Effects of cholesterol content on activity of P-glycoproteins and membrane physical state, and consequences for anthelmintic resistance in the nematode Haemonchus contortus

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MβCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.

Anthelminthic resistance of gastrointestinal nematodes in sheep to monepantel treatment in central region of Rio Grande do Sul, Brazil

ABSTRACT: Given the numerous reports of anthelminthic resistance of sheep nematodes to different anthelmintic compounds, this study aimed to evaluate the resistance status of gastrointestinal nematodes from naturally infected sheep to monepantel in the state of Rio Grande do Sul. Four farms that present extensive raising system and absence of anthelmintic treatment for 60 days were selected for the study. Lambs that present counts of eggs per gram of feces (EPG) ≥200 (sensitivity of 50 EPG) one day (D-1) before the treatment were select for the study and randomly separated into two groups, a control group and an experimental group treated with monepantel. Feces were collected 9 days after the treatment (D+9) for EPG counts and fecal culture. The monepantel was 100% effective only on 2. The efficacy found on farm 1, 3, and 4 were 2.82%, 25.8%, and 78.4%, respectably. There were no viable larvae post-treatment at farm 2, but the genera Haemonchus, Trichostrongylus, Cooperia, and Strongyloides were resistant to it at the other farms. This study shows the presence of parasites resistant to the treatment with monepantel, pointing to the importance of monitoring its efficacy in sheep flocks of Rio Grande do Sul, Brazil.

Anthelmintic resistance in a dairy cattle farm in the State of Minas Gerais

Eighty-four half-blood Gir × Holstein (F1) calves aged six months who were naturally infected by gastrointestinal helminths and maintained in rotational grazing received different anthelmintic treatments. Group A received anthelmintics according to the usual management in the property (eight treatments, seven including a macrocyclic lactone agent). Group B received strategic treatment (ivermectin 3.15%) at the beginning and at the end of the rainy period. Eggs per gram of feces (EPG) counts and genus of larvae from fecal cultures were determined on a monthly basis from April 2002 to December 2003. There was no significant reduction (p > 0.05) in EPG counts in any group after anthelminthic treatment, and the larvae in fecal cultures observed were Cooperia, Haemonchus, Oesophagostomum and a few Trichostrongylus. Cooperia was the most prevalent genus in the first four months of the experiment and Haemonchus in the following months. In 2003, tracer calves were introduced onto the pastures monthly and they showed high nematode burden many times throughout the year, and Cooperia punctata and Haemonchus contortus were the main species identified. The results suggest that there is anthelminthic resistance in this farm, mainly to macrocyclic lactones, and the development of immunity by crossbred animals was vital to reduce nematode burden.

ESTUDO DO TIPO DE HEMOGLOBINA COMO AUXILIAR NA SELEÇÃO DE OVINOS RESISTENTES E SUSCEPTÍVEIS AOS HELMINTOS GASTRINTESTINAIS

A criação de ovinos vem adquirindo, ao longo do tempo, importância econômica e social no Estado do Paraná. As parasitoses gastrintestinais tornam-se muitas vezes fatores limitantes desta cultura, principalmente quando associadas ao sistema intensivo de criação e à resistência dos parasitas aos diferentes princípios ativos. Com o objetivo de buscar alternativas ao controle da verminose ovina, estudou-se, em um rebanho do município de Curitiba-PR, a relação do tipo de hemoglobina (Hb) com a resistência dos animais aos helmintos gastrintestinais. A determinação do tipo de Hb foi feita por meio de eletroforese em gel espesso de amido. Dos 141 animais testados, 80,14% eram do tipo BB; 17,02% do tipo AB e apenas 2,84% dos ovinos eram do genótipo AA, resultando na freqüência alélica de 0,11 e 0,89 para os alelos A e B, respectivamente. Quando estes animais foram classificados em resistentes e susceptíveis aos helmintos gastrintestinais, através da contagem de ovos nas fezes, observou-se que esta classificação ocorreu independente do tipo de Hb. Abstract Sheep production has been showing, these days, a greater economical and social importance in the State of Paraná, Brazil. The gastrointestinal nematodes are the major problem causing widespread clinical disease and productivity losses, mainly when associated with the high intensive husbandry system and the critic situation of anthelminthic resistance. In order to find alternative or complementary control methods to combat parasitism, one sheep flock of 64 ewes and 77 lambs was monitored to study whether there is a relationship between haemoglobin type and genetic resistance to gastrointestinal nematodes. Haemoglobin typing was performed by starch gel electrophoresis. In 141 animals examined, it was found that 80,14% were haemoglobin BB type; 17,02% were AB type; and only 2,84% showed a genotype AA, resulting in an allelic frequency of 0,11 and 0,89 for the alleles A and B, respectively. When these animals were classified into resistant and susceptible to gastrointestinal nematodes, using faecal egg counts, it was observed that this classification was done independently of the haemoglobin type.