Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-26T17:25:10Z No. of bitstreams: 1 LediaWanderley.pdf: 1766339 bytes, checksum: 1ff559d6faf9483c7037afc6b631b271 (MD5) / Made available in DSpace on 2017-06-26T17:25:10Z (GMT). No. of bitstreams: 1 LediaWanderley.pdf: 1766339 bytes, checksum: 1ff559d6faf9483c7037afc6b631b271 (MD5) Previous issue date: 2016-12-21 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Helminthes and bacterial infections are a major challenge for livestock production because they cause loss of productivity in herds. To reduce losses, producers control parasites and bacteria mainly using synthetic chemicals, but the resistance to the active principles of these products calls for the continuous search for biologically active molecules. This research was conducted with the objective of obtaining protein fractions and protease from the latex of Ficus benjamina, in addition to evaluating the potential use of these protein samples in the control of the parasite nematode of small ruminants, Haemonchus contortus, as well as on the pathogenic bacteria Salmonella enterica, Staphylococcus aureus and Enterobacter aerogenes. Latex was obtained from an incision made at the apex of the plant limb and collected in sodium phosphate buffer (75 mM, pH 7.0). The latex was centrifuged (12,000 x g at 4 ° C for 15 min), dialysed against water (14 kDa cut-off), again centrifuged under the same conditions as before and then referred to as EPL (Latex Protein Extract). The EPL was fractionated with ammonium sulphate at 30-60% and 60-90% saturation to obtain the fractions denominated F30-60 and F60-90, respectively. F60-90 was fractionated by ion exchange chromatography on CM-Cellulose matrix and the fraction with the highest proteolytic activity obtained was denominated FbP (Ficus benjamina Protease). With these fractions, protein analysis, proteolytic activity and FbP identification were carried out. In addition, inhibition of larval development of the nematode H. contortus and inhibition of the growth of S. enterica, S. aureus and E. aerogenes in a liquid medium were carried out. The total protein levels obtained were, respectively, 121.5, 21.2, 75.7 mg for EPL, F30-60 and F60-90. The proteolytic activity of the protein samples was 43,471, 13,245 and 57,857 UA/mL/mgP for EPL, F30-60 and F60-90, respectively. The FbP presented proteolytic activity of 36,393.8 AU/mL/mgP. Gel analysis showed FbP with a protein band with mass between 20 and 30 kDa and by mass spectrometry the FbP was identified as cysteine protease, with a molecular weight of 23.97 kDa. FbP maintained stable proteolytic activity in the pH range between 6 and 10 and over a wide temperature range, with optimum activity at 60 °C. In tests for inhibition of larval development, EPL, F30-60, F60-90 and FbP presented IC50 of 0.22, 1.38, 0.42 and 0.26 mg/mL, respectively. It was verified that EPL, F30-60 and F60-90 did not exert an inhibitory action on the growth of bacteria, but FbP inhibited the growth of S. aureus and S. enterica with minimum inhibitory concentration (MIC) of 3.12 μg/mL. It is concluded that FbP obtained from latex of F. benjamina has promising biological properties for the development of products that can be used in the control of the gastrointestinal nematode, H. contortus, and the pathogenic bacteria S. aureus and S. enterica. / Helmintoses e infecções bacterianas são um grande desafio para a produção pecuária porque provocam perda de produtividade nos rebanhos. Para reduzir prejuízos, os produtores realizam o controle de parasitos e bactérias principalmente utilizando produtos químicos sintéticos, mas a resistência aos princípios ativos desses produtos insta a busca contínua por moléculas biologicamente ativas. Essa pesquisa foi conduzida com o objetivo de obter frações proteicas e protease a partir do látex de Ficus benjamina, além de avaliar uso potencial dessas amostras proteicas no controle do nematóide parasito de pequenos ruminantes, Haemonchus contortus, bem como sobre as bactérias patogênicas Salmonella enterica, Staphylococcus aureus e Enterobacter aerogenes. O látex foi obtido a partir de uma incisão feita no ápice do galho das plantas e coletado em tampão fosfato de sódio (75 mM, pH 7,0). O látex foi centrifugado (12.000 x g a 4 °C por 15 min), dialisado contra água (cut-off 14 kDa), novamente centrifugado nas mesmas condições anteriores e, então, denominado EPL (Extrato Proteico do Látex). O EPL foi fracionado com sulfato de amônio a 30-60% e 60-90% de saturação para a obtenção das frações denominadas F30-60 e F60-90, respectivamente. A F60-90 foi fracionada por cromatografia de troca iônica em matriz de CM-Cellulose e a fração com maior atividade proteolítica obtida foi denominada FbP (Ficus benjamina Protease). De posse dessas frações, foram conduzidos ensaios de análise de proteínas, atividade proteolítica e identificação da FbP. Adicionalmente foram realizados ensaios de inibição do desenvolvimento larvar do nematódeo H. contortus e inibição do crescimento das bactérias S. enterica, S. aureus e E. aerogenes em meio líquido. Os teores de proteína totais obtidos foram, respectivamente, 121,6, 21,2, 75,7 mg para EPL, F30-60 e F60-90. A atividade proteolítica das amostras proteicas foi de 43.471, 13.245 e 57.857 UA/mL/mgP para EPL, F30-60 e F60-90, respectivamente. A FbP apresentou atividade proteolítica de 36.393,8 UA/mL/mgP. A análise em gel mostrou a FbP com uma banda proteica com massa entre 20 e 30 kDa e por espectrometria de massa identificou-se a FbP como protease cisteínica, com massa molecular de 23,97 kDa. FbP manteve atividade proteolítica estável na faixa de pH entre 6 e 10 e em uma ampla faixa de temperatura, com atividade ótima à 60 °C. Em testes de inibição do desenvolvimento larvar, EPL, F30-60, F60-90 e a FbP apresentaram IC50 de 0,22, 1,38, 0,42 e 0,26 mg/mL, respectivamente. Foi verificado que EPL, F30-60 e F60-90 não exerceram ação inibitória sobre o crescimento das bactérias, mas FbP inibiu o crescimento de S. aureus e S. enterica com mínima concentração inibitória (MIC) de 3,12 μg/mL. Conclui-se que a FbP obtida a partir do látex de F. benjamina possui propriedades biológicas promissoras para o desenvolvimento de produtos que possam ser utilizados no controle do nematóide gastrointestinal, H. contortus, e das bactérias patogênicas S. aureus e S. enterica.

Thesis (M. Agricultural Management (Animal Production)) -- University of Limpopo, 2018 / Ethno veterinary medicine practices are popular among the resource constrained small-holder farmers. Nonetheless, the effectiveness of traditional remedies particularly the indigenous plants has not been extensively documented. Hence, this study was conducted to determine efficacy of indigenous plant extracts used by small-holder farmers in Chief Albert Luthuli Local Municipality, Mpumalanga Province, South Africa for internal parasites control. Information on indigenous plants used for the control of internal parasites of livestock by local farmers in the study area was gathered through a questionnaire survey. A total of 188 livestock farmers (both males and females) of mixed ages were interviewed. Thirteen different plants were frequently mentioned by the respondents as remedies for livestock internal parasites. Of these, seven plants could be identified up to their families and species. Plant species collected were Dicerocaryum sp (50%), Pappeacapensis (61%), Aloe ferox (90%), Helichrysum sp (56%), Senecio congestus (83%), Senecio barbertonicus (67%) and Gardenia sp (73%). These plants were extracted using distilled water and analysed to determine their efficacy through in vitro assays; Egg hatch, larval development and larval mortality assays. All the assays were performed at different concentrations of 2.5mg/ml, 5.0mg/ml and 7.5mg/ml. The nematode third stage larvae were incubated for 24hr, 48hr and 72hr during the larval mortality assay. The present study showed that all the seven-plant species under investigation possessed some anthelmintic activities of varying strength. The highest egg hatch inhibition was observed from the extracts of Senecio barbertonicus with 100 % and the lowest from Dicerocaryum eriocarpum with 2.25 %, for larval development the highest was Gardenia sp 100 % and the lowest Helichrysum sp 26 % at concentration 7.5 mg/mL respectively. Whereas, the highest in larval mortality assay was Senecio barbertonicus and Gardenia sp achieved 100 % after 48hrs and the lowest was Dicerocaryum eriocarpum with49.89 % after 72hrs at concentration 7.5 mg/mL respectively. The use of other different forms of extraction media is recommended because different results can observe and be compared with the results of the present study. Toxicity studies on the indigenous plants observed to have stronger anthelmintic activities would assist in the future recommendation of these remedies for large scale or commercial use as anthelmintic drugs. Keywords: ethno veterinary medicine, gastrointestinal parasites, anthelmintic

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.

Vaccines and anthelmintics induce an inflammatory response in equids. Since they are commonly given concurrently, it is practical to study any interaction between them. This study evaluated whether IVM and PYR would modulate the acute phase inflammatory response, the systemic gene expression of pro-inflammatory cytokines, and vaccine-specific titers induced by WNV, EHV, and KLH vaccines. Naturally-infected, yearling ponies were sorted by gender, then fecal epgs. They were randomly assigned to three treatment groups: IVM, PYR, and control. All ponies received vaccinations intramuscularly on days 0 and 29. Whole blood, serum, and plasma samples were collected 1, 3, and 14 days post-vaccination. Samples were analyzed for inflammatory markers, cytokine, mRNA expression, and vaccine-specific IgG titers by ELISA. The acute-phase inflammatory marker data showed no statistical significance; they did show an increase in SAA, haptoglobin, and fibrinogen, and a decrease in iron after vaccination. The mRNA data showed that anthelmintics had a significant effect on interleukin mRNA levels, but not on TNF-α or IFN-γ levels. The ELISA assays showed no biologically significant reduction in IgG as compared to the control group. We conclude that deworming does not affect vaccine IgG titers; therefore, ceasing vaccinating and deworming concurrently is not necessary.

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 2016-08-15T20:31:35Z (GMT). No. of bitstreams: 1 WesleyACC_DISSERT.pdf: 1678480 bytes, checksum: 12ee28746910d56b43e6ba6b38d7495e (MD5) Previous issue date: 2009-01-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / For many centuries the Brazilian Northeast area is vocation in the production of goats, in the which the gastrointestinal nematodes is among the largest problems. Even with the appearance of anthelmintic drugs of wide action spectrum, the control of the parasites has been harmed by the anthelmintic resistance. The purpose of this study was to verify the sensibility of the goat nematodes the action of the substance ivermectin 1% and albendazole 10% as well as to determine the profile of the properties that with resistant. The work was accomplished in 30 creations of goats located into Mossoró city between January to November 2008, where each property was divided in three groups, a control and two treatments. The obtained data were analyzed by statistical program RESO. In each property 45 animals were selected, divided in three groups: group I treatment benzimidazole, I group II ivermectin and I Group III, without treatment. The animals was drugged and 10 days powder-treatment was collection of feces, directly of the rectal flask and processing of the samples for the method of OPG and larva culture. The geographical coordinates of all the properties were obtained by GPS. The obtained data were analyzed by statistical program RESO. In studied the Haemonchus contortus, was the more prevalence in all the treated groups, and the population of resistant helmints to albendazole was 90% of the properties, while, the ivermectina 36.6%. Already Trichostrongylus sp, was found in 70% of the properties and your percentile of the population of resistant helmintos the ivermectina was of 33.3%, while the albendazole, 42.8%. The reduction in the count of eggs in the feces (RCEF) after 10 days of the treatment with ivermectina and albendazole, varied among 43% to 100%, 29% to 100% respectively. In resistance order the gender Haemonchus contortus was larger index, followed for Trichostrongylus sp and Oesophagostomun sp. The data of GPS revealed that 27 properties resistance to the albendazole were broadly distributed for every municipal district, concentrating at the areas west of the city of Mossoró-RN. The 13 resistant properties the ivermectin were distributed in the municipal district, but in smaller proportion that albendazole. In all resistant properties the ivermectin were simultaneously resistant albendazole. The profile of the properties demonstrated that the anthelmintic used was groups of the benzimidazole (63.3%), ivermectin (33.3%), in smaller scale the imidazol (3.3%). In these properties were not adopted correct handling of the animals as well as ignorance of the correct dose e drugs anthelmintic. The profile of the properties demonstrated that the anthelmintic used was groups of the benzimidazóis (63.3%), ivermectin (33.3%), in smaller scale the imidazol (3.3%). In these properties were not adopted correct handling of the animals as well as ignorance of the correct dose e drugs anthelmintic. / Tendo assumido durante séculos área de vocação pecuária o Nordeste brasileiro atualmente destaca-se na caprinocultura, na qual as helmintoses gastrintestinais estão entre os maiores problemas. Mesmo com o surgimento de drogas anti-helmínticas de amplo espectro de ação, o controle dos parasitos vem sendo prejudicado pela resistência parasitária. O objetivo desse trabalho foi verificar a sensibilidade dos nematódeos de caprinos a ação dos vermífugos ivermectina 1% e albendazole 10%, como também determinar o perfil das propriedades que apresentou helmintos resistentes aos princípios ativos em questão. O trabalho foi realizado no período de janeiro a novembro de 2008 em 30 criações de caprinos localizadas no município de Mossoró-RN. Em cada propriedade, previamente positiva para OPG, foram selecionados 45 animais, os quais foram divididos em três grupos: grupo I tratamento albendazole, grupo II ivermectina e Grupo III, sem tratamento. Foram vermifugados e 10 dias pós-tratamento realizou-se a coleta de fezes, diretamente da ampola retal, e processamento das amostras pelo método de OPG e coprocultura. As coordenadas geográficas de todas as propriedades foram obtidas por GPS. Os dados obtidos foram analisados pelo programa estatístico RESO. Das propriedades estudadas o Haemonchus contortus, teve percentual de população de helmintos resistentes ao albendazole em 90%, enquanto que, à ivermectina 36,6%. Este por sua vez, foi o mais prevalente em todos os grupos tratados. Já o Trichostrongylus sp, foi encontrado em 70% das propriedades e seu percentual da população de helmintos resistentes a ivermectina foi de 33,3%, enquanto o albendazole, 42,8%. A redução na contagem de ovos nas fezes (RCOF) 10 dias pós-tratamento com ivermectina e albendazole, variou entre 43% a 100%, 29% a 100% respectivamente. Por ordem de resistência o Haemonchus contortus foi o que apresentou maior índice, seguido pelos genêros Trichostrongylus sp e Oesophagostomun sp. Os dados do GPS revelaram que as 27 propriedades resistência ao albendazole estão largamente distribuídas por todo município, concentrando-se nas regiões oeste da cidade de Mossoró-RN. As 13 propriedades resistentes a ivermectina estão distribuídas no município, mas em menor proporção que o albendazole. Todas as propriedades resistentes a ivermectina foram resistentes albendazole simultaneamente. O perfil das propriedades demonstrou que os anti-helmínticos mais utilizados pertenciam aos grupos dos benzimidazóis (63,3%), avermectinas (33,3%), em menor escala os imidazóis (3,3%). Nestas propriedades não se adotavam manejo correto dos animais como também havia pelo criador, desconhecimento da dose correta e uso dos vermífugos.

Les nématodes gastro-intestinaux (NGIs) sont une contrainte majeure en élevage des ruminants au pâturage. La méthode de lutte usuelle est l’utilisation de traitements anthelminthiques (AH) de synthèse. Cependant, la diffusion constante de résistances aux AHs dans les populations de NGIs chez les petits ruminants impose de rechercher des méthodes alternatives de maitrise. L’exploitation de plantes contenant des métabolites secondaires est une de ces options. Le sainfoin (Onobrychis viciifolia), une légumineuse riche en tanins condensés (TCs), est un modèle pour explorer le concept de nutricament = fourrage associant valeurs nutritionnelles et propriétés sanitaires (AH). Récemment, une filière de production de granulés déshydratés de sainfoin (GDS) s’est construite. Les avantages en sont : cultiver le sainfoin dans les meilleures conditions agronomiques possibles, favoriser la standardisation et la caractérisation des lots avant usage pour assurer leur distribution en élevages voire leur exportation. L’objectif général de cette thèse a été de fournir les informations scientifiques pour favoriser l’utilisation de GDS en élevage de petits ruminants. Il s’est décliné en 3 axes de recherche : Le premier axe visait à valider des méthodes simples de dosage des tanins et de leur activité biologique, pour caractériser des ressources avant usage, en se fondant sur des mesures biochimiques (méthode colorimétrique de Folin- Ciocalteu) et d’activité biologiques (complexation des protéines = méthode de diffusion radiale et activité AH = méthode du dégainement larvaire imitant le processus d’infestation in vivo). Les corrélations entre les diverses mesures montrent un lien entre la quantité de TCs et l’activité AH ainsi que l’intérêt de la méthode NIRS pour caractériser les différentes sources de sainfoin (GDS et fourrages). Le second axe visait à mieux définir sur la base d’études en conditions expérimentales contrôlées, les conditions optimales d’application des GDS en élevage. Une première série d’études a cherché à explorer 1) les effets AH combinés, liés à la concentration en TCs dans la ration et à la durée de distribution, contre divers stades (L3, vers adultes) d’espèces abomasales ou intestinales et 2) l’intérêt d’associer le sainfoin avec un AH de synthèse. Les résultats montrent 1) qu’un seuil minimum de TCs dans la ration doit être atteint et une période minimum de 15 jours de distribution est nécessaire pour altérer la biologie des nématodes (notamment la fertilité des vers femelles de Trichostrongylus colubriformis et le nombre d’Haemonchus contortus) et 2) qu’une utilisation dissociée d’un AH de synthèse (ivermectine par voie orale) est préférable en raison d’interactions négatives. Le dernier axe a visé à comparer la capacité d’ovins ou de caprins à moduler leur comportement alimentaire vis-à-vis de GDS en fonction de leur état parasitaire (capacité d'automédication). L’objectif était de vérifier les hypothèses suivantes : compte-tenu de leur moindre résistance envers les parasites et une plus grande propension à consommer des tanins, les caprins consommeront plus de sainfoin que les ovins ; les animaux fortement parasités exprimeront une préférence pour le sainfoin, et celle-ci augmentera au cours du temps. Les résultats de cet essai ont montré, dans le test de cafétéria, que 1) les chèvres préféraient le sainfoin et 2) une augmentation de la consommation en sainfoin au cours du temps chez les animaux parasités, sans toutefois devenir supérieure à celle des animaux sains. En revanche, ni les animaux parasités, ni les caprins n’ont montré de motivation plus importante pour obtenir la ressource riche en tanins dans le test de conditionnement opérant. Ces résultats montrent l’intérêt de l’utilisation de granulés de sainfoin en élevage, afin de compléter les AHs de synthèse et limiter le développement des résistances à ces molécules chez les NGIs. / Gastrointestinal nematodes (GINs) represent a major threat for grazing ruminants. The traditional mode of control of these parasites mainly relied on the use of synthetic anthelmintics (AHs). However, the repeated use of these molecules has led to resistance against all AH drugs in worm populations of small ruminants. Alternative solutions are needed. Among those, the use of plants that contain plant secondary metabolites, such as condensed tannins (CTs), has been studied for the last 20 years. These CT-containing resources, such as sainfoin (Onobrychis viciifolia) have AH properties and beneficial effects on health and also interesting nutritional values; hence they correspond to the definition of nutraceuticals. Recently, an organization has developed to produce dehydrated pellets of sainfoin. The advantages of such pellets are 1/ to grow sainfoin in the best agronomic conditions; 2/ to better standardize the product; 3/ to evaluate the pellet before use and to facilitate exportation. The general aim of this thesis was to optimize the use of sainfoin pellets in farm conditions. Our first objective was to characterize the CT content and AH properties of sainfoin pellets. For industrial purposes, simple, low cost and repeatable methods to evaluate the CT content and the AH activity are requested in order to rapidly qualify the pellets. The CT content was evaluated by biochemical measurements of total phenols and tannins (Folin-Ciocalteu). This was completed by the measure of the complexation of tannins with proteins (Radial Diffusion) and by development of prediction calibrations using near infra-red spectroscopy (NIRS). The AH evaluation was performed on samples before and after dehydration, using the Larval Exsheathment Inhibition Assay. Calculations of correlations between the different methods showed significant links between the quantity of CT and the AH activity, and the possible use of NIRS to measure the CT content and the AH activity of pellets and forages. The second objective aimed at defining the optimal conditions to use sainfoin pellets on farm, to improve the AH activity. We aimed at analyzing, in experimental conditions, 1) how the combination of 2 factors, namely the CT concentration in feed and the length of distribution, influence the AH efficacy of sainfoin pellets on intestinal and abomasal L3 larvae or adult worms; 2) whether or not the combined use of sainfoin with a chemical AH (oral ivermectine) is beneficial. Results showed that a minimum CT content in sainfoin and a minimum length of distribution of 15 days are mandatory to alter the adult worm and L3 larvae biology of nematodes, particularly the Trichostrongylus colubriformis fertility and the number of Haemonchus contortus. Moreover, the combined use of AH and sainfoin consumption should be avoided because of negative interactions. The last axis aimed at studying the behavior of sheep and goats in relation to sainfoin pellets and their ability to change the feeding behavior according to their parasitic status (self-medication ability). The objective was to verify the following hypotheses: due to their lower resistance against parasites and their higher propensity to eat CTs, goats will eat more sainfoin than sheep, parasitized animals will prefer sainfoin and this preference will increase overtime. The results showed that two of our hypothesis were verified in a cafeteria study: 1) goats preferred sainfoin while sheep preferred alfalfa and 2) the sainfoin consumption increased overtime for infected animals, without being higher than nonparasitized ones. However, nor goat, nor parasitized animals, showed a higher motivation in the operant-conditioning trial to feed on CT rich resources. All the present results showed the interest to exploit sainfoin pellets in farms, as one alternative to AHs molecules in order to limit the development of resistances to these drugs in GIN populations.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2018-06-19T14:21:45Z No. of bitstreams: 1 PDF - Allana Brunna Sucupira Duarte.pdf: 18551366 bytes, checksum: 4964f2ac412172ef2e8ca648e00e4758 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2018-07-03T17:48:01Z (GMT) No. of bitstreams: 1 PDF - Allana Brunna Sucupira Duarte.pdf: 18551366 bytes, checksum: 4964f2ac412172ef2e8ca648e00e4758 (MD5) / Made available in DSpace on 2018-07-03T17:48:01Z (GMT). No. of bitstreams: 1 PDF - Allana Brunna Sucupira Duarte.pdf: 18551366 bytes, checksum: 4964f2ac412172ef2e8ca648e00e4758 (MD5) Previous issue date: 2017-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Intestinal parasites caused by helminths are among the most frequent public health problems and affect mainly children. Conventional treatment of such diseases is carried out with antiparasitics having various adverse effects and are available in the dosage forms of tablets, syrups and suspensions which have disadvantages in pediatric use such as inability to swallow and unpleasant taste. Among the various forms of treatment, phytotherapy has been highlighted by the use of medicinal plants as an excellent alternative for the treatment of helminthes. In this way, this study proposed to obtain a formulation in the form of suppository containing nebulized extract of the tuber Operculina macrocarpa. Eighty-one extracts were produced using different extraction techniques, hydroalcoholic concentrations and quantities of plant material, following a Factorial Planning 3^3 , in order to determine the best yield extract. All extracts were characterized for their composition of polyphenols, condensed tannins and flavonoids. The extract with the highest amount of phytochemicals was selected for the other studies and development of the formulation, since there was no statistical difference in relation to yield. Thus, the extract produced by maceration was selected using 10 g of vegetal material dissolved in 100 mL of 50% (v/v) ethanol/water solution. The extract did not present cytotoxicity in erythrocytes. The anti-helminth activity test performed in goats showed that 14 days after treatment administration there was a reduction in the mean number of eggs per gram of faeces (OPG) of 79,40% compared to day zero. Thermal data demonstrated that the pharmaceutical excipients cocoa butter, EDTA and the mixture of methylparaben and propylparaben did not present incompatibility with the O.macrocarpa nebulizer extract and its binary mixtures, and these were therefore selected for the manipulation of suppositories. Therefore, the proposed anti-helminthic pediatric formulation was successfully obtained and had adequate organoleptic characteristics, average weight and disintegration time. / As parasitoses intestinais causadas por helmintos estão entre os mais frequentes agravos de saúde pública e afetam principalmente as crianças. O tratamento convencional dessas doenças é realizado com antiparasitários que apresentam diversos efeitos adversos e estão disponíveis nas formas farmacêuticas de comprimidos, xaropes e suspensões, as quais apresentam desvantagens no uso pediátrico como a incapacidade de deglutição e sabor desagradável. Dentre as diversas formas de tratamento a fitoterapia tem se destacado pela utilização de plantas medicinais como uma excelente alternativa para o tratamento de helmintoses. Desta forma, esse estudo se propôs a obter uma formulação na forma de supositório contendo extrato nebulizado do tubérculo Operculina macrocarpa. Oitenta e um extratos foram produzidos utilizando-se diferentes técnicas de extração, concentrações hidroalcoólicas e quantidades de material vegetal, seguindo um Planejamento Fatorial 3^3 , de modo a se determinar o extrato de melhor rendimento. Todos os extratos foram caracterizados quanto à sua composição de polifenois, taninos condensados e flavonoides. O extrato com maior quantidade de fitoconstituintes foi selecionado para os demais estudos e desenvolvimento da formulação, uma vez que não houve diferença estatística em relação ao rendimento. Assim, foi selecionado o extrato produzido por maceração, utilizando 10 g de material vegetal dissolvido em 100 mL de solução de etanol/água a 50% (v/v). O extrato não apresentou citotoxicidade em eritrócitos. O teste de atividade anti-helmíntica realizado em caprinos demonstrou que 14 dias após a administração do tratamento houve uma redução na média do número de ovos por gramas de fezes (OPG) de 79,40% comparado ao dia zero. Os dados térmicos demonstraram que os excipientes farmacêuticos manteiga de cacau, o EDTA e a mistura de metilparabeno e propilparabeno não apresentaram incompatibilidade com o extrato nebulizador de O.macrocarpa e suas mistura binárias, sendo estes, portanto, selecionados para a manipulação dos supositórios. Sendo assim, a formulação pediátrica anti-helmíntica proposta foi obtida com êxito e apresentou características organolépticas, peso médio e tempo de desintegração adequados.

The aim of this work was to assess the bioactivity of extracts of the tannin-rich plants Acacia mearnsii, Myracrodruon urundeuva, Caesalpinea bracteosa and Leucaena leucocephala against egg and infective larvae stages of Haemonchus contortus. Two in vitro assays were held: an Egg Hatch Assay (EHA) and a Larval Exsheathment Inhibition Assay (LEIA). The EHA consists of the incubation of previously recovered eggs from infected animal\'s faeces in a solution of plant extract for 24 hours and later differentiation between larvae and nonhatched eggs. The concentrations used were 50.00, 25.00, 12.50, 6.25, 3.12, 1.56, 0.78 and 0.39 mg/mL for A. mearnsii; 1.56, 0.78, 0.39, 0.19, 0.09 and 0.04 mg/mL for M. urundeuva; 6.25, 3.12, 1.56, 0.78, 0.39 and 019 mg/mL for C. bracteosa; and 6.25, 3.12 and 1.56 mg/mL for L. leucocephala. The LEIA consists in the artificial exsheathment of infective larvae, obtained by previous coproculture, after a three hour incubation period with plant extract solution in the concentrations 1,200, 600, 300 and 150 \'mü\'g/mL. The 50 and 99 lethal doses (LD) were calculated for both tests. A dose-dependent effect was found in the two tests, except for L. leucocephala in EHA, where it was not possible to calculate DL50 and DL99 with the chosen doses. The DL50 results for EHA were 0.18, 0.32, and 7.20 mg/mL and for DL99 were 4.31, 5.41, and 187.26 mg/mL, respectively for M. urundeuva, C. bracteosa, and A. mearnsii. For LEIA, the DL50 were 0.40, 0.52, 1.24, and 2.24 mg/mL and for DL99 these were 2.37, 2.28, 19.99 and 2.53 x 103 mg/mL respectively for M. urundeuva, A. mearnsii, L. leucocephala and C. bracteosa. The two highest concentrations of A. mearnsii and the three highest for C. bracteosa were effective (more than 90% of bioactivity); the three highest concentrations of M. urundeuva and the 0.78 mg/mL level of C. bracteosa were moderately effective (between 80 and 90% of bioactivity); the concentrations with low effectiveness (between 60 and 80% of bioactivity) were the 12.50 mg/mL (A. mearnsii), 0.19 mg/mL (M. urundeuva), and 0.39 mg/mL (C. bracteosa); all other concentrations were ineffective (less than 60% of bioactivity). For the LEIA, only the highest dose from A. mearnsii and M. urundeuva were effective; the 600 \'mü\'g/mL were moderately effective and all other doses were ineffective. It should be observed that even if the dose is ineffective against exsheathment, some concentrations were able to significantly delay the process. It was observed that the extracts had bioactivity in vitro within the chosen doses against the hatchability of eggs and exsheathment of larvae of H. contortus, except for L. leucocephala, which was not able to block the hatching of eggs / Objetivou-se com o presente trabalho verificar a bioatividade dos extratos das plantas taniníferas Acacia mearnsii, Myracrodruon urundeuva, Caesalpinea bracteosa e Leucaena leucocephala contra os estágios de ovo e de larva L3 infectante de Haemonchus contortus. Para isso, dois ensaios in vitro foram realizados: o Teste da inibição da eclodibilidade dos ovos (TIEO) e o teste da Inibição do desembainhamento larvar (TIDL). O TIEO consiste na incubação de ovos recém recuperados das fezes de animais infectados em solução liquida de extrato de planta por 24 horas e posterior diferenciação entre larvas e ovos não eclodidos. As concentrações utilizadas para A. mearnsii foram de 50,00, 25,00, 12,50, 6,25, 3,12, 1,56, 0,78 e 0,39 mg/mL; 1,56, 0,78, 0,39, 0,19, 0,09 e 0,04 mg/mL para M. urundeuva; 6,25, 3,12, 1,56, 0,78, 0,39 e 0,19 mg/mL para C. bracteosa; e 6,25, 3,12 e 1,56 mg/mL para L. leucocephala. O TIDL consiste no desembainhamento artificial de larvas infectantes, obtidas através de coprocultura, que passaram por período de incubação de três horas em solução liquida de extratos de plantas nas concentrações 1.200, 600, 300 e 150 \'mü\'g/mL. As doses letais (DL) 50 e 99 foram calculadas para ambos testes. Um efeito dose-dependente foi encontrado para os dois testes, exceto para a L. leucocephala no TIEO, onde não foi possível calcular o valor da DL50 e DL99 para o respectivo teste nas doses escolhidas. Os resultados da DL50 para TIEO foram de 0,18, 0,32 e 7,20 mg/mL e da DL99 foram de 4,31, 5,41 e 187,26 mg/mL respectivamente para M. urundeuva, C. bracteosa e A. mearnsii. Para o TIDL, a DL50 foi de 0,40, 0,52, 1,24 e 2,24 mg/mL e da DL99 foi de 2,37, 2,28, 19,99 e 2,53 x 103 mg/mL respectivamente para M. urundeuva, A. mearnsii, L. leucocephala e C. bracteosa. Para o TIEO, as duas maiores concentrações de A. mearnsii e as três maiores de C. bracteosa foram efetivas (mais de 90% de bioatividade); as três maiores concentrações de M. urundeuva e a 0,78 mg/mL de C. bracteosa foram moderadamente efetivas (entre 80 e 90% de bioatividade); as concentrações de pouca efetividade (entre 60 e 80% de bioatividade) foram a 12,50 mg/mL (A. mearnsii), 0,19 mg/mL (M. urundeuva), e 0,39 mg/mL (C. bracteosa); as demais concentrações foram consideradas ineficientes (menos de 60% de bioatividade). Para o TIDL, apenas a maior concentração de A. mearnsii e M. urundeuva foram efetivas; a concentração de 600 \'mü\'g/mL de M. urundeuva foi moderadamente efetiva; todas as outras concentrações foram ineficientes. Entretanto, deve-se observar que apesar de ineficientes em bloquear o desembainhamento, algumas concentrações atrasaram significativamente o desembainhamento das larvas, sendo esse um resultado de interesse. Assim, foi observado que os extratos, excetuando o extrato de L. leucocephala quanto a eclodibilidade, possuem bioatividade in vitro nas doses utilizadas contra a eclosão dos ovos e contra o desembainhamento das larvas de H. contortus

A bananeira é cultivada pelo homem há pelo menos 6.500 anos. O início desse processo foi marcado pela manifestação espontânea dos fenômenos de partenocarpia, hibridização, poliploidia e pela sua combinação, gerando diferentes cultivares, cujos clones foram selecionados por agricultores primitivos e se disseminaram pelos trópicos, exclusivamente pela ação humana. A banana é hoje a principal fruta cultivada no mundo, existindo farta documentação sobre a utilização dos resíduos da cultura para alimentação de animais e sobre o uso medicamentoso de diferentes partes da planta. No âmbito da Medicina Veterinária, folhas, inflorescências masculinas, pseudocaules e rizomas já foram testados in vitro e in vivo para avaliação de seu efeito anti-helmíntico. Entretanto, em escala comercial, toda essa biomassa deve permanecer no bananal, para cobertura e fertilização do solo, inviabilizando seu aproveitamento como forragem. Por outro lado, no momento da colheita, os cachos de bananas são levados para unidades de triagem denominadas “packing-houses”, onde as frutas são selecionadas e acondicionadas para expedição, restando os engaços ou pedúnculos dos cachos de bananas disponíveis para o aproveitamento na alimentação animal, com o benefício adicional potencial de controlar infecções verminóticas. O presente trabalho demonstrou que o extrato dos engaços do cultivar “Nanica”, Subgrupo Cavendish AAA, inibiu significativamente a eclosão in vitro de ovos de Haemonchus contortus, da mesma forma que extratos de folhas e pseudocaules. Transpondo esse achado para um modelo in vivo, 24 cordeiros inteiros, ⅞ Dorper, foram experimentalmente infectados com larvas de Haemonchus contortus e alocados em blocos a cada um de quatro tratamentos, de acordo com as contagens de ovos nas fezes. Os ovinos receberam, conforme o grupo, 0% (controle), 10%, 20% ou 30% de engaços frescos de bananeira picados, calculados em matéria seca, sendo a dieta complementada com feno de braquiária ad libitum e sal mineral. Os animais foram tratados durante 14 dias consecutivos. Glucose de milho e melaço de cana de açúcar foram utilizados para melhorar a aceitação dos engaços pelos animais. Verificou-se um aumento significativo da oviposição em todos os grupos tratados na primeira semana de tratamento, fato possivelmente relacionado com um dos efeitos farmacológicos da bananeira, que promove elevação da serotonina e, por consequência, aumenta a taxa de reprodução dos nematódeos. Na segunda semana obteve-se redução significativa das contagens de ovos nas fezes, sem diferença estatística entre os grupos tratados, sugerindo que o uso continuado dos engaços de bananeira na alimentação pode promover um controle das infecções helmínticas em ruminantes. Demonstrou-se ainda que os taninos condensados exercem papel marginal quando a bananeira é testada in vitro, pois, nesse caso, a adição de polivinilpolipirrolidona, substância capaz de precipitar e suprimir a atividade desses taninos, proporcionou o mesmo grau de inibição da eclosão de ovos de Haemonchus contortus que os extratos puros. Embora relatos recentes tenham sugerido a presença de alcalóides e saponinas na bananeira, tais compostos não foram identificados nas amostras estudadas. Observou-se, todavia, a presença de siringina, um fenilpropanóide, cuja via metabólica engloba as defesas bioquímicas dos cultivares de bananas contra infecções por nematódeos parasitos de plantas. Não obstante, a presença de catecolaminas e ação antiparasitária de esteróis e triterpenos sugerem fortemente que a bananeira atua como um fitocomplexo quando fornecida aos ruminantes como alimento e tratamento contra endoparasitos. / The banana plant has been cultivated by men for at least 6,500 years. The beginning of this process was marked by the spontaneous occurrence of the phenomena of partenocarpy, hybridization, polyploidy and their combination, generating different cultivars, which clones were selected by primitive farmers and were disseminated throughout the tropics, by exclusive human action. Nowadays bananas are the main fruit crop cultivated in the world, and there is plenty documentation regarding the use of crop residues to feed animals and concerning the medicinal use of different parts of the plant. In the field of Veterinary Medicine, leaves, masculine inflorescences, pseudostems and rhizomes have been tested both in vitro and in vivo for the evaluation of their anthelminthic effect. However, in commercial scale, all this biomass has to be kept in the plantation, to cover and fertilize the soil, frustrating its use as forage. On the other hand, at the moment of harvesting, banana bunches are carried to screening facilities called “packing-houses”, where the fruits are selected and boxed for shipment, leaving the stalks or peduncles of the bunches available to be used as animal fodder, with the potential additional benefit of controlling worm infections. The current work has shown that the extract of the cultivar “Nanica”, Subgroup Cavendish AAA, has significantly inhibited the in vitro eclosion of Haemonchus contortus eggs, in the same manner as extracts of leaves and pseudostems. Transposing this finding to an in vivo model, 24 non-castrated ⅞ Dorper lambs were experimentally infected with Haemonchus contortus larvae and allocated in blocks to each of four treatments, according to their faecal egg counts. The sheep were fed, according to the group, 0% (control), 10%, 20% or 30% fresh chopped banana plant stalks, calculated as dry matter, while the diet was complemented with Brachiaria hay ad libitum and mineral salt. The animals were treated during 14 consecutive days. Maize glucose and sugar cane molasses were used to improve the acceptance of the stalks by the lambs. There was a significant increase of the oviposition in all treated groups during the first week of treatment, fact possibly related to one of the pharmacological effects of the banana, which promotes an increase of serotonin levels, consequently enhancing the reproductive tax of the nematodes. On the second week a significant reduction of the faecal egg counts was noted, without statistical difference between the treated groups, suggesting the continuous use of the banana plant stalks as feed may promote a control of helminthic infections in ruminants. It was further shown that condensed tannins play a marginal role when the banana plant is tested in vitro, because the addition of polyvinylpolypyrrolidone, a substance capable of precipitating and supressing the activity of these tannins, resulted in the same level of Haemonchus contortus egg hatch inhibition obtained with the pure extracts. In spite of recent reports suggesting the presence of alkaloids and saponins in the banana plant, these compounds were not detected in any of the samples tested. Otherwise, screening identified syringing, a phenylpropanoid, which metabolic pathway comprises the biochemical defences of the banana cultivars against infections by nematode plant parasites. The presence of catecholamines and the anti-parasitic action of sterols and triterpenes strongly suggest the banana plant acts as a phytocomplex when administered to ruminants as feed and treatment against endoparasites.