| Abstract | Ptičji reovirusi su ubikvitarni mikroorganizmi u domaće peradi i ostalih ptičjih vrsta, te se često izdvajaju i u zdrave i u bolesne peradi iz gastrointestinalnog i respiratornog trakta. Oni se mogu širiti vertikalno i horizontalno feko – oralnim i respiratornim putem. Između sojeva postoje značajne razlike s obzirom na sposobnost horizontalnog načina širenja. Virusni artritis – tenosinovitis je najteža klinička i gospodarska bolest uzrokovana reovirusima, a opisana je u kokoši, prvenstveno tovnih pilića, i purana. Virusni artritis – tenosinovitis može se dijagnosticirati na temelju kliničke slike, makroskopskih i mikroskopskih promjena, ali za definitivnu dijagnozu potrebno je izolirati i identificirati reovirus. Molekularna metoda qPCR danas se koristi kao univerzalna metoda za identifikaciju ptičjih reovirusa. Serološki se reovirusni antigen može utvrditi u smrznutim uzorcima tetivnih ovojnica ili drugih tkiva pomoću fluorescentnih protutijela. Komercijalni ELISA testovi koriste se za procjenu razine reovirusnih protutijela unutar jata.
Zbog ubikvitarnosti reovirusa i modernog, intenzivnog načina peradarske proizvodnje teško je održavati jata slobodna od reovirusa. Na tržištu su prisutna komercijalna cjepiva za rasplodne kokoši, koja osiguravaju dostatnu zaštitu, ali zbog varijabilnosti sojeva ni roditelji ni potomstvo nisu uvijek potpuno zaštićeni, te stoga postoji potreba za primjenom autogenih cjepiva protiv lokalnih sojeva.
Svrha ovog istraživanja bila je provesti monitoring na roditeljskoj farmi teške linije peradi, na kojoj je već serološki dokazana prisutnost reovirusa koristeći molekularnu metodu qPCR.
Iz uzoraka crijeva pilića, membrana i ovojnica kokoši prethodnog jata, te korioalantoisne membrane SPF zametaka nakon inokulacije uzoraka tetiva i ovojnica ovog jata dobi 14 tjedana, izdvojena je ukupna DNK/RNK, te je do daljnje analize čuvana na -80°C. Za preliminarni dokaz reovirusa u uzorku korištena je qPCR metoda kao vrlo osjetljiva i specifična primjenom početnica i Taqman probe. Rezultati qPCR reakcije pokazali su prisustvo virusa u 3 od 14 uzoraka crijeva pilića, no uzorci tetiva i membrana od klinički manifestnih jedinki prethodnog jata bili su negativni. Analizom pozitivnih uzoraka crijeva pilića klasičnom PCR reakcijom dobili smo negativne rezultate. Nakon inokulacije na zametke došlo je do uginuća dva od pet zametka 3. i 4. dan od inokulacije, no i navedeni i uzorci korioalantoisne membrane ostalih inokuliranih zametaka su qPCR analizom bili negativni.
Poznato je da u gotovo u 100% slučajeva izostaje dokaz virusa klasičnim PCR postupkom. Smatra se da je najprije potrebno virus umnožiti u SPF zametcima, a potom dokazati klasičnom PCR reakcijom. U našem slučaju negativni nalaz qPCR reakcije nakon umnažanja na SPF zamecima vjerojatno je rezultat uznapredovalog stadija bolesti u kojim je došlo do iščezavanja virusa iz područja promjena.
Sukladno rezultatima potrebno je monitoring provesti kroz duži period, osobito prije očekivane pojave simptoma, uz obveznu inokulaciju na zametke. Dobiveni rezultati omogućili bi izradu autogenih cjepiva kojim bi unaprijedili imunoprofilaksu na farmi, a protokol bi se koristio i za kontrolu reovirusa na drugim farmama. |
| Abstract (english) | Avian reoviruses are ubiquitous microorganisms in domestic poultry and other avian species, and are often isolated in both healthy and sick poultry from the gastrointestinal and respiratory tract. They can be spread vertically and horizontally by the feco - oral and respiratory routes. There are significant differences between strains when it comes to the ability of horizontal transmission. Viral arthritis - tenosynovitis is the most severe clinical and economic disease caused by reoviruses, and has been described in chickens, primarily broilers, and turkeys. Viral arthritis - tenosynovitis can be diagnosed based on the clinical signs, macroscopic and microscopic changes, but for a definitive diagnosis it is necessary to isolate and identify the reovirus. The q PCR is now used as a universal method for the identification of avian reoviruses.
Reovirus antigen can be detected in frozen samples of tendon sheaths or other tissues using fluorescent antibodies. Commercial ELISA assays are used to assess the level of specific reovirus antibodies within a flock.
Due to the ubiquitous nature of the reovirus and the modern, intensive way of poultry production, it is difficult to keep flocks free of reovirus. There are commercial vaccines for breeder hens, which provide sufficient protection, but due to the variability of strains, neither parents nor offspring are always fully protected, and therefore there is a need for the use of autogenous vaccines.
The purpose of this study was to conduct monitoring on the broiler breeder farm, where the presence of reovirus has already been serologically proven, using qPCR, and later type it using classical PCR method.
Total DNA / RNA was extracted from chicken gut samples, membranes and envelopes from previous flock, and the chorioallantoic membrane of SPF embryos inoculated with membranes and envelopes from this flock at age 14 weeks, and stored at -80 ° C until further analysis. For preliminary evidence of reovirus in the sample, the qPCR was used as very sensitive and specific method using primers and Taqman probes. In the case of a positive qPCR reaction, the samples were also analyzed by the classical PCR with the aim of amplifying the specific section and subsequent sequencing.
The results of the qPCR reaction showed the presence of virus in 3 out of 14 chicken gut samples, but tendon and membrane samples from clinically infected individuals were negative. By analyzing positive chicken gut samples by traditional PCR, we obtained negative results. After inoculation on embryos, two out of five embryos died on the 3rd and 4th day after inoculation, but the chorioallantoic membrane samples of all embryos were qPCR negative.
It is known that in almost 100 % cases, the virus was not detected by traditional PCR. It is thought that the virus should first be propagated in SPF embryos and then detected by a traditional PCR. In our case, the negative finding of the qPCR reaction after multiplication on SPF embryos is probably the result of an advanced stage of the disease in which the virus has disappeared from the infected area.
According to obtained results, it is recommended to perform monitoring over longer period, especially before expected clinical manifestation, with obligatory inoculation into SPF chicken embryos. The obtained results would enable the development of autogenous vaccines that would improve immunoprophylaxis on the farm, and the protocol would also be used to control reoviruses on other farms. |
