A simple method for identifying and quantifying gastrointestinal nematodes of cattle

Authors: Zarlenga, Dante; BARONE, CARLY - Bia Diagnostics, Llc; Hebert, Deborah - Van Hook; Santin-Duran, Monica; NEWCOMB, HAROLD - Merck Animal Health

Submitted to: Parasitology Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/27/2021
Publication Date: 10/14/2021
Citation: Zarlenga, D.S., Barone, C., Hebert, D.A., Santin, M., Newcomb, H. 2021. A simple method for identifying and quantifying gastrointestinal nematodes of cattle. Parasitology Research. 120(12):3979-3986. https://doi.org/10.1007/s00436-021-07340-3.
DOI: https://doi.org/10.1007/s00436-021-07340-3

Interpretive Summary: Control of gastrointestinal nematodes (GIN) has historically been achieved using complex management systems that, among other things, limited the exposure of the animals to infected pastures and kept stocking rates low. The advent of broad spectrum anthelmintics altered the mindsets of producers and therefore parasite management programs. Current programs have come to rely exclusively on drug treatment leading to overuse, abuse and eventually anthelmintic resistance. Thus, mainstream producers are encouraged to reduce unnecessary drug intervention and alter the herd mentality in drug treatment by delineating the more pathogenic parasite species such as Ostertagia ostertagi which require abatement, from those less likely to affect production traits. This will help producers control escalating costs associated with treatment and control. The goal of this work was to develop a simple, sensitive and rapid PCR-based test that can simultaneously differentiate and quantify important genera of GIN infecting cattle without the need for external controls, standard curves and the multitude of manipulations required by other techniques. In this test, a single set of conserved primers, one of which is fluorescently labeled, is used to amplify a region common among GIN but with length polymorphism. The PCR products are analyzed using Capillary Electrophoresis – Fragment Length Analysis which generates a chromatogram that allows one to qualitatively and quantitatively define the contents of the samples. Since peak heights are proportional to peak area in the chromatogram, quantification can be achieved by simply comparing the amplitudes of each fragment. Results demonstrate good agreement with DNA sequencing and expose the inconsistencies and poor diagnostic character of morphological examination. The availability of such a test could help diagnostic labs to quickly identify the contents of parasite samples. It would also help curtail the number of drug treatments used by producers by targeting only those animals harboring the most pathogenic parasites. In so doing, it would also reduce the potential for drug residues in consumer products, decrease producer costs, and decrease the risk of drug resistance by reducing selective pressures on the worms. Such a test will provide an important and a much-needed tool for studies on parasite epidemiology and may be applicable to infections in both synanthropic and wildlife hosts and to common sheep GIN.

Technical Abstract: Classic approaches for antemortem identification of gastrointestinal nematodes (GIN) require egg cultivation to infective L3 followed by morphological examination. While adequate for diagnosis, many PCR techniques fall short of easily quantifying 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 fluorescently-labeled PCR products followed 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 and HPLC purified 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 0.90-1.1. When tested on eggs from naturally-infected animals, the assay showed superior results on two farms when compared to morphological identification of cultured L3. 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 can be used on common sheep GIN.