Abstract
Microsatellites are short repetitive DNA sequences of 2–6 repeats interspersed in the genome that display a rapid mutation rate and consequently show high variation between individuals or populations. They have been used to characterize population diversity and structure and the level of variation between different isolates of a number of different organisms, including apicomplexan protozoa. Currently nothing is known about the genetic variability and population structure of Cystoisospora suis (Apicomplexa: Coccidia), the causative agent of piglet coccidiosis, and we made use of the recently available genome of C. suis (strain Wien-I) to amplify microsatellite regions (ca. 300–550 bp) and evaluate the applicability of fluorescence-labeled primers to investigate amplicon length variation at high resolution using capillary electrophoresis (CE). Two phenotypically characterized isolates (Wien-I, toltrazuril susceptible; Holl 1 toltrazuril resistant) and six field isolates from Europe were compared by conventional PCR followed by agar-gel electrophoresis, Sanger sequencing, and CE (fluorescence labeling and fragment length analysis) to evaluate the applicability of the method. Four primer pairs could be identified that amplified bands of the expected size and were labeled for CE analysis. High resolution CE for size determination of PCR amplicons proved to be a reliable and simple method. It revealed high diversity of the analyzed strains, with marked differences even between two strains from neighboring swine farms. In follow-up studies, adaptation of the PCR assay to multiplexing and amplification of small DNA quantities will provide a cost-effective tool to analyse field strains to reveal geographic diversity that could be mapped to phenotypic traits.
Highlights
Members of the class Coccidia comprise a large number of species parasitizing a wide range of hosts including domestic animals and humans with subsequent impact on human and animal health [1]
Since molecular tools to address the genetic diversity of C. suis and geographical and temporal strain distribution have not been developed, we evaluated the usefulness of microsatellite-based DNA analysis to address these issues in C. suis
Coccidian species with large genomes such as T. gondii, N. caninum, or Eimeria falciformis have a high percentage (>80%) of genes which contain introns and a high percentage of microsatellites (0.29–0.99% in these three species) in their genomes [31, 32], and it can be assumed that this is the case for C. suis as the sister taxon to T. gondii and N. caninum [29]
Summary
Members of the class Coccidia (subphylum Apicomplexa) comprise a large number of species parasitizing a wide range of hosts including domestic animals and humans with subsequent impact on human and animal health [1]. Intestinal coccidia from the genera Eimeria and Cystoisospora have been described, among which Cystoisospora suis Isospora suis) has the most significant clinical and economic impact [2]. It causes diarrhea and reduced weight gain in affected suckling piglets and, in combination with other enteropathogens, can cause considerable mortality [3,4,5,6,7,8,9]. Transmission is direct and after ingestion of oocysts the parasites undergo rapid development in intestinal cells (mostly of the jejunum) followed by excretion of oocysts after a. The fast life cycle and high excretion rates in piglets favor the spread of the parasite within the farrowing unit [12]. Nothing is known about the phenotypic and genetic diversity and phenotype and genotype distribution of C. suis in and between farms and geographical regions
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