Abstract
Classic approaches for antemortem identification of gastrointestinal nematodes (GIN) require coproculture of eggs and morphological examination. While adequate for diagnosis, many PCR techniques cannot easily quantify mixed infections without controls and/or standard curves. Herein, we developed a simple and rapid test for differentiating and quantifying mixed infections of GIN using PCR products separated by capillary electrophoresis. Among the cattle GIN, the ITS2 region is sufficiently distinct in length to delineate among the most common infecting genera, Ostertagia ostertagi = 373 bases (b), Haemonchus contortus (placei) = 366b, Cooperia punctata (oncophora) = 376b, Trichostrongylus axei = 372b, and Oesophagostomum radiatum = 357b. Conserved primers were synthesized that span the ITS2 where one primer was fluorescently labeled with 6-FAM. DNAs from infective L3 were PCR amplified then loaded onto an ABI 3130 sequencer adapted for size fragment analysis. Resulting peak amplitudes were both diagnostic and quantitative on a relative basis. As proof of principle, quantification was performed on PCR fragments from mixed species pairs of Ostertagia ostertagi, Cooperia punctata, and Haemonchus contortus and analyzed using Gene Marker V1.85 software. In all cases, linear responses were observed where R2 > 0.97 and line slopes ranged between 0.90 and 1.1. When tested on eggs from naturally infected animals, the assay showed superior results on two farms when compared to coproculture and morphological identification. Using wildlife-derived samples, results coincided well with deep amplicon sequencing. The assay is adaptable to large-scale studies, does not require comparative PCR controls, and should be compliant with GIN from small ruminant livestock.
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Acknowledgements
We would like to recognize the prior work of Dr. John Gilleard and supporting lab members at the University of Calgary for foundational studies on nemabiome sequencing
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The research was funded under the auspices of Agricultural Research Service Project No. 8042-32000-105-00D. The authors have no relevant financial or non-financial interests to disclose. Experiments involving animals were performed under protocol no. 20-010 that was approved by the Institutional Animal Care and Use Committee.
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436_2021_7340_MOESM1_ESM.pdf
Supplementary file1 Comparison of CE-FLA and nemabiome sequencing data from wildlife samples As additional proof of principle, CE-FLA was performed as described and compared to Nemabiome sequencing data from Barone et al. (2020) using wildlife samples. Results showed a high level of congruence among the two techniques. Minor peaks in CE-FLA migrating below 355b are not shown. Lanes highlighted in green present a high level of divergence between the two techniques that was not further investigated. Using the conditions and primers described herein, fragments migrating at or near 372b could not unequivocally be called by CE-FLA when O. ostertagi or O. gruehneri and Trichostrongylus spp. were present in the same sample; however, this occurrence is less probable in domestic livestock. (PDF 441 KB)
436_2021_7340_MOESM2_ESM.pdf
Supplementary file2 Alternative primer sets This table describes an alternative primer set (1687/1690) along with the appropriate call sizes of amplified fragments, that is capable of differentiating O. ostertagi and O. gruehneri from Trichostrongylus spp. The primer set is diagnostic but not quantitative. (PDF 503 KB)
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Zarlenga, D., Barone, C., Hebert, D. et al. A simple molecular method to identify and quantify genera of gastrointestinal nematodes of cattle. Parasitol Res 120, 3979–3986 (2021). https://doi.org/10.1007/s00436-021-07340-3
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DOI: https://doi.org/10.1007/s00436-021-07340-3