Internal parasites are economically costly to the sheep industry and are responsible for an estimated $665 million in losses in Australia alone (Shephard et al., 2022). Among these internal parasites the trichostrongylids, in particular Haemonchus contortus, are considered the most economically and clinically relevant. Haemonchus contortus late L4 through adult stages feed on blood and predominantly cause anemia, which can be fatal, especially in young animals (Zajac, 2006, Flay et al., 2022). Adult H. contortus are prolific egg layers, producing between 5,000 and 15,000 eggs daily; they also have a short life cycle, which means parasite burdens can increase rapidly (Emery et al., 2016). Due to its fecundity, large effective population sizes, high levels of genetic diversity and the overuse of anthelmintics, H. contortus has rapidly developed anthelmintic resistance to every product introduced to control it (including multiple drug resistance) in many geographical areas (Kaplan, 2004, Kotze and Prichard, 2016), although prevalence of resistance depends upon region. For example, in the United States (USA), H. contortus is highly prevalent across many regions and anthelmintic resistance is widespread. In one study conducted in the southeastern USA between 2002 and 2006, resistance to the three main drug classes was detected in 48% of surveyed farms (Howell et al., 2008). In the mid-Atlantic region, multi-drug resistance was detected on 18% of farms, with complete drug failure on 12% of them (Crook et al., 2016), whereas in the western region of the country, anthelmintic resistance is less prevalent (Kaplan, 2020).

Due to the widespread and rapid development of resistance, parasite control programs must be carefully planned in order to reduce the number of treatments as much as possible while also keeping animals healthy and productive. Faecal egg counts (FECs) can be used to monitor which drugs are efficacious on a given farm and make treatment decisions depending on the local prevalence of nematode species and their resistance status (Besier et al., 2016, Sabatini et al., 2023). Doing this, however, requires knowing which species is predominant, which is not possible using traditional FECs due to the inability to reliably differentiate trichostrongylid eggs based solely on morphology (Zajac, 2006). There are a few methods presently available that are suited to this purpose, the most common of which is coproculture, where faeces are cultured for 10-14 days for subsequent identification of L3s. Molecular tests using PCR are also available (Besier et al., 2016). However, both of these methods require sending samples out to a laboratory with the proper and extensive specialized training in order to complete analysis, both of which can be inconvenient to local veterinary practitioners.

Lectins are proteins that bind to specific carbohydrates, and it has been previously shown that fluorescently labelled peanut agglutinin (PNA) specifically binds the eggs of H. contortus and can be used to unambiguously identify their eggs via fluorescent microscopy or flow cytometry (Palmer and McCombe, 1996, Colditz et al., 2002, Hillrichs et al., 2012; Umair, 2016). This method has been validated by comparison to coproculture for determining the percentage of H. contortus eggs in a given faecal sample (Jurasek et al., 2010) and good agreement has been reported between traditional light microscopy, PNA staining, PCR, and loop-mediated isothermal amplification (LAMP) methods (Ljungström et al., 2018). Some studies have also been conducted to determine the best fluorescence detection system (Abbas and Hildreth, 2019) and to develop a high-throughput system (Douanne et al., 2019) for fluorescent detection of H. contortus.

Despite all of this research, the PNA method for H. contortus identification has not been widely adopted; this could partly be due to the need for costly and bulky laboratory equipment and specialized technical training that may not be practical for use within a veterinary practice or on a farm. Furthermore, previous studies required egg isolation and utilized at least an hour-long incubation for staining the ova (Jurasek et al., 2010; Umair, 2016; Ljungström et al., 2018, Abbas and Hildreth, 2019). These lengthy processes may have contributed to the lack of implementation of this procedure, which has been criticized as being “too cumbersome” for practical use (Ljungström et al., 2018). Presently, only a few laboratories offer the PNA test for H. contortus and it is therefore unavailable to most farmers.

The ParasightTM System (PS; ParasightTM System, Inc., Lexington, Kentucky) is an automated FEC system that fluorescently stains and images faecal samples (Slusarewicz et al., 2016) and then counts parasite eggs using a custom deep-learning algorithm. The system has been validated for the detection and quantification of common parasite eggs in both horses (Scare et al., 2017, Cain et al., 2020) and small ruminants (Slusarewicz et al., 2021). In this study, the PS fluorescence imaging capability was leveraged to develop a simple, automated method for the determination of the proportion of H. contortus eggs in ovine faeces that can produce results on-site in approximately 20 min.

Herein we describe the validation of the automated system by comparison to the most commonly used coproculture (CO) method as well as to manual counting (MC) under a fluorescence microscope. While numerous other automated FEC systems exist (Elghryani et al., 2020, Nagamori et al., 2020, Cringoli et al., 2021, Nagamori et al., 2021), PS is unique in that it is the only one that utilizes a fluorescence detection system and so can be used to differentiate ova using various fluorescent dyes. This work, therefore, describes the first automated FEC system to be validated for genera-specific parasite identification between parasites that produce morphologically indistinguishable eggs. This system provides a simple and rapid method for the identification of H. contortus infections that requires no specialized training and holds the potential to improve parasite control programs in small ruminants globally.