Identification of critical enzymes in the salmon louse chitin synthesis pathway as revealed by RNA interference-mediated abrogation of infectivity

Treatment of infestation by the ectoparasite Lepeophtheirus salmonis relies on a small number of chemotherapeutant treatments that currently meet with limited success. Drugs targeting chitin synthesis have been largely successful against terrestrial parasites where the pathway is well characterised. However, a comparable approach against salmon lice has been, until recently, less successful, likely due to a poor understanding of the chitin synthesis pathway. Post-transcriptional silencing of genes by RNA interference (RNAi) is a powerful method for evaluation of protein function in non-model organisms and has been successfully applied to the salmon louse. In the present study, putative genes coding for enzymes involved in L. salmonis chitin synthesis were characterised after knockdown by RNAi. Nauplii I stage L. salmonis were exposed to double-stranded (ds) RNA specific for several putative non-redundant points in the pathway: glutamine: fructose-6-phosphate aminotransferase (LsGFAT), UDP-N-acetylglucosamine pyrophosphorylase (LsUAP), N-acetylglucosamine phosphate mutase (LsAGM), chitin synthase 1 (LsCHS1), and chitin synthase 2 (LsCHS2). Additionally, we targeted three putative chitin deacetylases (LsCDA4557, 5169 and 5956) by knockdown. Successful knockdown was determined after moulting to the copepodite stage by real-time quantitative PCR (RT-qPCR), while infectivity potential (the number of attached chalimus II compared with the initial number of larvae in the system) was measured after exposure to Atlantic salmon and subsequent development on their host. Compared with controls, infectivity potential was not compromised in dsAGM, dsCHS2, dsCDA4557, or dsCDA5169 groups. In contrast, there was a significant effect in the dsUAP-treated group. However, of most interest was the treatment with dsGFAT, dsCHS1, dsCHS1+2, and dsCDA5956, which resulted in complete abrogation of infectivity, despite apparent compensatory mechanisms in the chitin synthesis pathway as detected by qPCR. There appeared to be a common phenotypic effect in these groups, characterised by significant aberrations in appendage morphology and an inability to swim. Ultrastructurally, dsGFAT showed a significantly distorted procuticle without distinct exo/endocuticle and intermittent electron dense (i.e. chitin) inclusions, and together with dsUAP and dsCHS1, indicated delayed entry to the pre-moult phase.publishedVersio

Salmon louse labial gland enzymes: implications for host settlement and immune modulation

Salmon louse (Lepeophtheirus salmonis) is a skin- and blood-feeding ectoparasite, infesting salmonids. While feeding, labial gland proteins from the salmon louse may be deposited on the Atlantic salmon (Salmo salar) skin. Previously characterized labial gland proteins are involved in anti-coagulation and may contribute to inhibiting Atlantic salmon from mounting a sufficient immune response against the ectoparasite. As labial gland proteins seem to be important in the host–parasite interaction, we have, therefore, identified and characterized ten enzymes localized to the labial gland. They are a large group of astacins named L. salmonis labial gland astacin 1–8 (LsLGA 1–8), one serine protease named L. salmonis labial gland serine protease 1 (LsLGSP1), and one apyrase named L. salmonis labial gland apyrase 1 (LsLGAp1). Protein domain predictions showed that LsLGA proteins all have N-terminal ShK domains, which may bind to potassium channels targeting the astacins to its substrate. LsLGA1 and -4 are, in addition, expressed in another gland type, whose secrete also meets the host–parasite interface. This suggests that LsLGA proteins may have an anti-microbial function and may prevent secondary infections in the wounds. LsLGAp1 is predicted to hydrolyze ATP or AMP and is, thereby, suggested to have an immune dampening function. In a knockdown study targeting LsLGSP1, a significant increase in IL-8 and MMP13 at the skin infestation site was seen under LsLGSP1 knockdown salmon louse compared to the control, suggesting that LsLGSP1 may have an anti-inflammatory effect. Moreover, most of the identified labial gland proteins are expressed in mature copepodids prior to host settlement, are not regulated by starvation, and are expressed at similar or higher levels in lice infesting the salmon louse-resistant pink salmon (Oncorhynchus gorbuscha). This study, thereby, emphasizes the importance of labial gland proteins for host settlement and their immune dampening function. This work can further contribute to anti-salmon louse treatment such as vaccine development, functional feed, or gene-edited salmon louse-resistant Atlantic salmon

Dystoni - Klassifisering, utredning og genetikkens rolle

Objective: The aim of this review is to investigate whether or not the current classification systems of dystonia is appropriate, and if not to present suitable alternatives. Additional goals are to investigate if and how the different sub groups of dystonia can be separated clinically, when to suspect a genetic cause and identify when further investigations are indicated. Finally we want to illuminate the importance and the possibilities achieved by understanding the genetic background of the disease. Background: Dystonia is a rare syndrome characterised by involuntary muscle contractions, producing twisting and repetitive movements in a predictable pattern, or abnormal postures. In many cases an underlying genetic cause is identified. Dystonia is commonly classified by age of onset, distribution of symptoms and aetiology. Method: Literature searches in McMaster Plus, PubMed, Ovid, Cochrane, Google Scholar and Medline performed mainly in the period between January 2012 and August 2012. Only articles written in English and Norwegian are included. Searches were based on relevant key words concentrating on the correlation between dystonia and genetics. A total of 40 articles were reviewed. Result and Conclusion: Are the current classification systems appropriate? The classification by age into early onset ( 26 years) dystonia is appropriate from a clinical point of view. The categorisation by means of distribution into focal, segmental and generalized dystonia is also useful for the clinician. The genetic classification, i.e. various types of DYTs, should be reserved for specialists in genetics. Is it possible to separate the different sub groups of dystonia clinically? In many cases of dystonia, an investigation based on age of onset, symptoms and the development of the disease is sufficient for the diagnosis. Genetic testing is sometimes necessary in finding an exact diagnosis, but should be reserved for specialists in genetics. Specific guidance for various clinical situations is presented in the article. Importance of a genetic diagnosis An understanding of genetics is crucial to drive the field forward. Specialized centers should be responsible for this.

RNAi-mediated treatment of two vertically transmitted rhabdovirus infecting the salmon louse (Lepeophtheirus salmonis)

Abstract Rhabdoviruses are a family of enveloped negative-sense single-stranded RNA viruses infecting a variety of hosts. Recently, two vertically transmitted salmon louse (Lepeophtheirus salmonis) rhabdoviruses (LsRV) have been identified. The prevalence of these viruses was measured along the Norwegian coast and found to be close to 100%, and with the present lack of suitable cell lines to propagate these viruses, it is challenging to obtain material to study their host impact and infection routes. Thus, virus free lice strains were established from virus infected lice carrying one or both LsRVs by treating them with N protein dsRNA twice during development. The viral replication of the N protein was specifically down-regulated following introduction of virus-specific dsRNA, and virus-free lice strains were maintained for several generations. A preliminary study on infection routes suggested that the LsRV-No9 is maternally transmitted, and that the virus transmits from males to females horizontally. The ability to produce virus free strains allows for further studies on transmission modes and how these viruses influences on the L.salmonis interaction with its salmonid host. Moreover, this study provides a general fundament for future studies on how vertically transmitted rhabdoviruses influence the biology of their arthropod hosts

RNAi-mediated treatment of two vertically transmitted rhabdovirus infecting the salmon louse (Lepeophtheirus salmonis)

Rhabdoviruses are a family of enveloped negative-sense single-stranded RNA viruses infecting a variety of hosts. Recently, two vertically transmitted salmon louse (Lepeophtheirus salmonis) rhabdoviruses (LsRV) have been identified. The prevalence of these viruses was measured along the Norwegian coast and found to be close to 100%, and with the present lack of suitable cell lines to propagate these viruses, it is challenging to obtain material to study their host impact and infection routes. Thus, virus free lice strains were established from virus infected lice carrying one or both LsRVs by treating them with N protein dsRNA twice during development. The viral replication of the N protein was specifically down-regulated following introduction of virus-specific dsRNA, and virus-free lice strains were maintained for several generations. A preliminary study on infection routes suggested that the LsRV-No9 is maternally transmitted, and that the virus transmits from males to females horizontally. The ability to produce virus free strains allows for further studies on transmission modes and how these viruses influences on the L.salmonis interaction with its salmonid host. Moreover, this study provides a general fundament for future studies on how vertically transmitted rhabdoviruses influence the biology of their arthropod hosts

Mindre gass, færre eksplosjoner : Implementering av CO2-insufflasjon ved koloskopi ved gastroenterologisk avdeling på Arendal sykehus

Problemstilling Det gjennomføres omtrent 50 000 koloskopier i Norge årlig. Tradisjonelt har man brukt romluft til å utvide tarmlumen, men de siste årene har det vist seg at insufflering med kun CO2 gir færre pasientplager. Likevel utføres fortsatt koloskopiene ved mange norske sykehus med romluft. Vi kommer i denne oppgaven med et forslag til hvordan implementering av CO2-insufflering kan gjennomføres ved Arendal sykehus. Kunnskapsgrunnlag Når koloskopiene utføres med CO2-insufflering, opplever pasientene mindre smerter både under og etter undersøkelsen, og er mindre plaget av flatus, enn når det benyttes romluft. Det er ikke vist noen signifikant forskjell i komplikasjoner. I 2012 ble det publisert EU-retningslinjer der bruk av CO2 anbefales som førstevalg ved koloskopi. Dagens praksis og utfordringer Over halvparten av koloskopiene i Norge utføres med romluft, og ikke CO2. I Norge brukes det i tillegg mindre sedasjon og smertestillende under koloskopi enn i mange andre land. Det skulle tilsi at behovet for å bruke den minst smertefulle undersøkelsesmetoden er enda større her i landet. Ved Arendal sykehus er det innført CO2-insufflering utelukkende ved det endoskopilaboratoriet som brukes i et screeningsprosjekt for cancer coli, mens de tre ordinære skopilaboratoriene benytter romluft. Utfordringene til implementering av CO2 er å overbevise de budsjettansvarlige om at det er nødvendig å bevilge midler til innkjøp av utstyr, og å lære opp de ansatte om fordelene til CO2-insufflering, slik at de følger nødvendige rutinene for at CO2 skal bli benyttet ved så mange koloskopier som mulig. Prosess, ledelse og organisering Dette er et tofaset kvalitetsforbedringsprosjekt, der fase 1 er innføring av CO2-insufflering, og fase 2 er sikring av korrekt og hyppig bruk av de nye insufflatorene. Som utgangspunkt for gjennomføring av prosjektet, har vi brukt Langley og Nolans metode for kvalitetsforbedring med individuelle PUKK-sirkler for hver fase. Diskusjon og konklusjon Vi tror at en hovedutfordring ligger i å få frem behovet for en slik kvalitetsforbedring blant de ansatte, både ved laboratoriene og blant dem som har ansvar for bevilgning av midler. Det blir viktig å få frem at plager under og etter koloskopi er et problem, selv om helsepersonellet ikke legger så mye merke til det, og at disse plagene enkelt kan reduseres. Vi konkluderer med at dette prosjektet bør gjennomføres for å sikre norske pasienter et best mulig helsetilbud. Totalkostnaden er ikke større enn at fordelene med CO2-insufflering bør veie tyngre enn det økonomiske aspektet

Identification of critical enzymes in the salmon louse chitin synthesis pathway as revealed by RNA interference-mediated abrogation of infectivity

Treatment of infestation by the ectoparasite Lepeophtheirus salmonis relies on a small number of chemotherapeutant treatments that currently meet with limited success. Drugs targeting chitin synthesis have been largely successful against terrestrial parasites where the pathway is well characterised. However, a comparable approach against salmon lice has been, until recently, less successful, likely due to a poor understanding of the chitin synthesis pathway. Post-transcriptional silencing of genes by RNA interference (RNAi) is a powerful method for evaluation of protein function in non-model organisms and has been successfully applied to the salmon louse. In the present study, putative genes coding for enzymes involved in L. salmonis chitin synthesis were characterised after knockdown by RNAi. Nauplii I stage L. salmonis were exposed to double-stranded (ds) RNA specific for several putative non-redundant points in the pathway: glutamine: fructose-6-phosphate aminotransferase (LsGFAT), UDP-N-acetylglucosamine pyrophosphorylase (LsUAP), N-acetylglucosamine phosphate mutase (LsAGM), chitin synthase 1 (LsCHS1), and chitin synthase 2 (LsCHS2). Additionally, we targeted three putative chitin deacetylases (LsCDA4557, 5169 and 5956) by knockdown. Successful knockdown was determined after moulting to the copepodite stage by real-time quantitative PCR (RT-qPCR), while infectivity potential (the number of attached chalimus II compared with the initial number of larvae in the system) was measured after exposure to Atlantic salmon and subsequent development on their host. Compared with controls, infectivity potential was not compromised in dsAGM, dsCHS2, dsCDA4557, or dsCDA5169 groups. In contrast, there was a significant effect in the dsUAP-treated group. However, of most interest was the treatment with dsGFAT, dsCHS1, dsCHS1+2, and dsCDA5956, which resulted in complete abrogation of infectivity, despite apparent compensatory mechanisms in the chitin synthesis pathway as detected by qPCR. There appeared to be a common phenotypic effect in these groups, characterised by significant aberrations in appendage morphology and an inability to swim. Ultrastructurally, dsGFAT showed a significantly distorted procuticle without distinct exo/endocuticle and intermittent electron dense (i.e. chitin) inclusions, and together with dsUAP and dsCHS1, indicated delayed entry to the pre-moult phase