Identification and characterization of epicuticular proteins of nematodes sharing motifs with cuticular proteins of arthropods.

Specific collagens and insoluble proteins called cuticlins are major constituents of the nematode cuticles. The epicuticle, which forms the outermost electron-dense layer of the cuticle, is composed of another category of insoluble proteins called epicuticlins. It is distinct from the insoluble cuticlins localized in the cortical layer and the fibrous ribbon underneath lateral alae. Our objective was to identify and characterize genes and their encoded proteins forming the epicuticle. The combination between previously obtained laboratory results and recently made available data through the whole-genome shotgun contigs (WGS) and the transcriptome Shotgun Assembly (TSA) sequencing projects of Ascaris suum allowed us to identify the first epicuticlin gene, Asu-epic-1, on the chromosome VI. This gene is formed of exon1 (55 bp) and exon2 (1067 bp), separated by an intron of 1593 bp. Exon 2 is formed of tandem repeats (TR) whose number varies in different cDNA and genomic clones of Asu-epic-1. These variations could be due to slippage of the polymerases during DNA replication and RNA transcription leading to insertions and deletions (Indels). The deduced protein, Asu-EPIC-1, consists of a signal peptide of 20 amino acids followed by 353 amino acids composed of seven TR of 49 or 51 amino acids each. Three highly conserved tyrosine motifs characterize each repeat. The GYR motif is the Pfam motif PF02756 present in several cuticular proteins of arthropods. Asu-EPIC-1 is an intrinsically disordered protein (IDP) containing seven predicted molecular recognition features (MoRFs). This type of protein undergoes a disorder-to-order transition upon binding protein partners. Three epicuticular sequences have been identified in A. suum, Ascaris lumbricoides, and Toxocara canis. Homologous epicuticular proteins were identified in over 50 other nematode species. The potential of this new category of proteins in forming the nematode cuticle through covalent interactions with other cuticular components, particularly with collagens, is discussed. Their localization in the outermost layer of the nematode body and their unique structure render them crucial candidates for biochemical and molecular interaction studies and targets for new biotechnological and biomedical applications.

Correlates of parasites and pseudoparasites in wolves (Canis lupus) across continents: A comparison among Yellowstone (USA), Abruzzo (IT) and Mercantour (FR) national parks.

Little is known about the impact of infectious diseases on large carnivores. We investigated factors structuring the helminth and protozoan infections of wolves (Canis lupus) by using coprological analyses. Faecal samples (n = 342) were analysed from 11 wolf packs belonging to three different geographical and ecological settings in Italy (Abruzzo, Lazio e Molise National Park, PNALM: 4 packs, 88 samples), in France (Mercantour National Park, PNM: 4 packs, 68 samples) and in the U.S.A. (Yellowstone National Park, YNP: 3 packs, 186 samples). Parasites were found in 29.4%-88.6% of the samples and parasite taxa ranged from four to ten in each study area. Taeniidae (Taenia/Echinococcus), Sarcocystis spp. and Toxascaris leonina were most common in faecal samples from YNP, whereas Capillaria spp., Taeniidae and Uncinaria stenocephala were predominant in PNALM. We used generalised linear mixed models to assess the relationship between parasite infection or the number of parasite taxa and selected ecological drivers across study areas. Significant effects illustrated the importance of the ecological factors such as occurrence of free-ranging dogs, diet composition and wolf density, as well as the ancestry of the wolf populations, in shaping parasite-wolf communities. Additional investigations are needed to elucidate the impact of parasitic infections on wolf populations, as well as the role of anthropogenic factors in facilitating parasitic diffusion to apex predators.

Environmental and Intrinsic Correlates of Stress in Free-Ranging Wolves.

BACKGROUND: When confronted with a stressor, animals react with several physiological and behavioral responses. Although sustained or repeated stress can result in severe deleterious physiological effects, the causes of stress in free-ranging animals are yet poorly documented. In our study, we aimed at identifying the main factors affecting stress levels in free-ranging wolves (Canis lupus). METHODOLOGY/PRINCIPAL FINDINGS: We used fecal cortisol metabolites (FCM) as an index of stress, after validating the method for its application in wolves. We analyzed a total of 450 fecal samples from eleven wolf packs belonging to three protected populations, in Italy (Abruzzo), France (Mercantour), and the United States (Yellowstone). We collected samples during two consecutive winters in each study area. We found no relationship between FCM concentrations and age, sex or social status of individuals. At the group level, our results suggest that breeding pair permanency and the loss of pack members through processes different from dispersal may importantly impact stress levels in wolves. We measured higher FCM levels in comparatively small packs living in sympatry with a population of free-ranging dogs. Lastly, our results indicate that FCM concentrations are associated with endoparasitic infections of individuals. CONCLUSIONS/SIGNIFICANCE: In social mammals sharing strong bonds among group members, the death of one or several members of the group most likely induces important stress in the remainder of the social unit. The potential impact of social and territorial stability on stress levels should be further investigated in free-ranging populations, especially in highly social and in territorial species. As persistent or repeated stressors may facilitate or induce pathologies and physiological alterations that can affect survival and fitness, we advocate considering the potential impact of anthropogenic causes of stress in management and conservation programs regarding wolves and other wildlife.

Comparative survey of canine parvovirus, canine distemper virus and canine enteric coronavirus infection in free-ranging wolves of central Italy and south-eastern France.

Diseases likely affect large carnivore demography and can hinder conservation efforts. We considered three highly contagious viruses that infect a wide range of domestic and wild mammals: canine parvovirus type 2 (CPV-2), canine distemper virus (CDV) and canine enteric coronaviruses (CECoV). Infection by either one of these viruses can affect populations through increased mortality and/or decreased general health. We investigated infection in the wolf populations of Abruzzo, Lazio e Molise National Park (PNALM), Italy, and of Mercantour National Park (PNM), France. Faecal samples were collected during one winter, from October to March, from four packs in PNALM (n = 79) and from four packs in PNM (n = 66). We screened samples for specific sequences of viral nucleic acids. To our knowledge, our study is the first documented report of CECoV infection in wolves outside Alaska, and of the large-scale occurrence of CPV-2 in European wolf populations. The results suggest that CPV-2 is enzootic in the population of PNALM, but not in PNM and that CECoV is episodic in both areas. We did not detect CDV. Our findings suggest that density and spatial distribution of susceptible hosts, in particular free-ranging dogs, can be important factors influencing infections in wolves. This comparative study is an important step in evaluating the nature of possible disease threats in the studied wolf populations. Recent emergence of new viral strains in Europe additionally strengthens the need for proactive monitoring of wolves and other susceptible sympatric species for viral threats and other impairing infections.

Distribution patterns of three sodium channel mutations associated with pyrethroid resistance in Rhipicephalus (Boophilus) microplus populations from North and South America, South Africa and Australia.

Resistance to synthetic pyrethroids (SP) in the cattle tick Rhipicephalus (Boophilus) microplus is widespread throughout its distribution area. Three single nucleotide substitutions identified in Domains II and III of the sodium channel gene of R. (B.) microplus are known to be associated with target site pyrethroid resistance. We developed a multiplex PCR using allele-specific primers to amplify wild type or mutated genotypes of the three mutations simultaneously. This assay was used to screen tick samples originating from Brazil, Argentina, Mexico, South Africa and Australia whose phenotype to flumethrin and cypermethrin had been determined by the use of the Larval Tarsal test (LTT) or the Larval Packet Test (LPT). These mutations were found to have distinct geographical distributions and result in different resistance phenotypes. The L64I Domain II mutation conferring resistance to several SP compounds was found in all the Brazilian, Argentinean and Australian populations and in one South African population, with frequencies between 38% and 100% in flumethrin and cypermethrin resistant populations. In contrast, this mutation was not found in samples from Mexico, while the Domain III mutation was found exclusively in this country. The G72V Domain II flumethrin-specific mutation was found in a single Australian population, with a very low resistant allele frequency (3%). The homozygous resistant RR genotype of the L64I Domain II mutation correlated significantly with the survival rates at the discriminating doses of flumethrin and cypermethrin. This survey shows the widespread distribution of the L64I Domain II mutation and provides evidence of its geographic separation from the Domain III mutation.

Quantifying the evolution and impact of antimalarial drug resistance: drug use, spread of resistance, and drug failure over a 12-year period in Papua New Guinea.

BACKGROUND. Antimalarial use is a key factor driving drug resistance and reduced treatment effectiveness in Plasmodium falciparum malaria, but there are few formal, quantitative analyses of this process. METHODS. We analyzed drug usage, drug failure rates, and the frequencies of mutations and haplotypes known to be associated with drug resistance over a 12-year period (1991-2002) in a site in Papua New Guinea. This period included 2 successive treatment policies: amodiaquine (AQ) or chloroquine (CQ) from 1991 through 2000 and their subsequent replacement by sulfadoxine-pyrimethamine (SP) plus AQ or SP plus CQ. RESULTS. Drug use approximated 1 treatment per person-year and was associated with increasing frequencies of pfcrt and pfmdr1 mutations and of treatment failure. The frequency of pfdhfr mutations also increased, especially after the change in treatment policy. Treatment failure rates multiplied by 3.5 between 1996 and 2000 but then decreased dramatically after treatment policy change. CONCLUSIONS. With high levels of resistance to CQ, AQ, and SP, the deployment of the combination of both drugs appears to increase clinical effectiveness but does not decelerate growth of resistance. Our estimates of mutation and haplotype frequencies provide estimates of selection coefficients acting in this environment, which are key parameters for understanding the dynamics of resistance.

Protein expression profile of Gasterophilus intestinalis larvae causing horse gastric myiasis and characterization of horse immune reaction.

BACKGROUND: Little information is available on the immunological aspect of parasitic Gasterophilus intestinalis (Diptera, Oestridae) larvae causing horse gastric myiasis. The objectives of this research were to analyze the protein content of larval crude extracts of the migrating second and third larvae (L2 and L3) of G. intestinalis in order to characterize the immune response of horses. RESULTS: The proteomic profile of L2 and L3, investigated by using one and two dimensional approaches, revealed a migration pattern specific to each larval stage. Furthermore, Western blots were performed with horse sera and with sera of Balb/c mice immunised with the larval crude extracts of L2 or L3, revealing a different immune reaction in naturally infected horses vs. artificially induced immune reaction in mice. The comparisons of the immunoblot profiles demonstrate that the stage L2 is more immunogenic than the stage L3 most likely as an effect of the highest enzymatic production of L2 while migrating through the host tissues. Fifteen proteins were identified by mass spectrometry. CONCLUSION: This work provides further information into the understanding of the interaction between G. intestinalis and their host and by contributing a novel scheme of the proteomic profile of the main larval stages.

A study on the seasonal epidemiology of Anoplocephala spp.-infection in horses and the appropriate treatment using a praziquantel gel (Droncit 9% oral gel).

In a study on the seasonal dynamics of the gastro-intestinal nematode egg production in horses, one breeding farm also revealed a particularly high prevalence of Anoplocephala spp. infection. Consequently, this farm was chosen for analysing the seasonal pattern of the tapeworm egg excretion over a one year period in order to establish the most favourable periods for an appropriate and successful cestocidal treatment. The seasonal analysis showed a significantly higher (p < 0.05) Anoplocephala spp. egg excretion between July and October, i.e. during the second part of the grazing period. This result clearly underlines the importance of a cestocidal treatment during that period of the year. Subsequently, horses of this farm and of a second farm with a high prevalence of Anoplocephala spp. were used to evaluate the efficacy of praziquantel in a specific oral gel formulation for horses under field conditions. The efficacy of praziquantel was tested in a total of 33 horses from the two farms harbouring a coproscopically detected Anoplocephala spp. infection prior to treatment. Praziquantel (Droncit 9%, oral gel, 1 mg/kg bodyweight) was administered to the horses according to their body weight. The efficacy of the drug was evaluated ten days after treatment by a double faecal analysis. Thereby, no Anoplocephala spp. eggs were found in the faeces of 32 horses (97%). The single horse remaining positive for Anoplocephala spp. eggs did not completely swallow the anthelmintic gel and consequently, did not receive the appropriate dose of the drug.