Determination of acaricide resistance in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) field populations of Argentina, South Africa, and Australia with the Larval Tarsal Test.

Infestations with ticks have an important economic impact on the cattle industry worldwide and resistance to acaricides has become a widespread phenomenon. To optimize their treatment strategy, farmers need to know if and against which classes potential acaricide-resistance does occur. Bioassays are used to assess the resistance level and pattern of Rhipicephalus (Boophilus) microplus populations. The objective of the current study was to assess the susceptibility of field populations originating from Argentina (8), South Africa (3), and Australia (2) using the Larval Tarsal Test. Nine acaricidal compounds from five major classes were tested: organosphosphates, synthetic pyrethroids (SP), macrocyclic lactones, phenylpyrazols, and amidines. The resistance ratios at concentrations inducing 50 and 90% mortality were used to detect established and emerging resistance. This study confirmed the newly reported presence of amitraz resistance in populations from Argentina In addition, resistance to SP appeared to be widespread (88%) in the Argentinean farms, which had been selected based on the observation of lack of treatment efficacy by farmers. In South Africa one of the three populations was found to be resistant to SP and to a phenylpyrazol compound (pyriprol). Furthermore, resistance to organosphosphates and SP was observed in Australia. Finally, the Larval Tarsal Test proved to be a suitable test to evaluate the susceptibility of R. microplus field populations to the most relevant acaricidal classes.

Use of the Larval Tarsal Test to determine acaricide resistance in Rhipicephalus (Boophilus) microplus Brazilian field populations.

Acaricide resistance of the cattle tick Rhipicephalus (Boophilus) microplus is widespread in most of the countries where this parasite is present. Bioassays are used to diagnose the level and pattern of resistance in tick populations. In the present study, we describe a detailed protocol of the Larval Tarsal Test (LTT) using simplified equipment and data on the resistance of 17 tick field populations originating from 5 Brazilian states. Nine acaricidal compounds from 5 major classes were tested: organophosphates (OP), synthetic pyrethroids (SP), macrocyclic lactones (ML), phenylpyrazols (PYZ) and amidines. For comparison, four of the tick populations were also tested with the Larval Packet Test (LPT) with one compound per class. The most common resistances were to SP, amitraz and OP, with frequencies of 94%, 88% and 82%, respectively. Resistance to PYZ was also found to be widespread (65%), suggesting a rapid development of fipronil resistance in Brazil. One case of ML resistance and 2 cases of suspected ML resistance were identified with the LTT. The LTT led to higher resistance ratios to all compounds than the LPT, reflecting its high sensitivity to detect resistance. Finally, the LTT allowed testing a larger number of compounds and doses with reduced labour in comparison to the LPT and turned out to be a reliable bioassay to detect resistance in field populations.

A new in vitro test to evaluate the resistance level against acaricides of the cattle tick, Rhipicephalus (Boophilus) microplus.

In this article we present a new bioassay to assess the resistance status of ticks to acaricides. The Larval Tarsal Test (LTT) is a sensitive, highly time-effective in vitro test. It allows the investigation of a large number of compounds and doses on the cattle tick Rhipicephalus (Boophilus) microplus in a short period of time. The ability of the LTT to assess the lethal concentration at 50% mortality (LC(50)) and resistance ratios (RRs) of a susceptible and a resistant R. microplus strain was compared with the FAO-recommended Larval Packet Test (LPT). Representative compounds of the carbamate, organophosphate (OP), synthetic pyrethroid (SP), formamidine (FOR), macrocyclic lactone and pyrazole classes were used for this comparison. The resistance status against OP, SP and FOR of the resistant R. microplus strain was confirmed in vivo. The LTT resulted in resistance ratios comparable to those obtained with the LPT. However, the lethal concentrations were up to 150-fold lower in the LTT than in the LPT. The advantage of the LTT is to simplify the methodology by avoiding the handling of larvae and using multi-well plates. The LTT is therefore a suitable test for the assessment of the level of resistance of R. microplus and is very promising to evaluate the resistance profile of field strains. Additionally, the LTT is also suitable to test other ixodid species.

Light and electron microscopy studies of the midgut and salivary glands of second and third instars of the horse stomach bot, Gasterophilus intestinalis.

A morphological study of the midgut and salivary glands of second and third instars of Gasterophilus intestinalis (De Geer) (Diptera: Oestridae) was conducted by light, scanning and transmission electron microscopy. The midgut is anteriorly delimited by a proventriculus, without caeca, and is composed of posterior foregut and anterior midgut tissue from which a double-layered peritrophic matrix is produced. The midgut can be divided into anterior, median and posterior regions on the basis of the structural and physiological variations of the columnar cells which occur along its length. Two other types of cell were identified: regenerative cells scattered throughout the columnar cells, and, more rarely, endocrine cells of two structural types (closed and open). Different secretion mechanisms (merocrine, apocrine and microapocrine) occur along the midgut epithelium. Abundant microorganisms are observed in the endoperitrophic space of the anterior midgut. The origin and nature of these microorganisms remain unknown. No structural differences are observed between the second and third instar midguts. The salivary glands of G. intestinalis second and third instars consist of a pair of elongated tubular structures connected to efferent ducts which unite to form a single deferent duct linked dorsally to the pharynx. Several intermediate cells, without cuticle, make the junction with the salivary gland epithelium layer. Cytological characteristics of the gland epithelial cells demonstrate high cellular activity and some structural variations are noticed between the two larval stages.

[Cattle farmers' perception of the nuisances caused by insects and ticks to cattle in the Canton Jura]. / Perception par les éleveurs des nuisances causées par les in-sectes et les tiques aux bovins dans le Canton du Jura.

During summer 2005, a survey of 172 farmers from the Canton Jura was carried out to determine their perception of the nuisances caused by insects and ticks to cattle. The presence of ticks was significantly more often spontaneously mentioned by farmers in the Clos-du-Doubs and in the district of Delémont (95% and 72%, respectively) than in Ajoie and in the Franches-Montagnes (29% and 19%, respectively, p < 0.0001). 20% of the farmers perceived the tick populations to be increasing. The following diseases were spontaneously listed: keratoconjunctivitis (59%), mastitis related to flies (31%), hypodermosis (15%), ehrlichiosis (12%) and babesiosis (10%). Suspicion of ehrlichiosis was significantly more often mentioned in the district of Delémont than in the rest of the canton (p < 0.001) which was associated with the presence of ticks observed by farmers (p < 0.001). Cases of clinical babesiosis, which is common in the Clos_du_Doubs, were mentioned by farmers outside this area: Delémont (3/57) and Ajoie (1/26). In this study babesiosis depends on the production area, on the presence of ticks observed by farmers and on the presence of water in the pasture (p < 0.05). This study shows that cattle farmers are well aware of the presence of the diseases linked to insects and ticks and that the perception of their distribution is in accordance with the epidemiological data. An overview of the epidemiology of tick- and insect-related diseases can therefore be established by surveying farmers' perception, but should then be confirmed by a biomedical study. Due to their accurate observations, farmers are key participants at the first level of surveillance systems of animal diseases.

Prospect for anthelminthic plants in the Ivory Coast using ethnobotanical criteria.

Sixty plant species were collected in the Ivory Coast on the basis of an ethnobotanical literature using the following three criteria: activity against worms, diarrhoea and/or abdominal pain. Eighty six plant extracts were prepared using 90% ethanol and tested for potential anthelminthic activities with a larvicidal test of Haemonchus contortus. 25.6% of the extracts showed a high activity, 12.8% were active or marginally active and the remaining 61.6% were inactive. The fact that a relatively high percentage of the plants species (50%) had an activity can be explained by the initial preselection of the plants on the basis of ethnobotanical indications.

Prophylaxis failure due to probable mefloquine resistant P falciparum from Tanzania.

Failures of mefloquine prophylaxis in travellers returning from Africa have been reported repeatedly. Non-compliance to chemoprophylaxis is considered to be a major factor for failure. Only few reports on mefloquine prophylaxis failure in sub-Saharan Africa were able to report blood levels of the drug that were sufficient for prophylactic effectiveness. We report the case of a 44-year-old German female who travelled to Tanzania for 3 weeks. The patient reported that she never missed a dose of mefloquine during her weekly prophylaxis schedule. Four weeks after returning from Tanzania, the patient presented with fever, headache and myalgia. Only a few trophozoites of Plasmodium falciparum were found in a thick film. Blood levels of mefloquine at that stage were at 1400 ng/ml, thus largely excluding non-compliance and malabsorption. To our knowledge, this is the first case of confirmed prophylaxis failure due to mefloquine resistance in East Africa.

Seasonal epidemiology of ticks and aspects of cowdriosis in N'Dama village cattle in the Central Guinea savannah of Côte d'Ivoire.

In the Central Guinea savannah of Côte d'Ivoire, cattle breeding started only approximately 30 years ago. The impact of parasitism on the overall health status and productivity of the trypanotolerant N'Dama cattle in this area is unknown. In close collaboration with national veterinary institutions and local farmers, we studied spectrum, burden and seasonal dynamics of ticks (including aspects of cowdriosis) on N'Dama village cattle. In a longitudinal study, three randomly selected cattle herds (traditional farming type) of one village were examined repeatedly for ticks. Spectrum, burden, seasonal epidemiology of ticks were assessed. In these traditional herds (which lack (ecto)parasite management), all animals were infested by ticks at monthly counts. Five different tick species were identified; the four genera in order of frequency were: Amblyomma (overall prevalence 96%), Boophilus (47%), Hyalomma (<1%) and Rhipicephalus (<1%). Amblyomma variegatum was the most-abundant tick on cattle in all seasons. Seroprevalence of Cowdria ruminantium was 31% (95% CI: 26, 36%). Most of the animals typically carried low tick burdens. N'Dama cattle seem well adapted to their environment and can resist the tick burdens under this traditional farming system.

Isolation, characterisation and organisation of histone H1 genes in African trypanosomes.

A tandemly arranged gene family coding for histone H1 in African trypanosomes is described. Many variants, differing by some substitutions and/or deletions in their monotone and repetitive amino acid sequences, are found. The different variants can be sorted into three subtypes using their N-terminal region. PCR amplification experiments with primers specific to these three H1 subtypes suggest that the genes may be separated into two transcriptional units. Heterogeneity among species, subspecies and even strains was found. The transcripts are polyadenylated and the trans-splicing site is located very closely to the start codon. The intergenic regions are typical when compared to other polycistronic clusters described in trypanosomes. Amino acid sequence motifs may explain differences seen in chromatin compaction patterns between African and American trypanosomes.

The synthetic, oxidized C-terminal fragment of the Plasmodium berghei circumsporozoite protein elicits a high protective response.

A polypeptide of 69 amino acids (PbCS 242-310) encompassing the C-terminal region of the circumsporozoite protein of Plasmodium berghei (PbCS) was generated using solid-phase peptide synthesis. The immunological and protective properties of peptide PbCS 242-310 were studied in BALB/c mice (H-2d). Two subcutaneous injections, in the presence of IFA at the base of the tail, generated (i) high titers of anti-peptide antibodies which also recognized the native P. berghei CS protein, (ii) cytolytic T cells specific for the Kd-restricted peptide PbCS 245-253 and (iii) partial CD8+-dependent protection against sporozoite-induced malaria. The same frequencies of peptide PbCS 245-253 specific CD8+ T cells were found by IFN-gamma ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245-253 or with the polypeptide 242-310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as the short CTL peptide. Interestingly, higher levels of IFN-gamma producing CD8 T cells and protection were observed when the four cysteine residues present in the C-terminal peptide were fully oxidized. These findings underline the potential importance of the chemical nature of the C-terminal fragment on the activation of the immune system and concomitant protection.

Isoniazid plus sulphadoxine-pyrimethamine can reduce morbidity of HIV-positive patients treated for tuberculosis in Africa: a controlled clinical trial.

An annual 20% excess mortality rate is observed in HIV-seropositive patients after treatment for tuberculosis. An affordable secondary prophylaxis against main opportunistic diseases is needed, i.e. against tuberculosis, toxoplasmosis, pneumocystosis and other infections occurring in this target population. This open prospective randomized study assessed morbidity and mortality in 2 cohorts of HIV-seropositive patients having recently recovered from pulmonary tuberculosis: 134 patients assigned to prophylactic treatment with isoniazid (INH, 300 mg once daily) plus sulphadoxine-pyrimethamine (S, 500 mg/P, 25 mg once weekly), and 129 were controls, comparable for sex, age, weight and HIV-serology. Patients were followed-up for up to 2 years: 192 person-years (PY) in the prophylaxis group and 142 PY in the control group. Four patients developed tuberculosis and 20 patients died in the prophylaxis group, compared to 10 and 23 controls, respectively. Sick days were reported by 22 patients in the prophylaxis group and by 77 patients in the control group. This prophylaxis was associated with a moderate decrease of mortality (log rank test: p = 0.1736), a significant decrease of tuberculosis incidence (log rank test: p = 0. 0234), a highly significant reduction of adverse events and sick days, and a prevention of wasting (p = 0.008) and anaemia (p = 0. 045). No death from toxoplasmosis occurred in the prophylaxis group as compared to 2 possible cases among controls; toxoplasmosis IgG levels declined in treated patients, but increased in controls (p = 0.01). There was no adverse drug reaction due to SP (10,006 doses) or to INH. Compliance with SP intake was good, but moderate as with INH intake. We conclude that a secondary prophylaxis with INH+SP represents a cost-effective measure to improve health conditions of HIV-infected adults in Côte d'Ivoire, following a full treatment course against tuberculosis.

DNA extraction from Ascaris suum muscle tissue.

A new method for the extraction of DNA from Ascaris suum muscle has been developed. It combines a standard SDS-based extraction with a plant DNA extraction procedure. The use of SDS and proteinase K allows the elimination of proteins, while CTAB and polyclar AT eliminate glycogen and polyphenols. The DNA thus obtained can easily be digested by endonucleases and amplified by PCR.

Elimination of P. berghei liver stages is independent of Fas (CD95/Apo-I) or perforin-mediated cytotoxicity.

Immunization of mammals with irradiated malaria sporozoites protects from a subsequent contact with the parasite. Protective immunity is directed against the pre-erythrocytic stages of the parasite, sporozoites and liver stages. Specific antibodies neutralize part of the infectious sporozoites infected by the mosquito vector, while liver stages are the target of a cellular immune response which is mediated by T cells. In this study, we evaluated the T-cell dependent protection induced by the infection of P. berghei irradiated sporozoites and the contribution of perforin and of the receptor/ligand system CD95/CD95L, two T cell-dependent mechanisms known to mediate elimination of target cells. Wild type, perforin deficient, CD95 mutant, CD95L mutant and perforin deficient/CD95L mutant mice were immunized with P. berghei irradiated sporozoites and submitted to a challenge with infectious sporozoites. All mice immunized with P. berghei irradiated sporozoites were protected against a sporozoite challenge, including perforin deficient/CD95L mutant animals. These results indicate that T cells do not kill malaria-infected hepatocytes via one of the known pathways, but rather that activated parasite-specific T cells produce cytokines which activate in cascade other mechanisms responsible for the intracellular elimination of the parasite.

Ascaris suum: molecular cloning of an intermediate filament.

It has been proposed that intermediate filament proteins are involved in force transduction from the muscle cells through the hypodermis to the cuticle of nematodes. An additional role of intermediate filaments as excretory/secretory components of parasitic nematodes is under discussion. We report on the molecular characterization of the cDNA clone AsIF of the intestinal nematode parasite Ascaris suum, encoding a member of the intermediate filament protein family by sequence comparison with intermediate filaments of other nematodes. We also show the precise location of the product encoded by AsIF within the organism by immunoelectron microscopy.

Repetitive peptide motifs in the cuticlin of Ascaris suum.

The cuticle of parasitic nematodes is composed of extracellular structural proteins. Over 90% of these proteins are collagenous molecules in the basal and median layers of the cuticle. The outermost layers of the cuticle, the epicuticle, is composed of non-collagenous proteins, that represent the structural surface of nematodes. In Ascaris these proteins have been termed 'cuticlins'. While cuticular collagens have been well studied by both biochemical and genetic means, knowledge of the molecular structure of cuticlin components of parasitic nematodes is scarce. In the present paper we report on the production of monoclonal antibody 8.1, which is specific for cuticlin, but does not recognize collagen epitopes. We have screened a cDNA library derived from adult Ascaris suum mRNA of the hypodermal tissue underlying and synthesizing the cuticle. One positive cDNA clone encodes alanine-rich repetitive motifs, which are part of the insoluble cuticlin of the outermost layers of the epicuticle of Ascaris suum. This was shown in immunocytochemical experiments using specific polyclonal antisera raised against these motifs, expressed as fusion protein with glutathione S-transferase of the helminth Schistosoma japonicum. Comparison of the repetitive amino acid sequence with structural proteins of the nematode Caenorhabditis elegans and the insects Locusta migratoria and Ceratitis capitata revealed a minimal consensus motif.

Identification and sequence comparison of a cuticular collagen of Brugia pahangi.

The cuticle of filarial nematodes is a specialized extracellular matrix that covers the parasite and protects it from adverse conditions of the environment. As a surface structure it is in direct contact with the host defence mechanisms and therefore plays an important role in the molecular host-parasite relationship. Using polyclonal antisera raised against the insoluble components of the cuticle of the adult filarial parasite Brugia pahangi, we have isolated cDNA clones encoding collagen molecules of the cuticle. The protein domain structure of cDNA clone Bpcol-1 was compared with the known structures of cuticular collagens of the nematodes Brugia malayi, Caenorhabditis elegans, Ascaris suum and Haemonchus contortus, confirming interspecies similarities. Using affinity-purified anti-Bpcol-1 antibodies we identified Bpcol-1 antigenic determinants in different nematode extracts, and determined the localization of such epitopes within the cuticle of B. pahangi.

Properties of the histones and functional aspects of the soluble chromatin of epimastigote Trypanosoma cruzi.

The amino acid composition of all histones of Trypanosoma cruzi was analyzed, and the terminology of the histones of higher eukaryotes adopted. One chromatin associated protein, previously considered to be a variant of histone H1, could not be clearly identified, and shows features of core histones as well as of histone H1. An improved method for the isolation of intact nuclei and the production of soluble chromatin in T. cruzi was established. The chromatin of T. cruzi is relatively instable and histone H1 is easily lost during experimental manipulations. Histone H1 dissociates completely at a relatively low NaCl concentration of 380 mM, leading to an open nucleosome filament which does not condense. The influence of histone H1 of T. cruzi and of rat liver on the compaction pattern of the chromatin was investigated by homologous and heterologous reconstitution experiments, and analysed by electron microscopy. It could be shown that histone H1 of T. cruzi induces nucleosome filaments of T. cruzi as well as those of rat liver to condense. The same is true for histone H1 of rats. It can be concluded that T. cruzi has a functional histone H1.

Trypanosoma brucei brucei and high-density lipoproteins: old and new thoughts on the identity and mechanism of the trypanocidal factor in human serum.

Nature has provided humans with a surprising means of protection against the African trypanosome Trypanosoma brucei brucei There is consensus, in that this singular trypanocidal factor is serum high-density lipoproteins (HDL). which the trypanosomes engulf through a physiological, receptor-mediated pathway for delivery to acidic intracellular vesicles. There is also controversy, however, in that the active particles and their essential cytotoxic elements are disputed, in part reflecting the ill-defined mechanism by which the parasites are finally killed. Here Patrick Lorenz, Bruno Betschart and Jim Owen discuss the possibilities for resolving these discrepancies and speculate on the prospects of exploiting this unexpected property of human HDL for protecting livestock.

CD8+ T-cell protective immunity induced by immunization with Plasmodium berghei CS protein-derived synthetic peptides: evidence that localization of peptide-specific CTLs is crucial for protection against malaria.

Immunization of BALB/c mice (H-2d) with a mixture of major histocompatibility complex (MHC) class I- and MHC class II-restricted synthetic peptides emulsified in incomplete Freund's adjuvant (IFA) induced a high level of specific cytotoxic T lymphocyte (CTL) activity. Peptides 249-260 or 252-260, derived from the circumsporozoite protein of Plasmodium berghei and representing a H-2Kd-restricted CTL epitope, were injected twice subcutaneously or intraperitoneally in BALB/c mice in combination with the tetanus toxin-derived universal T-helper peptide P30 in IFA. No protection was observed after exposure of immunized mice to infected mosquitoes. In contrast, when peptide 252-260-specific CTLs were expanded in vitro and adoptively transferred into naive recipient, mice were partially protected (64%) against a subsequent sporozoite challenge. Furthermore, direct transfer of lymph nodes or spleen cells from mice immunized with the peptide PbCS 252-260 also conferred protection to recipient mice. This protection was long-lasting and similar to that obtained with irradiated sporozoites.