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Haemonchus contortus, otherwise known as the “Barber Pole Worm”, “The Wire Worm” or simply as the large stomach worm of sheep, has been the subject of much scientific interest for the past quarter of a century. Considerable work has been done which has established this parasite as a factor of economic importance and its presence as a pathological agent in the abomasum of ruminants is generally well recognized. This worm is one of the largest of the Strongyloid group, measuring sometimes an inch in the females and three-quarters inch in the males, and its general anatomy is fairly well known. The literature does not, however, reveal any work which has been done in connection with the development of the reproductive cells of this species. In the present work a study has been made of spermatogenesis and oogenesis with some attention directed to methods of copulation, the passage of the sperm cells upon entrance into the vulva and the anatomical location in the female where the sperms are collected for the purpose of fertilization of the ova. No histologic section, depicting the entrance of the sperm into the ova, has been observed but the developmental stages immediately following this penetration of the ova are shown in this paper. Even though a considerable amount of material has been examined which has afforded opportunity for studying the entire reproductive organs of both male and female worms, several developmental stages which have been reported as occurring in nematodes, have not been observed in this study. Sufficient evidence, however, is herein presented which indicated very clearly and definitely the normal chromosome number. / Master of Science

Widespread anthelmintic resistance in trichostrongyle nematodes of ruminants has created an urgent need for alternatives to commercial anthelmintics. The bacterium Bacillus thuringiensis (Bt) can produce crystal proteins during sporulation, which can be lethal to insects in multiple orders when ingested. One protein, Cry5B, has demonstrated effectiveness against multiple parasitic nematodes. We hypothesized that Cry5B would be effective against Haemonchus contortus, a highly pathogenic parasite, in sheep. Two experiments tested efficacy of Cry5B in sheep experimentally infected with H. contortus. In the first, a live genetically modified, asporogenous strain of B. thuringiensis expressing cytosolic Cry5B protein (BaCC) was administered orally daily for four days (~40mg/kg Cry5B/day). The mean fecal egg count (FEC) of treated animals was reduced by 94% three days after treatment, and at necropsy the female worm burden was significantly reduced by 98%. In the second experiment inactivated, asporogenous Bt expressing cytosolic Cry5B (IBaCC) was used. Treated animals received 60mg/kg Cry5B, administered daily for three days. By 72 hours after the first treatment FEC was reduced by 91%. Mean total worm burden of treated sheep at necropsy was significantly reduced, with female worms reduced by 95%. A third study tested the effect of BaCC and IBaCC on development of eggs to infective larvae in feces under laboratory and outdoor environmental conditions. Cry5B (15mg) added to feces (10g) reduced numbers of infective larvae by 99% in both environments within 12 days. Cry5B appears to have potential for controlling H. contortus in sheep. All protocols approved by VT IACUC and IBC. / Master of Science / Many animals and humans can be infected with roundworm, also called nematode, parasites. Infection of animals and humans by parasitic nematodes can result in disease. Some animals like ruminants (cows, sheep, and goats) can be infected with multiple species at once with few effects on the host. However, certain species can cause major disease, and even kill their ruminant host. Younger animals like lambs can easily become overwhelmed by these parasites. Anthelmintics are the type of drug used to treat those infected with these parasitic worms. However, just like bacteria are becoming resistant to antibiotics, these worms are also becoming resistant to anthelmintics. Because of this, researchers are looking for new compounds and materials that are lethal to the parasite and can be used to treat infected animals. One species of bacterium, Bacillus thuringiensis, is usually found in the soil. This bacterium can produce a large crystal structure that is made up of proteins. These crystal (Cry) proteins can be lethal to pest insects like beetles, caterpillars, and mosquitos. When the insect eats the protein, it binds to cells in the insect intestine, creating holes in the insect gut. These proteins can be lethal to nematodes as well when they are eaten by the worms. Because of this, these proteins are being investigated as potential alternative treatments for parasitic nematodes. One type of protein, Cry5B, has been tested in hamsters, mice, and pigs. We hypothesized that Cry5B would also be effective against a sheep stomach worm called Haemonchus contortus. We tested the Cry5B in two different formulations and found that the protein was effective against both the adult worm in the stomach, and the young worms in the feces of the host. This protein could potentially be used to treat parasitic nematodes that have become resistant to anthelmintics.

Resumo: Este trabalho objetivou avaliar o efeito do genótipo e da dieta protéica na resistência e no desempenho de cordeiros experimentalmente infectados por Haemonchus contortus. Foram utilizados 24 cordeiros provenientes do cruzamento das raças Dorper x Santa Inês (DSI) e 24 Santa Inês (SI), isentos de nematódeos gastrintestinais, distribuídos em quatro grupos de seis animais. De acordo com o genótipo, os cordeiros foram subdivididos em dois grupos infectados e dois mantidos como controle, recebendo dieta com menor teor (12%) e maior teor de proteína bruta (20%), respectivamente. Os cordeiros infectados artificialmente receberam um inóculo contendo 1.000 larvas infectantes (L3) de H. contortus por dose, duas vezes por semana, durante todo o experimento. Os cordeiros foram mantidos em baias individuais, com controle diário do alimento fornecido e de sobras. Semanalmente, foram realizadas contagens de ovos de nematódeos por grama de fezes (OPG), hemograma, dosagens bioquímicas, além de pesagens individuais. Ao atingirem 35 kg de peso corporal, os cordeiros foram abatidos e necropsiados, objetivando mensurar a carga parasitária, avaliar as características quantitativas das carcaças e qualitativas da carne. O desempenho (consumo de matéria seca, ganho de peso diário, conversão alimentar e eficiência alimentar) foi superior (P<0,05) para o genótipo DSI, para a dieta com teor mais elevado de proteína e para os cordeiros mantidos sem infecção por H. contortus. O efeito do genótipo, da suplementação protéica e da hemoncose também influenciou as principais variáveis hematológicas e bioquímicas, cujos valores foram menores em cordeiros infectados e mantidos com alimentação menos protéica. Contagens médias superiores (P<0,05) de OPG (520,06) e H. contortus (200,38) foram observadas no genótipo DSI. Observou-se influência do genótipo DSI (P<0,05) nos pesos médios... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work aimed to evaluate the effect of genotype and dietary protein on the resistance and performance of lambs to experimentally infected by Haemonchus contortus. Twenty-four Dorper x Santa Inês (DSI) crossbreed lambs and 24 pure Santa Inês (SI), without gastrointestinal nematodes, were distributed into four groups of six lambs. Within each genotype, the lambs were subdivided into two infected and two control groups, which received a low protein (12%) or high protein diet (20%), respectively. The experimentally infected lambs received inoculate containing 1,000 infecting larvae (L3) of H. contortus per dose, twice weekly, throughout the entire experimental period. The lambs were maintained in individual stalls, with daily control of feed provided and remaining. Weekly, nematode egg per gram of feces (EPG) counts were performed, together with hemograms, biochemical concentrations and individual weighing. Once they achieved 35 kg, the lambs were slaughtered and necropsied to determine the parasite load and evaluate the quantitative characteristics of the carcass and the qualitative characteristics of the meat. For the DSI genotype, the performance (dry material consumption, daily weight gain, feed conversion and feed efficiency) was greater (P<0.05) for the diet containing higher protein content and for lambs not infected by H. contortus. The effect of genotype, dietary protein content and parasitic condition also influenced the principal hematological and biochemical variables, showing lower values in infected lambs and those fed with low protein feed. The highest mean EPG (520.06) and H. contortus (200.38) counts were observed in the DSI genotype (P<0.05). Observation verified the influence of the DSI genotype (P<0.05) on the mean warm and (17.03 kg) cold (16.48 kg) carcass weights and of H. contortus infection on weight-loss due to cooling (6.2%) ...(Complete abstract click electronic access below) / Orientador: Alvimar José da Costa / Coorientador: Américo Garcia da Silva Sobrinho / Banca: Fernando de Almeida Borges / Banca: Gilson Pereira de Oliveira / Banca: Mauro Dal Secco de Oliveira / Banca: Vando Edésio Soares / Doutor

In recent years has increased the search for alternative methods to combat gastrointestinal parasites, and in this context, biological control achieved by the fungus Duddingtonia flagrans draws attention. In this study, we analyze the possibility of the temperature have influence on the nematophagic activity of D. flagrans against the parasites Haemonchus contortus of sheep. We used four sheep, two parasitized with H. contortus and two dewormed. One dewormed sheep received, for three consecutive days before collection of feces, 1 x 106 chlamydospores of D. flagrans, lyophilized, in gelatin capsules. After feces collection, with the aid of collectors bags, they were mixed, resulting in samples with (fungus) and without fungus (control). Each sample was incubated at seven different temperatures (5, 10, 15, 20, 25, 30 and 35 º C) for 21 days. After the incubation period recovery and counting of larvae was performed. The best temperature for fungus action was 30 º C. No larvae were recovered at 5 ºC. From 10 ºC, the fungus action was detected, but there was no significant difference in the percentage of larval reduction between the temperatures, demonstrating that the presence of larvae seems to be the main factor affecting its nematophagic action. It can be concluded that the temperature does not seem to be a limiting factor in the action of Duddingtonia flagrans used for biological control of Haemonchus contortus. Even at lower temperatures, the lyophilized D. flagrans reduced the number of H. contortus larvae, showing, once again, the potentiality of this species in biological control, even in non-ideal conditions for their development and the larvae development, so that it can be used in colder climates, as in Rio Grande do Sul state. / Nos últimos anos vem crescendo a busca por métodos alternativos para o combate a parasitas gastrintestinais e, nesse contexto, o controle biológico realizado pelo fungo Duddingtonia flagrans destaca-se. Neste trabalho, analisamos a possibilidade de a temperatura influenciar a atividade nematofágica de D. flagrans contra o parasita de ovinos Haemonchus contortus. Utilizou-se 04 ovelhas, sendo duas parasitadas com H. contortus e duas isentas de parasitas. Uma das ovelhas desparasitadas recebeu, durante 03 dias consecutivos, antes da coleta, 1 x 106 clamidósporos de D. flagrans, liofilizados, em cápsulas gelatinosas. Após coleta das fezes com auxílio de sacos coletores, as fezes foram misturadas, resultando amostras com fungo e sem fungo (controle). Cada amostra foi incubada em sete diferentes temperaturas (5, 10, 15, 20, 25, 30 e 35 ºC), durante 21 dias. Após foi realizada a recuperação e contagem larval. A melhor temperatura para ação do fungo foi a de 30ºC. Não foram recuperadas larvas na temperatura de 5 ºC. A partir da temperatura de 10 ºC, a ação do fungo foi detectada, porém não houve diferença significativa na percentagem de redução larval entre as temperaturas, significando que a presença de larvas parece ser o principal fator a influenciar sua ação nematofágica. Pode-se concluir que a temperatura não parece ser um fator limitante no controle biológico de Haemonchus contortus de ovinos realizado pelo fungo Duddingtonia flagrans. Mesmo em temperaturas mais baixas, o liofilizado de D. flagrans reduziu o número de larvas H. contortus demonstrando, mais uma vez, a potencialidade dessa espécie no controle biológico, mesmo em condições não ideais para o seu desenvolvimento e das larvas, podendo ser utilizado em climas mais amenos, como no Estado do Rio Grande do Sul. Palavras-chave: Duddingtonia flagrans, Haemonchus contortus, temperatura, controle biológico

The present study describes the evaluation of the effect of supplementation with selenium and vitamin E on the hemogram and protein and oxidative profiles of lambs experimentally infected with Haemonchus contortus (HC). Thirty female lambs were divided into four groups as follows: G1 (n = 10): infected animals; G2 (n =10): infected and supplemented; G3 (n = 5): control; G4 (n = 5): non-infected and supplemented. Groups 1 and 2 received 500 HC larvae (L3) orally for a period of 20 days, with 2-day intervals between doses. Supplementation in groups 2 and 4 was performed on day zero by injecting 0.1 mg kg-1 of sodium selenite (1.67%) and 2,000 IU vitamin E through the intramuscular (IM) route. Vitamin E alone was injected once again on day 30. In addition to blood collections for performing the hemograms, the numbers of eggs per grams of feces (EPG) were also determined. Considering the number of erythrocytes, volume of globular proteins, hemoglobin level, total leukocytes and lymphocytes, the main differences were observed when the parasitized groups were compared to the supplemented, non-parasitized group, the latter exhibiting higher values. It is concluded that supplementation of lambs with vitamin E and selenium has no influence on their erythrogram when they are parasitized with HC. The leukogram showed higher segmented neutrophil numbers in the supplemented groups. On the other hand, vitamin E and selenium increased hematological parameters in animals not infected with HC. In considering the levels of total proteins, albumin, beta and gamma globulins, the main differences were observed when the parasitized groups were compared to the supplemented, non-infected group, the latter exhibiting higher values. It is concluded that supplementation of lambs with vitamin E and selenium has no influence on their blood protein levels when they are parasitized with HC. The non-parasitized, supplemented animals sustained better glutathione peroxidase (GSH-Px) values up to the 60th day of the experiment. No oxidative stress state took place since an increase in GSH-Px levels exerted a compensatory effect when thiobarbituric acid reactive substances (TBARS) levels rose. / O presente estudo descreve a avaliação da influência da suplementação com selênio e vitamina E sobre o hemograma, perfil protéico e oxidativo de cordeiros infectados experimentalmente pelo Haemonchus contortus (HC). Trinta cordeiros, fêmeas, foram distribuídos em quatro grupos, sendo: G1 (n = 10): animais infectados; G2 (n =10): infectados e suplementados; G3 (n = 5): controle; G4 (n = 5): não infectados e suplementados. Os grupos 1 e 2 receberam 500 larvas de HC (L3), via oral, por um período de 20 dias, com intervalo de dois dias entre as doses. A suplementação nos grupos 2 e 4 foi realizada no dia zero com 0,1mg kg-1 de Selenito de sódio (1,67%) e com 2.000 UI de vitamina E por via intramuscular (IM). Somente a vitamina E foi reaplicada no dia 30. Além das coletas de sangue para a realização do hemograma, foram determinados números de ovos por grama de fezes (OPG). Em relação ao número de hemácias, volume globular, hemoglobina, leucócitos totais e linfócitos, as principais diferenças foram observadas quando os grupos parasitados foram comparados com o grupo somente suplementado, este, mantendo valores mais elevados. Conclui-se que não há influência da suplementação dos cordeiros com vitamina E e selênio sob os parâmetros de eritrograma quando estes se encontram parasitados por HC. No leucograma pôde-se observar valores mais elevados de neutrófilos segmentados nos grupos suplementados. No entanto, a vitamina E e o selênio aumentaram parâmetros hematológicos em animais não infectados com HC. Em relação aos valores de proteínas totais, albumina, betaglobulina, gamaglobulina, as principais diferenças foram observadas quando os grupos parasitados foram comparados com o grupo somente suplementado, este, mantendo valores mais elevados.Conclui-se que não há influência da suplementação dos cordeiros com vitamina E e selênio sob os parâmetros protéicos quando estes se encontram parasitados por HC. Os animais suplementados e não parasitados mantiveram melhores valores de Glutationaperoxidase (GSH-Px) até o 60º dia de experimento. Não houve situação de estresse oxidativo visto que, quando aumentou os teores de TBARS houve um efeito compensatório gerado pelo aumento da GSH-Px.