A study was undertaken to evaluate and compare the effectiveness of three anthelmintics, ivermectin, fenbendazole, and a combination of ivermectin and pyrantel pamoate, on fecal egg count reductions of cyathostomes and Parascaris equorum in 30 foals at the Texas A&M Horse Center. The foals were reared under standard horse center practices and were naturally infected with both cyathostomes and Parascaris. The foals were randomized into three treatment groups with individuals being rerandomized after each eight week observation period. The treatments of ivermectin and fenbendazole were given at the manufacturer's recommended doses and the pyrantel treatment was given at two times the manufacturer's recommended dose. All doses were based on weights taken prior to treatment. Fecal egg counts were performed at the time of treatment and at two week intervals after treatment for a total of eight weeks. Each foal received a total of three treatments during the course of the study along with the most effective treatment at the conclusion of the study. Fecal egg counts were performed by a modified McMaster's test with a sensitivity of 25 eggs per gram of feces and by the modified Wisconsin double centrifugal floatation with a sensitivity of 0.2 eggs per gram of feces. Fecal egg reduction percentages were calculated for each two week interval. Analysis of the results showed that ivermectin, either used alone or with pyrantel was a more effective anthelmintic for cyathostome (small strongyle) control than fenbendazole. Fenbendazole and pyrantel showed a higher initial reduction in Parascasris eggs when compared to the ivermectin only treated group, but ivermectin showed improved egg reduction over time. At the conclusion of this study, a primary treatment of ivermectin at the manufacturer's recommended dose and treatment of pyrantel at two times the manufacturer's recommended dose was recommended to control cyathostome egg production and severely reduce the initial number of Parascaris adults in the foals at this facility. Subsequent monthly does of ivermectin at the manufacturer's recommended dose was also recommended to continue to control both parasites. Follow up fecal examinations were also recommended to test the continued effectiveness of the recommended treatment protocol.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-06-28T17:37:30Z No. of bitstreams: 1 texto completo.pdf: 720039 bytes, checksum: be71c87081da88ff51ed92fcf3cb714c (MD5) / Made available in DSpace on 2018-06-28T17:37:30Z (GMT). No. of bitstreams: 1 texto completo.pdf: 720039 bytes, checksum: be71c87081da88ff51ed92fcf3cb714c (MD5) Previous issue date: 2018-03-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Arthrobotrys musiformis é um fungo formador de armadilhas do tipo redes adesivas tridimensionais, capazes de capturar e predar larvas infectantes (L3) de nematoides. Nos equinos, os nematoides gastrintestinais podem levar a condições patológicas que interferem no desenvolvimento corporal e podem, inclusive, levar os animais a óbito. Dessa forma, o objetivo deste trabalho foi avaliar a eficácia do fungo nematófago Arthrobotrys musiformis (isolado A144) no controle de L3 de nematoides estrongilídeos que acometem equinos. Inicialmente foi analisado a capacidade do isolado A144 de passar pelo trato gastrintestinal de equinos e manter a sua atividade predatória sobre larvas infectantes de ciatostomíneos. Coletas de fezes foram feitas 12h, 24, 36, 48 e 72h após a administração de péletes contendo micélio fúngico. Os péletes recuperados na última coleta foram colocados em placas de Petri contendo meio ágar-ágar 2% para germinarem, e larvas infectantes de ciatostomíneos foram adicionadas, para avaliar a atividade do fungo sobre essas. Paralelamente, placas contendo o isolado A144 crescido a partir de colônias mantidas em laboratório também receberam L3 de ciatostomíneos e ao final de sete dias de interação, foram obtidos os percentuais de redução de 96.61% e 96.18% dos grupos contendo o fungo mantido em laboratório e o fungo recuperados dos péletes, respectivamente, demonstrando que o fungo é capaz de passar pelo trato gastrintestinal de equinos e manter a sua capacidade predatória. Diferentes doses de conídios do isolado A144 foram avaliadas em placas, em coproculturas e em bolos fecais depositados em pastagem e o que se observou é que a redução de L3 nas dosagens de conídios testadas apresentou pouca variação entre elas, mas todas apresentaram diferença estatisticamente significativa quando comparadas ao grupo controle. As doses iniciais de esporos avaliadas neste trabalho não foram determinantes na capacidade do isolado A144 em reduzir o número de larvas infectantes de estrongilídeos, tanto em condições controladas, quanto em condições à campo. O isolado A144 do fungo nematófago Arthrobotrys musiformis é capaz de manter sua viabilidade após passagem pelo trato gastrintestinal de equinos e predar larvas infectantes de estrongilídeos. Os resultados encontrados indicam que o fungo Arthrobotrys musiformis (isolado A144) é uma alternativa para o controle das fases de vida livre dos estrongilídeos nematoides de equinos. / Arthrobotrys musiformis is a trap-forming fungus of three-dimensional adhesive nets, capable of capturing and predating infective (L3) nematode larvae. In horses, gastrointestinal nematodes can cause pathological conditions that interfere with body development and can even cause death. The objective of this work was to evaluate the efficacy of the nematophagous fungus Arthrobotrys musiformis (isolate A144) in the control of L3 of strongylid nematodes of horses. Initially, the ability of the isolate A144to pass through the gastrointestinal tract of horses and to maintain its predatory activity on infective Cyathostomin larvae was analyzed. The mycelium of the fungus was incorporated into pellets of sodium alginate and administered orally, in a single dose, to horses. Fecal collections were performed 12h, 24, 36, 48 and 72h after administration of pellets. Pellets recovered in the last collection were placed in Petri dishes containing 2% agar-agar medium to germinate, and infective larvae of cyathostomins were added to evaluate fungus activity on them. In parallel, plaques containing the A144 isolate grown from colonies kept in the laboratory also received L3 from cyathostomins and at the end of seven days of interaction, reduction rates of 96.61% and 96.18% of the L3 in group containing the fungus were kept in the laboratory and the fungus recovered from the pellets, respectively, demonstrating that the fungus is able to pass through the gastrointestinal tract of horses and maintain its predatory capacity. Different doses of conidia of isolate A144 were evaluated in plaques, coprocultures and fecal pats deposited on pasture and was observed that the reduction of L3 in the dosages of conidia tested showed little variation among them, but all presented a statistically significant difference when compared to the control group. The initial doses of spores evaluated in this work were not determinant in the ability of isolate A144 to reduce the number of infectious larvae of strongyles under both controlled and field conditions. The A144 isolate of the nematophagous fungus Arthrobotrys musiformis is able to maintain its viability after passage through the gastrointestinal tract of horses and predate infectious larvae of strongyles. The results indicate that the fungus Arthrobotrys musiformis (isolate A144) is an alternative to the control of free life stages of equine nematodes strongyles.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-04T15:33:20Z No. of bitstreams: 1 texto completo.pdf: 633073 bytes, checksum: 9f589b3983c89a40a93296fd92fcfb0f (MD5) / Made available in DSpace on 2015-11-04T15:33:20Z (GMT). No. of bitstreams: 1 texto completo.pdf: 633073 bytes, checksum: 9f589b3983c89a40a93296fd92fcfb0f (MD5) Previous issue date: 2014-02-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O Brasil possui o maior rebanho de equinos da América Latina e o quarto mundial. Somente a produção de cavalos movimenta R$ 7,3 bilhões anuais. Com relação aos helmintos que geram prejuízos à equideocultura mundial, o Parascaris equorum pode ser destacado, em função dos prejuízos permanentes sobre a capacidade funcional de órgãos como pulmão e fígado, causados pela migração das larvas. Além disso, pesquisas sobre o controle biológico de helmintos de interesse veterinário vêm ocorrendo ao longo dos anos, na tentativa de se encontrarem medidas que venham somar forças ao controle convencional, na busca por melhores condições de sanidade e produtividade animal. Assim, os objetivos do presente trabalho foram: avaliar a ação, in vitro, de dois isolados do fungo helmintófago Pochonia chlamydosporia (isolados VC1 e VC4) crescidos em diferentes meios de cultura, sobre ovos de Parascaris equorum; avaliar a ação do extrato bruto enzimático do fungo P. chlamydosporia sobre ovos de P. equorum; analisar a produção enzimática do fungo P. chlamydosporia (isolados VC1 e VC4) após 21 dias de interação com ovos de P. equorum. Foram utilizados dois isolados do fungo P. chlamydosporia (VC1 e VC4), provenientes da micoteca do Laboratório de Parasitologia do Departamento de Veterinária da Universidade Federal de Viçosa. Os ovos de P. equorum foram obtidos por meio de dissecção de exemplares fêmeas adultos, recuperados de fezes de potros naturalmente infectados, após a administração de citrato de piperazina tetrahidratado. Ao final de um período de 21 dias de interação, foram observados percentuais de destruição dos ovos de P. equorum de 41,9% e 48,3%, para os isolados VC1 e VC4, respectivamente. A ação do extrato bruto enzimático do fungo P. chlamydosporia (isolado VC4) apresentou um percentual de destruição de 36,5, após um período de 48 horas de interação. Os resultados apresentados no presente trabalho sugerem que o fungo Pochonia chlamydosporia, bem como o seu extrato bruto enzimático, podem ser utilizados para auxiliar no controle de ovos embrionados do ascarídeo Parascaris equorum. / Brazil has the largest herd of horses in Latin America and the fourth worldwide. Only the production of horses moves $ 7.3 billion annually. With helminths that generate losses to world equideoculture the Parascaris equorum can be highlighted based on the permanent damage on the functional capacity of organs such as lung and liver, caused by the migration of these larvae. Furthermore , research on the biological control of nematodes in veterinary interest have occurred over the years in an attempt to find measures that will add strength to the conventional control, the search for better conditions of animal health and productivity. Thus, research on the biological control of nematodes in veterinary interest have occurred over the years in an attempt to find measures that will add strength to the conventional control, the search for better conditions of animal health and productivity. The objectives of this study were to assess the efficacy, in vitro, of two isolates (VC1 and VC4) of helmintophagous fungus Pochonia chlamydosporia grown in different culture media on Parascaris equorum eggs; evaluate the action of a crude enzymatic extract of fungus P. chlamydosporia on eggs P. equorum; analyze the enzymatic production of fungus P. chlamydosporia (isolates VC1 and VC4) after 21 days of interaction with P. equorum eggs. Was used two isolates of the fungus P. chlamydosporia (VC1 and VC4) arising from mycology collection Laboratory of Parasitology, Department of Veterinary Medicine of the Federal University of Viçosa. The P. equorum eggs were obtained by dissection of adult female specimens recovered from feces of foals naturally infected after administration piperazine citrate tetrahydrate. At the end of a period of 21 days of interaction, were observed percentage of P. equorum eggs destruction of 41.9 % and 48.3 % for isolates VC1 and VC4, respectively. The action of a crude enzymatic extract of the fungus P. chlamydosporia (isolated VC4) showed a percentage of 36.5% of destruction, after a 48 hours period of interaction. The results presented in this work suggest that the fungus Pochonia chlamydosporia, and its crude enzymatic extract, can be used to assist in the control of embryonated eggs of the ascarid Parascaris equorum.

A scaris is a genus of parasitic worms (helminths) found in the small intestine of various mammalian hosts, including Ascaris lumbricoides in humans, Parascaris equorum and P univalens in horses, Ascaris suum in pigs, Toxocara cati in cats and Toxocara canis in dogs. To date, Parascaris spp. are the only Ascaris worms that have developed resistance to anthelmintic drugs. The mechanisms of resistance in Parascaris spp are incompletely understood, partly due to the absence of robust in-vitro models. Further complicating in-vitro studies, Parascaris spp lack a free-living larval stage as their larva only hatch within the host. The aim of this study was to develop in-vitro methods for hatching, scoring the viability of Parascaris L3 larvae and exposing them to the anthelmintic drugs ivermectin, pyrantel, thiabendazole, and the herbal extract carvacrol. This study shows that mechanical Ascaris egg breaking using a homogenizer resulted in a hatching rate of 98%. Our viability scoring system could distinguish an ivermectin resistant larvae from an ivermectin susceptible larvae derived from different farms. This indicates that this method may have utility for the screening of larvae ivermectin resistance on the level of farm populations. Interestingly, a highly paralytic effect observed after carvacrol exposure. Carvacrol shows direct paralytic effects on Parascaris larvae in a dose-dependent manner, as higher concentrations were lethal to all exposed larvae. This result presents a potential future opportunity for carvacrol used in the treatment of Ascaris infections. To conclude our results, we have successfully developed an in-vitro model as well as a scoring system for the viability of Parascaris L3 stage larvae, which can be used for assaying the effect on larvae after drug exposure

In den vorliegenden Studien wurden verschiedene diagnostische Verfahren zum Nachweis von Protozoen und Nematoden im Hinblick auf Sensitivität, Arbeitsaufwand und Kosten miteinander verglichen. Zudem wurde die Eignung der PCR zur molekularen Charakterisierung der Cryptosporidium spp. exemplarisch an Igelkotproben getestet. Bei der Untersuchung von 90 Ferkelkotproben auf I. suis war die Sensitivität eines Kotausstriches mit nachfolgender Autofluoreszenzmikroskopie (AM) signifikant höher als bei einem Flotationsverfahren (FV) mit NaCl-Zucker-Lösung und bei dem kombinierten Sedimentations-Flotations-Verfahren (KSFV) mit verschiedenen Flotationslösungen (NaCl, ZnSO4, NaCl-Zucker-Lösung) mit nachfolgender Lichtmikroskopie. Zudem ist der Arbeitsaufwand für die AM deutlich geringer als bei dem FV und KSFV. Die höheren apparativen Kosten für die AM sind bei hohem Probendurchsatz durch den geringeren Zeitaufwand und der höheren Sensitivität gerechtfertigt. Die Anzahl Kryptosporidien-positiver Proben war bei der Untersuchung von 103 Kälberkotproben auf Cryptosporidium sp. mittels Enzymimmunoassays (EIA; ProSpecT® Cryptosporidium Microplate Assay) im Vergleich zur Karbolfuchsin-Färbung (CF) nach HEINE (1981) und der modifizierten-Ziehl-Neelsen-Färbung (MZN) nach HENRIKSEN u. POHLENZ (1982) am höchsten und signifikant höher als bei der Anwendung der MZN, wenn 10 Blickfelder durchmustert wurden. Bei der Untersuchung von 74 Igelkotproben auf Cryptosporidium sp. mittels EIA (ProSpecT®), einem immunochromatographischen Verfahren (FASTest® CRYPTO Strip), der MZN nach HENRIKSEN u. POHLENZ (1981) und einem direkten Immunfluoreszenz-Test (IFA; MERIFLUOR Cryptosporidium/Giardia) wurden in 9 (EIA), 10 (FASTest®), 11 (MZN) und 12 (IFA) Proben Cryptosporidium sp. nachgewiesen. Der Arbeitsaufwand des FASTest® und der CF ist mit dem EIA vergleichbar, während der IFA und die MZN mehr Zeit benötigen. Die Anwendung des FASTest®, des IFA und des EIA ist mit höheren Kosten verbunden als bei den Färbemethoden, können aber gut in den Arbeitsablauf eines diagnostischen Labors eingefügt werden und sind einfach auszuwerten. Darüber hinaus wurden 45 Kotproben, welche bis zu 27 Tage bei verschiedenen Temperaturen (+6 °C, +16 °C, +30 °C, +40 °C) gelagert wurden, untersucht, um einen Einfluss der Temperatur auf das Untersuchungsergebnis von EIA, CF und MZN zu ermitteln. Während sich die Anzahl positiver Proben bei der Untersuchung mit den Färbemethoden temperatur- und zeitabhängig reduzierte, wurde das Untersuchungsergebnis mittels EIA von der Lagerungstemperatur nicht beeinflusst, so dass ungekühlt transportierte Proben vorzugsweise mit dem EIA untersucht werden sollten. Dagegen ist die CF aufgrund ihrer einfachen und preiswerten Durchführung zur Untersuchung einer hohen Anzahl an Proben geeignet, sofern eine ununterbrochene Kühlung der Proben gewährleistet ist und diese innerhalb von drei Tagen untersucht werden. Der FASTest® ist zur Anwendung in Tierarztpraxen und Ställen geeignet, da zur Untersuchung kein Mikroskop benötigt wird und die Resultate schnell vorliegen. Die Verwendung des IFA, der Kryptosporidien-Oozysten und Giardien-Zysten nachweist, bietet sich vor allem bei Proben an, die auf beide Protozoen untersucht werden sollen. Das Vorkommen der Kryptosporidiose bei unterentwickelten und geschwächten Igeln, welche zum Überwintern in Igelstationen aufgenommen werden, ist hoch. Von 188 untersuchten Igelkotproben konnten in 29,8 % der Proben Cryptosporidium spp. nachgewiesen werden. Durch die Genotypisierung der Kryptosporidien aus 15 positiven Igelkotproben mittels RFLP-PCR basierend auf dem 18S rRNA-Gen konnte in allen untersuchten Proben die Präsenz von C. parvum gezeigt werden. Mit Hilfe der Multilocus-Sequenz-Typisierung der Fragmente des 60kDa Glycoprotein-Gens, des 18S rRNA-Gens, des Actin-Gens und des 70 kDa Hitzeschockprotein-Gens konnten drei verschiedene Subtypen-Familien (IIa, IIc und eine neue als VIIa vorgeschlagene Subtypen-Familie) erkannt werden. Die von den Igeln ausgeschiedenen Kryptosporidien-Oozysten mit zum Teil nachgewiesenem zoonotischen Potential (IIa Subtypen-Familie) könnten eine Infektionsquelle für den Menschen sein, aber auch ein antropozoonotisches Potential (IIc Subtypen-Familie) sollte in Betracht gezogen werden, so dass die Hygiene in den Igelstationen einen hohen Stellenwert einnehmen sollte. Die Untersuchungsergebnisse zum Nachweis von Eimeria-Arten beim Kalb von 70 Sammelkotproben, hergestellt aus 10 Einzelkotproben (SKP10), bzw. von 30 Sammelkotproben, zusammengesetzt aus 5 Einzelkotproben (SKP5), wurden mit denen der zugehörigen Einzelkotproben (EKP) verglichen. Die Resultate der EKP (arithmetischer Mittelwert) und der zugehörigen SKP weisen mit den signifikant häufigeren Abweichungen im Bereich von bis zu 100 Oozysten pro Gramm Kot (OpG) eine geringe Differenz zwischen den beiden Verfahren auf. Durch den sicheren Nachweis von Eimeria-Oozysten bei einem erwarteten Oozystengehalt von nur 202 OpG (SKP10) und 122 OpG (SKP5) ist die Untersuchung von Kälbersammelkotproben, eine Methode mit geringem Arbeitsaufwand und geringen Untersuchungskosten, zum Nachweis einer klinischen oder subklinischen Kokzidiose geeignet. Bei 51 Pferdekotproben wurde jeweils dreimal das kombinierte Sedimentations-Flotations-Verfahren (KSFV), wobei die Entnahme von verschiedenen Lokalisationen der Kotprobe (aus der Randregion, dem Inneren oder aus beiden Lokalisationen) erfolgte, und jeweils dreimal das KSFV mit vorheriger Homogenisierung einer größeren Kotmenge zum Nachweis von Nematodeneier durchgeführt. Eine Anhäufung der Strongyliden- und Ascarideneier in einem bestimmten Bereich der Proben konnte durch die Untersuchungen der verschiedenen Lokalisationen (á 10 g Kot) nicht nachgewiesen werden, so dass eine weitgehend homogene Verteilung dieser Nematodeneier in einer Pferdekotprobe wahrscheinlich ist. Zudem konnten die Untersuchungsergebnisse des KSFV, bei welchem 10 g Kot untersucht werden, durch die vorherige Homogenisierung einer größeren Probenmenge nicht verbessert werden. Zum Nachweis von Nematoden beim Pferd sollte dem Labor eine ausreichende Probenmenge (ca. 50 g) zugesandt werden. Die Homogenisierung einer größeren Probenmenge vor der Durchführung einer diagnostischen Methode, bei der Aliquote von mindestens 10 g Kot Verwendung finden, ist unnötig. / The studies presented were carried out to compare different diagnostic methods for detection of protozoa and nematodes regarding sensitivity, expenditure of human labour and costs. Besides, the ability of the PCR for the molecular characterization of the Cryptosporidium spp. was tested exemplarily in faecal samples of hegdehogs. The examination of ninety faecal samples of suckling piglets showed a significantly higher sensitivity of faecal smears examined by autofluorescence microscopy (AM) compared to the flotation method (FV) using NaCl-sucrose solution and the combined sedimentation-flotation method (KSFV) using different flotation solutions (NaCl, ZnSO4, NaCl-sucrose) scanned by bright field microscopy. Moreover the expenditure of human labour by AM is considerably lower than FV and KSFV. The costs related to equipment for AM is justified in case of high sample throughput and by superior sensitivity. The enzyme immunoassay (EIA; ProSpecT® Cryptosporidium Microplate Assay) was the most sensitive method for diagnosis of cryptosporidiosis in calves (n = 103) compared to the carbol fuchsin (CF; HEINE 1981) and modified Ziehl-Neelsen (MZN; HENRIKSEN a. POHLENZ 1982) staining techniques. The sensitivity of the EIA was significantly higher than the MZN, if ten fields of view were scanned. 74 faecal samples of hedgehogs were examined with the EIA (ProSpecT®), an immunochromatographic method (FASTest® CRYPTO Strip), the MZN (HENRIKSEN u. POHLENZ (1981)) and a direct immunofluorescent assay (IFA; MERIFLUOR Cryptosporidium/Giardia). Cryptosporidium sp. were detected in 9 (EIA), 10 (FASTest®), 11 (MZN) und 12 (IFA) faecal samples. The hands on time of the FASTest® and CF is comparable to EIA while the IFA and MZN are more time-consuming. The examination of the FASTest®, IFA and EIA is combined with higher costs than the staining techniques, but they can be integrated in the work flow of a routine diagnostic laboratory easily and evaluation is simple. Moreover 45 faecal samples stored up to 27 days at different temperature (+6 °C, +16 °C, +30 °C, +40 °C) were examined to evaluate the influence of temperature on the results of EIA, CF and MZN. While the number of the positive samples of stained smears decreased in a temperature and time-dependent manner, the results of the EIA were not influenced by sample storage at any temperature, so that samples transported without cooling should be examined preferably by EIA. Nevertheless the CF due to its simplicity and low costs is suited for scanning of a high number of samples, if they were cooled continuously and examined within three days. The FASTest® is qualified for use in veterinary practice and stables, because the examination requires no microscope and the results are obtained immediately. The IFA, which can detect Crypotsporidium oocysts as well as Giardia cysts, is suited especially for faecal samples suspected to contain both protozoa. Cryptosporidial infections are very frequent in hedgehogs which are admitted for hibernation to hedgehog rehabilitation centres because of their insufficient body weight and weakness. Cryptosporidium spp. were detected in 29.8 % of 188 faecal samples of hedgehogs. The genotyping of Cryptosporidium spp. by PCR and RFLP-PCR based on the 18S ribosomal RNA gene were performed on 15 faecal samples of hedgehogs positive for Cryptosporidium spp. and suggested the presence of C. parvum in all samples. Multilocus sequence typing on partial 60 kDa glycoprotein gene, 18S rRNA gene, actine gene, 70 kDa heat shock protein gene sequences revealed 3 different subtype families: IIa, IIc and a new proposed as VIIa subtype family. Some of the Cryptosporidium oocysts excreted from hedgehogs are zoonotical (IIa subtype family) or anthropozoonotic(IIc subtype family). Thus hygienic measurements to avoid transmission are essential in hedgehog rehabilitation centres. The results of examination of 70 pooled faecal samples originating from 10 calves (SKP10) and 30 pooled faecal samples originating from 5 calves (SKP5) for detection of Eimeria spp. were compared with the arithmetic means of opg (oocysts per gram of faeces) counts of the respective single 10 or 5 samples. A low difference between both methods of less than 100 opg was significantly more frequently observed than higher differences. Low values of 202 opg and 122 opg were reliably detected in SKP10 und SKP5, respectively, and thus examination of pooled faecal samples appears to be suitably sensitive and cost effective to detect clinical and subclinical coccidiosis in calves. 51 faecal samples of horses were examined three times by KSFV for nematode eggs by taking aliquots from different locations of the same faecal samples (from the margin, from inside and from both locations). Thereafter the KSFV with the homogenisation of a larger amount of faeces was also carried out three times. The examination of samples from the different locations (each 10 g of faeces) delivered no evidence for accumulation of nematode eggs (strongyles and Parascaris equorum) in the faeces and thus the distribution of the nematode eggs appears sufficiently homogeneous in faecal samples of horses. Homogenisation of a larger amount of faeces did not improve the results of coproscopy. For diagnostic purposes 50 g faeces per sample should be shipped to the laboratory. The homogenisation of a larger amount of faeces before using a diagnostic method is dispensable, if aliquots of 10 g faeces are examined.

Parascaris equorum is a nematode parasite that infects young horses, sometimes causing unthriftiness, respiratory signs, or intestinal impaction in severe cases. Infection can be diagnosed by detection of eggs in feces, but this is only possible after the worms are fully mature. The goal of this study was to develop an antibody-based test for prepatent diagnosis of P. equorum infection. To produce western blot (WB) antigen, P. equorum larvae were cultured for collection of excretory-secretory antigens (ESA). Sera from 18 pregnant broodmares, their subsequent foals, and a group of 12 older mares and geldings were analyzed. In order to check for cross-reactivity between P. equorum and other ascarid species and equine parasites, additional sera were analyzed. Sera from a horse with monospecific P. equorum infection was compared to horses with monospecific Strongyloides westeri or cyathostome infections, rabbits inoculated with Baylisascaris procyonis or Toxocara canis eggs, dogs naturally infected with T. canis, and rabbits immunized with B. procyonis or P. equorum ESA. Molecular weights of silver-stained P. equorum larval ESA ranged between 12 to 94 kDa. In WB analysis, sera from 94% of broodmares contained IgG(T) antibody that recognized multiple P. equorum larval ESA. Foals showed no IgG(T) antibodies pre-suckle, but antibodies similar to their dams were observed post-suckle and thereafter. Of the older mares and geldings, 58% had IgG(T) antibodies recognizing larval ESA. Serum IgG(T) antibodies against P. equorum larval ESA were also found in parasite-free and monospecific infection equine sera. Ascarid positive foals did not produce detectable amounts of IgE or IgM antibodies against larval ESA. When P. equorum, T. canis, and B. procyonis antibody reactivity was compared, antigens at 19 kDa and 34 kDa had the highest potential for identification of larval P. equorum infections. When immature adult P. equorum ESA was examined, IgG(T) antibody recognition was demonstrated in 50% of broodmares and 17% of the older horses, and appeared several weeks prior to patency in foal serum. Results indicate that IgG(T) antibodies against P. equorum ESA are common in mature horses, and are transferred from mare to foal, limiting the diagnostic potential of an antibody-based test.

Anthelmintic resistance of parasites infecting livestock animals is a global problem resulting in decreased animal welfare and production losses. Horses are not exempt from this issue as wide-spread anthelmintic resistance exists among the equine cyathostomins and Parascaris spp. Of the three drug classes available for treating equine intestinal helminths anthelmintic resistance, defined as less than 90-95% drug efficacy, exist to all three. New pharmaceutical control regimens and the elucidation of parasite drug response mechanisms are needed. Two studies were carried out evaluating combination deworming regimens. A population of cyathostomins with known resistance to the benzimidazole (BZ) and pyrimidine drug classes maintained in a herd of Shetland ponies was used. Fecal egg counts were performed every two weeks and used to evaluate drug efficacy. The first study evaluated the combination of a BZ and pyrimidine drug for four consecutive treatments, and compared the individual drug efficacies before and after combination use. The first combination treatment exhibited an additive effect at 76.6%, but the subsequent three combination treatments decreased to approximately 40%. There was no significant difference between the initial and final efficacies of individual drugs (BZ, p=0.4421; pyrimidine, p=0.8361). It appears the combination treatment selected for double-drug resistant adult parasites. The timeframe of this study (1 year) and the one year lifespan of adult cyathostomins prevented observations of combination treatment on subsequent generations, however given the sustainability of resistance in this cyathostomin population, it seems unlikely efficacy would improve over time. The second study examined the combination of a BZ drug with a macrocyclic lactone (ML) drug. This parasite population was 100% naïve to the ML drug class. This study was carried out in a similar manner to the first, except only two combination treatments were given. ML exhibited 100% efficacy when it was used alone, or in combination. The initial and final BZ efficacy did not significantly differ (p=0.9890). In summary, the results described herein do not support the use of combination treatments where resistance is prevalent, but more long term studies are needed to fully understand the long-term effects on subsequent generations. The in vitro maintenance of Parascaris spp. provides opportunity for various molecular analyses. An objective motility scoring assessment allowed for continuous monitoring of worm viability. In this study, several saline solutions, nutrient supplements, environmental conditions, and Roswell-Park Memorial Institute medium 1640 (RPMI-1640) were evaluated for the longevity and viability of adult Parascaris spp. Overall, RPMI-1640 resulted in better longevity (168 hours) and significantly better viability (pParascaris spp. to in vitro drug exposure. Oxibendazole at 10 µg/mL for 24 hours and ivermectin at 1 µg/mL for three hours were employed, and worms were used for transcriptomic analyses to identify drug response mechanisms. The top four genes which were significantly different between drug treated and control groups were: cyp4504C1, sup-9, frmd4a, and klhdc10. It is hypothesized that cyp4504C1 and klhdc10 are drug detox mechanisms, while sup-9 and frmd4a may be indirect response related to the drug effects. Their expression was further evaluated using quantitative RT-PCR, however there was no significant difference in any gene expression between groups. It should be noted that there are several limitations associated with the qPCR method, and the lack of significance should not rule out the possible involvement of these genes and more research on drug response mechanisms is needed. In summary, there is very little research regarding combination deworming in horses, and their current use is largely due to some success for ruminant parasites, but the current work summarized herein does not support their use. Finally, until now the lack of in vitro methods for equine helminths has significantly delayed the elucidation of drug response mechanisms. This was the first whole-transcriptome approach for any ascarid parasite and uncovered proteins with possible involvement in drug metabolism or compensate for the toxic effects Overall, the research surrounding anthelmintic resistance in livestock helminths, particularly in horses, is lacking and the resistance crisis demands further investigation.

In den vorliegenden Studien wurden verschiedene diagnostische Verfahren zum Nachweis von Protozoen und Nematoden im Hinblick auf Sensitivität, Arbeitsaufwand und Kosten miteinander verglichen. Zudem wurde die Eignung der PCR zur molekularen Charakterisierung der Cryptosporidium spp. exemplarisch an Igelkotproben getestet. Bei der Untersuchung von 90 Ferkelkotproben auf I. suis war die Sensitivität eines Kotausstriches mit nachfolgender Autofluoreszenzmikroskopie (AM) signifikant höher als bei einem Flotationsverfahren (FV) mit NaCl-Zucker-Lösung und bei dem kombinierten Sedimentations-Flotations-Verfahren (KSFV) mit verschiedenen Flotationslösungen (NaCl, ZnSO4, NaCl-Zucker-Lösung) mit nachfolgender Lichtmikroskopie. Zudem ist der Arbeitsaufwand für die AM deutlich geringer als bei dem FV und KSFV. Die höheren apparativen Kosten für die AM sind bei hohem Probendurchsatz durch den geringeren Zeitaufwand und der höheren Sensitivität gerechtfertigt. Die Anzahl Kryptosporidien-positiver Proben war bei der Untersuchung von 103 Kälberkotproben auf Cryptosporidium sp. mittels Enzymimmunoassays (EIA; ProSpecT® Cryptosporidium Microplate Assay) im Vergleich zur Karbolfuchsin-Färbung (CF) nach HEINE (1981) und der modifizierten-Ziehl-Neelsen-Färbung (MZN) nach HENRIKSEN u. POHLENZ (1982) am höchsten und signifikant höher als bei der Anwendung der MZN, wenn 10 Blickfelder durchmustert wurden. Bei der Untersuchung von 74 Igelkotproben auf Cryptosporidium sp. mittels EIA (ProSpecT®), einem immunochromatographischen Verfahren (FASTest® CRYPTO Strip), der MZN nach HENRIKSEN u. POHLENZ (1981) und einem direkten Immunfluoreszenz-Test (IFA; MERIFLUOR Cryptosporidium/Giardia) wurden in 9 (EIA), 10 (FASTest®), 11 (MZN) und 12 (IFA) Proben Cryptosporidium sp. nachgewiesen. Der Arbeitsaufwand des FASTest® und der CF ist mit dem EIA vergleichbar, während der IFA und die MZN mehr Zeit benötigen. Die Anwendung des FASTest®, des IFA und des EIA ist mit höheren Kosten verbunden als bei den Färbemethoden, können aber gut in den Arbeitsablauf eines diagnostischen Labors eingefügt werden und sind einfach auszuwerten. Darüber hinaus wurden 45 Kotproben, welche bis zu 27 Tage bei verschiedenen Temperaturen (+6 °C, +16 °C, +30 °C, +40 °C) gelagert wurden, untersucht, um einen Einfluss der Temperatur auf das Untersuchungsergebnis von EIA, CF und MZN zu ermitteln. Während sich die Anzahl positiver Proben bei der Untersuchung mit den Färbemethoden temperatur- und zeitabhängig reduzierte, wurde das Untersuchungsergebnis mittels EIA von der Lagerungstemperatur nicht beeinflusst, so dass ungekühlt transportierte Proben vorzugsweise mit dem EIA untersucht werden sollten. Dagegen ist die CF aufgrund ihrer einfachen und preiswerten Durchführung zur Untersuchung einer hohen Anzahl an Proben geeignet, sofern eine ununterbrochene Kühlung der Proben gewährleistet ist und diese innerhalb von drei Tagen untersucht werden. Der FASTest® ist zur Anwendung in Tierarztpraxen und Ställen geeignet, da zur Untersuchung kein Mikroskop benötigt wird und die Resultate schnell vorliegen. Die Verwendung des IFA, der Kryptosporidien-Oozysten und Giardien-Zysten nachweist, bietet sich vor allem bei Proben an, die auf beide Protozoen untersucht werden sollen. Das Vorkommen der Kryptosporidiose bei unterentwickelten und geschwächten Igeln, welche zum Überwintern in Igelstationen aufgenommen werden, ist hoch. Von 188 untersuchten Igelkotproben konnten in 29,8 % der Proben Cryptosporidium spp. nachgewiesen werden. Durch die Genotypisierung der Kryptosporidien aus 15 positiven Igelkotproben mittels RFLP-PCR basierend auf dem 18S rRNA-Gen konnte in allen untersuchten Proben die Präsenz von C. parvum gezeigt werden. Mit Hilfe der Multilocus-Sequenz-Typisierung der Fragmente des 60kDa Glycoprotein-Gens, des 18S rRNA-Gens, des Actin-Gens und des 70 kDa Hitzeschockprotein-Gens konnten drei verschiedene Subtypen-Familien (IIa, IIc und eine neue als VIIa vorgeschlagene Subtypen-Familie) erkannt werden. Die von den Igeln ausgeschiedenen Kryptosporidien-Oozysten mit zum Teil nachgewiesenem zoonotischen Potential (IIa Subtypen-Familie) könnten eine Infektionsquelle für den Menschen sein, aber auch ein antropozoonotisches Potential (IIc Subtypen-Familie) sollte in Betracht gezogen werden, so dass die Hygiene in den Igelstationen einen hohen Stellenwert einnehmen sollte. Die Untersuchungsergebnisse zum Nachweis von Eimeria-Arten beim Kalb von 70 Sammelkotproben, hergestellt aus 10 Einzelkotproben (SKP10), bzw. von 30 Sammelkotproben, zusammengesetzt aus 5 Einzelkotproben (SKP5), wurden mit denen der zugehörigen Einzelkotproben (EKP) verglichen. Die Resultate der EKP (arithmetischer Mittelwert) und der zugehörigen SKP weisen mit den signifikant häufigeren Abweichungen im Bereich von bis zu 100 Oozysten pro Gramm Kot (OpG) eine geringe Differenz zwischen den beiden Verfahren auf. Durch den sicheren Nachweis von Eimeria-Oozysten bei einem erwarteten Oozystengehalt von nur 202 OpG (SKP10) und 122 OpG (SKP5) ist die Untersuchung von Kälbersammelkotproben, eine Methode mit geringem Arbeitsaufwand und geringen Untersuchungskosten, zum Nachweis einer klinischen oder subklinischen Kokzidiose geeignet. Bei 51 Pferdekotproben wurde jeweils dreimal das kombinierte Sedimentations-Flotations-Verfahren (KSFV), wobei die Entnahme von verschiedenen Lokalisationen der Kotprobe (aus der Randregion, dem Inneren oder aus beiden Lokalisationen) erfolgte, und jeweils dreimal das KSFV mit vorheriger Homogenisierung einer größeren Kotmenge zum Nachweis von Nematodeneier durchgeführt. Eine Anhäufung der Strongyliden- und Ascarideneier in einem bestimmten Bereich der Proben konnte durch die Untersuchungen der verschiedenen Lokalisationen (á 10 g Kot) nicht nachgewiesen werden, so dass eine weitgehend homogene Verteilung dieser Nematodeneier in einer Pferdekotprobe wahrscheinlich ist. Zudem konnten die Untersuchungsergebnisse des KSFV, bei welchem 10 g Kot untersucht werden, durch die vorherige Homogenisierung einer größeren Probenmenge nicht verbessert werden. Zum Nachweis von Nematoden beim Pferd sollte dem Labor eine ausreichende Probenmenge (ca. 50 g) zugesandt werden. Die Homogenisierung einer größeren Probenmenge vor der Durchführung einer diagnostischen Methode, bei der Aliquote von mindestens 10 g Kot Verwendung finden, ist unnötig. / The studies presented were carried out to compare different diagnostic methods for detection of protozoa and nematodes regarding sensitivity, expenditure of human labour and costs. Besides, the ability of the PCR for the molecular characterization of the Cryptosporidium spp. was tested exemplarily in faecal samples of hegdehogs. The examination of ninety faecal samples of suckling piglets showed a significantly higher sensitivity of faecal smears examined by autofluorescence microscopy (AM) compared to the flotation method (FV) using NaCl-sucrose solution and the combined sedimentation-flotation method (KSFV) using different flotation solutions (NaCl, ZnSO4, NaCl-sucrose) scanned by bright field microscopy. Moreover the expenditure of human labour by AM is considerably lower than FV and KSFV. The costs related to equipment for AM is justified in case of high sample throughput and by superior sensitivity. The enzyme immunoassay (EIA; ProSpecT® Cryptosporidium Microplate Assay) was the most sensitive method for diagnosis of cryptosporidiosis in calves (n = 103) compared to the carbol fuchsin (CF; HEINE 1981) and modified Ziehl-Neelsen (MZN; HENRIKSEN a. POHLENZ 1982) staining techniques. The sensitivity of the EIA was significantly higher than the MZN, if ten fields of view were scanned. 74 faecal samples of hedgehogs were examined with the EIA (ProSpecT®), an immunochromatographic method (FASTest® CRYPTO Strip), the MZN (HENRIKSEN u. POHLENZ (1981)) and a direct immunofluorescent assay (IFA; MERIFLUOR Cryptosporidium/Giardia). Cryptosporidium sp. were detected in 9 (EIA), 10 (FASTest®), 11 (MZN) und 12 (IFA) faecal samples. The hands on time of the FASTest® and CF is comparable to EIA while the IFA and MZN are more time-consuming. The examination of the FASTest®, IFA and EIA is combined with higher costs than the staining techniques, but they can be integrated in the work flow of a routine diagnostic laboratory easily and evaluation is simple. Moreover 45 faecal samples stored up to 27 days at different temperature (+6 °C, +16 °C, +30 °C, +40 °C) were examined to evaluate the influence of temperature on the results of EIA, CF and MZN. While the number of the positive samples of stained smears decreased in a temperature and time-dependent manner, the results of the EIA were not influenced by sample storage at any temperature, so that samples transported without cooling should be examined preferably by EIA. Nevertheless the CF due to its simplicity and low costs is suited for scanning of a high number of samples, if they were cooled continuously and examined within three days. The FASTest® is qualified for use in veterinary practice and stables, because the examination requires no microscope and the results are obtained immediately. The IFA, which can detect Crypotsporidium oocysts as well as Giardia cysts, is suited especially for faecal samples suspected to contain both protozoa. Cryptosporidial infections are very frequent in hedgehogs which are admitted for hibernation to hedgehog rehabilitation centres because of their insufficient body weight and weakness. Cryptosporidium spp. were detected in 29.8 % of 188 faecal samples of hedgehogs. The genotyping of Cryptosporidium spp. by PCR and RFLP-PCR based on the 18S ribosomal RNA gene were performed on 15 faecal samples of hedgehogs positive for Cryptosporidium spp. and suggested the presence of C. parvum in all samples. Multilocus sequence typing on partial 60 kDa glycoprotein gene, 18S rRNA gene, actine gene, 70 kDa heat shock protein gene sequences revealed 3 different subtype families: IIa, IIc and a new proposed as VIIa subtype family. Some of the Cryptosporidium oocysts excreted from hedgehogs are zoonotical (IIa subtype family) or anthropozoonotic(IIc subtype family). Thus hygienic measurements to avoid transmission are essential in hedgehog rehabilitation centres. The results of examination of 70 pooled faecal samples originating from 10 calves (SKP10) and 30 pooled faecal samples originating from 5 calves (SKP5) for detection of Eimeria spp. were compared with the arithmetic means of opg (oocysts per gram of faeces) counts of the respective single 10 or 5 samples. A low difference between both methods of less than 100 opg was significantly more frequently observed than higher differences. Low values of 202 opg and 122 opg were reliably detected in SKP10 und SKP5, respectively, and thus examination of pooled faecal samples appears to be suitably sensitive and cost effective to detect clinical and subclinical coccidiosis in calves. 51 faecal samples of horses were examined three times by KSFV for nematode eggs by taking aliquots from different locations of the same faecal samples (from the margin, from inside and from both locations). Thereafter the KSFV with the homogenisation of a larger amount of faeces was also carried out three times. The examination of samples from the different locations (each 10 g of faeces) delivered no evidence for accumulation of nematode eggs (strongyles and Parascaris equorum) in the faeces and thus the distribution of the nematode eggs appears sufficiently homogeneous in faecal samples of horses. Homogenisation of a larger amount of faeces did not improve the results of coproscopy. For diagnostic purposes 50 g faeces per sample should be shipped to the laboratory. The homogenisation of a larger amount of faeces before using a diagnostic method is dispensable, if aliquots of 10 g faeces are examined.

The diploma work The occurrence and the seasonal dynamic of the intestinal parasite by racing horses deals with the occurrence of intestinal parasites by individual horse-age categories, parasites{\crq} prevalence in comparison to annual seasons. The helminthes with highest prevalence {--} small and large Strongylus, Strongyloides westeri, Parascaris equorum, Oxiuris Equi {--} are described in the work.