Serology mainly achieves the purpose of detection and diagnosis by detecting antibodies in serum, plasma, saliva, semen, or cerebrospinal fluid of the host (29, 30). In some clinical settings, serological testing remains the gold standard for diagnosing parasitic infections when biological samples cannot be directly obtained. Although serological analysis is similar to microscopic examination in terms of operational convenience and test turnaround time, for parasites such as Babesia and Theileria, which are difficult to accurately identify on blood smears, or some horses infected with Trypanosoma cruzi, these individuals may be mild or asymptomatic, serological analysis can show higher sensitivity and specificity, which is crucial for the diagnostic process (31). The parasitic stage in the host triggers both nonspecific and specific immune responses, which are influenced by the location of the parasite within the host, the host's health condition, and the characteristics of the parasite antigens. Therefore, a deep understanding of the host's immune response is critical in the development of serological diagnostic methods (32). Equine parasites are typically obligate parasites that remain in the host for extended periods. Their life cycles primarily occur within the host, with eggs or adult parasites expelled in the feces. Among the current diagnostic methods, serological diagnostics serve as an effective alternative to parasitological examinations and autopsy diagnoses. During the larvae are in the migration stage in the host or fail to find signs of parasitic infection on the body surface, serological detection is a simple and effective method for rapid diagnosis of parasitic infection. As large animals, horses typically do not experience significant pain or noticeable damage to the host's body from internal parasitic infections, making it difficult for horse owners to determine whether their horses are infected with parasites through daily observation. However, during the early stages of parasitic infection (especially during the larval development stage), the establishment of early diagnostic methods could provide strong support for timely drug treatment and intervention, preventing or mitigating the development of resistance.

At present, studies on ruminants infected with liver flukes have shown that during the parasitic infection of the host, both humoral and cell-mediated immune responses occur, and there is a complex interaction between the parasite and the host's immune system after infection (33). Common equine parasitic nematodes such as P. equorum, Strongyloides westeri, and Gastrophilus spp. regulate the immune system by releasing soluble mediators. These mediators interact with the host's immune cells and molecules, leading to the expression of secretory proteins and carbohydrates. In nematode studies, it has been clarified that cysteine and serine protease inhibitors (cystatins and serpins) can block T-cell responses, regulate macrophage function, and alter the activity of eosinophils and neutrophils (34). The regulation of the host immune system is both rapid and sustained, allowing parasites to persist for extended periods within the host. During the process of maintaining immune system homeostasis, both parasites and host-derived damage-associated molecular patterns are crucial for protective immune responses and tissue repair mechanisms. In studies involving fly larvae parasites, the life cycle and parasitic stages of Hypoderma bovis (H. bovis) share many similarities with certain obligate equine parasites, such as the horse stomach bot fly. The larvae of H. bovis can penetrate the host's skin after hatching, triggering an immune response. Currently, immunological techniques detecting excretory-secretory antigens (ESA) specific to parasites can identify anti-parasitic antibodies within 3–5 weeks post-infection (35, 36). Although natural ESA are immunogenic, their accuracy and sensitivity are limited, especially in low-intensity infections, where they are particularly ineffective, especially in large animals like cattle and horses, where such methods provide only coarse diagnostic results. In contrast, recombinant antigens exhibit greater sensitivity and specificity than natural crude antigens. Their standardized production not only improves yield but also has significant cost-control implications (37). However, despite its diagnostic value, serological testing faces limitations in large-scale epidemiological surveys. For instance, after horses undergo systematic deworming treatment, antibody titers remain elevated for an extended period, which can significantly affect serological test results and lead to a considerable number of “false positives” (38).

Currently developed serological diagnostic methods include the complement fixation test (CFT), agglutination test (AT), indirect immunofluorescence assay (IFA), Western blot, and enzyme-linked immunosorbent assay (ELISA). These methods significantly enhance diagnostic sensitivity. A highly sensitive and specific ELISA method for detecting Fasciola hepatica has been developed, based on recombinant F. hepatica-specific antigens (39). These antigens include enzymes essential for the parasite's survival within the host, such as glutathione S-transferases (40–42). Research on has shown that after initial infection with H. bovis larvae, first-stage larvae (L1) produce trypsin and chymotrypsin. These proteases regulate the immune response by inhibiting both specific and nonspecific host reactions (43, 44). IgG antibodies in cattle typically appear around 45 days after H. bovis infection, peaking at approximately 18 weeks. This is critical for implementing early serological diagnostic strategies. Although the larvae emerge from the host's back skin during the third larval stage (L3), antibodies remain at a high level for about 4 months. Diagnosing during this period is essential for formulating an effective, scientifically informed deworming strategy (45). However, the prevalence of parasitic diseases is often associated with poor pasture management or prolonged exposure to parasitic-contaminated environments. Most horses on pastures or farms are wild and free-roaming, which presents significant challenges for tasks such as restraining horses and collecting blood samples. The collection process may result in varying degrees of injury to the horses. Additionally, these diagnostic methods also have inherent limitations, such as cross-reactivity, which can lead to false-positive results.

Since the advent of the optical microscope, microscopic examination has been considered the most commonly used and economical gold standard for clinical parasitic diagnosis. Parasitological research originated from taxonomic studies, where researchers identified species by examining the morphological characteristics of parasites under the microscope. Although simple and cost-effective, this method has several limitations. For instance, it is labor-intensive, time-consuming, requires highly trained personnel, and makes it difficult to distinguish subtle differences between closely related species (46), During the microscopic examination of roundworms and nematodes, FEC values do not always correlate with worm burden (47), leading parasitologists to increasingly explore molecular biology techniques, such as gene amplification. As previously mentioned, immunodiagnostic tests have significant limitations. Currently, there are no commercially available or FDA-approved antibody tests for diagnosing parasitic diseases, such as cryptosporidiosis, schistosomiasis, and African trypanosomiasis. Commonly used antigen preparations include crude proteins, recombinant purified proteins, adult parasites, eggs, and non-standardized test antigens, leading to significant variability in experimental outcomes. In the development of diagnostic methods for Theileria haneyi (T. haneyi), since T. equi and T. haneyi are closely related, the current equi merozoite antigen 1 -based competitive ELISA used in the U.S. detects T. equi but not T. haneyi. Previous studies on serological diagnostic methods have found that immunoblot results show antigen cross-reactivity between species within the same genus, especially in regions where more than one parasite is prevalent, leading to no results or false-positive results (48). Furthermore, when the number of parasites in the host is low, serological tests tend to have lower sensitivity, which poses significant challenges for detection. Studies have shown that circulating antigens may persist in the host for some time after systematic deworming treatment, leading to false-positive results when compared with parasitic test outcomes. Therefore, the most critical time to apply this diagnostic method is during the acute phase of parasitic infection, ensuring the accuracy and reliability of the test results (49–51).

With the ongoing development of molecular biology techniques, polymerase chain reaction (PCR) technology has been increasingly applied to parasite detection. In addition to traditional PCR, multiplex PCR and nested PCR techniques have also emerged. Multiplex PCR can detect multiple sequences in a single reaction, enabling the simultaneous detection of various parasites (52). Several molecular biology-based diagnostic methods, including amplified fragment length polymorphism, restriction fragment length polymorphism (RFLP), and real-time quantitative PCR (qPCR), have been utilized in parasitic detection. Loop-mediated Isothermal Amplification (LAMP) and Luminex Multi-Analyte Profiling (xMAP) technologies are emerging as novel methods for parasitic disease diagnosis. LAMP is a rapid, isothermal DNA amplification method that operates at a constant temperature (60–65 °C) using Bst DNA polymerase. It forms stem-loop structures containing repeated target DNA sequences, enabling the amplification of DNA to high copy numbers within 1 h. Nucleic acid-based molecular techniques provide superior sensitivity and specificity compared to traditional diagnostic tests. In microscopic parasitology, species identification based solely on egg observation is challenging, especially for certain parasite intermediate hosts. Equine fasciolosis is a significant re-emerging parasitic disease of horses, donkeys, and mules, caused by trematodes of the Fasciola genus, infecting animals through the ingestion of encysted metacercariae found on plants. To date, no diagnostic method is considered the gold standard for detecting equine fasciolosis (53). Molecular biology techniques can detect parasitic infections sensitively from small host samples, such as blood and saliva, even in asymptomatic hosts, making them highly valuable for early diagnosis and the detection of subclinical infections (54). qPCR uses fluorescently labeled probes or double-stranded DNA-specific fluorescent dyes to analyze the fluorescence signal of amplification products in real time, eliminating the need for complex electrophoresis steps (55). Due to its rapid speed, high specificity, excellent sensitivity, broad dynamic range, and low risk of cross-contamination, qPCR is widely used in the development of diagnostic methods for various diseases. It is particularly favored by researchers (56, 57). Additionally, RT-qPCR can be used to study parasite vector capability, host infectivity, diagnostics, and drug efficacy (22). TaqMan probe-based qPCR utilizes a non-extendable DNA probe that hybridizes to a specific region within the amplicon, ensuring high specificity and protecting quantitative precision by preventing the amplification of nonspecific molecules (58). This method enables accurate assessment of infection intensity by the same parasite in different hosts, with exceptional specificity and quantitative capability. This characteristic makes it an ideal tool for assessing host infection loads, providing a scientific basis for developing individualized deworming treatment plans. Molecular biology-based diagnostic techniques allow precise quantification of infection levels, providing important references for developing more effective and accurate deworming treatment plans, thereby playing a significant role in improving treatment efficacy and reducing drug misuse.

For most equine parasites, the humoral immune response typically precedes clinical symptoms during the initial infection. Horses often display notable behavioral changes only when they experience abdominal pain or more severe limb and hoof disorders. Parasitic infections are challenging to diagnose through routine visual inspection. During microscopic examination, parasites such as F. hepatica, Clonorchis sinensis, and Strongyloides can be detected in fecal samples. However, due to intermittent shedding or sampling limitations, eggs per gram (EPG) are typically very low when using the modified McMaster method (56). A retrospective study found that horses with EPG values below 500 had significantly fewer parasites in their intestines than those suggested by the fecal egg count (FEC). In summary, fecal egg tests have issues with missed diagnoses and instability. Serological methods may exhibit cross-reactivity between phylogenetically closely related species, and combining immunochroma-tographic tests with rapid immunoenzymatic assays (RIA) can greatly enhance the specificity and sensitivity of these tests, improving diagnostic accuracy (59, 60). Most serological methods for detecting equine parasites rely on ELISA (61).

Cyathostomins (small strongyles) are the most prevalent and significant parasites affecting horses (62). Over the past two decades, the Jacqueline B. Matthews team at the Moredun Research Institute in the UK has made significant progress in developing in vitro diagnostic methods for Cyathostomins. Early studies identified two diagnostic antigens (20 and 25 kDa) and developed an ELISA method based on serum-specific IgG(T) responses (63–65). Although this method exhibits low cross-reactivity with S. edentatus and S. vulgaris, it remains valuable as a diagnostic antigen. Subsequent studies involved constructing a cDNA library from mixed Cyathostomins species and immunoscreening, resulting in the identification of the gut-related larval antigen Cy-GALA protein, which acts as an immune marker for the developmental larval stage. This antigen is exclusively expressed during the larval stage and is the first diagnostic method capable of detecting the latent phase of Cyathostomins (66). Further research focused on recombinantly expressing two proteins from 14 Cyathostomins species, leading to the selection of an antigen cocktail (Cocktail 3; CT3), which, based on specific recombinant antigen IgG(T), provides a standard curve to generate a “serum score” for each horse. The results demonstrated excellent detection performance (receiver operating characteristic area under the curve values >0.9), making it suitable for diagnosing horses infected with over 1,000–10,000 Cyathostomins in the mucosa and intestines, with sensitivity and specificity greater than 90 and 70%, respectively, making it commercially viable (67).

Early studies on A. perfoliate involved serological research on scolex antigens (68), whole worm extracts, and excretory-secretory (ES) antigens (69). However, crude protein antigens, although easy to prepare, complicate the protein composition, with at least 14 different proteins identified, predominantly low molecular weight proteins in the 31–45 kDa range. Among the crude antigen mixture, two highly immunogenic proteins (12 and 13 kDa) were identified. Researchers measured the negative threshold values for anti-12/13 kDa IgG and IgG(T) ELISA systems, with critical OD values of 0.123 and 0.103, and sensitivity values of 56 and 62%, respectively. This suggests that anti-12/13 kDa IgG(T) may more accurately predict infection intensity in A. perfoliate (70). However, this method also yielded negative results in horses with low infection intensity, suggesting that diagnostic factors in parasitic diseases are not solely based on the parasite presence (especially in large animals) but also depend on the host's infection intensity (71). Among common equine parasitic infections, Anoplocephala spp. exhibit mixed infections, with A. perfoliata having significantly higher infection levels than other species. Immunological studies have yet to resolve antigen cross-reactivity between A. perfoliata and A. magna (72). No cross-reactivity has been observed between 12/13 kDa ES antigens and other parasite antigens such as A. mamillana and Gastrophilus spp. (70). Currently, commercial diagnostic kits for equine tapeworm, including the Tapeworm Blood Test and Saliva Test launched in 2014 by Austin Davis Biologics Ltd., are based on these 12/13 kDa ES antigens. The saliva test has a sensitivity of 83% and specificity of 85%, with no misdiagnoses when detecting infections with more than 20 adult tapeworms, yielding results comparable to serological tests (73).

Jin et al. developed an indirect ELISA detection method using crude L3 antigens from Gastrophilus pecorum and salivary gland antigens. Only salivary gland antigens could be used for fecal sample detection, exhibiting high specificity (100%) and sensitivity (83.33%), while crude protein may non-specifically bind with other substances in the feces, hindering direct antigen detection (74).

P. equorum, a nematode that threatens foal health, begins shedding eggs approximately 10–15 weeks after the initial infection (75). Serological diagnostic tools are crucial for assessing the likelihood of latent P. equorum infections. Currently, many parasite ES products from Ascaris species are utilized in antigen-based diagnostic development (76), including those from Ascaris. Lumbricoides (77) and Ascaris suum (78). In previous studies on P. equorum ES products, the fifth stage (L5) of P. equorum has a molecular weight range of 12–189 kDa, while L2/L3 products range from 12 to 94 kDa. Blood samples from two naturally infected foals showed antigen recognition at approximately 19, 22, 26, and 34 kDa for ES products in Silver-stained SDS-PAGE analysis (79). However, IgG(T) antibodies were not detected in foal serum prior to colostrum ingestion.

According to the performance assessment by Gasser et al. (80), ES antigens, when used as the capture layer in ES-ELISA, show significantly higher sensitivity than the other two antigens, although this increased sensitivity compromises antigen specificity. Acquiring a detailed treatment history is especially important for serological testing. Common deworming drugs, such as MLs, do not provide prolonged deworming effects, and IgG levels typically decrease over a period of 6 months following treatment (81, 82). It is generally recommended to conduct serological testing at least 4 months after treatment. A key issue in serology is the potential for antibody cross-reactivity. Although current serological diagnostic methods have achieved acceptable specificity, identifying and accurately expressing target antigens, as well as ensuring their high quality, are critical for their diagnostic application (83). We hope that equine parasite serology will advance to the same level of advancement as virology and bacteriology. Table 1 summarizes the advantages of various serological detection methods in parasite diagnosis and the main challenges faced by the current technology. Additionally, the current research progress and factors influencing future developments are summarized.

In recent years, significant progress has been made in parasitic research of great public health and veterinary importance, particularly in phylogenetic systematics and diagnostics, due to ongoing advancements in life sciences and technology. The application of molecular biology has introduced new approaches and methods for the early diagnosis and precise control of parasites. Studies aimed at differentiating A. magna and A. perfoliata have shown that both parasites, whether in crude or recombinant protein form, contain low molecular weight immunoreactive components in their antigens. These components are recognized by Anoplocephala-positive sera at the genus level, but not at the species level (84). Currently, no reliable serological assays are available to differentiate A. magna from A. perfoliata, primarily due to the absence of species-specific antigens. This diagnostic limitation highlights the critical importance of molecular-based approaches in differentiating these closely related species. Several PCR-based diagnostic methods have been developed for equine parasite detection, primarily utilizing ITS1 and ITS2 sequence regions. These two sequences, employed as phylogenetic molecular markers in molecular classification and phylogenetic diagnosis, have significantly corroborated taxonomic frameworks derived from traditional morphological and life history analyses (85). To confirm infections in equines caused by S. vulgaris, S. equinus, A. perfoliata, and Parascaris species, fecal egg counts are commonly conducted, but the identification of the parasite species causing the infection cannot be determined by egg morphology alone. Accurate diagnosis often requires culturing the collected feces for a certain period to allow development to the third larval stage (L3). DNA fingerprinting techniques, such as restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), and random amplified polymorphic DNA (RAPD), have been widely used in the development of molecular diagnostic methods for equine parasites, due to their speed, cost-effectiveness, and the fact that they do not require complete genome information. The fundamental principle of RFLP involves the digestion of DNA fragments from target genes using specific restriction endonucleases. Genotypes are identified based on the electrophoretic patterns of the resulting digestion products, eliminating the need for further sequencing or sequence alignment analysis (86). In 1995, Campbell et al. first used PCR-RFLP technology to amplify the ITS2 region of Strongylus spp., and the results indicated that the ITS2 sequence, as a diagnostic target gene, exhibits low intraspecific variation and high interspecific variation. This study laid the foundation for the ITS2 region as the basis for molecular identification. Additionally, RFLP technology in this study also provided a means of separating amplified products using agarose gel based on fragment size, allowing for rapid identification of Strongylus spp. without sequencing or sequence analysis (87). In subsequent studies, Gasser and Monti optimized PCR-RFLP and PCR-SSCP methods to distinguish 16 species of equine strongyles (88, 89). PCR-RAPD technology has been widely used in the classification of numerous parasites (90, 91). In equine parasitic detection, this technology has been applied to define species of two Cylicocyclus species, such as C. insigne and C. elongatus, which are morphologically very similar but can be accurately distinguished, further demonstrating that this method is suitable for closely related species. Over the past 30 years, researchers have primarily developed diagnostic methods for equine parasites based on ribosomal RNA genes, particularly the ITS and IGS (Intergenic Spacer) regions. In studies of equine strongylids, by comparing the performance of two molecular barcodes (COI gene and ITS-2 rDNA gene) in different biological type samples, it was found that although the results of the two barcodes were fairly consistent, the PCR amplification efficiency of the COI gene was lower than that of ITS2, particularly in certain strongyle species (e.g., Cylicostephanus calicatus and Cylicostephanus longibursatus), where the COI barcode amplification efficiency was less than 70%. In group identification, the COI gene, due to its higher genetic diversity and larger sequence variation, can lead to PCR amplification bias, making it difficult to identify specific life cycle stages of some species and affecting the final sequencing results (92).

Hodgkinson et al. (93, 94) used Southern blot to validate the design of digoxigenin (DIG)-labeled probes targeting the IGS conserved region of four highly prevalent species (C. ashworthi, C. nassatus, C. longibursatus, C. goldi), achieving molecular identification of single eggs, L3 larvae, and L4 larvae. The research team later developed a PCR-ELISA method, which was completely consistent with the Southern blot results and could identify low-abundance species (e.g., C. catinatum, which accounted for only 1.7%). This method was used to assess the therapeutic efficacy in horses treated with BZDs. Compared to the RFLP probe method, it was more suitable for analyzing polymorphic fragments (95). The widely used Reverse Line Blot (RLB) detection method for diagnosing strongyloid species currently involves non-radioactive hybridization of PCR amplicons with different oligonucleotide probes in a single reaction, allowing for the detection and identification of more than 16 strongyloid species simultaneously (96–98). This method has also been widely applied to study the relationship between drug resistance and changes in Cyathostomin population structure, thereby exploring the potential mechanisms behind the shortened egg reappearance period after treatment (99, 100). Martins amplified the ITS2 DNA fragment of S. vulgaris by PCR and compared it with the results of fecal culture L3, demonstrating a lower consistency, further confirming that PCR diagnostic methods have become the most effective tool for diagnosing S. vulgaris (101). Nielsen developed a dual-labeled TaqMan qPCR assay specifically targeting S. vulgaris, which exhibited no cross-reactivity with other equine parasitic nematodes. This assay demonstrated a detection limit of 0.5 egg equivalents, providing a precise and efficient tool for diagnosing S. vulgaris infections. Similarly, Zhou et al. developed a novel real-time quantitative PCR assay based on the TaqMan-MGB probe, specifically for detecting T. haneyi, a pathogen causing equine piroplasmosis. The assay demonstrated excellent specificity and sensitivity, with a detection limit of 1 × 10² copies/μl, and was validated by comparison with nested PCR. This new diagnostic method promises to enhance early detection and control of T. haneyi infections, complementing existing PCR techniques for Theileria equi (T. equi), Babesia caballi (B. caballi). The LAMP-based diagnostic method for T. equi and B. caballi has been developed, providing rapid on-site results within 1 h (102). Fecal samples, the most accessible for on-site diagnosis, contain more potential inhibitors than blood samples, which can reduce the sensitivity and accuracy of the LAMP assay. Furthermore, fecal samples often require more rigorous pretreatment and ultrapure DNA extraction (103, 104). The advancement of microfluidic chip technology has enabled the successful application of isothermal amplification methods for detecting various veterinary parasites (105–107). Chen et al. (108) developed a microfluidic LAMP system that can identify five parasites. This method integrates sample pretreatment, biological separation, biochemical reactions, and signal analysis into microchips just a few square centimeters in size, achieving both miniaturization and automation in parasite detection (109, 110).

With the ongoing development of high-throughput sequencing technology, next-generation sequencing (NGS) has become widely applied in the diagnosis and research of equine parasites. Mitchell used NGS technology to conduct qualitative and quantitative analysis of the species and egg abundance in equine fecal samples, replacing traditional larval culturing and necropsy methods. This also marks the first recorded instance of the equine “nemabiome” (111). Ghazanfar et al. combined NGS technology with the modified McMaster technique to study the prevalence and diversity of gastrointestinal nematodes in Australian racehorses. Compared to traditional microscopic examination or PCR methods, NGS proved more efficient and precise, identifying 23 strongylid species, including 18 cyathostomins and five large strongyle species. Using high-throughput data from NGS, the species abundance of various parasites in fecal samples was analyzed. The three most abundant species of small strongyles were Cylicostephanus longibursatus (28%), Cylicocyclus nassatus (23%), and Coronocyclus coronatus (23%), which together accounted for 95.8% of the entire parasite community. The study also revealed the diversity of nematode communities across different climatic zones and age groups. In foals and weanlings, the most common strongylid species was Cylicostephanus longibursatus, with relative abundances of 35.3% in foals and 34.1% in weanlings. Nematode community diversity in the Non-Seasonal Rainfall Zone and Winter Rainfall Zone was significantly higher than in the Mediterranean Rainfall Zone (112). Recently, Hamad et al. employed ITS-2 rDNA metabolic barcoding sequencing technology with the Illumina MiSeq platform to study the strongylid nematode populations on a large equine farm in Thailand. By analyzing fecal samples from 57 horses on the farm, they successfully identified 14 strongylid species, including Cylicocyclus nassatus and Cylicostephanus longibursatus. The study revealed the complexity of the strongylid nematode community and the relative abundance of species, while also validated the quantitative potential of high-throughput technology in equine parasite detection. It provides an important molecular data foundation for future drug resistance monitoring and parasite management (113, 114). Table 2 summarizes the advantages of widely used molecular biological detection methods in parasite diagnosis, along with the main challenges faced by current technologies. It also outlines the current research progress and factors influencing future developments.