Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zdeňka Čermáková Supervisor: Prof. RNDr. Jiří Lamka, CSc. Title of diploma thesis: The progression and results of the model parasite culturing liver fluke (Fasciola hepatica) in sheep (Ovis aries) Fasciolosis is a parasitic disease caused by liver fluke (Fasciola hepatica). This parasitosis is a significant disease in livestock, wild animals and in some parts of the world in humans. It causes a decrease in livestock performance and fertility, weight loss and high economic losses in livestock. The aim of this work was to describe in detail the course of artificial infection of sheep infected with F. hepatica metacercaria. Specifically, changes in blood count, egg excretion, and response of the immune system of infected individuals were monitored. The observed values were compared with the studies already performed. The demonstration of fasciolosis in artificially infected sheep was performed using the coprological sedimentation method, the differential determination of leukocytes from the blood layers, the determination of hematocrit and the immunological method ELISA (enzyme-linked immunosorbent assays). Three lambs of Texel breeds infected with 200 metacercaria were used for the...

Fasciolosis, caused by the trematode parasite Fasciola hepatica, is a multi-host parasitic disease affecting many countries worldwide. It is a well-recognized clinically and economically important disease of food producing animals such as cattle and sheep. In the UK, the incidence and distribution of fasciolosis has been increasing in the last decade while the timing of acute disease is becoming more variable and the season suitable for parasite development outside the mammalian host has been extended. Meanwhile control is proving increasingly difficult due to changing weather conditions, increased animal movements and developing anthelmintic resistance. Forecasting models have been around for a long time to aid health planning related to fasciolosis control, but studies identifying management related risk factors are limited. Moreover, the lack of information on the accuracy of meat inspection and available liver fluke diagnostic tests hinders effective monitoring of disease prevalence and treatment. So far, the evaluation of tests available for the diagnosis of the infection in cattle has mainly been carried out using gold standard approaches or under experimental settings, the limitations of which are well known. In cattle, the infection mainly manifests as a sub-clinical disease, resulting in indirect production losses, which are difficult to estimate. The lack of obvious clinical signs results in these losses commonly being attributed to other causes such as poor weather conditions or bad quality forage. This further undermines establishment of appropriate control strategies, as it is difficult to convince farmers to treat without demonstrating clear economic losses of sub-clinical disease. This project explores the value of slaughterhouse data in understanding the changing epidemiology of fasciolosis, identifying sustainable control measures and estimating the effect of infection on production parameters using data collected at one of the largest cattle and sheep abattoirs in Scotland. Data used in this study include; a) abattoir data routinely collected during 2013 and 2014, b) data collected during 3 periods of abattoir based sampling, c) data collected through administration of a management questionnaire and d) climatic and environmental data from various online sources. A Bayesian extension of the Hui Walter no gold standard model was used to estimate the diagnostic sensitivity and specificity of five diagnostic tests for fasciolosis in cattle, which were applied on 619 samples collected from the abattoir during three sampling periods; summer 2013, winter 2014 and autumn 2014. The results provided novel information on the performance of these tests in a naturally infected cattle population at different times of the year. Meat inspection was estimated to have a sensitivity of 0.68 (95% BCI 0.61-0.75) and a specificity of 0.88 (95% BCI 0.85-0.91). Accurate estimates of sensitivity and specificity will allow for routine abattoir liver inspection to be used as a tool for monitoring the epidemiology of F. hepatica as well as evaluating herd health planning. Linear regression modelling was used to estimate the delay in reaching slaughter weight in beef cattle infected with F. hepatica, accounting for other important factors such as weight, age, sex, breed and farm as a random effect. The model estimated that cattle classified as having fluke based on routine liver inspection had on average 10 (95% CI 9-12) days greater slaughter age, assuming an average carcass weight of 345 kg. Furthermore, estimates from a second model indicated that the increase in age at slaughter was more severe for higher fibrosis scores. More precisely, the increase in slaughter age was 34 (95% CI 11-57) days for fibrosis score of 1, 93 (95% CI 57-128) days for fibrosis score 2 and 78 (95% CI 30-125) days for fibrosis score 3. Similarly, in a third model comparing different burden categories with animals with no fluke burden, there was a 31 (95% CI 7-56) days increase in slaughter age for animals with 1 to 10 parasites and 77 (95% CI 32-124) days increase in animals with more than 10 parasites found in their livers. Lastly, a multi-variable mixed effects logistic regression model was built to estimate the association between climate, environmental, management and animal specific factors and the risk of an animal being infected by F. hepatica. Multiple imputation methodology was employed to deal with missing data arising from skipped questions in the questionnaire. Results of the regression model confirmed the importance of temperature, rainfall and cattle movements in increasing the risk for fasciolosis, while it indicated that the presence of deer can increase the risk of infection and that male cattle have a reduced risk of infection. Overall, this project has used slaughterhouse data to fill important knowledge gaps regarding F. hepatica infection in cattle. It has provided valuable information on the accuracy of routine abattoir meat inspection, as well as other diagnostic tests. It has also provided estimates of the effect of infection on the time cattle take to reach slaughter weight at different levels of infection and identified relevant risk factors related to the infection. In conclusion, knowledge of the effect of infection on slaughter age, as well as regional risk factors for F. hepatica infection, along with an improved use of abattoir inspection results in the evaluation of treatment strategies, can provide farmers and veterinarians with better incentives and tools to improve their herd health strategies and in the longer term help reduce the incidence of liver fluke in cattle.

Made available in DSpace on 2016-12-23T13:53:36Z (GMT). No. of bitstreams: 1 Cintia das Chagas Bernardo.pdf: 617196 bytes, checksum: 4fffe6c1253ee4daf431fe54065375a6 (MD5) Previous issue date: 2012-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Methods diagnostics of hepatic fascioliasis are being studied to propose more accurate techniques, easy perform, less costly if they have applicability in the field. The aim of this study was to validate the commercial® ELISA kits for detection of coproantigens and antibodies in serum and milk from cattle naturally infected by Fasciola hepatica. At first, sample of feces, blood and milk of cattle naturally infected with F. hepatica were collected. Fecal samples from 577 animals were processed according to sedimentation fecal technique, and 92 and 43 serum and milk samples , were processed according to manufacturer s instructions of a commercial® ELISA kit. Were used McNemar chi-square for statistical comparison, and calculated sensitivity, specificity, predictive values and kappa, and the sedimentation fecal technique was used as standard. Second, were evaluated 81 slaughter cattle livers of whom 45 were condemned by fasciolosis. Count was conducted the parasites in the livers condemned and collected fecal samples of these animals, 36 fecal samples were collected from animals without condemnation. The samples were separated in two aliquots and the first part of samples were processed by sedimentation fecal technique, and the other samples according to manufacturer s instructions of commercial® ELISA kit for coproantigens detection. The indicators of validity, reproducibility, Spearman correlation and McNemar chi-square were calculated and used as the gold standard the livers condemnation at slaughterhouse. With the results obtained in these studies, became clear that commercial® ELISA kits showed higher sensitivity to sedimentation fecal technique for diagnosis of bovine fasciolosis, however for the diagnosis of the disease in the field should take into account also the operation of the methods, not ruling out the use of fecal test, which is less labor intensive and less expensive compareted to ELISA kits tested / Métodos de diagnóstico da fasciolose hepática veem sendo estudados a fim de se propor técnicas mais acuradas, de fácil execução e de menor custo, que tenham aplicabilidade a campo. O objetivo do presente estudo foi validar kits comerciais® de ELISA para detecção de coproantígenos e anticorpos em soro e leite de bovinos infectados naturalmente por Fasciola hepatica. Numa primeira etapa, foram coletadas amostras de fezes, sangue e leite de bovinos naturalmente infectados por F. hepatica. Amostras de fezes de 577 animais foram processadas segundo a técnica coproparasitológica de sedimentação fecal para ovos de F. hepatica, e 92 amostras de soro e 43 de leite foram processadas segundo instruções do fabricante de um kit ELISA comercial®. Utilizou-se o Qui-quadrado de McNemar para comparação estatística, e calculou-se a sensibilidade e especificidade, valores preditivos e kappa dos kits®, sendo o exame coproparasitológico usado como padrão. Numa segunda etapa, foram avaliados ao abate 81 fígados bovinos dos quais 45 foram condenados por fasciolose. Foi realizada a contagem dos parasitos nos fígados condenados e coletada as amostras de fezes desses animais, além de 36 amostras fecais provenientes de animais que não tiveram os fígados condenados para nenhuma enfermidade. Das amostras de fezes foram separadas duas alíquotas sendo a primeira parte das amostras processadas pela técnica coproparasitológica de sedimentação e a outra, segundo instruções do fabricante de um kit ELISA comercial® para detecção de coproantígenos. Foram calculados os indicadores de validade e reprodutibilidade, e realizado o teste de correlação de Spearman e Qui-quadrado de McNemar, sendo utilizada como padrão ouro a condenação de fígados ao abate. Com os resultados obtidos nesses estudos, ficou claro que os kits comerciais® de ELISA apresentaram maior sensibilidade em relação ao exame coproparasitológico de sedimentação para o diagnóstico da fasciolose bovina, porém, para o diagnóstico da enfermidade a campo além da eficácia, deve-se levar em consideração também a operacionalização das técnicas, não descartando assim, o uso do exame coproparasitológico, sendo este menos trabalhoso e de menor custo em relação aos kits de ELISA testados / 619

Les interactions hôte – parasites sont des systèmes qui affectent probablement la totalité des êtres vivants et constituent un facteur clé dans la compréhension de la dynamique des maladies infectieuses. On a abordé cette problématique en utilisant le système Fasciola hepatica/Lymnaeidae dans sur l'île de Cuba. Cette thèse utilise une approche basée sur différentes disciplines de la biologie comme l’écologie des populations (distribution et abondances de mollusques hôtes intermédiaires), l'écologie parasitaire (données de prévalences naturelles de parasites chez les hôtes), la génétique des populations en utilisant des marqueurs microsatellites (tant pour le parasite que pour les mollusques hôtes), et des études de susceptibilité et compatibilité douve/limnée.En ce qui concerne la diversité génétique du parasite, une très haute diversité et une forte probabilité d’allofécondation ont été observées. En revanche, on n’a pas trouvé de différentiation significatives entre souches. Les taux d’infection chez le bétail sont très élevés.Pour ce qui est de la biologie des populations des limnées hôtes, on a cartographié la distribution des deux espèces présentes à Cuba : Galba cubensis qui est très répandue et Pseudosuccinea columella qui n'est présente que dans la partie centre-occidentale. Nous avons mis en évidence des différences concernant les types d’habitats préférés pour chaque espèce : G. cubensis est plus plastique écologiquement et se retrouve beaucoup plus dans les sites anthropisés.L'étude des compatibilités douve/mollusque a révélé l’existence de populations avec une résistance naturelle à l’infection par F. hepatica chez la limnée P. columella. Ces populations sont génétiquement différenciées des populations sensibles. La plupart des populations sensibles sont monomorphes avec le même haplotype très répandu. Par contre, on a observé une diversité génétique plus importante chez G. cubensis qui suggère un temps évolutif plus ancien à Cuba. L’échantillonnage fait dans une aire de où la fasciolose sévit fortement chez le bétail a révélé un très faible taux d’infection naturel chez les limnées, mais avec de fortes variations d'intensités. Différentes combinaisons douve/limnée sympatriques et allopatriques testées expérimentalement ont montré une compatibilité supérieure de G. cubensis qui suggère une meilleure adaptation aux souches de F. hepatica cubaines.Les résultats obtenus montrent que la compréhension de la dynamique des maladies infectieuses ainsi que leur contrôle doivent s’appuyer sur des connaissances très solides de la biologie, écologie, génétique et évolution des systèmes hôtes – parasites. / Host – parasite interactions are biological systems that probably affect every living being. It also constitute a key factor in the understanding of infectious diseases. This subject has been studied using the Fasciola hepatica/Lymnaeidae system in the insular environment of Cuba. This thesis makes a biological multidisciplinary approach through population ecology (distribution and abundance of intermediary hosts snails), infection rates estimations of parasites in their hosts, population genetics using microsatellites markers (for both parasite and snails hosts), and studies of susceptibility and compatibility fluke/snail.The genetic diversity of the parasite is characterized by high values of diversity as well as probability of cross fertilization. However, we failed to detect significant differences between the strains but a highly infection rate is shared in cattle.Regarding the lymnaeid snails, the distribution of the two occurring species is mapped showing that Galba cubensis is widely distributed while Pseudosuccinea columella only exists in central-western Cuba. Both snail species differ in preferred habitat types and G. cubensis displays a higher ecological plasticity commonly observed in heavily human-transformed sites.Differences in host-parasite compatibilities have been revealed. It has been noted the existence of some populations of P. columella with a natural resistance to F. hepatica infection which display a marked differentiation of their population genetic structure compared to susceptible populations. Most susceptible populations are monomorphic with a very common haplotype in Cuba. Conversely, we observed a higher diversity in G. cubensis indicating a larger evolutionary time in Cuba. The sampling conducted in a fasciolosis endemic area revealed very low prevalences in the snail populations. However, strong variation in parasite mean intensity within individuals supports the high infection rates observed in cattle. Different sympatric and allopatric fluke/snail combinations were experimentally tested in which G. cubensis proved to be more compatible and suggest a better adaptation to the circulating F. hepatica in Cuba.Our results show that the understanding of infectious diseases’ dynamics and their effective control must strictly rely in a full knowledge of the biology, ecology, genetics and evolution of host – parasite systems.