Interactions between nutrition and infections with Haemonchus contortus and related gastro-intestinal nematodes in small ruminants

Interactions between host nutrition and feeding behaviour are central to understanding the pathophysiological consequences of infections of the digestive tract with parasitic nematodes. The manipulation of host nutrition provides useful options to control gastrointestinal nematodes as a component of an integrated strategy. Focused mainly on the Hameonchus contortus infection model in small ruminants, this chapter (i) illustrates the relationship between quantitative (macro- and micro-nutrients) and qualitative (plant secondary metabolites) aspects of host nutrition and nematode infection, and (ii) shows how basic studies aimed at addressing some generic questions can help provide solutions, despite the considerable diversity of epidemiological situations and breeding systems.

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole and ivermectin in vitro

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity.

Oscheius tipulae as an example of eEF1A gene diversity in nematodes

We characterized four eEF1A genes in the alternative rhabditid nematode model organism Oscheius tipulae. This is twice the copy number of eEF1A genes in C. elegans, C. briggsae, and, probably, many other free-living and parasitic nematodes. The introns show features remarkably different from those of other metazoan eEF1A genes. Most of the introns in the eEF1A genes are specific to O. tipulae and are not shared with any of the other genes described in metazoans. Most of the introns are phase 0 (inserted between two codons), and few are inserted in protosplice sites (introns inserted between the nucleotide sequence A/CAG and G/A). Two of these phase 0 introns are conserved in sequence in two or more of the four eEF1A gene copies, and are inserted in the same position in the genes. Neither of these characteristics has been detected in any of the nematode eEF1A genes characterized to date. The coding sequences were also compared with other eEF1A cDNAs from 11 different nematodes to determine the variability of these genes within the phylum Nematoda. Parsimony and distance trees yielded similar topologies, which were similar to those created using other molecular markers. The presence of more than one copy of the eEF1A gene with nearly identical coding regions makes it difficult to define the orthologous cDNAs. As shown by our data on O. tipulae, careful and extensive examination of intron positions in the eEF1A gene across the phylum is necessary to define their potential for use as valid phylogenetic markers.

Susceptibility of mosquito vectors to Dirofilaria immitis on Madeira Island, Portugal

Dirofilaria immitis (Leidy, 1856), an agent of heartworm disease, is an important parasite from both the veterinary standpoint and as a model to study human filariasis. It is a mosquito-borne filarial nematode which inhabits the right ventricle and pulmonary arteries of dogs. D. immitis is an important disease agent on Madeira Island with about 30% of dogs testing positive for this worm. Nevertheless, the vectors of this parasite in Madeira have never been studied, nor has the interaction between pathogen and vector, or the environmental variables that might influence heartworm transmission. Innate susceptibility to infection is only one component of vector competence, and field isolation of naturally infected mosquitoes has shown the capability of D. immitis to exploit a great diversity of vector species under natural conditions. The purpose of this work was to determine which mosquitoes are vectors of heartworm disease, the relation between population density and environment, and the association between immune response of the vector to the filarial parasite. Seasonal abundance of Culex theileri and Culex pipiens molestus was studied. Correlation and canonical correspondence analysis were performed using abundance data of these two species with selected weather variables, including mean temperature, relative humidity and accumulated precipitation. The most important factor determining Cx. theileri abundance was accumulated precipitation, while Cx. pipiens molestus abundance did not have any relationship with weather variables. Field studies were performed to verify whether Cx. theileri Theobald functions as a natural vector of D. immitis on Madeira Island, Portugal. Cx. theileri tested positive for D. immitis for the first time. The same study was made regarding Cx. p. molestus. Two abnormal L2 stage filarial worms were found in Malpighian tubules in field caught Cx. p. molestus. In the laboratory, two strains of Cx. p. molestus were studied for their susceptibility to D. immitis. None presented infective-stage larvae. Finally, because Cx. p. molestus is an autogenous mosquito, we evaluated the reproductive costs when this mosquito mounts an immune response against D. immitis in the absence of a blood meal. This mosquito showed an active immune response when inoculated intrathoracically with microfilariae (mf) of the heartworm. The ovaries from mosquitoes undergoing melanotic encapsulation developed more eggs than those which could not melanize the mf. This fact is contradictory with some previous studies of reproductive costs in Armigeres subalbatus and Ochlerotatus trivittatus, and it was the first time that an autogenous mosquito was used to study this subject.

Prácticas culturales para el manejo de la Sigatoka negra en bananos y plátanos

Black sigatoka (Mycosphaerella fijiensis) control consists in the continuous use of fungicide sprays and cultural practices. Cultural control reduces inoculum sources of the pathogen and favorable environmental conditions for its development, as well as it increases the vigor of the plants. In order to manage the disease it is necessary to know its behavior through the time, its relationship with the weather and the crop management. The most important cultural practice to reduce the inoculum source is the removal of the whole affected leaves or only the diseased portions of the leaves. The removed tissue is placed over the ground and the use of urea is feasible to accelerate its decomposition. An alternative practice is the "mini-composting" that consists in forming small heaps with the dead leaves and harvested plants for its quick degradation, which allows to reduce the pathogen inoculum and to incorporate nutrients and organic matters into the soil. The early pruning of the tips of the Young leaves before they show symptoms of the disease and the quick elimination of harvested plants reduce the inoculum. Also, the agronomic management of the crop helps to reduce the favorable conditions and to increase the vigor of the plants: this management considers plantation density, drainage systems, irrigation methods, control of weeds, chemical-biological fertilization, and nematode control.

Aspectos sanitários do cultivo da alface americana, irrigada com águas recptoras de efluentes urbanos

Avaliou-se a influência da disposição de mangueiras gotejadoras nos canteiros e a injeção ou não de cloro na água de irrigação, nas condições sanitárias do solo e da alface americana irrigada (Lactuca sativa L.) com águas receptoras de efluentes urbanos. Foram realizadas análises microbiológicas de amostras de água do solo e da alface, no decorrer de todo o ciclo de cultivo. Objetivou-se determinar a possível existência de Salmonella spp. e de formas evolutivas de parasitos humanos e a quantidade de coliformes fecais, em pontos e épocas diferentes do experimento, impedindo assim o consumo da alface. Os resultados não indicaram a presença dos dois primeiros em nenhuma das amostras, mas sim de parasitos não humanos (nematóides) de vida livre no solo. em relação à quantidade de coliformes fecais (NMP ml-1), o valor encontrado na cultura atende às exigências da Secretaria de Vigilância Sanitária do Ministério da Saúde brasileiro, porém a presença dos nematóides em quantidades superiores ao permitido pela Organização Mundial de Saúde (OMS) inviabiliza o seu consumo.

First record of molluscs naturally infected with Angiostrongylus cantonensis (Chen, 1935) (Nematoda: Metastrongylidae) in Brazil

Seeking the identification of Angiostrongylus cantonensis as a potential etiological agent of three clinical cases of eosinophilic meningitis, mollusc specimens were collected in the state of Espírito Santo, Brazil. The snails were identified as Sarasinula marginata (45 specimens), Subulina octona (157), Achatina fulica (45) and Bradybaena similaris (23). Larvae obtained were submitted to polymerase chain reaction and restriction fragment length polymorphism diagnosis. Their genetic profile were corresponded to A. cantonensis. Rattus norvegicus experimentally infected with third-stage larvae, developed menigoencephalitis, and parasites became sexually mature in the lungs. Additionally, larvae obtained from A. fulica snails, from São Vicente, state of São Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.

Geostatistical analysis of the spatial distribution of Rotylenchulus reniformis on cotton cultivated under crop rotation

The spatial distribution of Rotylenchulus reniformis on cotton cultivated in crop rotation with sorghum-peanut-velvetbean was studied using geostatistics. The experimental field, which had been continuously cropped with cotton for 20 years, comprised two 32 x 48 m-grids, each divided in sixty-four 4 x 6 in sampling plots. For all crops, 300 cm(3) soil samples were taken at the center of each plot at crop germination (Pi) and again at harvest (Pf), from which the numbers of nematodes were determined. The results revealed that the spatial distribution of R. reniformis was highly aggregated and with the aid of geostatistical techniques the nematode intensities were mapped and the risk areas accurately identified. Consequently, geostatistics is here considered a useful tool for planning nematode control strategies, particularly in precision agriculture.

Estudo morfométrico comparativo de 58 populações brasileiras de Rotylenchulus reniformis (Nemata: Rotylenchulinae)

Foram recuperadas 58 populações de Rotylenchulus reniformis de amostras de solo e raízes de diferentes culturas e inoculadas em plantas de algodoeiro (Gossypium hirsutum) cv. COODETEC 402 e de mamona (Ricinus communis), mantidas em vasos de argila em casa de vegetação do Departamento de Fitossanidade da UNESP/FCAV, Campus de Jaboticabal - São Paulo. Foi realizado estudo morfométrico comparativo das populações ao microscópio óptico composto, seguido da ordenação das populações segundo análises multivariadas de agrupamento e de componentes principais. Foram avaliadas 11 variáveis morfométricas em dez fêmeas jovens de cada população e sete variáveis derivadas. A amplitude de variação de caracteres morfométricos em populações brasileiras desse nematóide tais como, comprimento do estilete, V e forma da cauda, é maior que em populações da mesma espécie de outras regiões do mundo. Os dados obtidos confirmam que o comprimento do estilete, presença de machos e V são suficientes para identificação de R. reniformis e que esta é a espécie do grupo predominante nos agroecossistemas brasileiros.

Description and prevalence of Thynnascaris sp. larvae Dollfus, 1933 (Nematoda: Anisakidae) in Plagioscion squamosissimus Heckel, 1840 from Volta Grande Reservoir, State of Minas Gerais, Brazil

O presente trabalho teve por objetivo o estudo da helmintofauna da corvina de água doce Plagioscion squamosissimus, do reservatório de Volta Grande, MG, Brasil. Foram colhidos 68 peixes com comprimento médio de 25,2 cm e peso médio de 180,9 g, com rede de espera, bimestralmente no período de dezembro de 1995 a dezembro de 1996. Os parasitos foram cuidadosamente retirados de seus cistos que estavam aderidos ao mesentério intestinal. Os espécimes foram fixados em AFA a 65°C e conservados em álcool 70°GL contendo 5% de glicerina. Após diafanização com ácido acético ou lactofenol de Amann, 21 nematóides foram desenhados em câmara clara. As larvas foram identificadas como Thynnascaris sp. (Nematoda: Anisakidae). Das 68 corvinas examinadas, 30 estavam infectadas por esses parasitos com uma prevalência de 44,1%. O número médio de parasitos por hospedeiro foi de 0 a 13,8 e a intensidade média, de 0 a 16. A análise estatística, de acordo com o Teste Exato de Fisher, mostrou que a sazonalidade foi dependente da pluviosidade e temperatura do ar.

Levantamento de Meloidogyne exigua na cultura da seringueira em São José do Rio Claro, MT, Brasil

Este trabalho foi realizado com o objetivo de efetuar um levantamento da ocorrência de Meloidogyne exigua em seringueira em São José do Rio Claro, MT. Foram amostradas 191 propriedades agrícolas, totalizando cerca de 18.000ha. Os nematóides foram identificados no Laboratório de Nematologia do Departamento de Fitossanidade da FCAV/UNESP, em Jaboticabal, SP. Foram encontrados níveis populacionais de M. exigua entre 0 e 61.824 juvenis/5g de raízes.

Ocorrência de Meloidogyne enterolobii em muricizeiro (Byrsonima cydoniifolia)

A entrada de agentes fitopatogênicos em novas localidades através de mudas infectadas constitui uma das principais formas de disseminação. Meloidogyne enterolobii é uma espécie de nematoide altamente virulenta que tem causado sérios danos a plantas cultivadas no Brasil. Neste trabalho é relatada a primeira ocorrência de M. enterolobii em mudas de muricizeiro (Byrsonima cydoniifolia), uma planta nativa da Amazônia e em mudas de goiabeira (Psidium guajava), no Estado de Mato Grosso. Com base nos caracteres morfológicos do padrão perineal de fêmeas, região labial dos machos e no fenótipo isoenzimático de esterase, foi confirmado que a espécie encontrada tanto nas mudas de muricizeiro quanto nas de goiabeira é M. enterolobii.

Reação de clones de umezeiro (Prunus mume sieb. et zucc.) e cultivares de pessegueiro a Meloidogyne javanica (treub, 1885) Chitwood, 1949

Um amplo projeto de estudos sobre a utilização do umezeiro como porta-enxerto para pessegueiro está sendo desenvolvido na FCAV/UNESP, Câmpus de Jaboticabal-SP, devido, especialmente, às promissoras características para uso como redutor de vigor da copa e sua boa qualidade de frutos. Alguns trabalhos na literatura citam o umezeiro como resistente ao nematóide das galhas, entretanto dispõe-se de poucas informações. Neste trabalho, teve-se por objetivo estudar a reação de clones de umezeiro e cultivares de pessegueiro a Meloidogyne javanica. O experimento foi conduzido em casa de vegetação, com 6 tratamentos (Clones 05; 10 e 15 de umezeiro e as cultivares Okinawa, Aurora-1 e Dourado-1 de pessegueiro) e 9 repetições. As plantas foram mantidas em vasos de cerâmica contendo uma mistura de solo e areia (1:1, v/v), previamente autoclavada a 121ºC e 1kgf.cm-2 por 2 horas. Aos sessenta dias após o plantio, cada planta foi inoculada com 3.000 ovos e juvenis de segundo estádio de Meloidogyne javanica. Aos 100 dias após a inoculação, as plantas foram colhidas para avaliação da massa de matéria fresca do sistema radicular, número de galhas por sistema radicular, número de ovos e juvenis por 10 g de raízes, número de ovos e juvenis por sistema radicular e fator de reprodução. Verificou-se que todos os clones e cultivares de umezeiro e pessegueiro, respectivamente, mostraram-se resistentes a Meloidogyne javanica.

Análise da variabilidade genética de acessos dePsidium spp. (Myrtaceae) avaliados quanto à reação a Meloidogyne enterolobii

This study aimed to characterize molecular of 13 accessions of Psidium spp. (Myrtaceae) that was been identified for the reaction to rootknot guava nematode. The DNA extraction of the samples was carried according to the protocol of Shillito & Saul (1988). The molecular markers type fAFLP, were obtained from fAFLP Regular Plant Genomes Fingerprinting Kit' (Applied Biosystems from Brasil Ltda.) and were tested 24 selectives combinations of primers, of which 18 showed amplification that produced 272 polymorphic markers. To the analysis of the markers were employed the softwares GeneScan (ABI Prism versao 1.0) and Genotyper (ABI Prism version 1.03), and the data collected were transformed into a binary matrix that was analyzed in the software PAUP (Phylogenetic Analysis Using Parcimony - version 3.01). Were calculated genetic distance index intra and interespecific between the genotipes. It was found that the AFLP markers were efficient in the discrimination between accessions, as well as in showing genetic similarity among accessions identified as resistant to the nematode Meloidogyne enterolobii, which could be discussed in the future.

Ocorrência de Meloidogyne enterolobii Yang & Eisenback, no município de Uberlândia, Minas Gerais, Brasil

Meloidogyne enterolobii Yang & Eisenback (Sin.: M. mayaguensis Rammah & Hirschmann), poliphagous plant parasitic nematode, has been reported causing hard damage in several plant species in Brazil. This communication represents the first occurrence of this nematode in the municipality of Uberlandia, Minas Gerais State, Brazil.