Tilapia lake virus (TiLV) : utvikling av PCRbaserte diagnostiske metoder og studier av infeksjonsmekanismer

Tilapia lake virus (TiLV) is an emerging virus of wild and farmed tilapiines responsible for causing mortalities and significant economic losses to the aquaculture industry. Since its first report in Israel, the virus has been reported in four continents (Asia, Africa, South and North America) to cause mortalities ranging from the lower extreme of 5-20% and higher extremes of up to 90%. The infection status in many countries is not yet known and implementation of surveillance programs has been recommended. Lake Victoria was selected for TiLV surveillance because it contains a large number of tilapiine species of economic importance to the surrounding East African countries. Well-optimized tools for rapid detection and quantification of the virus are also needed. Moreover, the mode of virus uptake in cells and importance for replication is not known. Therefore, this thesis aimed at understanding the possible presence of TiLV in Lake Victoria in East Africa, to develop tools for detection and quantification of the virus and to shed light of virus uptake mechanisms in permissive cells in vitro. In this thesis, a standard RT-PCR and a quantitative real-time RT-PCR (qRT-PCR) were developed based on all the ten segments of TiLV. The standard PCR was used to screen Nile tilapia (Oreochromis niloticus) from Lake Victoria in Eastern Africa. The quantitative real-time RT-PCR was developed based on virus supernatant of known titre and against samples of unknown virus titre originating from infected TFC #10 cells and fish organs. The virus’ ability to hemagglutinate avian and piscine erythrocytes was assessed, and the modulation of ammonium chloride on uptake and replication of TiLV in E-11 cells was studied. The findings reported in study I showed that primers designed from segment two of TiLV for a standard RT-PCR were the best at detecting TiLV in the infected cells and Nile tilapia organs. TiLV genome was detected in 28 Nile tilapias (14. 66%, N = 191) in which 17.78% (N=45) were from wild fish and 13.70% (N=146) from farmed fish (cage farming). The genomes of circulating TiLV in Nile tilapia from Lake Victoria were identical to those detected from Israel (98%), Ecuador (98%), Thailand (96%), Peru (96%) and USA (97%). TiLV was not grown from infected fish and thus its ability to cause disease in Nile tilapia was not studied. Therefore, I am recommending further studies to fulfil Koch’s postulates. The data reported in study II showed that the developed and optimized quantitative real-time RT-PCR detected TiLV in virus supernatants of known titre and in organs of unknown titre from infected Nile tilapia. The developed assay is sensitive and specific to TiLV with all the primers efficiency being within the range of 95-105%, except primers targeting segment ten that gave an efficiency of 93%. The intra- and inter-assay coefficient of variation ranged between 0.00% ~ 2.63% and 0.00% ~ 5.92%, respectively, which is within the recommended range (below 5%) for an assay to be repeatable and reproducible. The detection limit of 2 TCID50/ml was found for primers targeting segments 1, 2, 3, 4 and 9 while lower detection limit of 20 TCID50/ml was found for primers targeting segment 5, 6, 7, 8 and 10. Overall primers targeting segment 3 had the highest detection limit and primers targeting segment 7 had the lowest detection limit. Interestingly, despite the two primer sets (for segment 3 and 7) having different TiLV detection limits they had an equal amplification efficiency of 98%. Therefore, primer optimization for qRT-PCR is important to optimize assay sensitivity. The study reported in paper III was directed at understanding the hemagglutination property of TiLV using avian and piscine erythrocytes, and the infection mechanisms in E-11 cells. TiLV did not hemagglutinate erythrocytes from any of the species tested indicating that the virus lack hemagglutinin. Further, the study has shown that ammonium chloride does not affect the replication of TiLV in E-11 cells indicating that the virus is not using the endocytic pathway during internalization. Taken together, the two observations suggest that, TiLV is not taken up by receptor-mediated endocytosis during internalization into E-11 cells. Thus, further studies are needed to unravel the uptake mechanism(s), which is the important information for controlling the virus by antiviral agents or immunoprophylaxis.Tilapia lake virus (TiLV) er et fremvoksende virus som infiserer ville arter og oppdrettsarter av tilapia og gir dødelighet og betydelige økonomiske tap i oppdrett. Siden den første beskrivelsen av sykdommen fra Israel, har viruset blitt påvist på fire kontinenter (Asia, Afrika, Sør- og Nord-Amerika) og gir dødelighet fra 5-20% opp mot 90%. Forekomst av viruset er ikke kjent i mange av de landene som driver tilapiaoppdrett, og det er nødvendig å etablere bedre overvåknings- og kontrollprogrammer i disse landene. I denne studien ble Lake Victoria valgt for TiLV-screening fordi den inneholder et stort antall tilapia-arter som er av økonomisk betydning for de omkringliggende østafrikanske landene. Det er også behov for optimaliserte metoder for rask deteksjon og kvantifisering av viruset. Hvordan viruset tas opp i cellene og hvordan det replikerer er ikke kjent. I denne avhandlingen ble det gjennomført studier for å forstå forekomst av TiLV i Lake Victoria i Øst-Afrika, det ble etablert verktøy for påvisning og kvantifisering av viruset med molekylærbiologiske metoder, og det ble gjennomført studier for å bedre forstå opptaksmekanismer i celler under infeksjonen. Det ble utviklet en standard RT-PCR og en kvantitativ RT-PCR (qRT-PCR) basert på alle de ti segmentene til viruset. Standard PCR ble brukt til å undersøke Nile tilapia (Oreochromis niloticus) fra Victoriasjøen. Den kvantitative RT-PCR metoden ble testet mot kjent og ukjent virustiter fra henholdsvis infiserte TFC# 10 celler og organer fra infiserte fisk. Hemagglutinering av røde blodlegemer fra hønsefugl og fisk ble også undersøkt, samt effekten av ammoniumklorid på replikasjonen av TiLV i E-11-celler. Resultatene i studie I viste at primere designet fra segment 2 benyttet for påvisning med standard RT-PCR var best egnet til å påvise TiLV i infiserte cellene og organer fra infisert fisk. TiLV fra Victoriasjøen ble påvist i 28 fisk (14. 66%, N = 191) hvor 17,78% (N = 45) var fra villfisk og 13,70% (N = 146) fra oppdrettsfisk. De sekvensene som ble påvist i Nil-tilapia fra Victoriasjøen var tilnærmet identiske med de som ble påvist i Israel (98%), Ecuador (98%), Thailand (96%), Peru (96%) og USA (97%). TiLV ble ikke dyrket eller testet med tanke på virulens/evne til å forårsake sykdom i Nil-tilapia, og derfor anbefaler jeg videre studier for å oppfylle Kochs postulater. I studie II ble det etablert en ny og optimalisert kvantitativ RT-PCR metode for påvisning av TiLV genom i prøver fra infiserte celler med kjent titer (mengde virus) og fra organer fra infisert Nil-tilapia uten kjent titer. Metoden som ble utviklet er sensitiv og spesifikk for TiLV, og primer-effektiviteten var innenfor et akseptabelt område, 95-105%, bortsett fra primere rettet mot segment 10 (93%). Variasjonskoeffisienten for intra- og inter-analyse varierte mellom henholdsvis 0,00% ~ 2,63% og 0,00% ~ 5,92%, som er innenfor det anbefalte området (under 5%) for at en analyse skal anses som repeterbar og reproduserbar. Sensitiviteten til metoden var 2 TCID50/ml, og primere spesifikke for segmentene 1, 2, 3, 4 og 9 gav samme resultat. En nedre deteksjonsgrense på 20 TCID50/ml ble påvist for primere rettet mot segment 5, 6, 7, 8 og 10. Primere spesifikke for mot segment 3 gav høyest sensitivitet og primere segment 7 den laveste. Begge primersettene hadde en effektivitet på 98%. I artikkel III var målsettingen å forstå hemagglutinasjonsegenskapen til TiLV ved bruk av erythrocytter fra hønsefugl, tilapia og laks, samt betydningen av endocytose i tidlig fase av infeksjonen i E-11-celler. TiLV gir ikke hemagglutinering av erytrocytter fra noen av de testede artene, noe som indikerer at viruset mangler hemagglutinin. Studien har også vist at ammoniumklorid ikke påvirker, dvs. hemmer eller forsinker replikasjonen av TiLV i E-11-celler, noe som indikerer at viruset ikke tas opp ved endocytose. Samlet antyder de to observasjonene at TiLV ikke blir tatt opp av reseptormediert endocytose i E-11-celler. De gjennomførte studiene viser at det er behov for å forstå opptaksmekanismen(e) til virus, som er en viktig informasjonen for å kontrollere virusinfeksjonen med anti-virale midler eller vaksiner

Describing new species of Cryptosporidium in fish

The protozoan parasite Cryptosporidium (class Gregarinomorphea, subclass Cryptogregaria) causes a range of symptoms in humans and clinical signs in animals from asymptomatic to severe diarrhoea and death. However, relatively little information is available regarding the taxonomy, zoonotic potential and host relationships of Cryptosporidium in fish. Previous studies have indicated that extensive genetic diversity exists with piscine Cryptosporidium species and genotypes. The present study screened fish from two sources in Perth, Western Australia; Water Garden Life Fish Farm (n=233) and Vebas Aquarium (n=234) for Cryptosporidium. Intestinal and gastric tissue was dissected out and screened by PCR and Sanger sequencing using Cryptosporidium specific primers that amplify DNA at the 18S and actin loci. Samples that were positive by PCR were also screened by histology. The overall prevalence of Cryptosporidium was 4.3% (20/467, 95% CI: 2.6-6.5). Phylogenetic analyses of 18S sequences identified C. huwi (n=11), piscine genotype 2 (n=3), piscine genotype 4 (n=1) and piscine genotype 7 (n=5). In addition, ten novel sequences most genetically similar to species from the genus Goussia and a sequence from the non-parasitic alveolate Colpodella were identified. Sequences amplified at the actin locus were C. huwi (n=7), piscine genotype 2 (n=1), piscine genotype 7 (n=1) and one novel Cryptosporidium sequence. Piscine genotype 2 was most closely related to piscine genotype 4 (4.1% genetic distance) and exhibited 11.1-11.9%, 15.3% and 22.3% genetic distances from C. molnari, C. huwi and C. scophthtalmi, respectively. At the actin locus, piscine genotype 2 was again most closely related to piscine genotype 4 (7.2% genetic distance) and exhibited genetic distances ranging from 18.1% (C. molnari) to 20% (C. huwi) and 26.1% for C. scophthalmi, respectively, and 20.7%- 32% genetic distance from all other species. Phylogenetic analysis of concatenated 18S and actin sequences showed that piscine genotype 2 exhibited 14% (C. molnari) to 24.6% (C. canis) genetic distance from all other Cryptosporidium spp. Using concatenated sequences, piscine genotype 7 was most closely related to C. huwi at a genetic distance of 7.5% and exhibited 13.4% (C. molnari) to 23.1% (C. scophthalmi) genetic distances from other piscine Cryptosporidium species, with 17.9% (C. testudinis) to 22.6% (C. canis) genetic distance from all non-piscine Cryptosporidium species. Piscine genotype 2 exhibited 14.6% genetic distance from piscine genotype 7. These genetic distances at two separate loci confirm the genetic distinctness of piscine genotype 2 and piscine genotype 7 and indicate that they are likely novel species. Additionally, 10/467 (2.1%, 95% CI; 1.0-3.9) samples that were positive at the 18S locus, produced sequences most genetically similar to species from the genus Goussia, subclass Conoidasida, nine were novel sequences and were compared at the 18S locus to established species of Goussia and genetic distances between 1.9% and 14.8% were identified, adding to the diversity of this genus. Furthermore, Schyzocotyle acheilognathi, the invasive Asian fish tapeworm, was identified (n=2) by morphology infecting goldfish from a local fish farm. This is only the second report of S. acheilognathi in Western Australia as it was first discovered in 2018 by a Murdoch researcher in feral goldfish from a Lake in Joondalup. Analysis at additional loci or whole genome sequencing will shed more light on the evolutionary relationships between Cryptosporidium species, while next generation sequencing would elucidate the prevalence of mixed infections of Cryptosporidium in fish. The genetic data produced by the present study describes two piscine genotypes of Cryptosporidium (that are likely valid species) in detail and provides new genetic data on the diversity of Goussia spp. Keywords: Cryptosporidium, 18S, actin, Schyzocotyle acheilognathi, Goussi

Harnessing the Power of Genomics to Secure the Future of Seafood

Best use of scientific knowledge is required to maintain the fundamental role of seafood in human nutrition. While it is acknowledged that genomic-based methods allow the collection of powerful data, their value to inform fisheries management, aquaculture, and biosecurity applications remains underestimated. We review genomic applications of relevance to the sustainable management of seafood resources, illustrate the benefits of, and identify barriers to their integration. We conclude that the value of genomic information towards securing the future of seafood does not need to be further demonstrated. Instead, we need immediate efforts to remove structural roadblocks and focus on ways that support integration of genomic-informed methods into management and production practices. We propose solutions to pave the way forward.Peer reviewe

Deteksjon av antistoffer i atlantisk laks med et kulebasert multipleks immunoassay

Viral diseases are among of the main challenges in aquaculture in Norway today. Heart- and skeletal muscle inflammation, caused by Piscine orthoreovirus (PRV), and pancreas disease (PD), caused by Salmonid alphavirus (SAV), both cause huge losses of farmed Atlantic salmon. Vaccination is a possible solution that is already contributing to the control of bacterial diseases. Unfortunately, making effective vaccines against viral diseases has been challenging and information about which immune mechanisms that are involved in protection is lacking. Antibodies can mediate complete protection against some diseases, and antibody levels after vaccination can correlate with protection, even if the protection is mediated by other mechanisms. In addition, antibody detection can be used in disease surveillance to determine if a fish population is or has been infected with a virus. Unfortunately, measuring antibody levels in salmon is not straightforward. Such measurements have not been widely used, and often show high levels of background binding. An important reason for this is that fish antibodies are of the IgM type. IgM is less specific than IgG, the dominating antibody in mammals. Therefore, other methods, like PCR and histology, are more used in diagnostics in Atlantic salmon. In this work, we have for the first time used an assay based on microscopic magnetic beads conjugated with antigen to measure antibody levels in Atlantic salmon. This method has a high sensitivity and can be used to measure antibodies against several proteins simultaneously in the same sample. By using this method, we have detected antibodies against PRV and SAV. In paper I and II, plasma from two PRV challenge trials was used to detect antibodies against the PRV proteins μ1c, μNS and σ1. We also detected antibodies against PRV1- σ1 in plasma from PRV-3-infected rainbow trout. There have been no previous publications detecting antibodies against PRV. The peak antibody level coincided with decreased pathology in the heart. In addition to showing virus-specific antibodies, our results show an increase in non-specific antibodies in PRV-infected salmon. This unspecific binding, but not the virus-specific binding, was decreased by heat treatment of samples. The nonspecific antibodies could be so-called polyreactive antibodies. Polyreactive antibodies could be crucial in protecting the salmon against infections before the adaptive immune system has had time to react, but their function, as well as the function of virus-specific antibodies during a PRV infection is unclear. In paper III, we detected antibodies against whole SAV particles disrupted with Triton-X. The antibody binding increased from between week three and week six after the introduction of SAVinjected shedder fish. The SAV particles also worked well with little background binding when analyzing plasma from a PD outbreak in the field. Antibodies were detected in most fish from four weeks after the start of the outbreak, and the antibody level stayed elevated until the last sampling point at 15 weeks. At this time point, most samples were negative for virus when analyzed with RTqPCR, showing that serology has a longer window of detection compared to detection of viral RNA by RT-qPCR. These results show that both virus-specific and non-specific antibodies can be produced after infection in Atlantic salmon. The method used is well suited for antibody detection in salmon but can be complicated by the presence of non-specific antibodies. It is therefore crucial to optimize the antigens for detection of specific antibodies.Virussykdommer er en av de store utfordringene i oppdrettsnæringa i Norge i dag. Hjerte- og skjelettmuskelbetennelse, forårsaket av Piscine orthoreovirus (PRV), og pankreas sykdom (PD), forårsaket av Salmonid alphavirus (SAV), fører begge til store tap av atlantisk laks i oppdrett. Vaksinering er en mulig løsning som allerede har bidratt til god kontroll på bakterielle infeksjoner hos laks. Dessverre er det utfordrende å lage vaksiner som virker godt mot virusinfeksjoner. Informasjon om hvilke immunfunksjoner som er involvert i beskyttelse er også mangelfull. Antistoffer kan gi full beskyttelse mot noen sykdommer, og antistoffnivået etter vaksinering kan korrelere med beskyttelsen, også om beskyttelsen skyldes andre immunmekanismer. I tillegg kan antistoffdeteksjon brukes i sykdomsovervåkning for å finne ut om fiskepopulasjoner er eller har vært infisert med et virus. Dessverre er det ikke rett fram å måle antistoffnivå hos laks. Antistoffmåling er lite brukt og det er ofte mye bakgrunnsbinding. En viktig grunn til dette er at fiskens antistoff er av typen IgM, som er mindre spesifikk enn IgG i pattedyr. Derfor er andre metoder, som PCR og histologi, mest brukt til påvisning av virussykdom hos laks. I dette arbeidet har vi for første gang brukt et assay basert på mikroskopiske magnetiske kuler konjugert med antigen til å måle antistoffnivå i laks. Denne metoden har høy sensitivitet og gir muligheten til å måle antistoffer mot mange proteiner i samme prøve samtidig

Cryptosporidium in fish: Morphological and molecular characterisation

Cryptosporidium is an Apicomplexa protozoan parasite that causes gastrointestinal illness in a wide range of vertebrate hosts, including humans. Little is known of the epidemiology of Cryptosporidium in fish. This study investigated the prevalence of Cryptosporidium in goldfish (Carassius auratus) (n=216) and mullet (Mugil cephalus) (n=13). Goldfish can be host to a range of Cryptosporidium sp. and research has shown that mullet has been host to genotype 3, therefore sampling from these breeds of fish could provide further characterisation. The fish were acquired from three sources in Metropolitan Perth, Western Australia; Vebas Aquarium (n=16), Water Garden Life fish farm (n=200) and a local bait shop in Fremantle (n=13). Intestines and stomachs were dissected and half kept for histology and the remaining half used for DNA extraction. All samples were initially screened at the 18S locus by quantitative PCR (qPCR) and positives further analysed by nested PCR and sequencing at the 18S and actin loci. Further subtyping was conducted on human-infectious species at the glycoprotein 60 (gp60) locus. The overall prevalence by qPCR was 30.1% (69/229) (CI 24.2-36.1). Of these only 34 samples amplified at the 18S locus and 23 clean sequences were obtained, with the remaining 11 sequences exhibiting mixed chromatograms. At the actin locus, 6 samples were successfully amplified and 3 clean chromatograms were obtained. Sequencing and phylogenetic analysis at the 18S locus identified C. parvum (n=2), C. hominis (n=10) and a novel species (n=11), which was identical to a novel genotype identified in a single isolate from a goldfish from a previous Honours project. Phylogenetic analysis confirmed that this novel genotype was genetically distinct and most closely related to C. scopthalmi (10.4% genetic distance). At the actin locus, all three isolates sequenced belonged to the novel genotype, which again grouped with C. scopthalmi and exhibited 14.1% genetic distance at this locus. Subtyping of C. hominis and C. parvum isolates at the gp60 locus was successful for 3 C. hominis isolates and all were typed as 1bA10G2, which is the main subtype involved in human outbreaks of cryptosporidiosis. Unfortunately, the parasite could not be identified in histological sections, which may be due to the patchy distribution of Cryptosporidium infections and rapid tissue autolysis. This is the first characterisation of the novel genotype at the actin locus and provides further support for its species status. The identification of human-infectious species in these fish is of public health concern as it may enable control of cryptosporidiosis outbreaks. Future research should focus on analysis of a wider range of fish species as well as clinical signs and histology to better understand the host range and pathogenicity of the novel genotype and the prevalence of human-infectious species in fish

A Review of Some Parasite Diseases of African Fish Gut Lumen Protozoa, Coccidioses, Cryptosporidium Infections

Abstract: A review of some parasite diseases of African fish: Gut lumen protozoa, coccidioses, cryptosporidium infections, haemoprotozoa, haemosporidia was carried out from some existing literature to provide fish culturists and the public sector information on some challenges faced in culture fisheries. The Description, taxonomy, diagnosis Life cycles, biology, Epizootiology and control, Protozoans of the gut lumen, Coccidioses, Cryptosporidium Infections, Haemoprotozoa (Haemoflagellates), Haemosporidia -Dactylosoma and Hemogregarines are reviewed in this study

Bacterial diseases of tilapia, their zoonotic potential and risk of antimicrobial resistance

Tilapia culture is an important source of income and nutrition to many rural families. Since 2000, the production of tilapia increased and reached domestic and global markets. Major farmed species is Nile tilapia (Oreochromis niloticus), in earthen ponds and cage cultures. Intensification contributed to global tilapia disease outbreaks, with bacterial infections causing mortalities and morbidities, threatening sustainable production. At tilapia farms, high nutrient concentrations, water temperature and fish densities enhance bacterial growth including virulent bacterial clones and potential zoonotic bacteria. Global warming favours this. This review respectively provides a comprehensive overview of the most common and emerging bacterial pathogens, diseases, clinical presentations and diagnostics of tilapia, including bacteria and diseases with zoonotic potential. First, common bacterial disease outbreaks, including streptococcosis, motile Aeromonas septicaemia, francisellosis, columnaris disease and vibriosis are described. Then, information on emerging bacterial infections of concern for tilapia, like edwardsiellosis through Edwardsiella ictaluri and E. tarda, as well as Aeromonas schubertii is provided. Reports of infectious bacterial tilapia disease outbreaks from other bacteria, including Lactococcus garvieae, Aerococcus viridans, Pseudomonas spp., Mycobacterium marinum and Chlamydia spp., and others are reviewed. Furthermore, bacteria with zoonotic potential, like Streptococcus agalactiae ST283, S. iniae, Aeromonas sp., E. tarda, Vibrio vulnificus pathovar (pv) piscis and M. marinum are included in the review, to provide the most current overview of the disease risks affecting production and post-harvest stages. Additionally, the status and risks of antimicrobial resistance in bacteria from tilapia and other cultured fish through imprudent use of antibiotics, and its future at a global level are provided