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Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2010. / Full text also available online. Scroll down for electronic link.

Membros do gênero Rhinebothroides são parasitas exclusivos de potamotrigonídeos, elasmobrânquios de água doce endêmicos da região Neotropical. Atualmente, seis espécies são reconhecidas para este gênero dentre as oito espécies nominais disponíveis. A taxonomia de Rhinebothroides é convoluta, pois a maioria de suas espécies são diagnosticadas por caracteres morfométricos e merísticos definidos por estudos que desconsideram a variabilidade intraespecífica destas linhagens. A ampla distribuição de algumas espécies, bem como seus padrões generalistas de especificidade padrão este discrepante em relação à tetrafilídeos marinhos sugere a existência de complexos de espécies que requerem melhor refinamento taxonômico. Este estudo visa refinar a taxonomia de um destes complexos Rhinebothroides freitasi, no qual estão inseridas outras 3 especies nominais (R. campbelli, R. circularisi, R. venezuelensis) cujas circunscrições são ambíguas. Neste estudo, partiuse da premissa de que a conjunção de dados moleculares e morfológicos pode elucidar a taxonomia deste complexo. Com este objetivo, dados moleculares para os genes 28S, ITS1 e COI foram compilados para 57 haplótipos de Rhinebothroides representando todas as espécies válidas para o gênero e a ampla distribuição biogeográfica no gênero nas bacias hidrográficas brasileiras. A otimização direta das sequências nucleotídicas destes haplótipos concatenadas com outros 26 terminais que incluem linhagens de tetrafilídeos marinhos e de água doce, resultou em cinco clados de Rhinebothroides que possuem morfologia congruente com a série tipo de cinco espécies nominais. Desta forma, este estudo reconhece cinco espécies de Rhinebothroides como válidas: R. glandularis, R. freitasi, R. moralarai, R. scorzai e R. venezuelensis. Dentre as espécies do complexo R. freitasi, os dados morfológicos compilados para ~ 400 indivíduos permitiu delimitar os níveis de variabilidade morfológica de R. freitasi e R. venezuelensis. A representatividade biogeográfica e de hospedeiros contemplada neste estudo revela que, ao contrário das linhagens de tetrafilídeos marinhos, membros de Rhinebothroides possuem baixa especificidade aos seus hospedeiros. / Members of Rhinebothroides are parasites of the Neotropical freshwater stingrays of the family Potamotrygonidae. To date, six species are recognized for the genus within which there are eight nominal species available. The taxonomy of Rhinebothroides is confusing, since most of its species are currently diagnosed by morphometric and meristic characters that have been defined by studies that disregarded the intraspecific variability of its lineages. The widespread distribution of some species, as well as their relaxed host specificity pattern which differs from what has been documented for marine tetraphyllideans suggests the existence of species complexes that require taxonomic refinement. This study aims at refining the taxonomy of one of these complexes Rhinebothroides freitasi, in which are included other three nominal species (R. campbelli, R. circularisi, R. venezuelensis) circumscribed ambiguously. In this study, it has been assumed that the combination of molecular and morphological data can shed some light on the taxonomic status of this complex. Within this framework, molecular data were compiled for 28S, ITS1, and COI for 57 haplotypes of Rhinebothroides representing all currently valid species within the genus and their biogeographical distribution along the major Brazilian river basins. The direct optimization of nucleotide sequences from these haplotypes, simultaneously analised with 26 terminals which included marine and freshwater lineages of tetraphyllideans, generated a phylogenetic hypothesis that recognized five major clades within Rhinebothroides. Each of these clades are morphologically congruent with the type series of five nominal species. Therefore, this study recognizes five valid species within Rhinebothroides: R. glandularis, R. freitasi, R. moralarai, R. scorzai, and R. venezuelensis. Within the R. freitasi complex, the compiled morphological data for ~ 400 specimens provided a robust assessment of intraspecific variability for R. freitasi and R. venezuelensis. The biogeographic and host extensive sampling available for this study reveals that members of Rhinebothroides show low host specificity, as opposed to the marine tetraphyllidean lineages.

A prevalência de patógenos intestinais foi estudada entre os índios da etnia Tapirapé, da Amazônia mato-grossense, por meio de técnicas coproparasitológicas, imunológicas e moleculares. Do total de 1526 amostras, 83,35% apresentaram ao menos um parasito intestinal e 65% tinham mais de um parasito (poliparasitismo). Entamoeba coli foi o mais prevalente (827/1526 - 54,19%). Entamoeba histolytica/dispar (581/1526 - 38,07%), Giardia intestinalis (287/1526 - 18,81%), Blastocystis spp. (257/1526 - 16,84%) e Ancylostoma spp. (293/1526 - 19,20%) também foram freqüentes. Cistos de Giardia intestinalis foram seqüenciados utilizando os genes <font face=\"Symbol\">b-Giardina e gdh. Apenas os assemblages A e B foram encontrados, sendo que o assemblage A foi o mais prevalente. Análise molecular de Blastocystis spp. demonstrou que, por meio do gene SSU-rNA, o subtipo 1 foi o mais dominante entre os Tapirapé, seguido pelos subtipos 2 e 3. Com base nisso, G. intestinalis e Blastocystis spp. são potencialmente zoonóticos. Os resultados corroboram com outros estudos realizados na Amazônia brasileira. / The prevalence of intestinal pathogens was studied in indigenous of the Tapirapé ethnic from Amazon region of Mato Grosso State, using the coproparasitological, immunological and molecular. Of the total 1,526 fecal samples 83.35% had at least one intestinal parasite and 65% had more than one parasite (poliparasitism). The most prevalent parasite was Entamoeba coli (827/1526 - 54.19%). Entamoeba histolytica/dispar (581/1526 - 38, 07%), Giardia intestinalis (287/1526 - 18.81%), Blastocystis spp. (257/1526 - 16.84%) and Ancylostoma spp. (293/1526 -19.20%) were found too. Cysts of G. intestinalis were sequence by <font face=\"Symbol\">b-Giardina and GDH gene. Only assemblages A and B were found and assemblage A was the most prevalent. The molecular characterization of Blastocystis spp. by SSU-rRNA demonstrated that subtype 1 was dominant followed by subtypes 2 and 3. So, G. intestinalis and Blastocystis spp. are potentially zoonotic. The results are in agreement with previous studies conducted in the Brazilian Amazon.

A prevalência de patógenos intestinais foi estudada entre os índios da etnia Tapirapé, da Amazônia mato-grossense, por meio de técnicas coproparasitológicas, imunológicas e moleculares. Do total de 1526 amostras, 83,35% apresentaram ao menos um parasito intestinal e 65% tinham mais de um parasito (poliparasitismo). Entamoeba coli foi o mais prevalente (827/1526 - 54,19%). Entamoeba histolytica/dispar (581/1526 - 38,07%), Giardia intestinalis (287/1526 - 18,81%), Blastocystis spp. (257/1526 - 16,84%) e Ancylostoma spp. (293/1526 - 19,20%) também foram freqüentes. Cistos de Giardia intestinalis foram seqüenciados utilizando os genes <font face=\"Symbol\">b-Giardina e gdh. Apenas os assemblages A e B foram encontrados, sendo que o assemblage A foi o mais prevalente. Análise molecular de Blastocystis spp. demonstrou que, por meio do gene SSU-rNA, o subtipo 1 foi o mais dominante entre os Tapirapé, seguido pelos subtipos 2 e 3. Com base nisso, G. intestinalis e Blastocystis spp. são potencialmente zoonóticos. Os resultados corroboram com outros estudos realizados na Amazônia brasileira. / The prevalence of intestinal pathogens was studied in indigenous of the Tapirapé ethnic from Amazon region of Mato Grosso State, using the coproparasitological, immunological and molecular. Of the total 1,526 fecal samples 83.35% had at least one intestinal parasite and 65% had more than one parasite (poliparasitism). The most prevalent parasite was Entamoeba coli (827/1526 - 54.19%). Entamoeba histolytica/dispar (581/1526 - 38, 07%), Giardia intestinalis (287/1526 - 18.81%), Blastocystis spp. (257/1526 - 16.84%) and Ancylostoma spp. (293/1526 -19.20%) were found too. Cysts of G. intestinalis were sequence by <font face=\"Symbol\">b-Giardina and GDH gene. Only assemblages A and B were found and assemblage A was the most prevalent. The molecular characterization of Blastocystis spp. by SSU-rRNA demonstrated that subtype 1 was dominant followed by subtypes 2 and 3. So, G. intestinalis and Blastocystis spp. are potentially zoonotic. The results are in agreement with previous studies conducted in the Brazilian Amazon.

Infection of small ruminants by gastrointestinal nematodes (GIN) is a major health concern because they cause substantial economic losses, especially in the tropics and sub-tropics. For many years, control of GIN has been based upon use of anthelmintics. However, there is now a global challange because mutant GIN individuals can tolerate most of the widely used anthelmintics. Therefore, alternative control measures are needed. The objective of the study was to screen a number of plant species for their anthelmintic effects, and to evaluate selected strains of Bacillus thuringiensis (Berliner) and Clonostachys rosea (Schroers) for activity against sheep GIN. Subsequently, the combined treatments would test a dual control strategy for nematodes by using a combination of plant extracts with biocontrol agents. Ethanol extracts of 25 plant species were screened for their anthelmintic effects against Haemonchus contortus (Rudolphi 1803). Extracts of each plant were used in vitro at various concentrations (10, 20 and 30%) to treat 10 day faecal cultures. Five plants with high efficacies (Ananas comosus L. Merr., Aloe ferox Mill., Allium sativum Linn., Lespedeza cuneata Dum. Cours. and Warburgia salutaris Bertol.f. Chiov) were selected for further investigation, using ethanol, dichloromethane and water extracts at four concentrations (2.5, 5, 10 and 20%). Ethanol was the most effective solvent. Larval counts decreased as a result of increasing extract concentrations. An ethanolic extract of Lespedeza cuneata caused more than 70% mortality at all concentrations. In an in vivo study, the five plants A. comosus, A. ferox, A. sativum, L. cuneata and W. salutaris extracts were compared to a positive Control (Equimax®, a modern anthelmintic based on abamectin and praziquantel). Gender, eggs count (EPG₀) and initial body weights were used in assigning sheep (24 females and 24 males) to six groups. Each group was randomly assigned a treatment. Plant extracts were applied as an oral dose (100 mg kg⁻¹ BW), one dose per week per animal for 42 days (Phase 1). Subsequently, the same sheep were dosed for three consecutive days with the same treatments, keeping them in the same groups (Phase 2). Rectal faecal samples were taken for counting of eggs per gram of faeces (EPG) and L₃ larvae per gram (LPG) in faecal cultures. With application of plant extracts, the EPG count decreased with time (P<0.001), and the impact of the plant extracts increased (P<0.001) with time. Two extracts, from A. comosus and L. cuneata, were the most effective in Phase 1 (58% and 61% reduction of EPG, respectively,), and in Phase 2 (77% and 81% reduction of EPG, respectively). In a study on potential biocontrol agents, two strains of Bacillus thuringiensis (Bt) and one of Clonostachys rosea f. rosea (C. rosea), and compared with a diatomaceous earth (DE) product for their anthelmintic activity in sheep. Bacillus thuringiensis and C. rosea were fed to sheep at a rate of 1g kg⁻¹ BW, and DE was fed at 2% of sheep diet. The biocontrol treatments had no effect on EPG (P>0.05), but reduced GIN larvae per gram (LPG) (P<0.001) in faecal culture. Efficacy varied with time (P<0.001). By Day 7 Bt, C. rosea and DE had caused mortalities of GIN of 75.7, 86.9 and 60.6%, respectively. In addition, the efficacy of feeding 1g kg⁻¹ BW of C. rosea chlamydospores to sheep every day, every second day and every third day was tested. Daily feeding of fungal chlamydospores reduced LPG (a count of 12±1.67 GIN larvae) (P<0.001) more than feeding them the biocontrol agent every second day (39±0.77) or third day (58±1.77). By Day 12, feeding the biocontrol agent to sheep every day, every second day, or every third day caused mortality of GIN larvae of 90, 63 and 49%, respectively. Four dietary levels (treatments) of C. rosea (0.25g (F1), 0.5g (F2), 1g (F3) and control (C) of C. rosea product kg-1 BW) were tested. Treatments were each mixed with a complete diet and fed to sheep once daily for 10 weeks, according to body weights. Increased doses of the biocontrol agent reduced LPG (P<0.001), larval development (LD) (P<0.001), and increased efficacy (P<0.001). On Day 70, F1, F2, F3 and the Control controlled LD by 33.3, 72.3, 89.4 and 2.6%, respectively. Clonostachys rosea was effective in reducing third stage larvae (L₃) on pastures significantly (P<0.001) by Day 63 and Day 70. Ethanolic extracts of A. comosus, A. ferox, A. sativum, L. cuneata and W. salutaris all reduced egg production by GIN parasites of sheep. Feeding sheep cultured chlamydospores of a biocontrol fungus, Clonostachys rosea, reduced counts of nematode larvae in sheep; and 1g C. rosea chlamydospores kg⁻¹ BW daily was enough to reduce nematode infective larvae, therefore reducing the degree of pasture contamination. An initial trial showed that the combination of the two treatments of an A. comosus extract and C. rosea chlamydospores was more effective than either treatment on its own in controlling gastrointestinal nematodes in sheep. A long-term trial is being undertaken currently to confirm this finding. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Made available in DSpace on 2015-03-26T13:46:34Z (GMT). No. of bitstreams: 1 texto completo.pdf: 205677 bytes, checksum: 93ca0cf3833e543ffed5592e081337ba (MD5) Previous issue date: 2006-03-23 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The viability of the formulation with nematophagous fungi D. flagrans was evaluated in the biological control of cattle gastrointestinal nematodes. Two groups, with eight Holsteinzebu calves each, it was placed in two lots grazed with Brachiaria decumbens on four hectare each one. The experiment started in March of 2005 and turn off in September of 2005. In the control group (A), each animal received 20 grams without fungus of the pellets; in the treated group (B), the animals received 20 grams of with D. flagrans fungus pellets. The counting of the infective larvae recovered in the paddock of the group B, it harvested until 20 centimeters of the distance in the dung pats, it was lower than in the plot A (P<0.01) harvested until 20 centimeters in the distance in the dung pat), there was significative reduction in the larvae recovered in the pasture in 53, 71, 65 e 85 %, respectably, in the April, July, August and September months. The genera Cooperia was the most prevalence (59.9 %). The monthly averages of the weight were bigger in the group B (P<0.05), there was difference between the group B and A in relation in profit weight, in June, July, August and September in de 21, 13, 8,5 e 20 %, respectably. The accountings of the egg per grams of the feces were obtained by the of the Gordon & Whitlock; modified by Lima, and of the Wisconsin of the Cox & Todd, for dairy cattle, modified by Ito techniques and they present differences in the value of the eggs per grams counts for control and treated group (P<0.05). It was observed significative reduction in the count eggs obtained by Gordon & Whitlock technique in May, June, July and August months in treated group in the 35, 46, 36 e 30 % , respectably e do not significative reduction in September (26 %). By Wisconsin modified technique, it there were difference in the June, July and August months in 58, 47 e 51 %, in the treated group, in relation in the control group and do not significative reduction in May (26 %) and September (25 %). It can to conclude that the application of the sodium alginate pellets with nematophagous fungus D. flagrans is effective in the control of the infective larvae of the bovine. / A viabilidade de uma formulação de fungo nematófago Duddingtonia flagrans foi avaliada no controle biológico de nematóides parasitos gastrintestinais de bovinos. Dois grupos de oito bezerros cada, mestiços Holandês-zebu foram colocados em duas pastagens de Brachiaria decumbens de 4 hectares cada. O experimento foi realizado no período de março a setembro de 2005. No grupo controle (A), os animais receberam 20 gramas de péletes sem fungo; no grupo tratado (B), os animais receberam 20 gramas de péletes com o fungo D. flagrans via oral. As contagens de larvas infectantes encontradas na pastagem B, coletadas até 20 centímetros de distância do bolo fecal, foram significativamente menores (P < 0,01) que na pastagem A (até 20 cm), sendo que houve redução no número de larvas recuperadas nas pastagens de 53, 71, 65 e 85 %, respectivamente nos meses de abril, julho, agosto e setembro do grupo tratado em relação ao controle. sendo que o gênero Cooperia foi o mais prevalente (59,96 %). As médias mensais de peso foram maiores significativamente no grupo B (P < 0,05), sendo que houve diferença no ganho de peso entre os grupos tratado e controle nos meses de junho, julho, agosto e setembro de 21, 13, 8,5 e 20 %. As contagens de ovos por gramas de fezes (OPG) foram obtidas pelas técnicas de Gordon & Whitlock, modificada por Lima e de Wisconsin, de Cox & Todd, modificada por Ito e apresentaram diferenças para valores de contagens de ovos por gramas entre grupo tratado e controle (P<0,05). Foi observada redução significativa nos valores da contagem no grupo tratado obtidos pela técnica de Gordon & Whitlock nos meses de maio, junho, julho e agosto de 35, 46, 36 e 30 % respectivamente e redução não significativa no mês de setembro de 26 %. E os dados obtidos pela técnica de Wisconsin houve redução significativa nos meses de junho, julho e agosto respectivos de 58, 47 e 51 % no grupo tratado em relação ao controle e redução não significativa nos meses de maio (26 %) e setembro (25 %). Pode se concluir que a aplicação de péletes de alginato de sódio contendo o fungo nematófago D. flagrans é efetiva no controle de larvas infectantes de bovinos.

Gastrointestinal nematode parasites cause great losses in the production of small ruminants through reduced productivity and the cost of preventive and curative treatments. Because of the threat of anthelmintic resistance, biological control of sheep nematodes has been identified as an alternative to anthelmintic drugs. Bacillus thuringiensis (Bt) (Berliner) and Clonostachys rosea (Schroers) have been widely studied as biocontrol agents. B. thuringiensis has been used for the biocontrol of insects and C. rosea has been successfully used as biocontrol agent of Botrytis cinera (De Bary) in plants. B. thuringiensis and C. rosea strains were isolated from soil collected from the Livestock Section at Ukulinga Research Farm, University of KwaZulu Natal, Pietermaritzburg. Twenty-five strains of Bt and 10 strains of C. rosea were successfully isolated. The Bt colonies were identified by their circular, white, flat and undulate character, and the gram-positive and rod-shaped endospores. C. rosea was identified by white colonies on Potato-dextose agar and the characteristic conidiophores, which were branched and showed phialides at the tips. In vitro screening of the isolates was undertaken to select the best isolates. The isolates that caused significantly greater mortality were Bt isolate B2, B10 and B12 and C. rosea isolates P1, P3 and P8. These isolates caused substantial nematode mortality in both faeces and water bioassay. Nematode counts were reduced by 28.5% to 62% and 44% to 69.9% in faecal bioassay for Bt and C. rosea, respectively. In the water bioassay, nematode counts were reduced by 62% to 85% for Bt and by 62.7% to 89.3% for C. rosea. The best inoculum level at which the best isolates were most effective, and the optimum frequency of application were determined. The trial was conducted using bioassays with faeces and water. Inoculum levels of 10(6), 10(8), 10(10), 10(12) spores ml-1 for Bt and 10(6), 10(8) and 10(10) conidia ml-1 for C. rosea was used in the faecal bioassay. The inoculum levels tested in water bioassay were 10(6), 10(8), 10(10) and 10(12) spores ml-1 for Bt and 10(9), 10(10), 10(11), 10(12) conidia ml-1 for C. rosea. In the faecal bioassay, B2 was the most effective Bt isolate at an inoculum level of 10(10) spores ml-1. Isolate P3 was the best C. rosea isolate at 10(8) conidia ml-1. In the water bioassay, Isolate P3 caused a mortality of 85% at inoculum levels of 10(9), 10(10) and 10(11) conidia ml-1. The performance of biological control agents in the field is sometimes inconsistent. Combining different biocontrol agents may be a method of improving their reliability and performance. However, the combination of most of the isolates was antagonistic, with efficacy less than that of either individual biocontrol agent. In particular, Isolate P3 was more effective when used alone than when combined with any other isolates. Therefore, the combination of biocontrol agents does not always result in synergistic interaction. There were some additive interactions between two bacterial isolates, and with one bacterial and fungal combination. The effect of feeding the best of the biocontrol agents, or diatomaceous earth (DE), was evaluated in sheep. Two doses of Bt (1g and 2g kg-1BW) and C. rosea (1g kg-1BW) reduced the numbers of L3 nematode larvae in sheep faeces. The DE product (at 15% of feed) also reduced L3 numbers but it was less effective than either the Bt or the C. rosea products. Nematode counts were reduced by 74.6%, 75.1%, 84.6%, 68.5% and 27.5% for Bt 1g kg-1BW, Bt 2g kg-1BW, C. rosea (1g kg-1 BW), DE and control, respectively. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.