Validation of Intestinal Metabarcoded Sequencing Tool to Determine the Composition of Cryptosporidium Species and Giardia Intestinalis Assemblages
Faculty Mentor
Umer Chaudhry
Major
Veterinary Sciences
Description
INTRODUCTION: Cryptosporidium and Giardia are common intestinal parasites worldwide and are the causative agents of diarrhoea, with disease severity ranging from asymptomatic to intense and debilitating infections. There are currently over 40 recognised species of Cryptosporidium, with several host groups susceptible to more than one species. Giardia intestinalis, on the other hand, consists of eight distinct forms (A to H). These species or assemblages are present in a number of hosts, but reliable molecular diagnostic tools are needed to understand the composition of these complex parasite communities.
METHOD: Here, we describe, for the first time, the use of a deep amplicon sequencing platform to identify proportions of Cryptosporidium species and G. intestinalis assemblages. First, four positive control Cryptosporidium species (C. parvum, C. bovis, C. ryanae, and C. suis) were used to prepare test DNA pools derived from different numbers of parasites, and six G. intestinalis assemblages (A–F) were used to prepare test DNA pools derived from synthetic DNA. The method’s detection thresholds for each of the four Cryptosporidium species and six G. intestinalis assemblages were assessed to evaluate the accuracy of their proportional quantification.
RESULTS: Having demonstrated the accurate determination of species composition in Cryptosporidium and assemblage composition in G. intestinalis, the method was applied to determine its detection threshold using positive samples collected from humans, cattle, sheep, pigs, dogs, chinchillas, and sewage with confirmed Cryptosporidium and G. intestinalis assemblage infections. Each sample exhibited a distinct Cryptosporidium and G. intestinalis assemblage composition, as determined by the proportion of MiSeq reads.
DISCUSSION/CONCLUSION: The metabarcoding deep amplicon sequencing method and bioinformatics pathway have several potential applications in animal and human research, including the evaluation of species interactions during co-infections and the determination of host and geographic distribution of cryptosporidiosis and giardiasis in humans and livestock.
Validation of Intestinal Metabarcoded Sequencing Tool to Determine the Composition of Cryptosporidium Species and Giardia Intestinalis Assemblages
INTRODUCTION: Cryptosporidium and Giardia are common intestinal parasites worldwide and are the causative agents of diarrhoea, with disease severity ranging from asymptomatic to intense and debilitating infections. There are currently over 40 recognised species of Cryptosporidium, with several host groups susceptible to more than one species. Giardia intestinalis, on the other hand, consists of eight distinct forms (A to H). These species or assemblages are present in a number of hosts, but reliable molecular diagnostic tools are needed to understand the composition of these complex parasite communities.
METHOD: Here, we describe, for the first time, the use of a deep amplicon sequencing platform to identify proportions of Cryptosporidium species and G. intestinalis assemblages. First, four positive control Cryptosporidium species (C. parvum, C. bovis, C. ryanae, and C. suis) were used to prepare test DNA pools derived from different numbers of parasites, and six G. intestinalis assemblages (A–F) were used to prepare test DNA pools derived from synthetic DNA. The method’s detection thresholds for each of the four Cryptosporidium species and six G. intestinalis assemblages were assessed to evaluate the accuracy of their proportional quantification.
RESULTS: Having demonstrated the accurate determination of species composition in Cryptosporidium and assemblage composition in G. intestinalis, the method was applied to determine its detection threshold using positive samples collected from humans, cattle, sheep, pigs, dogs, chinchillas, and sewage with confirmed Cryptosporidium and G. intestinalis assemblage infections. Each sample exhibited a distinct Cryptosporidium and G. intestinalis assemblage composition, as determined by the proportion of MiSeq reads.
DISCUSSION/CONCLUSION: The metabarcoding deep amplicon sequencing method and bioinformatics pathway have several potential applications in animal and human research, including the evaluation of species interactions during co-infections and the determination of host and geographic distribution of cryptosporidiosis and giardiasis in humans and livestock.