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

Tilapia Lake Virus Does Not Hemagglutinate Avian and Piscine Erythrocytes and NH4Cl Does Not Inhibit Viral Replication In Vitro

Tilapia lake virus (TiLV) is a negative-sense single-stranded RNA (-ssRNA) icosahedral virus classified to be the only member in the family Amnoonviridae. Although TiLV segment-1 shares homology with the influenza C virus PB1 and has four conserved motifs similar to influenza A, B, and C polymerases, it is unknown whether there are other properties shared between TiLV and orthomyxovirus. In the present study, we wanted to determine whether TiLV agglutinated avian and piscine erythrocytes, and whether its replication was inhibited by lysosomotropic agents, such as ammonium chloride (NH4Cl), as seen for orthomyxoviruses. Our findings showed that influenza virus strain A/Puerto Rico/8 (PR8) was able to hemagglutinate turkey (Meleagris gallopavo), Atlantic salmon (Salmo salar L), and Nile tilapia (Oreochromis niloticus) red blood cells (RBCs), while infectious salmon anemia virus (ISAV) only agglutinated Atlantic salmon, but not turkey or tilapia, RBCs. In contrast to PR8 and ISAV, TiLV did not agglutinate turkey, Atlantic salmon, or tilapia RBCs. qRT-PCR analysis showed that 30 mM NH4Cl, a basic lysosomotropic agent, neither inhibited nor enhanced TiLV replication in E-11 cells. There was no difference in viral quantities in the infected cells with or without NH4Cl treatment during virus adsorption or at 1, 2, and 3 h post-infection. Given that hemagglutinin proteins that bind RBCs also serve as ligands that bind host cells during virus entry leading to endocytosis in orthomyxoviruses, the data presented here suggest that TiLV may use mechanisms that are different from orthomyxoviruses for entry and replication in host cells. Therefore, future studies should seek to elucidate the mechanisms used by TiLV for entry into host cells and to determine its mode of replication in infected cells

Tilapia lake virus does not hemagglutinate avian and piscine erythrocytes and NH4Cl does not inhibit viral replication in vitro

Tilapia lake virus (TiLV) is a negative-sense single-stranded RNA (-ssRNA) icosahedral virus classified to be the only member in the family Amnoonviridae. Although TiLV segment-1 shares homology with the influenza C virus PB1 and has four conserved motifs similar to influenza A, B, and C polymerases, it is unknown whether there are other properties shared between TiLV and orthomyxovirus. In the present study, we wanted to determine whether TiLV agglutinated avian and piscine erythrocytes, and whether its replication was inhibited by lysosomotropic agents, such as ammonium chloride (NH4Cl), as seen for orthomyxoviruses. Our findings showed that influenza virus strain A/Puerto Rico/8 (PR8) was able to hemagglutinate turkey (Meleagris gallopavo), Atlantic salmon (Salmo salar L), and Nile tilapia (Oreochromis niloticus) red blood cells (RBCs), while infectious salmon anemia virus (ISAV) only agglutinated Atlantic salmon, but not turkey or tilapia, RBCs. In contrast to PR8 and ISAV, TiLV did not agglutinate turkey, Atlantic salmon, or tilapia RBCs. qRT-PCR analysis showed that 30 mM NH4Cl, a basic lysosomotropic agent, neither inhibited nor enhanced TiLV replication in E-11 cells. There was no difference in viral quantities in the infected cells with or without NH4Cl treatment during virus adsorption or at 1, 2, and 3 h post-infection. Given that hemagglutinin proteins that bind RBCs also serve as ligands that bind host cells during virus entry leading to endocytosis in orthomyxoviruses, the data presented here suggest that TiLV may use mechanisms that are different from orthomyxoviruses for entry and replication in host cells. Therefore, future studies should seek to elucidate the mechanisms used by TiLV for entry into host cells and to determine its mode of replication in infected cells

Determination of the presence of Babesia species in blood samples of cattle, camel and sheep in Iran by PCR

Babesia species are protozoan parasites that parasitize the erythrocytes of domestic animals and humans, causing anemia in the host. The parasites cause a zoonotic disease known as babesiosis. Polymerase chain reaction (PCR) has proven to be very sensitive for detecting Babesia in blood samples of affected animals, particular in ruminants. The purpose of the current study was to determine the presence of Babesia spp. in blood samples obtained from 2 cattle, camel and sheep in Iran. In addition, the study aimed at establishing a rapid, reliable, specific and sensitive molecular tool, the polymerase chain reaction (PCR), for the detection of Babesia spp. in ruminants. Blood samples were collected from 372 ruminants (155 cattle, 95 sheep and 122 camel) kept at the Livestock Experimental Station. The animals came from randomly selected herds located in the important livestock-production regions of Iran of Isfahan and Chaharmahal va Bakhtiary during December 2012 to March 2013. PCR was used to detect Babesia spp. in the blood samples whereby an amplified band size of 428 bp was considered positive for Babesia spp. The results show that 7.10% (n= 155), 6.56% (n= 122) and 0.00% (n= 95) of the blood samples from cattle, camel and sheep, respectively, were positive for Babesia spp.. The findings from this study revealed that there were Babesia spp. in blood taken from cattle and camel. To our knowledge, this is the first report to show the presence of Babesia spp. in blood samples of Iranian ruminants in Chaharmahal Va Bakhtiari and Isfahan provinces by PCR. These findings justify the importance of control of and eradication plans for Babesia spp. infection in cattle and camel in Iran. As diagnosis of low-level infections by the parasite is important for epidemiological studies, our findings support the power of PCR test for Babesia spp. detection in blood samples and could be easily used for routine diagnosis

Knowledge, attitudes and practices on rift valley fever among pastoral and agropastoral communities of Ngorongoro in the rift valley ecosystem, Tanzania, conducted in 2021/2022.

Epidemics of Rift Valley fever (RVF), a mosquito-borne zoonotic disease caused by RVF virus, have been linked to exceptionally heavy rainfall and widespread flooding. The disease is endemic in most African countries and pose a major global health risk. Given that the disease was reported in various districts of Tanzania, we hypothesized a lack of knowledge about RVF epidemiology among agropastoral and pastoral communities. The research took place in a period of 7 months, from July, 2021 to January, 2022. The aim of this study was to assess the knowledge, attitudes, and practices (KAP) among the agropastoral and pastoral communities of Ngorongoro district towards RVF. The survey employed a mixed method system, which included 3 focus groups (each comprised 12 individuals), 20 key informant interviews and administration of questionnaire (N = 352) in agropastoral and pastoral community members of Ngorongoro district. The relationship between demographic characteristics and communities' knowledge, attitudes, and practices regarding RVF was observed using a multiple logistic regression model. A total of 352 participants were interviewed, with the majority (67.61%) being male and 32.39% being female, majority (39.5%) attending primary school, and majority (58.2%) being pastoralists. The findings showed that only 36.1%, 38.64% and 16.19% of participants had good knowledge, positive attitude and good practices regarding RVF respectively. Significant demographic factors related with knowledge included: gender (OR = 1.9, CI = 1.03-3.56, P = 0.041), education levels (primary: OR = 3.97, CI = 2-8.16, P = 0.000; secondary: OR = 15.27, CI = 5.5-46.23, P = 0.000 and college: OR = 34. 23, CI = 5.4-67.22, P = 0.000), and locality (Pinyinyi: OR = 0.14, CI = 0.05-0.38, P = 0.000 and Sale: OR = 0.14, CI = 0.04-0.44, P = 0.001). Male participants showed significant positive attitude towards RVF compared to female (OR = 2.37, CI = 1.35-4.17, P = 0.003). Individuals with formal education showed a significant positive attitude toward RVF compared to informal (OR>1, P<0.05). Agropastoral members showed a significant negative attitude toward RVF compared to pastoralists (OR = 0.51, CI = 0.26-0.99, P = 0.048). The calculated RVF prevention practices values were insignificantly (P = 0.853) correlated with knowledge values. The significant correlation between knowledge and attitude, as well as attitude and practice were found (P<0.05). In general, the study revealed poor knowledge, negative attitude and poor practices of communities towards RVF. The lack of regular education programs to make the communities aware of the disease was implicated for these findings. This recommends that provision of health education should be a long-term practice among agropastoral and pastoral communities in order to prevent further RVF outbreaks in Tanzania

Exploring Pathogenic and Zoonotic Bacteria from Wild Rodents, Dogs, and Humans of the Ngorongoro District in Tanzania Using Metagenomics Next-Generation Sequencing

Globally, zoonoses have serious consequences due to their socioeconomic impacts. Ngorongoro District is home to a diverse range of wildlife and domestic animals, including rodents and dogs, which often coexist in close proximity with humans. The aim of the study was to identify the zoonotic bacteria present in wild rodents, domestic dogs, and humans using metagenomics next-generation sequencing technology. A cross-sectional study was conducted in 2022. This study used both Illumina and Oxford Nanopore sequencing technologies to identify bacteria in 530 blood samples collected from humans (n = 200), wild rodents (n = 230), and dogs (n = 100). Several zoonotic airborne/contagious bacteria, including Mycobacterium spp., Mycoplasma spp., Bordetella spp., and Legionella spp., were detected in wild rodents, domestic dogs, and humans. Arthropod-borne zoonotic bacteria such as Bartonella spp., Borrelia spp., and Rickettsia spp. were detected in all three hosts, while Orientia spp. was found in wild rodents and domestic dogs. Yersinia pestis, Streptobacillus spp. and Anaplasma spp. were found only in wild rodents. Other zoonotic bacteria found shared among wild rodents, domestic dogs, and humans are Leptospira spp., Brucella spp., and Salmonella spp. Generally, wild rodents had the highest prevalence of zoonotic bacterial species when compared to domestic dogs and humans. The detection of zoonotic bacteria in rodents, dogs, and humans supports the hypothesis that infections can spread between animals and humans sharing the same environment