium mas patogena es Cryptosporidium parvum; esto se debe a que los esporozoitos invaden el epitelio intestinal luego de la exquistacion del ooquiste produciendo acortamiento y destruccion de las vellosidades, lo que disminuye la capacidad de absorcion, afectando parametros productivos como el crecimiento. Ademas de su baja dosis infectante, luego de la cual, los casos clinicos apareceran entre los 7 y 30 dias de edad del ternero, pudiendose observar desde diarrea acuosa, depresion, anorexia y dolor abdominal hasta la muerte por la deshidratacion y la falla cardiovascular (2).

Los estudios a nivel morfologico y fenotipico del parasito no permiten establecer diferencias taxonomicas (3); por lo tanto, en las ultimas dos decadas se han realizado estudios epidemiologicos haciendo uso de herramientas moleculares, generando como resultado el conocimiento de especies, genotipos y subtipos del parasito (4) facilitando el entendimiento de la epidemiologia, taxonomia y genetica evolutiva.

Diagnostico molecular

Dado que, la morfologia de los ooquistes no permite diferenciar entre las especies de Cryptosporidium spp., la identificacion microscopica resulta problematica para la determinacion de las especies que afectan a humanos o animales y el papel de las especies en la enfermedad o en la transmision. Ademas, la mayoria de las infecciones son subclinicas y para su reconocimiento se recomienda realizar metodos mas sensibles como la reaccion en cadena de la polimerasa (PCR), metodo de eleccion al momento del diagnostico de la enfermedad; por lo tanto, la identificacion y evaluacion de la prevalencia de las diferentes especies de Cryptosporidium spp. han sido realizada a traves de pruebas moleculares (5). Por esta razon, las herramientas moleculares se han convertido en la clave para la identification de especies (6) y son reconocidas como esenciales para determinar la taxonomia de Cryptosporidium, favoreciendo el entendimiento de la biologia, epidemiologia y salud relacionando la importancia de varias especies de Cryptosporidium y aquellas poblaciones conocidas como genotipos que no han sido reconocidos como especie (1). Los avances en las tecnicas de biologia molecular han mejorado significativamente el diagnostico de la criptosporidiosis, asi como la caracterizacion genetica de las especies de Cryptosporidium (7).

Para la categorizacion de las especies, genotipos y subgenotipos de Cryptosporidium se han empleado tecnicas basadas en PCR, empleando primers para la amplification selectiva de uno o mas loci geneticos (marcadores), seguido de una hidrolisis enzimatica o una secuenciacion (7). La PCR ha permitido la identification y subtipificacion de Cryptosporidium spp., facilitando la identification de las vias de transmision entre animales y humanos (8). Esta tecnica, tambien ha permitido ahondar en el campo de la epidemiologia molecular del parasito facilitando la reconstruction filogenetica y el analisis evolutivo del mismo, identificando genotipos y especies con especificidad y otras con amplia gama de hospedadores (9).

De forma general, para la determinacion de la especie, se han utilizado regiones de baja o moderada variabilidad. Con base en las diferencias encontradas en las secuencias de la subunidad pequena de ARNr, gen de la actina, la proteina de choque termico y en la identificacion de subgenotipos la glicoproteina GP60 han sido descritos diferentes genotipos de Cryptosporidium (4). Lo que hace que la secuenciacion directa de ADN sigue siendo el enfoque mas adecuado para la detection de estas variaciones o polimorfismos geneticos; entre los genes de baja variabilidad se encuentran aquellos que codifican como, por ejemplo, el gen de la subunidad menor del ARN ribosomal (ADNr 18S), la proteina de la pared del ooquiste (COWP), la proteina de choque termico de 70 KDa (HSP-70) o el gen de la actina. Entre las regiones de variabilidad moderada se han utilizado los genes de la p-tubulina, genes TRAP (C1, C2 y C4) o las regiones intergenicas ITS-1 e ITS-2. Estas regiones se utilizan tanto en estudios de taxonomia como diagnostico o epidemiologia; sin embargo, estas regiones unicamente identifican la especie y algunos genotipos (10). Para la identification de genotipos, subtipos o linajes, se utilizan regiones mas variables, como por ejemplo, la subunidad pequena del RNA ribososmal (SSUrRNA) que es usada para genotipificar Cryptosporidium a partir de muestras humanas, animales y aguas. Esto se debe a una multicopia natural del gen y la presencia de regiones semiconservadas y regiones hipervariables, que facilitan el diseno de primers genero especificos (11).

For the categorization of species, genotypes or subtypes of Cryptosporidium, PCR based techniques are used, employing primers for the selective amplification of one or more genetic loci (markers) followed by an enzymatic cleavage or sequencing (7). PCR has permitted the identification and subtyping of Cryptosporidium spp., facilitating identification of the routes of transmission between animals and humans (8). This technique also has deepened investigations in the field of molecular epidemiology of the parasite, facilitating the phylogenetic reconstruction and evolutionary analysis of it, specifically identifying genotypes and species, and others with a wide range of possibilities (9).

In general, for the determination of species, regions of low or moderate variability have been used. Approaches to species determination have been described based on differences encountered in the sequences of the small subunit of rRNA, the gen for actin and the heat shock protein, and in the identification of subtypes of glycoprotein GP60 (4). The direct sequencing of DNA continues to be the best approach for the detection of variations or genetic polymorphisms. Included among the genes of low variability used in these studies are, for example, the gene for the small subunit of rRNA (18S rDNA,) the Cryptosporidium oocyst wall protein (COWP) the 70KDa heat shock protein (HSP-70) or the gene for actin. Within the regions of moderate variability, the genes for p-tubulin, TRAP (C1, C2 and C4) or the intergenic regions ITS-1 and ITS-2 have been used. These genes are used as well in taxonomic studies such as diagnostics or epidemiology; however these regions uniquely identify the species and some genotypes (10). For example, the small subunit of ribosomal RNA (SSUrRNA) is used to genotype Cryptosporidium in human and animal tests and in tests of water. This is due to a natural multicopy gene and the presence of semiconserved and hypervariable regions that facilitate the design of type-specific primers (11).

The analysis of the GP60 gene is frequently used in the subtyping of Cryptosporidium because of the heterogeneity of the sequence and its relevance to the biology of the parasite (11). Therefore, in the identification of genotypes, subtypes or lineages, highly polymorphic regions are used (6), like the GP60 gene and mini or microsatellite regions like ML1 and ML2 (12).

Making use of nested PCR, sequence analysis of the gene for GP60 has found that its sequence is similar to a microsatellite, having repeats of a serine codon (TCA, TCG or TCT) at the extreme 5' terminus of the gene (6,11), finding a high degree of polymorphism in the sequence isolates from C. hominis, C. parvum, and C. meleagridis permitted determination of the genotype and the subtype. A Roman numeral and small case letter identify the subtype. Both represent the genotype of Cryptosporidium spp. For example, Ia and Ib are subtypes of C. hominis, while Ila and lib are subtypes that correspond to C. parvum (13). Various groups of subtypes have been identified in these two species: 7 groups of subtypes in C. hominis (Ia-Ig), 6 groups of subtypes in C. meleagridis and 11 subtypes of families in C. parvum (IIa-III) (4); the subtypes of the families IIa and IId have been recognized as zoonotics (14). Within each group of subtype, various subgenotypes exist principally based on the number of trinucleotide serine repeats (4). The name of the GP60 subtypes begins with the subtype of the designated family (Ia, Ib, Id, Ie, If, etc for C. hominis, and IIa, IIb, IIc, lid, etc for C. parvum) followed by the number of repetitions of TCA (represented by the letter A), TCG (represented by the letter G) or TCT (represented by the letter T). In the subtype of the family of C. parvum IIa, there are a few genotypes that possess two copies of the sequence ACATCA just before the trinucleotide repeat. These genotypes are represented as "R2" (R1 represents many subtypes) (11). For example, the subtype IIaA15G2R1 is a subtype of C. parvum (IIa) with 15 repeats of TCA (A) 2 TCG repeats (G) and one ACATCA (13). In humans, as well as, in farms animals, specially in cattle the subtype of the most prevalent family corresponds to the family IIa, specifically IIaA15G2R1 (11).

Due to the need in epidemiology and public health to characterize the populations and subtypes within the distinct species of the genera Cryptosporidium, the analysis of various hypervariables loci are frequently used (MLT, multilocus typing) that increase the precision of the genotyping. In this way, a few patterns of MLT are created depending on the genotype combinations for each loci analyzed. These studies can be performed by detection of differences in length of the amplified fragments (MLFT) on agarose gels, or by sequencing (MLST), permitting the use of markers with single nucleotide polymorphisms (SNPs) (12). Satellites are characterized by allelic variability, and are used to explore the genetic structure of a population such as in the analysis of lineage and in the construction of a genetic map. Micro and minisatellites have been frequently used in work with other parasites such as Plasmodium spp. and Trypanosoma spp. With the information generated, it has been possible to increase the knowledge of, or to understand the epidemiology of the genetic structure in the population of these parasites (10). Similar markers also have been important tools in the understanding of the structure of the population of C. parvum (15), and have been successfully used to study the population dynamics of Cryptosporidium, evaluating their routes of transmission and their zoonotic potential. In a recent review it was shown that to study the existing variation between C. parvum and C. hominis using multilocus analysis, 55 markers in various combinations have been used over different platforms (16). The markers most used are 5B12, CP47, GP60, hsp70, ML1, ML2, MS5-Mallon, MS9-Mallon, MSB, MSC 6-7 and TP14 (17).

El analisis de la secuencia del gen GP60 es ampliamente usado en la subtipificacion de Cryptosporidium debido a la heterogeneidad de la secuencia y la relevancia en la biologia del parasito (11). Por esto, en la identificacion de genotipos, subtipos o linajes, se utilizan regiones altamente polimorficas (6), como el gen de la GP60 y regiones mini o microsatelites como ML1 y ML2 (12).

Haciendo uso de una PCR anidada, se ha realizado analisis de la secuencia del gen de la GP60, se ha encontrado que esta presenta una secuencia similar a la de un microsatelite, ya que tiene repeticiones del codon serina (TCA, TCG o TCT) en el extremo 5' terminal del gen (6,11), encontrando alto grado de polimorfismo en la secuencia en aislados de C. hominis, C. parvum y C. meleagridis, permitiendo determinar el subtipo y el genotipo. El subtipo es identificado por un numero romano y una letra minuscula, ambos representan el genotipo de Cryptosporidium spp. Por ejemplo Ia y Ib son subtipos de C. hominis; en tanto que, IIa y IIb son subtipos que corresponden a C. parvum (13). Varios grupos de subtipos han sido identificados en estas dos especies: 7 grupos de subtipos en C. hominis (Ia-Ig), 6 grupos de subtipos en C. meleagridis y 11 subtipos de familias en C parvum (IIa - III) (4); los subtipos de las familias IIa y IId han sido reconocidos como zoonoticos (14). Dentro de cada grupo de subtipo, existe varios subgenotipos basados principalmente en el numero de trinucleotidos de serina repetidos (4). El nombre de los subtipos GP60 comienzan con el subtipo de familia designado (Ia, Ib, Id, Ie, If, etc para C. hominis, y IIa, IIb, IIc, IId, etc. para C. parvum) seguido por el numero de repeticiones de TCA (representado por la letra A), TCG (representado por la letra G), o TCT (representado por la letra R). En el subtipo de la familia de C. parvum IIa, son pocos los genotipos que poseen dos copias de las secuencias ACATCA justo antes de que el trinucleotido se repita, el cual esta representado por "R2" (R1 para muchos subtipos) (11). Por ejemplo, el subtipo IIaA15G2R1 es un subtipo de C. parvum (IIa) con 15 repeticiones TCA (A) 2 TCG repetidas (G) y un ACATCA (13). En humanos; asi como, en animales de granja, especialmente en ganado vacuno, el subtipo de familia mas prevalente corresponde a la familia Ila, especificamente, IIaA15G2R1 (11).

Thanks to tools that permit us to obtain biological and genetic data, some genotypes have been recognized as unique and different, taking the name and the status of species. For example, the canine genotype has begun to be called C. canis; the porcine genotype C. suis; the bovine genotype B C. bovis; and the deer-like genotype C. ryanae (1). Furthermore, the specific diagnosis of cryptosporidiosis through molecular tests allows precision in the identification and characterization of species of Cryptosporidium, a central condition for the control of this disease and the comprehension of the complexities of its epidemiology (10).

Molecular epidemiology

The tools of molecular biology have not only helped to resolve the taxonomy of Cryptosporidium, but also have made a valuable contribution in understanding the range of hosts of different species and genotypes (18). Additionally the molecular characterization of the circulating parasites can permit the evaluation of the distribution and zoonotic potential of species and subtypes as well as their routes of transmission to humans and animals under different epidemiological situations (2).

Por otro lado, y debido a la necesidad en epidemiologia y salud publica de caracterizar las poblaciones y subgenotipos dentro de las distintas especies del genero Cryptosporidium, se esta utilizando con frecuencia el analisis de varios loci hipervariables (MLT, multilocus typing) que aumentan la precision del subgenotipado. De este modo, se crean unos patrones de MLT dependiendo de las combinaciones de genotipo para cada loci analizado. Estos estudios se pueden realizar por deteccion de diferencias de longitud de los fragmentos amplificados (MLFT) en gel de agarosa o por secuenciacion (MLST), permitiendo el uso de marcadores con SNP (single nucleotide polymorphism) (12). Los satelites se caracterizan por variabilidad alelica, y son empleados para explorar la estructura genetica de una poblacion asi como el analisis de linaje y el mapeo genetico. Los micro y minisatelites han sido ampliamente usados para el trabajo de otros parasitos tales como Plasmodium spp. y Trypanosoma spp.; con la informacion generada, se ha logrado incrementar o entender la epidemiologia de la estructura genetica de la poblacion de estos parasitos (10); marcadores similares, tambien han sido herramientas importantes en el entendimiento de la estructura de la poblacion de C. parvum (15), y han sido usados exitosamente para estudiar la dinamica poblacional de Cryptosporidium, evaluando las rutas de transmision y su potencial zoonotico. Una revision reciente, revelo que para investigar la variacion existente entre C. parvum y C. hominis mediante el analisis multilocus, se han usado 55 marcadores en varias combinaciones sobre diferentes plataformas (16). Los marcadores mas usados son 5B12, CP47, GP60, hsp70, ML1, ML2, MS5-Mallon, MS9Mallon, MSB, MSC 6-7 y TP14 (17).

Gracias a estas herramientas que permiten obtener datos biologicos y geneticos, algunos genotipos comienzan a ser reconocidos como unicos y diferentes, recibiendo el nombre y el estatus de especie. Por ejemplo, el genotipo canino se comenzo a ser llamado C. canis; el genotipo porcino Cryptosporidium suis; el genotipo bovino B C. bovisy el genotipo deer-like C. ryanae (1). Ademas, el diagnostico especifico de la criptosporidiosis a traves de pruebas moleculares, permite tener precision en la identificacion y caracterizacion de especies de Cryptosporidium, condicion central para el control de esta enfermedad y la comprension de las complejidades de su epidemiologia (10).

At the start of the HIV/AIDS pandemic the reports of pathogenic opportunists focused attention on cryptosporidiosis in humans. A summary of the literature at that time found reports of 159 cases of cryptosporidiosis in immunocompetent patients and 71 cases in immunocompromised patients. In 26 cases a clear association was established between the bovine infection and humans, however the transmission from animals to humans was not confirmed in any of the 71 cases of immunodeficient patients. Additionally, the dissemination from person to person had been reported. The reports of urban transmission without evidence of zoonotic transmission provided support for the hypothesis of Casemore and Jackson, which indicated that the infection in humans was not necessarily zoonotic, leading to the recognition of two independent cycles of transmission. The molecular studies then have provided evidence that these two routes of infection for human were related through two genotypes - the "human genotype" transmitted from human to human and the "bovine genotype" transmitted from animals to humans with the bovine sources as principle reservoirs (1).

The investigations at the epidemiological level required techniques with a greater power of discrimination, that could differentiate an intraspecific level (19). The implementation of subtyping with the GP60 gene has permitted the identification of geographic and temporal differences in the transmission of Cryptosporidium spp., and a better appreciation of the implication of the parasite in public health (4).

In a review including databases from Elsevier, Scielo, PubMed, SpringerLink and Wiley Library, the reports related to subtyping based on GP60 of the parasite in 29 countries, 28 cities that reported 163 subtypes of C. parvum. Concordant with the report by Couto et al. (14) and Feng et al (20), it was found that around the world the family of C. parvum that appeared with the greatest frequency is IIa (41/163), followed by the family IId (17/163), two families that have been recognized for their zoonotic implications. The subtype reported with the greatest frequency and that as well is present on every continent, with the exception of Oceania, is IIaA15G2R1 (18/28), followed by IIaA16G1R1 and IIaA18G2R1 which have been reported in 9 of 28 (Table 1, Figure1).

In 18 of 29 countries, 6 families and 67 subgenotypes of C. hominis were reported. With relation to this species, the most frequent found in the articles consulted were family Ib (28/89), followed by the family Ia (24/89), the subtype IbA10G2 being most frequently reported in the countries (9/18), followed by the subtype IbA9G3 (Table 2, Figure 1).

Epidemiologia molecular

Las herramientas de biologia molecular no solo han ayudado a resolver la taxonomia de Cryptosporidium, sino que han hecho un contribucion valiosa para el entendimiento de los rangos de hospedadores de las diferentes especies y genotipos (18). Adicionalmente, la caracterizacion molecular de los parasitos circulantes puede permitir la evaluacion de la distribucion y potencial zoonotico de especies y subtipos, asi como, sus rutas de transmision en humanos y animales bajo diferentes situaciones epidemiologicas (2).

Al inicio de la pandemia del VIH - SIDA, las descripciones de patogenos oportunistas centraron la atencion en la criptosporidiosis en los humanos. Un resumen de la literatura de ese tiempo, encontro reportes de 159 casos de criptosporidiosis en pacientes inmunocompetentes y 71 casos en pacientes inmundeficientes. En 26 casos se establecio una clara asociacion entre la infeccion bovina y humana; sin embargo, la transmision de animales a humanos no fue confirmada en ninguno de los 71 casos de los pacientes inmunodeficientes. Adicionalmente, la diseminacion de persona a persona ha sido documentada, los reportes de transmision urbana sin evidencia de transmision zoonotica proveen un soporte para la hipotesis de Casemore y Jackson en la cual indican que la infeccion en humanos no necesariamente fue zoonotica, conduciendo al reconocimiento de dos ciclos de transmision independientes. Los estudios moleculares, entonces, han provisto evidencia de que estas dos rutas de infeccion para los humanos estuvieron relacionadas con dos genotipos - el "genotipo humano" transmitido de humano a humano y el "genotipo bovino" transmitido de animales a humanos, con los bovinos como principales reservorios (1).

Las investigaciones a nivel epidemiologico requieren tecnicas mas discriminatorias, que puedan diferenciar a nivel intraespecifico (19). La implementation de la sutipificacion con el gen de la GP60, ha permitido la identification de diferencias geograficas y temporales en la transmision de Cryptosporidium spp., y una mejor apreciacion de la implicacion del parasito en la salud publica (4).

Al realizar una revision en las bases de datos Elsevier, Scielo, PubMed, SpringerLink y Wiley Library, se encontraron reportes relacionados con la subtipificacion basada en la GP60 del parasito en 29 paises, reportando 163 subtipos de C. parvum. En concordancia con lo reportado por Couto et al. (14) y Feng et al (20), se encontro que alrededor del mundo la familia de C. parvum que se presenta con mayor frecuencia es la IIa (41/163), seguida por la familia IId (17/163), dos familias que han sido reconocidas por su implication zoonotica. El subtipo reportado con mayor frecuencia y que ademas esta presente en todos los continentes, con excepcion de Oceania, es IIaA15G2R1 (18/28), seguido por IIaA16G1R1 y IIaA18G2R1 los cuales han sido reportados en 9 de los 28 (Tabla 1, Figura 1).

Evolutionary genetics

Cryptosporidium spp. belongs to the phylum Apicomplexa, class Sporozoae, subclase Coccidia, order Eucoccidiida, suborder Eimeriina, family Cryptosporidiidae (4).

In 2003 the complete genome of C. parvum as well as C. hominis was published in CryptoDB[R] demonstrating a high degree of similarity ranging between 95 y 97%. The genome of Cryptosporidium parvum is about 9 million base pairs in 8 chromosomes (17).

En 18 de los 29 paises, reportaron C. hominis con una presencia de 6 familias, 67 subgenotipos. En relacion con esta especie, la familia que es reportada con mayor frecuencia en los articulos consultados es la familia Ib (28/89), seguida por la familia Ia (24/89); siendo el subtipo IbA10G2 el mas frecuentemente reportado en los paises (9/18), seguido por el subtipo IbA9G3 (Tabla 2, Figura 1).

The organisms belonging to the phylum Apicomplexa, like the majority of the protists, diverged relatively early in the eukaryotic lineage and have many biological characteristics that are not shared with the principle models of eukaryotic systems (intracellular parasitism for example, or the possession of secondary plastids). Several large-scale sequencing efforts have drastically increased the number of genes known from the Apicomplexa. However, assigning biological function to many of these genes continues to be a major challenge. The generation of loss-of-function mutants is greatly facilitated by the fact that the parasites have a haploid genome over the majority of their life cycles (21).

Genetica evolutiva

Cryptosporidium spp. pertenece al phylum Apicomplexa, clase Sporozoae, subclase Coccidia, orden Eucoccidiida, suborden Eimeriina, familia Cryptosporidiidae (4).

En 2003 el genoma completo de C. parvum asi como de C. hominis fue publicado en CryptoDB[R], con una alta similitud que oscila entre el 95 y 97%. El genoma de Cryptosporidium parvum tiene un tamano de aproximadamente 9 millones de pb y 8 cromosomas (17).

Los organismos pertenecientes al phylum Apicomplexa, al igual que la mayoria de los protistas, se separaron relativamente temprano en el linaje eucariota y tienen muchas caracteristicas biologicas que no son compartidas por los modelos principales de sistemas eucarioticos (por ejemplo, el parasitismo intracelular o la posesion de los plastidios secundarios). Varios esfuerzos de secuenciacion a gran escala han aumentado drasticamente el numero de genes conocidos de los apicomplexa. Sin embargo, la asignacion de las funciones biologicas de muchos de estos genes sigue siendo un gran desafio. La generacion de mutantes con perdida de la funcion, se ve facilitada en gran medida debido a que los parasitos mantienen un genoma haploide sobre la mayor parte de su ciclo de vida (21).

Similar to other parasites in the phylum Apicomplexa, the life cycle of Cryptosporidium spp. has a sexual phase during which recombination between genetically different strains facilitated not only the evolution and appearance of subtypes but also the adaptation of Cryptosporidium spp. (5) and generation of genetic variation between different populations in agreement with the ecological demands and epidemiological conditions of a region (22).

The species of C. parvum that infect humans and some animals could undergo meiotic recombination between different lineages. This could play an important role in the evolution of virulent subtypes (5). Genetic recombination appears to be associated with the high frequency of polymorphism in the gene for GP60. For this, standardized association indices have been used, measured between alleles, which is zero in panmitic populations and with positive values in non-panmitic populations, alternating these behaviors due to the presence of different genotypes and their subsequent recombination generating impact on the genetic equilibrium (22). Markers like the actin gene, heat shock gene and the small ribosomal subunit have been used for phylogenetic investigations and the construction of the current classification (10) however the comparison has been criticized for the lack of a system of genotypic standardization (22).

Recent studies have suggested that the telomeric/subtelomeric regions are highly polymorphic and could carry putative virulence factors. When a locus shows extraordinary levels of genetic differentiation in the population, compared with other loci, it could be interpreted as evidence of positive selection (23). The identification of the grade of intraspecies variation using multilocus methods depends on three factors: the characteristics of the sampling, the types of techniques and the structure of the local population of the parasite (17). Caccio et al (22) analyzed different geographic zones evaluating the relation between those zones and GP60 polymorphisms. This study produced no evidence consistent with geographic isolation and the presence of mutations (22). However Del Coco et al (24) found an association between subtypes and the location, possibly indicating a geographic segregation, concluding that is was necessary to do more studies to evaluate the degree of association of subtypes and pathogenicity, including the postulate that more genes may be associated with this condition, suggesting the evaluation of the relationship between different geographical situations where Cryptosporidum spp. is present (24).

Al igual que en otros parasitos del phylum Apicomplexa, el ciclo de vida de Cryptosporidium spp. tiene una fase sexual durante la cual puede ocurrir recombinacion entre cepas geneticamente diferentes, facilitando no solamente la evolucion y aparicion de subtipos sino la adaptacion de Cryptosporidum spp. (5) y generando la variacion genetica entre diferentes poblaciones de acuerdo con las exigencias ecologicas y condiciones epidemiologicas de una region (22).

Las especies de C. parvum que infectan humanos y algunos animales pueden realizar recombinacion meiotica entre diferentes linajes, lo que puede jugar un papel importante en la evolucion de los subtipos virulentos (5). La recombinacion genetica parece estar asociada con la alta frecuencia de polimorfismo en el gen de la GP60. Para esto, se han usado indices estandarizados de asociacion, medida entre alelos, la cual es cero en poblaciones pancmiticas y con valores positivos para poblaciones no pancmiticas, alterandose estos comportamientos por la presencia de diferentes genotipos y su posterior recombinacion generando impacto sobre el equilibrio genico (22). Marcadores como el gen de la actina, proteina de choque termico y la subunidad ribosomal pequena han sido usados para investigaciones filogeneticas y la construction de la clasificacion actual (10), sin embargo, la comparacion informativa ha sido amenazada por la falta de un esquema de genotipo estandarizado (22).

Recientes estudios han sugerido que las regiones telomericas/subtelomericas son altamente polimorficas y pueden llevar factores de virulencias putativos. Cuando un locus muestra niveles extraordinarios de diferenciacion genetica de la poblacion, comparado con otro loci, puede ser interpretado como evidencia de seleccion positiva (23). La identification del grado de variacion intraespecies usando metodos multilocus, depende de tres factores: las caracteristicas del muestreo, el tipo de tecnicas y la estructura de la poblacion del parasito locales (17). Caccio et al (22) analizaron diferentes zonas geograficas evaluando la relacion entre estas y el polimorfismo de la GP60, este estudio no mostro evidencia consistente entre el aislamiento geografico y presencia de mutaciones (22). Sin embargo, Del Coco et al (24) encontraron asociacion entre subtipos y la ubicacion, indicando posiblemente una segregacion geografica, concluyendo que eran necesarios mas estudios que evaluaran el grado de asociacion de subtipos y patogenicidad, incluso la postulacion de mas genes que puedan estar asociados con esa condicion, sugiriendo la evaluacion de la relacion entre diferentes situaciones geograficas en donde el Cryptosporidum spp. este presente (24).

Conclusion

Molecular diagnosis of the parasite allows the design of strategies to avoid contamination of the environment, human and animal populations of the studied region. This type of diagnosis can be the most successful tool in the preventive management of cryptosporidiosis.

The phylogenetic studies allow to know the distribution, zoonotic potential and the genetic variation of Cryptosporidium species and subtypes. The variations among the different subtypes of the GP60 gene often occur as a consequence of synonymous or silent mutations in the microsatellite region, where they do not affect the coding capacity of the codon for serine. These mutations must be the end result of a purifying process of negative, positive, or neutral selection and may or may not be related to the virulence of the parasite.

Geographic area and its specific environmental conditions could affect the genetic composition of parasite, acting as an inductor agent of mutations and differentiating subtypes worldwide.

Conflict of interest

The authors of this paper declare no conflict of interest that may jeopardize its validity.

Conclusiones

El diagnostico molecular del parasito, permite disenar estrategias para evitar la contaminacion del medio ambiente, las poblaciones humanas y animales de la region estudiada. Este tipo de diagnosticos puede ser la herramienta mas acertada en el manejo preventivo de la criptosporidiosis.

Los estudios filogeneticos permiten conocer la distribucion, el potencial zoonotico y la variacion genetica de especies y subtipos. Las variaciones entre los diferentes subgenotipos del gen de la proteina GP60, suelen producirse como consecuencia de mutaciones sinonimas o silenciosas en la region microsatelite, en donde estas no afectan la codificacion del codon para serina. Estas mutaciones, pueden deberse al sometimiento de una seleccion negativa, purificante, positiva o neutral y podrian o no estar relacionadas con la virulencia del parasito.

El area geografica y sus condiciones ambientales especificas posiblemente pueden influir en la composicion genetica del parasito, actuando como un agente inductor de mutaciones y diferenciando los subtipos alrededor del mundo.

Conflicto de intereses

Los autores del presente escrito declaran que no hay conflicto de intereses que pongan en riesgo la validez.

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Catalina Avendano V (1*) Esp, Alejandro Amaya M (1) Esp.

(1) Universidad de Ciencias Aplicadas y Ambientales U.D.C.A. Facultad Ciencias Pecuarias. Calle 222 No.55 - 37. Bogota - Colombia. (*) Correspondence: cavendano@udca.edu.co

Received: July 2016; Accepted: April 2017.

DOI: 10.21897/rmvz.1138
Table 1: Families and subtypes of C. parvum reported in cattle and
humans.

Country                   Family   Subtype

                                   IIaA15G2R1 (*)
                                   IIaA14G2R1
                                   IIaA17G2R1
                          IIa      IIaA18G2R1
Germany                            IIaA21G0R1
                                   IIaA22G1R1
                                   IIaA16G1R1
                          IId      IIdA22G1
                          IIa      IIaA21G1R1
                                   IIaA17G1R1
                                   IIaA18G1R1
                          IIa      IIaA20G1R1 (*)
                                   IIaA21G1R1
                                   IIaA22G1R1
Argentina                          IIaA23G1R1
                                   IIaA18G1R1
                                   IIaA20G1R1 (*)
                                   IIaA21G1R1
                          IIa      IIaA22G1R1
                                   IIaA23G1R1
                                   IIaA16G1R1
                                   IIaA19G1R1
                                   IIaA15G2R1
                                   IIaA17G2R1
                          IIa      IIaA18G3R1 (*)
                                   IIaA19G2R1
                                   IIaA19G3R1
Australia                 IIc      IIcA5G3a
                                   IIa15G2R1
                                   IIaA18G3R1
                          IIa      IIaA1/G2R1 (*)
                                   IIaA18G3R1
                                   IIaA20G3R1
                                   IIaA20G2R1
                                   IIaA20G2R2
                                   IIaA19G2R1
                          IIa      IIaA19G2R2
Brazil                             IIaA18G1R1
                                   IIaA18G2R2
                                   IIaA16G3R2
                                   IIaA14G2R2
                          IIa      IIaA15G2R1 (*)
                          IIa      IIaA15G1R1
Egypt                              IIaA15G2R1
                          IId      IIdA20G1 (*)
                                   IIaA15G2R1 (*)
                                   IIaA13G1R1
                                   IIaA15G1R1
Spain                     IIa      IIaA16G2R1
                                   IIaA16G3R1
                                   IIaA17G2R1
                                   IIaA18G3R1
                                   IIaA19G3R1
                          IIa      IIaA15G2R1 (*)
                                   IIaA13G1R1
                          IIc      IIcA5G3R2
                          IId      IIdA22G2R1 IIdA23G1
                          IIa      IIaA15G2R1 (*)
                                   IIaA15G2R2
United States                      IIaA11G2R1
                          IIa      IIaA17G2R1
                                   IIaA18G2R1
                                   IIaA19G2R1
                                   IIaA15G2R1 (*)
Ethiopia                  IIa      IIaA16G2R1
                                   IIaA16G1R1
France                    IIa      IIaA15G2R1 (*)
                                   IIaA18G1R1 (*)
                                   IIaA16G1R1 (*)
                          IIa      IIaA17G1R1
Hungary                            IIaA18G1R1
                          IId      IIdA22G1
                                   IIdA19G1
                                   IIaA15G2R1 (*)
                          IIa      IIaA13G2R2
                                   IIaA14G2R1a
                                   IIaA14G2R1b
India
                          IIc      IIcA5G3 (*)
                          IId      IIdA14G1
                                   IIdA15G1
                          IIe      IIeA7G1
                                   IIaA16G3R1
                          IIa      IIaA14G2R1
                                   IIaA19G1R1
                                   IIaA18G1R1
England and Wales                  IIaA20G3R1
                          IIa      IIaA19G3R1
                                   IIaA15G2R1
                                   IIaA17G1R1
                          IIa      IIaA15G2R1 (*)
                                   IIAA16G3R1
                                   IIdA15G1
Iran                               IIdA18G1
                          IId      IIdA20G1a
                                   IIdA21G1a
                                   IIdA26G1
                                   IIaA18G3R1 (*)
                                   IIaA15G2R1 (*)
                                   IIaA17G2R1
                                   IIaA19G4R1
Ireland                   IIa      IIaA20G3R1
                                   IIaA19G3R1
                                   IIaA17G3R1
                                   IIaA20G5R1
                                   IIaA18G2R1
                                   IIaA20G2R1
Jamaica                   IIc      IIcA5G3d
Japan                     IIa      IIaA15G2R1 (*)
                          IIa      IIaA15G1R1
                                   IIaA20G3R1
                          IIc      IIcA5G3a
Jordan                             IIdA14G1
                          IId      IIdA20G1 (*)
                                   IIdA24G1
                                   IIdA29G1
                          IIa      IIaA15G1R1
                                   IIaA15G2R1
                          IIc      IIcA5G3a
Kuwait                    IId      IIdA20G1 (*)
                                   IIdA18G1
                          IIf      IIfA6
Malasya                   IId      IIdA15G2R1
Mexico                    IIa      IIaA15G2R1
                                   IIaA16G1R1
Nigeria                   IIc      IIcA5G3h
                          IIe      IIeA10G1
                          IIa      IIaA15G2R1 (*) IIaA17G1R1
                                   IIdA15G1
                          IId      IIdA16G1
                                   IIdA18G1
                                   IIaA16G3R1
                                   IIaA13G2R1
                                   IIaA14G2R1
                                   IIaA17G2R1
                                   IIaA18G4R1
Netherlands                        IIaA18R1
                                   IIaA19G2R1
                          IIa      IIaA11G2R1
                                   IIaA16G1R1
                                   IIaA19G1R1
                                   IIaA18G1R1
                                   IIaA16G2R1
                                   IIaA18G3R1
                                   IIaA21G3R1
                                   IIaA12G2R1
                          IIj      IIjA24R2
                                   IIcA5G3a (*)
Peru                      IIc      IIcA5G3b
                                   IIcA5G3c
                          IIa      IIaA15G2R1 (*)
                                   IIaA16G2R1
                          IIb      IIbA14
Portugal                  IIc      IIcA5G3
                                   IIdA17G1
                          IId      IIdA19G1
                                   IIdA21G1
                                   IIdA22G1
                                   IIaA15G2R1 (*)
                                   IIaA16G1R1
Czech Replubic            IIa      IIaA22G1R1
                                   IIaA15G1R1
                                   IIaA18G1R1
Romania                   IIa      IIaA15G2R1 (*)
                                   IIaA16G1R1
                          IIa      IIaA18G3R1
Switzerland               IId      IIdA22G1
                          IIa      IIaA16G1R1 (*)
                                   IIaA18G1R1
United States of Serbia   IId      IIaA20G1R1
and Montenegro
                                   IIdA18G1b
                          IIj      IIJA16R2
                                   IIJA17R2

Country                   Host                Reference

                          Cattle              (25)
Germany
                          Cattle              (25)
                          Cattle              (26)
                          Cattle               (2)
Argentina
                          Cattle              (24)
                          Human               (27)
Australia                 Human               (27)
                          Cattle              (27)
                                              (28)
                          Cattle              (14)
Brazil
                          Cattle              (29)
                          Human & Cattle.      (8)
Egypt                     Human
                          Human & Cattle.   (8,29)
                          Cattle
                          Human
                          Cattle
Spain                     Cattle           (12,30)
                          Cattle
                          Cattle
                          Cattle
                          Cattle
                          Cattle              (30)
                          Cattle
                          Human               (12)
                          Human & Cattle      (12)
                          Bovine           (31,32)
United States
                          Cattle              (32)
Ethiopia                  Human               (33)
France                    Cattle              (34)
                          Cattle              (35)
Hungary
                          Cattle              (35)
                          Cattle              (31)
India
                          Human                (6)
                          Human                (6)
                          Human                (6)
                          Cattle              (19)
England and Wales
                          Cattle              (36)
                          Human & Cattle      (37)
Iran
                          Human & Cattle      (37)
Ireland                   Cattle              (38)
Jamaica                   Human               (39)
Japan                     Cattle              (40)
                          Human               (41)
                          Human               (41)
Jordan
                          Human               (41)
                          Human                (3)
                          Human                (3)
Kuwait                    Human                (3)
                          Human                (3)
Malasya                   Human               (42)
Mexico                    Human               (43)
Nigeria                   Human               (44)
                          Human               (45)
                          Human & Cattle.      (9)
                          Human                (9)
Netherlands
                          Cattle               (9)
                          Cattle               (9)
Peru                      Human               (46)
                          Human & Cattle      (47)
                          Human               (47)
Portugal                  Human               (47)
                          Human & Cattle
                          Human               (47)
                          Human
                          Human
Czech Replubic            Cattle              (48)
Romania                   Cattle              (49)
                          Human               (27)
Switzerland               Human               (27)
                          Cattle              (50)
United States of Serbia
and Montenegro
                          Cattle              (50)
                          Cattle              (50)

(*) Subtype of C. parvum found in the highest frequency in the study.

Table 2: Families and subtypes of C. hominis reported in humans.

Country          Family   Subtype                Reference

                 Ia       IaA23                  (27)
                          IbA5G2T3
                 Ib       IbA9G2                 (27)
                          IbA9G2T1
Australia                 IbA10G2 (*)
                          IdA15G1
                 Id       IdA16                  (27)
                          IdA25
                 If       IfA11G1T1              (27)
                          IfA12G1
                 Ia       IaA9R3                 (51)
                          IbA16G2
China            Ib       IbA19G2                (51)
                          IbA20G2 (*)
                 Id       IdA21                  (51)
Spain            Ia       IaA21G1R1              (12)
                 Ib       IbA10G2R2              (12)
Ethiopia         Ib       IbA9G3                 (33)
                 Id       IdA15G1 (Cattle)       (31)
                          IaA18R3
                          IaA19R3
                 Ia       IaA21R3                 (6)
                          IaA26R3
                          IaA27R3
                          IaA29G1T3R3
India            Ib       IbA9G3                  (6)
                          IdA14G1
                 Id       IdA15G11                (6)
                          IdA16G1
                 Ie       IeA11G3T2               (6)
                          IeA11G3T3 (*)
                 If       IfA13G1                 (6)
Iran             Id       IdA20                  (37)
                 If       IfIA22G1               (37)
Jamaica          Ib       IbA10G2 (*)            (39)
                 Ie       IeA12G3T3              (39)
                          IbA6G3
                 Ib       IbA9G3                 (41)
                          IbA10G2                (41)
Jordan                    IbA20G2
                 Id       IdA21
                          IdA24 (*)              (41)
                 Ib       IbA9G3                  (3)
                          IbA10G2                 (3)
Kuwait           Id       IdA14                   (3)
                 Ie       IeA11G3T3               (3)
                 Ia       IaA14R1                (42)
                 Ib       IbA10G2R2              (42)
Malasya          Id       IdA15R2                (42)
                 Ie       IeA11G2T3R1            (42)
                 If       IfA11G1R2              (42)
                          IaA15R3
                 Ia       IaA14R3 (*)            (43)
Mexico           Ib       IbA10G2                (43)
                 Id       IdA17                  (43)
                 Ie       IeA11G3T3 (*)          (43)
                          IaA14R3
                          IaA16R3
                 Ia       IaA24R3            (44)(45)
                          IaA25R3
Nigeria                   IaA23R3
                          IaA25R3
                 Ib       IbA13G3                (44)
                 Ie       IeA11T3G3              (44)
                 Ib       IbA10G2 (*)             (9)
Netherlands      Id       IdA17                   (9)
                          IdA14
                 Ic       IcA5G3R2                (9)
                          IaA11R4
                          IaA12R4
                 Ia       IaA13R4                (46)
                          IaA13R7
                          IaA14R6
                          IaA15R3
Peru             Ib       IbA10G2 (*)            (46)
                          IdA10
                 Id       IdA15                  (46)
                          IdA20
                 Ie       IeA11G3T3              (46)
                 Ia       IaA19R3                (47)
                 Ib       IbA10G2 (*)            (47)
Portugal         Id       IdA15                  (47)
                 Ie       IeA11G3T3              (47)
                 If       IfA14G1                (47)
Switzerland      Ib       IbA10G2                (27)
                 Id       IdA15G1                (27)
                          IbA10G2
United Kingdom   Ib       IbA9G3                 (27)
                          IbA12G3T3
                          IbA10G2

(*) Subtype of C. hominis found in the highest frequency in the study.
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Author:Catalina, Avendano V.; Alejandro, Amaya M.
Publication:Revista MVZ (Medicina Veterinaria y Zootecnia)
Date:Sep 1, 2017
Words:7923
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