Enhanced detection of Giardia duodenalis mixed assemblage infections in pre-weaned dairy calves using next generation sequencing

Authors: HUBLIN, JOSEPHINE - US Department Of Agriculture (USDA); Maloney, Jenny; George, Nadja; Molokin, Aleksey; LOMBARD, JASON - Animal And Plant Health Inspection Service (APHIS); URIE, NATALIE - Animal And Plant Health Inspection Service (APHIS); SHIVLEY, CHELSEY - Animal And Plant Health Inspection Service (APHIS); Santin-Duran, Monica

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/24/2022
Publication Date: 3/28/2022
Citation: Hublin, J.S., Maloney, J.G., George, N.S., Molokin, A., Lombard, J.E., Urie, N.J., Shivley, C., Santin, M. 2022. Enhanced detection of Giardia duodenalis mixed assemblage infections in pre-weaned dairy calves using next generation sequencing. Veterinary Parasitology. 304:109702. https://doi.org/10.1016/j.vetpar.2022.109702.
DOI: https://doi.org/10.1016/j.vetpar.2022.109702

Interpretive Summary: Giardia is a common intestinal parasite of humans and animals worldwide. It frequently infects cattle, producing a high number of Giardia cysts. Cattle can harbor both animal-adapted assemblage E and human pathogenic assemblages A and B. Conventional molecular methods lack the sensitivity required for detecting mixed assemblage infections and that their occurrence in cattle are likely underestimated. To investigate the presence of mixed assemblage infections in cattle, 2,539 dairy calves from the United States were screened for the presence of Giardia using molecular tools. Next generation sequencing (NGS) was then performed for a subset of around 30% of positive samples (n=314) and the ability of NGS and conventional sequencing to detect mixed assemblage infections was compared. Giardia was detected in 1013 of the 2539 samples examined (39.9%). Only assemblage A and E were identified, with assemblage E as the predominant assemblage. Out of the 314 samples examined by both conventional sequencing and NGS, 9 samples (2.9%) were identified as mixed A/E infections by conventional sequencing while NGS identified 56 samples (17.8%), which was six-times more mixed infections compared to conventional sequencing. NGS demonstrated superior sensitivity to Sanger in detecting assemblages present in low abundances. The percentage of mixed A/E infections found in the sampled dairy calves was higher than previously reported. Findings highlight the advantages of NGS application in molecular epidemiological studies to uncover mixed assemblage infections and better understand Giardia epidemiology, establish routes of transmission, and assess the potential role of cattle and other animals as a source of environmental contamination with zoonotic assemblages. This information will be of interest to other scientists, veterinarians and public health agencies.

Technical Abstract: Giardia duodenalis is one of the most common parasitic causes of gastrointestinal illness in humans worldwide with widespread infections in mammalian hosts. It frequently infects cattle, producing a high number of cysts. Cattle can harbor both host-adapted assemblage E and human pathogenic assemblages A and B. Previous studies have demonstrated that conventional molecular methods lack the sensitivity required for detecting mixed infections and that the occurrence of mixed infections in cattle are likely underestimated. To investigate the presence of mixed assemblage infections in cattle, 2,539 pre-weaned dairy calves from the United States were screened for the presence of G. duodenalis using molecular tools. Next generation sequencing (NGS) was then performed for a subset of around 30% of positive samples (n=314) and the ability of NGS and Sanger sequencing to detect mixed assemblage infections was compared. The overall prevalence of G. duodenalis in pre-weaned dairy calves in the sample using PCR was high (1013/2539; 39.9%). Molecular genotyping identified only assemblage A and E, with assemblage E as the predominant assemblage. Out of the 314 samples examined by both Sanger and NGS, 9 samples (2.9%) were identified as mixed A/E infections by Sanger while NGS identified 56 samples (17.8%), which was six-times more mixed infections compared to Sanger sequencing. NGS demonstrated superior sensitivity to Sanger in detecting assemblages present in low abundances. The percentage of mixed A/E infections found in the sampled dairy calves was higher than was hypothesized using values from the literature. This underestimation could be present in the wider cattle population as well, though further exploration would be needed to verify that claim. These findings highlight the advantages of NGS application in molecular epidemiological studies of Giardia. To better understand Giardia epidemiology, establish routes of transmission, and assess the potential role of cattle and other animals as a source of environmental contamination with zoonotic assemblages it is necessary to uncover mixed assemblage infections.