Krog, Jesper Schak

I denne PhD-afhandling undersøges tilstedeværelsen af hepatitis E virus I Danmark. Der findes fire forskellige genotyper af HEV sammen med et stadigt øgende antal artsspecifikke varianter. Ikkezoonotiske genotyper af HEV findes i udviklings lande med lavere sanitære forhold, hvor de forårsager epidemier ved fækal kontaminering af vandforsyninger. De zoonotiske genotyper er fundet i grise fra hele verden inklusiv Danmark. Mennesker har for det meste et asymptomatisk sygdomsforløb, men det kan udvikle sig til en klinisk leverbetændelse der er akut og selvbegrænsende. Klinisk sygdom er ikke beskrevet for grise, men små læsioner på leveren er beskrevet fra grise inficeret med HEV. I de vestlige lande rapporteres der om stadigt flere sporadiske tilfælde af lokalt erhvervet HEV infektioner inklusiv i Danmark, hvor HEV også er stærkt udbredt i svinebesætningerne.Denne afhandling indeholder en gennemgang af litteraturen om HEV for nogle udvalgte emner. Derudover bliver resultaterne præsenteret i form af fire manuskripter.I manuskript I bliver undersøgelsen af om HEV findes I andre dyrearter end svin in Danmarkbeskrevet. Undersøgelsen blev fokuseret mod mink (Neovison vison), et økonomisk vigtigt avlsdyr i Danmark, hvor en tidligere ubeskrevet HEV variant blev fundet. De klassiske genotyper blev ikke påvist i mink. Virusset blev fundet på fire forskellige lokalisationer i prøver indsamlet i 2008 og frem til 2011 hvilket indikerer at virusset er udbredt og har cirkuleret i en længere periode. Der var ingen indikationer på at HEV varianten i mink forårsagede klinisk sygdom, men dette bør undersøges nærmere.Manuskript II beskrives udvaskningen af HEV samt rotavirus (RV), porcine circovirus type 2(PCV2), somatiske coliphager, E. coli og Enterococcus spp gennem jord på en mark og ud i drænvand under naturlige omstændigheder ved gødning af jorden. Mikroorganismerne var naturligt tilstede i svinegyllen der blev spredt på marken. Resultaterne viste at HEV sammen med de fem andre mikroorganismer findes i drænvandet efter spredning af gylle, hvilket potentielt kan kontaminere vand reservoirer i naturen hvortil drænvandet bliver ledt ud. Rotavirus blev endvidere detekteret i nogle dybere grundvandsboringer, hvilket viste at grundvandet også var i fare for at blive kontamineret af virus. Dette kan resultere i problemer med kvaliteten af drikkevand i Danmark. Manuscript III fokuserer på kontamineringen af muslinger dyrket til konsum I Danmark. Muslingerne blev undersøgt for tilstedeværelsen af HEV og RV, der tidlige er beskrevet i udbrud forbundet med indtagelse af muslinger. Derudover inkluderede vi en undersøgelse for PCV2, der skulle fungere som indikator for kontaminering af svine gylle. Alle prøver blev testet negative for HEV og RV, men en stor del af prøverne var positiv for PCV2. Dette er den første undersøgelse der beskriver potentialet i at bruge PCV2 som viral indikator i skaldyr. At der ikke blev fundet HEV stemmer overens med tidligere studier i andre lande, selvom skaldyr er sat i forbindelse med flere sporadiske udbrud af HEV.I manuscript IV præsenteres et fortløbende studie af 104 grise, hvor udviklingen af HEV blev fulgtfra farring til 17 uger. Grisene blev inddelt i tre grupper baseret på antistofniveauet af modersoen. Under forsøget blev overførsel af maternelle antistoffer observeret for grise født af søer med høje niveauer af antistoffer og en signifikant reducering af grise der udskilte HEV fra 70% for grise uden maternelle antistoffer til 50% med maternelle antistoffer. Ti af de grise, der udskilte HEV i uge 17, blev obduceret i uge 20 og tre af grisene viste sig stadig at udskille HEV. Fra de tre grise der udskilte HEV fækalt fandtes HEV i forskellige organer f.eks. lever, mandler og lunge, men ikke i muskler og dermed kød.Baseret på disse studier kan det konkluderes, at HEV tilhører en mangfoldig familie af virusser med varianter i mange forskellige arter, hvor der er stor mulighed for at vi kun har fundet de første. Dog har HEV kun forårsaget klinisk erkendelig sygdom i mennesker og fjerkræ. Tilstedeværelsen af HEV i vandmiljøet er velbeskrevet i lande med dårlige sanitære forhold. Vores studie viser vigtigheden af en øget forståelse af de processer, der foregår når virus udvaskes gennem jord, samt at kontrol med vores drikkevandsressourcer med henblik på virus, kan blive nødvendigt for at sikre en fortsat høj drikkevandskvalitet. Vi fandt ingen tegn på HEV og RV i skaldyr, men fandt dog tegn på fækal kontaminering med svine gylle. Dette kan udnyttes fremadrettet til at overvåge skaldyrsområderne i Danmark og sætte mere fokus på viral kontrol af skaldyr, hvilket endnu ikke er implementeret. For at mindske eksponeringen af befolkningen for HEV vil en begrænsning af de svin der indgår i fødevare produktionen have en stor effekt. Vores resultater indikerer at man kan gøre netop dette ved at vaccinere søer og derved bringe deres HEV antistof niveau op.I appendix 1 (Sektion 12) findes en dansk oversigtartikel der forklarer nogle af de zoonotiske aspekter.In this thesis the epidemiology of hepatitis E virus (HEV) in Denmark was explored. Globally, four genotypes of HEV are recognized along with several species specific variants. Non-zoonotic genotypes of the virus are found in the developing world, where they cause epidemics due to fecal contaminated water resources. Zoonotic genotypes have a primary reservoir in pigs worldwide. Humans infected with HEV are often asymptomatic, but can experience an acute self-limiting hepatitis. Pigs are asymptomatic and only mild lesions have been observed in the liver of infected animals. In the developed world, sporadic cases of HEV induced disease have been more frequently reported as awareness of the disease increases, including human cases in Denmark where HEV is also prevalent in pigs.The thesis consists of an introduction into HEV where the literature on specific subjects is reviewed. The results obtained during the work of this PhD are presented in the form of four manuscripts. Finally, the four manuscripts are discussed in a broader context.In Manuscript I, the work performed to investigate if HEV was found in other animal reservoirs than pigs in Denmark is presented. The research focused on mink (Nevison vison), an economically important livestock in Denmark. The main finding was the discovery of a novel variant of HEV. The virus was found in four different locations in samples collected in 2008 and in samples collected in 2011 indicating that the virus is widespread and has been circulating in minks for years. The virus did not seem to cause clinical disease in mink, however, this should be investigated further.Manuscript II describes the leaching of HEV along with rotavirus (RV), porcine circovirus type 2 (PCV2), somatic coliphages, E. coli and Enterococcus spp. through field soil into tile drains under natural conditions of field fertilization. The microorganisms were naturally present in the pig slurry applied to the field. The results showed that HEV along with the other five microorganisms were found in water drained from the field, indicating potential contamination of water reservoirs in connection with the untreated drainage runoff. Rotavirus was detected in deeper groundwater screen indicating the possibility of groundwater contamination of viruses originating from manure, posing a risk for the contamination of important drinking water reservoirs in Denmark. Manuscript III focuses on the viral contamination of mussels farmed in Denmark. The mussels was assayed for HEV and Rotavirus, known to occur in shellfish and cause disease. Furthermore, PCV2was also assayed as an indicator of porcine waste contamination. All samples analyzed were negative for HEV and RV, however, a large proportion of the samples tested positive for the PCV2. This is the first report that shows the potential of PCV2 as an indicator organism. The lack of HEV in the mussels is in accordance with previous studies, although shellfish has been reported as the source of multiple sporadic cases of HEV infection.Manuscript IV report the results of a longitudinal study performed in a multi-site farm, where HEV development in 104 pigs were followed from farrowing to 17 weeks of age. The pigs were divided into three groups according to the level of antibody titers of their sows. During the study, successful transfers of maternal antibodies were observed only for the pigs born to sows with the highest antibody level. Furthermore, a significant reduction of the number of pigs shedding HEV from approx. 70% to 50% was observed in the group that received maternal antibodies. Ten of the pigs that were shedding HEV at week 17 were necropsied at week 20 and three of these were still shedding HEV. Correspondingly, HEV was found in different tissues, e.g. liver, tonsils and lungs, of these three pigs. HEV was, however, not found in muscles. In addition, high level of HEV was found in 1 out of 73 Danish livers purchased at grocery stores in the larger Copenhagen areaBased on these studies it could be concluded that HEV belongs to a diverse family of viruses with variants in multiple species, and there is a possibility that many more of these will be identified during the coming years. However, clinical illness have so far only been described in humans and birds. HEV is known to be present in the water environment of poorly sanitized regions of the world, however, our study also emphasized the need for better understanding of viral leaching as well as the need for diagnostic tools enabling survey of water to maintain high drinking water quality also in the developed part of the world. We did not find HEV or RV in shellfish although indications of porcine waste contamination of shellfish occurring were observed in though the detection of the porcine specific virus PCV2. This is an interesting finding and suggest that PCV2 may be utilized to monitor viral contamination of shellfish, which is completely lacking today. Lastly, high levels of maternal antibodies can diminish the number of pigs shedding or carrying infectious HEV when entering the food supply chain. This could be utilized by vaccinating sows prior to farrowing, ensuring transfer of maternal antibodies and thereby reducing human exposure to HEV

Online Research Database In Technology

Zoonotic Aspects of Hepatitis E Virus in Denmark

http://orbit.dtu.dk/ws/files/74610678/Ph.d._afhandling_Jesper_Schak_Krog_.PDF

Zoonotic Aspects of Hepatitis E Virus in Denmark

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