Comparison of morphological and molecular Strongylus spp. identification in equine larval cultures and first report of a patent Strongylus asini infection in a horse.
Abstract: Surveillance of Strongylus vulgaris and other Strongylus spp. in equids is important for targeted intervention in parasite control, requiring reliable routine diagnostic methods. Objective: Comparing morphological examination and PCR analyses of larval cultures to identify Strongylus spp. species based on German diagnostic samples from 2018. Methods: Method comparison. Methods: During the routine diagnostic investigations, in total 712 strongyle-egg positive equine faecal samples were cultured. Third-stage larvae (L3) were morphologically differentiated. For molecular validation, samples were examined using S. vulgaris real-time PCR and Strongylus edentatus/Strongylus equinus/Strongylus asini high-resolution melting PCRs. Results: Based on 28S rRNA PCR, 594 samples positive for nematode DNA were included in the study. The inter-rater reliability to compare morphological and molecular species identification was fair for Strongylus spp. without species identification and for S. edentatus, slight for S. equinus and poor for S. vulgaris. The frequency based on morphological and molecular data in this study were for S. vulgaris 0% and 0.8%, respectively, for S. edentatus 0.3% and 1.5%, respectively, and for S. equinus 2.0% and 0.2%, respectively. Based on molecular analyses, one sample obtained from a domestic horse contained S. asini DNA, which was confirmed by sequencing. Conclusions: For many samples, no or only incomplete data regarding clinical history, the exact geographical location and whether samples were obtained on individual or farm level, were available. Conclusions: Results of morphological and molecular examination methods of strongyle L3 from equine samples can differ substantially. Further evaluation of these methods is required to provide reliable and cost-effective methods of screening equine parasites. Further studies using approaches suitable to detect S. asini are needed to evaluate its clinical and epidemiological relevance. Unassigned: Die Überwachung von Strongylus vulgaris und anderen Strongylus spp. bei Equiden ist wichtig für gezielte Maßnahmen zur Parasitenbekämpfung und erfordert zuverlässige routinemäßige Diagnosemethoden. Unassigned: Vergleich der morphologischen Untersuchung und PCR‐Analysen von Larvenkulturen zur Identifizierung von Strongylus spp. anhand deutscher Diagnostikproben aus dem Jahr 2018. Unassigned: Vergleich zweier Diagnosemethoden hinsichtlich der Erkennungshäufigkeit verschiedener Strongylus‐Arten. Methods: Während der routinemäßigen diagnostischen Untersuchungen wurden insgesamt 712 Stronygliden‐Ei‐positive equine Kotproben kultiviert. Die dritten Larvenstadien (L3) wurden morphologisch differenziert. Zur molekularen Validierung wurden die Proben mittels S. vulgaris Echtzeit‐PCR und S. edentatus/S. equinus/Strongylus asini high‐resolution melting (HRM) PCRs untersucht. Results: Basierend auf einer 28S rRNA PCR wurden 594 Proben, die positiv auf Nematoden‐DNA getestet wurden, in die Studie einbezogen. Die Übereinstimmung zwischen morphologischer und molekularer Speziesidentifikation war für Strongylus spp. ohne Speziesidentifikation und für S. edentatus zufriedenstellend, für S. equinus gering und für S. vulgaris schlecht. Die Häufigkeit basierend auf morphologischen und molekularen Daten in dieser Studie betrug für S. vulgaris jeweils 0% und 0.8%, für S. edentatus jeweils 0.3% und 1.5% und für S. equinus jeweils 2.0% und 0.2%. Basierend auf molekularen Analysen enthielt eine Probe, die von einem Pferd stammte, Strongylus asini DNA, was mittels Sequenzierung bestätigt wurde. WICHTIGSTE EINSCHRÄNKUNGEN: Für viele Proben waren keine oder nur unvollständige Metadaten bezüglich Anamnese, des genauen geografischen Standorts und ob die Proben auf Einzeltier‐ oder Betriebsebene entnommen wurden, verfügbar. Unassigned: Die vorliegende Studie zeigte, dass die Ergebnisse morphologischer und molekularer Untersuchungsmethoden von Strongyliden L3 aus equinen Proben erheblich unterschiedlich sein können. Eine weitere Evaluierung dieser Methoden ist erforderlich, um zuverlässige und kostengünstige Methoden zum Screening von equinen Parasiten bereitzustellen. Weitere Studien, die geeignete Methoden zum Nachweis von Strongylus asini anwenden, sind erforderlich, um die klinische und epidemiologische Relevanz dieser Spezies zu bewerten.
© 2024 The Author(s). Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.
Publication Date: 2024-07-16 PubMed ID: 39012065PubMed Central: PMC11807925DOI: 10.1111/evj.14134Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Comparative Study
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
Overview
- This study compared traditional morphological methods with molecular PCR techniques to identify Strongylus parasite species in horse fecal samples from Germany, and it reported the first confirmed case of Strongylus asini infection in a domestic horse.
Background and Importance
- Strongylus species are parasitic worms (nematodes) that infect equids (horses and related animals) and can cause serious health issues.
- Strongylus vulgaris, in particular, is a significant parasite requiring targeted control measures to reduce horse illnesses.
- Accurate identification of Strongylus species in infected horses is essential for effective parasite management and treatment.
- Two primary diagnostic approaches exist: morphological examination of larvae from fecal cultures and molecular diagnostic methods such as PCR.
- This study evaluated how well these two diagnostic methods correspond and explored their reliability.
Study Objective
- To compare the accuracy and consistency of morphological examination versus PCR-based molecular identification of Strongylus larvae cultured from equine fecal samples collected in Germany in 2018.
- To report the first case of patent (active) infection with Strongylus asini in a domestic horse.
Methods
- Study Population: 712 equine fecal samples that tested positive for strongyle eggs underwent larval culturing.
- Larval Culture: Larvae were grown to the third-stage (L3), which is suitable for species identification.
- Morphological Analysis: Larvae were identified and differentiated by physical characteristics under the microscope.
- Molecular Methods:
- Samples were tested with real-time PCR specific for Strongylus vulgaris DNA.
- High-resolution melting (HRM) PCR assays were conducted to detect and differentiate Strongylus edentatus, Strongylus equinus, and Strongylus asini DNA.
- 28S rRNA PCR was used to confirm the presence of nematode DNA, resulting in 594 usable samples.
- Data Analysis: The agreement between morphological and molecular identifications was assessed using inter-rater reliability statistics.
Results
- Of the 594 nematode DNA-positive samples:
- Strongylus spp. without specific species identification and S. edentatus showed fair agreement between the two methods.
- S. equinus showed slight agreement.
- S. vulgaris showed poor agreement between morphological and molecular methods.
- Species Frequency Comparison:
- S. vulgaris was identified in 0% of samples by morphology but 0.8% by PCR.
- S. edentatus was found in 0.3% of samples morphologically and 1.5% molecularly.
- S. equinus appeared in 2.0% morphologically but only 0.2% by PCR.
- Strongylus asini Detection:
- One sample from a domestic horse tested positive for S. asini DNA through PCR.
- This finding was confirmed by DNA sequencing, marking the first reported patent infection in a domestic horse.
- Limitations:
- Many samples lacked complete metadata such as clinical history, precise geographic location, or whether collected from an individual horse or a farm.
Conclusions
- Morphological and molecular methods for identifying Strongylus larvae often yield substantially different results, highlighting potential weaknesses in relying on a single method.
- PCR-based diagnostics may detect species missed by morphological examination, such as S. vulgaris and particularly S. asini.
- Further development and evaluation of these diagnostic methods are necessary to establish reliable, cost-effective screening tools for equine parasitic infections.
- Additional studies are required to assess the clinical importance and epidemiology of S. asini infections in horses, using detection methods capable of accurately identifying this species.
Implications for Practice and Research
- Veterinary parasitology diagnostics might benefit from integrating molecular techniques alongside or instead of traditional morphology to improve detection accuracy for Strongylus species.
- Improved diagnostics will support more informed parasitic control programs in equine populations.
- The detection of S. asini in a domestic horse suggests its potential wider presence and highlights the need to understand its role in equine health.
- Collecting comprehensive metadata alongside samples in future studies will enhance epidemiological understanding.
Cite This Article
APA
Diekmann I, Blazejak K, Krücken J, Strube C, von Samson-Himmelstjerna G.
(2024).
Comparison of morphological and molecular Strongylus spp. identification in equine larval cultures and first report of a patent Strongylus asini infection in a horse.
Equine Vet J, 57(2), 522-529.
https://doi.org/10.1111/evj.14134 Publication
Researcher Affiliations
- Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.
- Veterinary Centre for Resistance Research (TZR), Freie Universität Berlin, Berlin, Germany.
- Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, Hannover, Germany.
- Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.
- Veterinary Centre for Resistance Research (TZR), Freie Universität Berlin, Berlin, Germany.
- Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, Hannover, Germany.
- Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.
- Veterinary Centre for Resistance Research (TZR), Freie Universität Berlin, Berlin, Germany.
MeSH Terms
- Animals
- Horses
- Larva / classification
- Larva / anatomy & histology
- Strongylus / classification
- Strongylus / genetics
- Strongylus / anatomy & histology
- Strongyle Infections, Equine / parasitology
- Strongyle Infections, Equine / diagnosis
- Horse Diseases / parasitology
- Horse Diseases / diagnosis
- Feces / parasitology
- Real-Time Polymerase Chain Reaction / veterinary
Grant Funding
- 251133687/GRP2046 / Deutsche Forschungsgemeinschaft
Conflict of Interest Statement
The authors declare no conflict of interest.
References
This article includes 25 references
- nKester WO. n—the horse killer. Mod Vet Pract. 1975;56(8):569–572.n
- ESCCAP . A guide to the treatment and control of equine gastrointestinal parasite infections. Worcestershire, UK: European Scientific Counsel Companion Animal Parasites (ESCCAP); 2019.
- Deplazes P, Eckert J, Mathis A, von Samson‐Himmelstjerna G, Zahner H. Parasitology in veterinary medicine. Wageningen, The Netherlands: Wageningen Academic Publishers; 2016.
- Santos DW, Madeira de Carvalho LM, Molento MB. Identification of third stage larval types of cyathostomins of equids: an improved perspective. Vet Parasitol. 2018;260:49–52.
- Cernea M, de Carvalho LM, Cozma V. Atlas of diagnosis of equine strongylidosis. Cluj‐Napoca: Editura AcademicPres; 2008.
- Russell A. The development of helminthiasis in Thoroughbred foals. J Comp Pathol Ther. 1948;58(2):107–127.
- Beelitz P, Gobel E, Gothe R. Spectrum of species and incidence of endoparasites in foals and their mother mares from breeding farms with and without anthelmintic prophylaxis in upper Bavaria. Tierarztl Prax. 1996;24(1):48–54.
- nGreite L. Untersuchungen zur Verbreitung von im Rahmen der selektiven Entwurmung bei Pferden in Süddeutschland: LMU. 2013.
- nKaspar A, Pfister K, Nielsen MK, Silaghi C, Fink H, Scheuerle MC. Detection of in equine faecal samples by real‐time PCR and larval culture—method comparison and occurrence assessment. BMC Vet Res. 2017;13(1):19.n
- Schneider S, Pfister K, Becher AM, Scheuerle MC. Strongyle infections and parasitic control strategies in German horses—a risk assessment. BMC Vet Res. 2014;10:262.
- nBracken MK, Wohlk CB, Petersen SL, Nielsen MK. Evaluation of conventional PCR for detection of on horse farms. Vet Parasitol. 2012;184(2‐4):387–391.n
- nNielsen MK, Peterson DS, Monrad J, Thamsborg SM, Olsen SN, Kaplan RM. Detection and semi‐quantification of DNA in equine faeces by real‐time quantitative PCR. Int J Parasitol. 2008;38(3‐4):443–453.n
- nCampbell AJ, Gasser RB, Chilton NB. Differences in a ribosomal DNA sequence of species allows identification of single eggs. Int J Parasitol. 1995;25(3):359–365.n
- Gasser R, Stevenson L, Chilton N, Nansen P, Bucknell D, Beveridge I. Species markers for equine strongyles detected in intergenic rDNA by PCR‐RFLP. Mol Cell Probes. 1996;10(5):371–378.
- nJürgenschellert L, Krücken J, Bousquet E, Bartz J, Heyer N, Nielsen MK, et al. Occurrence of strongylid nematode parasites on horse farms in Berlin and Brandenburg, Germany, with high seroprevalence of infection. Front Vet Sci. 2022;9:892920.n
- nNielsen MK, Vidyashankar AN, Olsen SN, Monrad J, Thamsborg SM. n associated with usage of selective therapy on Danish horse farms‐is it reemerging?nVet Parasitol. 2012;189(2‐4):260–266.n
- nTyden E, Enemark HL, Franko MA, Hoglund J, Osterman‐Lind E. Prevalence of in horses after ten years of prescription usage of anthelmintics in Sweden. Vet Parasitol X. 2019;2:100013.n
- Kaplan RM, Nielsen MK. An evidence‐based approach to equine parasite control: it ain't the 60s anymore. Equine Vet Educ. 2010;22(6):306–316.
- Demeler J, Ramünke S, Wolken S, Ianiello D, Rinaldi L, Gahutu JB, et al. Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor‐resistant conventional and real‐time PCR. PLoS One. 2013;8(4):e61285.
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–410.
- Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33:159–174.
- Roeber F, Kahn L. The specific diagnosis of gastrointestinal nematode infections in livestock: larval culture technique, its limitations and alternative DNA‐based approaches. Vet Parasitol. 2014;205(3‐4):619–628.
- Rossanigo C, Gruner L. The length of strongylid nematode infective larvae as a reflection of developmental conditions in faeces and consequences on their viability. Parasitol Res. 1996;82:304–311.
- Nielsen MK, Kaplan RM, Thamsborg SM, Monrad J, Olsen SN. Climatic influences on development and survival of free‐living stages of equine strongyles: implications for worm control strategies and managing anthelmintic resistance. Vet J. 2007;174(1):23–32.
- nMalan FS, De Vos V, Reinecke RK, Pletcher JM. Studies on . I. Experimental infestation of equines. Onderstepoort J Vet Res. 1982;49(3):151–154.n
Citations
This article has been cited 5 times.- Klass LG, Krücken J, Mbedi S, Sparmann S, Schenk T, Andreotti S, von Samson-Himmelstjerna G. Characterizing mixed strongyle infections in foals and broodmares using cytochrome c oxidase subunit I deep amplicon sequencing.. Parasit Vectors 2026 Jan 3;19(1):65.
- Diekmann I, Supali T, Fischer K, Iskandar E, Sugianto N, Destani Y, Alfian R, Weil GJ, Fischer PU. Brugia malayi and other filarial parasite species in animals in areas endemic for lymphatic filariasis in Belitung District, Indonesia.. PLoS Negl Trop Dis 2025 Oct;19(10):e0013593.
- Wang T, Chen X, Yan X, Su Y, Gao W, Liu C, Wang W. Progress in serology and molecular biology of equine parasite diagnosis: sustainable control strategies.. Front Vet Sci 2025;12:1663577.
- Nielsen MK, Pyatt A, Perrett J, Tydén E, van Doorn D, Pihl TH, Schmidt JS, von Samson-Himmelstjerna G, Beasley A, Abbas G, Jabbar A. Global equine parasite control guidelines: Consensus or confusion?. Int J Parasitol Drugs Drug Resist 2025 Aug;28:100600.
- Fakheri A, Esmaeilnejad B, Akbari H, Molaei R. In vitro evaluation of anthelmintic activity of biocompatibile carbon quantum dot nanocomposite against egg and larval stages of equine strongyles.. BMC Vet Res 2025 Jan 22;21(1):32.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
