Epidemiology of common equine intestinal parasites and proteomics of Anoplocephala perfoliata

Gastrointestinal parasites are common in horses worldwide. Parascaris spp. is pathogenic to foals, small and large strongyles infect all age groups and Anoplocephala perfoliata tapeworm is a risk for horses having access to pasture. For effective parasite control it is important to understand the epidemiology, including the risk factors, of these infections. Anthelmintic resistance is a growing problem for equine gastrointestinal parasites making parasite control more difficult than in the past. Routine administration of anthelmintics to all horses without proper diagnostics has enhanced the development of resistance. To combat against anthelmintic resistance, the overall use of anthelmintics needs to be reduced and better targeted by proper diagnosis and evidence-based assessment of the antiparasitic treatment needs in each horse. The fecal egg count techniques commonly used for gastrointestinal parasite diagnostics have low sensitivity in diagnosing equine tapeworm infections. Therefore, an improved method for A. perfoliata diagnostics is needed. The aims of this thesis were to estimate the prevalence of strongyle, Parascaris spp. and A. perfoliata infections in Finnish horses as well as risk factors for strongyle and ascarid egg shedding. In addition, this work aimed to evaluate fenbendazole and pyrantel embonate efficacy against Parascaris spp. infection and an association between owner-reported clinical signs and patent Parascaris spp. infection. Lastly, the aim was to quantitatively identify A. perfoliata excretory/secretory (E/S) proteins and to screen for potential diagnostic antigens. A total of 506 fecal samples collected from under three-year-old horses were quantitatively analysed by flotation based methods for helminth eggs in two separate studies. The first study (Study I) used the McMaster method while the Mini-Flotac technique was applied by the second study (Study II). Data were collected via an online survey from 466 respondents and statistically analysed to identify the risk factors for Parascaris spp. and strongyle infections. Horses excreting more than 200 ascarid eggs per gram were included in the anthelmintic efficacy trial and allocated to the fenbendazole (n=31) or pyrantel embonate (n=26) treatment group, or to the control group (n=7). The fecal egg count reduction test (FECRT) was applied to evaluate the efficacy of fenbendazole and pyrantel embonate against Parascaris spp. In Study III, Anoplocephala perfoliata tapeworms were collected at an abattoir and cultured in vitro. Excretory/secretory proteins produced by A. perfoliata tapeworms were identified by label-free quantitative proteomics using a database composed of proteins of related species, namely Hymenolepis diminuta, Echinococcus multilocularis/granulosus, and Taenia aseatica proteins. Immunoblotting was applied to find immunoreactive E/S proteins of A. perfoliata. Strongyle infections were the most prevalent parasitic infections. In Study I, the strongyle egg shedding prevalence was 58%, while in Study II, there was a 73% prevalence for patent strongyle infection. The patent Parascaris spp. prevalence was 12% in Study I and 37% in Study II. We found A. perfoliata worms in 33% of the slaughtered horses (n = 84). Moreover, we identified two new risk factors for Parascaris spp. egg shedding: the breeding farm size (p = 0.028) and the frequency of horse movements (p = 0.010). Horses born on large breeding farms were more likely to shed Parascaris spp. eggs also after moving to training stables compared with horses born in small breeding farms. Horses living on farms with frequent horse movements to other locations had higher odds of a patent Parascaris spp. infection compared with farms with less frequent horse movements. Horses were less likely to shed strongyle eggs during spring compared with winter. There was an association between owner-observed clinical signs and a patent Parascaris spp. infection in foals (p = 0.002). Fenbendazole was efficacious against Parascaris spp., but pyrantel embonate showed reduced efficacy. This is the second report of reduced pyrantel embonate efficacy in Europe. All together 509 excretory-secretory proteins of Anoplocephala perfoliata were isolated and identified. Of these proteins, heat shock protein 90 and Sj-Ts4 hold the greatest potential as diagnostic targets because they were produced by both small- and large tapeworms cultivated in this study. Complementary immunoproteomics revealed several antigenic/immunogenic E/S proteins that elicit an IgG-mediated response in horses. The risk factors for gastrointestinal parasite infections revealed in this study assist horse farms in developing parasite control strategies and reducing the use of anthelmintics to mitigate the development of anthelmintic resistance. Moreover, information about the Parascaris spp. resistance situation in Finland will guide horse owners in selecting efficacious medications. The A. perfoliata E/S proteins identified in this study will assist future research in developing novel diagnostic tests for equine tapeworm. Complementary immunoproteomics indicated several antigenic proteins, which may help to understand the worm-to-worm communication, host adaptation, and immunogenicity of A. perfoliata.

Suolistoloisia esiintyy lähes kaikilla hevosilla. Suolinkainen on varsojen tärkein loinen. Pienet ja suuret strongylukset tarttuvat kaikkiin ikäryhmiin ja Anoplocephala perfoliata -heisimato on riski laiduntaville hevosille. Tartuntojen leviämisreittien ja riskitekijöiden ymmärtäminen on tärkeää loistorjunnassa. Hevosten rutiininomainen matolääkitseminen ilman diagnostiikkaa on johtanut loislääkeresistenssin lisääntymiseen, ja siksi hevosen uloste tulisi tutkia ennen lääkityspäätöstä. Heisimatotartunnat todetaan ulostetutkimuksella vain satunnaisesti ja A. perfoliata -diagnostiikkaan tarvitaankin parempi menetelmä. Tässä väitöskirjatyössä tutkittiin 506 alle kolmivuotiaiden hevosten lantanäytettä loismunien toteamiseksi. Hevosten tiedot kerättiin kyselytutkimuksella 466 vastaajalta sisäloistartuntojen riskitekijöiden tunnistamiseksi. Fenbendatsolin ja pyranteelin tehoa suolinkaistartuntojen hoidossa testattiin lääkitsemällä suolinkaisen munia erittäviä varsoja näillä lääkkeillä. Hevosen heisimatoja kerättiin teurastamossa ja kasvatettiin laboratoriossa niiden tuottamien proteiinien (E/S-proteiinit) tunnistamiseksi. Pienet ja suuret strongylukset olivat hevosten yleisimpiä suolistoloisia. Suolinkaistartuntoja esiintyi eniten alle 1-vuotiailla. Heisimatoja löytyi joka kolmannesta teurashevosesta, vaikka heisimadon munia todettiin ulostenäytteistä vain harvoin. Tutkimuksessa tunnistettiin useita riskitekijöitä sekä strongylus- että suolinkaistartunnoille. Siittoloissa ja isoilla kasvattajilla syntyneissä varsoissa todettiin useammin suolinkaistartuntoja kuin pienillä kasvattajilla syntyneissä varsoissa. Strongylustartunnat olivat yleisempiä hevosilla, joilla laidunala hevosta koti oli alle puoli hehtaaria verrattuna hevosiin, jotka laidunsivat laajemmalla alueella. Suolinkaistartuntoihin liittyviä sairauden oireita omistajat havaitsivat vain alle vuoden ikäisillä hevosilla. Fenbendatsoli todettiin tehokkaaksi lääkkeeksi suolinkaisia vastaan, mutta pyranteelin teho oli heikentynyt yksittäisillä talleilla. Kaikkiaan 509 A. perfoliata -heisimadon tuottamaa E/S-proteiinia eristettiin ja tunnistettiin. Näistä seulottiin kaksi, Hsp90 ja Sj-Ts4, heisimadon diagnostiikan kehittämisessä hyödynnettäviksi proteiineiksi. Tämän väitöskirjatyön löydökset auttavat talleja loiskontrolliohjelmien suunnittelussa, loislääkkeiden käytön paremmassa kohdentamisessa ja tehokkaan suolinkaislääkkeen valinnassa. Suunnitelmallinen loiskontrolli auttaa vähentämään matolääkkeiden käyttöä ja ehkäisemään loislääkeresistenssin lisääntymistä. Tutkimuksessa tunnistetut A. perfoliatan E/S -proteiinit edistävät uusien diagnostisten testien kehittämistä hevosen heisimadolle.

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