Pharyngeal Pumping and Tissue-Specific Transgenic P-Glycoprotein Expression Influence Macrocyclic Lactone Susceptibility in Caenorhabditis elegans

Macrocyclic lactones (MLs) are widely used drugs to treat and prevent parasitic nematode infections. In many nematode species including a major pathogen of foals, Parascaris univalens, resistance against MLs is widespread, but the underlying resistance mechanisms and ML penetration routes into nematodes remain unknown. Here, we examined how the P-glycoprotein efflux pumps, candidate genes for ML resistance, can modulate drug susceptibility and investigated the role of active drug ingestion for ML susceptibility in the model nematode Caenorhabditis elegans. Wildtype or transgenic worms, modified to overexpress P. univalens PGP-9 (Pun-PGP-9) at the intestine or epidermis, were incubated with ivermectin or moxidectin in the presence (bacteria or serotonin) or absence (no specific stimulus) of pharyngeal pumping (PP). Active drug ingestion by PP was identified as an important factor for ivermectin susceptibility, while moxidectin susceptibility was only moderately affected. Intestinal Pun-PGP-9 expression elicited a protective effect against ivermectin and moxidectin only in the presence of PP stimulation. Conversely, epidermal Pun-PGP-9 expression protected against moxidectin regardless of PP and against ivermectin only in the absence of active drug ingestion. Our results demonstrate the role of active drug ingestion by nematodes for susceptibility and provide functional evidence for the contribution of P-glycoproteins to ML resistance in a tissue-specific manner

Requirement of Caprine Arthritis Encephalitis VirusvifGene forin VivoReplication

AbstractReplication ofvif-caprine arthritis encephalitis virus (CAEV) is highly attenuated in primary goat synovial membrane cells and blood-derived macrophages compared to the wild-type (wt) virus. We investigated the requirement for CAEV Vif forin vivoreplication and pathogenicity in goats by intra-articular injection of either infectious proviral DNA or viral supernatants. Wild-type CAEV DNA or virus inoculation induced persistent infection resulting in severe inflammatory arthritic lesions in the joints. We were unable to detect any sign of virus replication invif-CAEV DNA inoculated goats, whilevif-CAEV virus inoculation resulted in the seroconversion of the goats. However, virus isolation and RT-PCR analyses on blood-derived macrophage cultures remained negative throughout the experiment as well as in joint or lymphoid tissues taken at necropsy. No pathologic lesions could be observed in joint tissue sections examined at necropsy. Goats inoculated with thevif-virus demonstrated no protection against a pathogenic virus challenge. These results demonstrate that CAEV Vif is absolutely required for efficientin vivovirus replication and pathogenicity and provide additional evidence that live attenuated lentiviruses have to establish a persistent infection to induce efficient protective immunity

A Nuclear Localization of the Infectious Haematopoietic Necrosis Virus NV Protein Is Necessary for Optimal Viral Growth

The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the 32EGDL35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the 32EGDL35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of 32EGDL35 responsible for nuclear localization are important for the inhibitory activity of NV

A Virulent Bioluminescent and Fluorescent Dual-Reporter Marek's Disease Virus Unveils an Alternative Spreading Pathway in Addition to Cell-to-Cell Contact.

International audienceMarek's disease virus (MDV) is a growing threat for the poultry industry. Unfortunately, despite successful vaccination against the disease, MDV remains in circulation within vaccinated flocks, leading to the selection of increasingly virulent pathotypes. Detailed knowledge of the virus biology and the host-virus interaction is required to improve the vaccine efficiency. In the present study, I engineered an original, dual-reporter MDV to track and quantify virus replication in vitro and in vivo

Identification of first-stage dorsal-spined lungworm larvae of Tunisian barbary red deer: First report of Varestrongylus sagittatus and Elaphostrongylus cervi in Africa

International audienceBarbary red deer (Cervus elaphus barbarus) is a protected rare subspecies of red deer. The study of its Protostrongylidae fauna based only on sporadic necropsy of naturally dead animals is difficult. Therefore diagnosis of lungworms rely mainly on the identification of the first stage larvae (L1). The L1 of the different species are not readily diagnosed on morphological basis since much variation is recorded within and among dorsal-spined larvae belonging to various species. The aim of this study was to identify the dorsal-spined lungworm larvae of the Barbary red deer. A discriminant function was established, using the measurements of L1 lungworms recorded from red deer in the literature, then applied to identify 220 dorsal-spined larvae extracted from 25 Tunisian Barbary red deer fresh fecal samples. Also the ITS2 region of rDNA of four pools of larvae (n = 25-60) were amplified, sequenced and analyzed. Using discriminant analysis of morphological traits, Elaphostrongylus cervi and Varestongylus sagittatus were identified. Molecular identification confirmed the presence of E. cervi which is the most prevalent species. This study represents the first identification of V. sagittatus and E. cervi in North Africa

Bioluminescence Imaging of Live Infected Salmonids Reveals that the Fin Bases Are the Major Portal of Entry for Novirhabdovirus

Although Novirhabdovirus viruses, like the Infectious hematopietic necrosis virus (IHNV), have been extensively studied, limited knowledge exists on the route of IHNV entry during natural infection. A recombinant IHNV (rIHNV) expressing the Renilla luciferase gene was generated and used to infect trout. A noninvasive bioluminescence assay was developed so that virus replication in live fish could be followed hours after infection. We provide here evidence that the fin bases are the portal of entry into the fish. Confirmation was brought by the use of a nonpathogenic rIHNV, which was shown to persist in fins for 3 weeks postinfection

Monitoring anthelmintic resistance: from molecular markers to phenotypic assays

COST ActionInternational audienceThe control of parasitic nematode infections in humans, livestock and companion animals is critically dependent on anthelmintic treatments. However, the indiscriminate use of anthelmintic drugs has inevitably led to the selection of resistant parasites. In this context, diagnosis tools are of particular interest to monitor the resistance spreading and refine strategies for the control of resistant parasites. In this presentation, recent advances in automated phenotypic assays performed on larval stages of the parasite will be discussed. In addition, based on the example of the imidazothiazoles/tetrahydropyrimidines anthelmintics, the identification and functional validation of molecular markers associated with resistance will be presented, opening a discussion about the advantages and limitations of the use of C. elegans as a model for parasitic species

Nouvelles approches pour le contrôle des parasites résistants aux vermifuges

National audienceThe control of parasitic nematode infections in humans, livestock and companion animals is critically dependent on anthelmintic treatment. However, the indiscriminate use of anthelmintic drugs has inevitably led to the selection of resistant parasites. In this respect there is currently an urgent need to increase our knowledge on the mode of action of available anthelmintics as well as to identify novel targets for the development of next generation anthelmintic drugs.Les méthodes de luttes employées contre les nématodes parasites qui impactent la santé humaine ou animale sont remises en cause par l’émergence d’isolats capables de développer des résistances aux vermifuges (anthelminthiques). Dans ce contexte, la compréhension des mécanismes moléculaires à l’origine de ce phénomène ainsi que l’identification de nouvelles cibles pharmacologiques pour la découverte de nouveaux anthelminthiques sont indispensables au développement de stratégies de gestion du parasitisme efficaces et plus durables

Studies on glc-3, a potential target of Ivermectin in parasitic nematodes

International audienceThe free-living nematode Caenorhabditis elegans has been used for many years as an expression system for genes from parasitic species. We wished to further develop and improve this system by using CRISPR/Cas9 to delete specific genes from C. elegans and replace them with single copies of orthologous genes from the parasite, Haemonchus contortus. Initial experiments focussed on glc-3 which encodes a subunit of the glutamate-gated chloride channels, the target of the avermectin/milbemycin family of anthelmintics. We cloned the promoters from the glc-3 genes of both species and compared the expression patterns of mCherry under the control of both promoters. The C. elegans glc-3 promoter drove expression in a subset of head interneurons, as previously reported whereas the H. contortus promoter drove expression in a pharyngeal motoneuron, M4. We were able to generate heterozygous worms in which one copy of glc-3 was deleted, but we could never obtain homozygous knock-outs. Further investigation of the mRNAs encoded by glc-3 revealed a novel transcript, glc-3T, which encodes a severely truncated form of GLC-3. The presence of such truncated transcripts may explain the unexpected difficulties encountered in attempting to knock out ion channel genes in C. elegans