Uporabnost ustne tekočine prašičev v laboratorijski diagnostiki prašičjega reprodukcijskega in respiratornega sindroma ter njena učinkovitost pri kontroliranem prekuževanju mladic

Plut, Jan (author), Štukelj, Marina (mentor), Jamnikar Ciglenečki, Urška (comentor)

Prašičji reprodukcijski in respiratorni sindrom (PRRS) povzroča istoimenski virus (PRRSV). Je ena izmed najpomembnejših bolezni prašičev in zaradi velike gospodarske škode je nujno uvesti nekatere ukrepe za omilitev posledic PRRS. Prvi izmed možnih ukrepov je kontrola bolezni, pri kateri so ključna kategorija mladice. Te po prihodu na farmo s prisotnim PRRSV aklimatiziramo. Cilj aklimatizacije je imunizacija mladic proti homolognem sevu virusa ter njihova zaščita in zaščita njihovih gnezd pujskov pred kliničnimi posledicami bolezni. Uspešnost aklimatizacije ugotavljamo z molekularnimi in serološkimi diagnostičnimi metodami. Namen dela je primerjati vzorce seruma in ustne tekočine (angl. oral fluid; OF) in z ugotavljanjem prisotnosti RNA PRRSV in specifičnih protiteles spremljati potek aklimatizacije pod različnimi pogoji glede na različne skupine mladic. V raziskavi smo spremljali aklimatizacijo pri preliminarni skupini in šestih različnih skupinah mladic, od I do VI. V preliminarni skupini mladic in v skupinah I in II smo uporabili metodo naravne prekužitve in aklimatizacije mladic. Pri skupinah III in IV smo poleg naravne prekužitve uporabili še bombažne vrvi, prepojene z OF s PRRSV okuženih prašičev. Pri skupinah V in VI pa smo za prekuževanje uporabili le bombažne vrvi, prepojene z OF s PRRSV okuženih prašičev. Vpliv uporabe bombažnih vrvi, prepojenih z OF s PRRSV okuženih prašičev, na hitrost in uspešnost aklimatizacije mladic doslej še ni bil opisan v znanstvenih publikacijah. Od uhlevitve smo tedensko ugotavljali prisotnost RNA PRRSV z verižno reakcijo s polimerazo s predhodno reverzno transkripcijo (RT-PCR) in specifičnih protiteles z encimsko imunskim testom (ELISA) v individualnih vzorcih seruma in skupinskih vzorcih OF. Pri analizi rezultatov prisotnosti RNA PRRSV in specifičnih protiteles smo ugotovili statistično značilne razlike med skupinama I in II ter III in IV. V primerjavi s skupinama I in II lahko pri skupinah III in IV virusno RNA in specifična protitelesa v krvi ugotovimo hitreje. Prav tako je delež živali z ugotovljeno RNA PRRSV v vzorcih krvi v skupinah III in IV značilno manjši 28 dni po okužbi (angl. days post infection, DPI) v primerjavi s skupinama I in II. Uporaba vrvi, prepojenih z OF s PRRSV okuženih prašičev, pospeši okužbo in tvorbo specifičnih protiteles. Zadnji dan poskusa, 56 DPI, smo pri skupinah mladic od I do IV ugotovili, da je pri nekaterih mladicah PRRSV RNA v krvi še zaznavna. Pri skupinah V in VI samo z uporabo vrvi, prepojenih z OF s PRRSV okuženih prašičev, nismo mogli izzvati aktivne klinične okužbe. Pri eni mladici iz skupine VI smo ugotovili, da je pozitivno reagirala na prisotnosti specifičnih protiteles od 7 DPI dalje. Primerjava individualnih vzorcev seruma in skupinskih vzorcev OF je pokazala, da lahko RNA PRRSV ugotavljamo s primerljivo verjetnostjo. Pri določanju specifičnih protiteles v skupinskih vzorcih OF ugotavljali prisotnost specifičnih protiteles že pred izpostavitvijo mladic PRRSV. Komercialni komplet za dokaz specifičnih protiteles v OF je medtem proizvajalec umaknil iz redne prodaje.

• Language: Slovenian
• Keywords: prašičji reprodukcijski in respiratorni sindrom, klinične laboratorijske tehnike, protitelesa proti virusom, slina, encimsko imunski test, reverzna transkripcija in verižna reakcija s polimerazo, adaptacija
• Work type: Doctoral dissertation
• Organization: VF - Veterinary Faculty
• Year: 2022
• PID: 20.500.12556/RUL-137680
• Publication date in RUL: 26.06.2022

Secondary language

• Language: English
• Title: The applicability of pig oral fluid in laboratory diagnostics of Porcine Reproductive and Respiratory Syndrome and its effectiveness in controlled exposure of gilts.

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRS is one of the most important economic pig diseases. Due to severe economic losses, it is mandatory to take measures to limit the negative disease effects. One of the possible measures against PRRS is control of the disease focusing on naïve replacement gilts as the key pig category in the process. PRRS-free replacement gilts are acclimated as soon as entering the PRRS-positive farm. The goal of acclimatization is homologue protection of them and their offspring against clinical PRRS. The outcome of acclimatization is monitored with molecular and serological diagnostic methods. The aim of this study is to compare sera and oral fluid (OF) samples and to assess the success of different approaches for gilt acclimatization in different gilt groups with detection of PRRSV RNA and specific antibodies in both samples. The gilts in this study were divided into 7 groups: preliminary group and groups I–VI. The method of natural acclimatization through contact with infected pigs and their secrets was used in preliminary group ad groups I and II. In groups III and IV, the same method was applied as in groups I and II with addition of ropes soaked with OF from PRRS-positive pigs. In groups V and VI, only ropes soaked with OF from PRRS-positive pigs were used for acclimatization. The impact of additional ropes contaminated with infective material for gilt acclimatization has not been yet assessed in peer-reviewed literature up until today. Gilts were monitored weekly, since the day of arrival at the PRRS-positive farm (day 0), both individual serum samples and group OF samples were tested for presence of PRRSV RNA and specific antibodies with reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Statistically significant differences were detected in detection of PRRSV RNA and specific antibodies between groups with different treatment, I and II and III and IV. In blood samples from groups III and IV, PRRSV RNA and specific antibodies were detectable sooner compared to groups I and II. The percentage of PRRSV RNA-positive blood samples was significantly lower in groups III and IV compared to groups I and II 28 days post infection (DPI). The implication of cotton ropes soaked with PRRSV-positive OF accelerated the disease course, causing faster emergence of PRRSV RNA and specific antibodies in gilts. At the last day, 56 DPI, there was a smaller number of gilts with detectable PRRSV RNA in the organism. We were unable to induce a clinical PRRS using only peroral contact with ropes containing PRRSV-positive OF in gilt groups V and VI. However, ELISA indicated possible presence of specific antibodies in one of the gilts from group VI since 7 DPI. Some group samples of OF were tested positive for specific antibody presence even before exposure of gilts to the virus. The commercial kit was removed from the regular product list by the manufacturer shortly thereafter due to troubleshooting.

• Keywords: pigs, porcine reproductive and respiratory syndrome, saliva, enzyme-linked immunosorbent assay, reverse transcriptase polymerase chain reaction, acclimatization