Dokaz virusa newcastleske bolesti molekularnim postupkom FTA-PCR

The feasibility of using Flinders Technology Associates (FTA) filter papers to store the Newcastle disease virus (NDV), infected allantoic fluid (AF) and tissue samples, for the molecular detection of NDV by reverse transcriptase - polymerase chain reaction (RT-PCR) - was investigated. An FTA card is a cotton based cellulose membrane, with lyophilized chemicals that lyse the viruses and bacteria. The viral RNA was detectable from FTA cards up to a concentration of 107.6 EID50/100 μL (a 100 times dilution of 109.6 EID50/100 μL of initial stock). The inactivated virus remained stable on the cards for up to 30 days, both at room temperature and 4 ºC. NDV was detected by RT-PCR from all the FTA imprints of the caecal tonsils, kidney, proventriculus, spleen, trachea, faecal swabs and intestinal lesions of NDV-suspected birds. NDV was inactivated upon contact with FTA, as shown by the inability of the virus to propagate in embryonated eggs and its inability to infect chicken embryo fibroblast culture. In conclusion, FTA cards are suitable for collecting and transporting NDV infected samples, without cold storage. The virus inactivated in FTA cards, however, is a suitable source of viral RNA for molecular detection and characterization.Istražena je mogućnost uporabe Flinders tehnologije s filtrirnim papirom za pohranjivanje alantoisne tekućine zaražene virusom newcastleske bolesti i uzoraka tkiva radi molekularnog dokazivanja toga virusa lančanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Flinders kartica celulozna je membrana pripravljena na osnovi pamuka s liofiliziranim kemikalijama koje liziraju viruse i bakterije. Virusna RNA mogla se na takvoj kartici dokazati u količini od 107,6 EID50/100 μL (100 puta manje razrjeđenje od 109,6 EID50/100 μL osnovne suspenzije). Inaktivirani virus bio je stabilan na karticama tijekom 30 dana pri sobnoj temperaturi i 4 ºC. Virus je bio dokazan RT-PCR-om u svima Flinders tehnologijom pripravljenim otiscima cekalnih tonzila, bubrega, voljke, slezene, dušnika, obriscima fecesa i crijevnih lezija ptica sumnjivih na newcastlesku bolest. Virus je bio inaktiviran nakon dodira s FT fifi ltrirnim papirom što je bilo potvrđeno činjenicom da se nije više mogao uzgojiti u kokošjim embrijima ni na kulturi fibroblasta podrijetlom od kokošjih embrija. Zaključuje se da su FT papirići prikladni za uzimanje i prijenos uzoraka koji sadrže virus newcastleske bolesti bez potrebe pohranjivanja u hladnom prostoru. Virus inaktiviran na FT papiriću (kartici) odgovarajući je izvor virusne RNA za njegov molekularni dokaz i identifikaciju

Genotypic and Pathotypic Characterization of Newcastle Disease Viruses from India

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry in most parts of the world. The susceptibility of a wide variety of avian species coupled with synanthropic bird reservoirs has contributed to the vast genomic diversity of this virus as well as diagnostic failures. Since the first panzootic in 1926, Newcastle disease (ND) became enzootic in India with recurrent outbreaks in multiple avian species. The genetic characteristics of circulating strains in India, however, are largely unknown. To understand the nature of NDV genotypes in India, we characterized two representative strains isolated 13 years apart from a chicken and a pigeon by complete genome sequence analysis and pathotyping. The viruses were characterized as velogenic by pathogenicity indices devised to distinguish these strains. The genome length was 15,186 nucleotides (nt) and consisted of six non-overlapping genes, with conserved and complementary 3′ leader and 5′ trailer regions, conserved gene starts, gene stops, and intergenic sequences similar to those in avian paramyxovirus 1 (APMV-1) strains. Matrix gene sequence analysis grouped the pigeon isolate with APMV-1 strains. Phylogeny based on the fusion (F), and hemagglutinin (HN) genes and complete genome sequence grouped these viruses into genotype IV. Genotype IV strains are considered to have “died out” after the first panzootic (1926–1960) of ND. But, our results suggest that there is persistence of genotype IV strains in India

Dokaz virusa newcastleske bolesti molekularnim postupkom FTA-PCR

The feasibility of using Flinders Technology Associates (FTA) filter papers to store the Newcastle disease virus (NDV), infected allantoic fluid (AF) and tissue samples, for the molecular detection of NDV by reverse transcriptase - polymerase chain reaction (RT-PCR) - was investigated. An FTA card is a cotton based cellulose membrane, with lyophilized chemicals that lyse the viruses and bacteria. The viral RNA was detectable from FTA cards up to a concentration of 107.6 EID50/100 μL (a 100 times dilution of 109.6 EID50/100 μL of initial stock). The inactivated virus remained stable on the cards for up to 30 days, both at room temperature and 4 ºC. NDV was detected by RT-PCR from all the FTA imprints of the caecal tonsils, kidney, proventriculus, spleen, trachea, faecal swabs and intestinal lesions of NDV-suspected birds. NDV was inactivated upon contact with FTA, as shown by the inability of the virus to propagate in embryonated eggs and its inability to infect chicken embryo fibroblast culture. In conclusion, FTA cards are suitable for collecting and transporting NDV infected samples, without cold storage. The virus inactivated in FTA cards, however, is a suitable source of viral RNA for molecular detection and characterization.Istražena je mogućnost uporabe Flinders tehnologije s filtrirnim papirom za pohranjivanje alantoisne tekućine zaražene virusom newcastleske bolesti i uzoraka tkiva radi molekularnog dokazivanja toga virusa lančanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Flinders kartica celulozna je membrana pripravljena na osnovi pamuka s liofiliziranim kemikalijama koje liziraju viruse i bakterije. Virusna RNA mogla se na takvoj kartici dokazati u količini od 107,6 EID50/100 μL (100 puta manje razrjeđenje od 109,6 EID50/100 μL osnovne suspenzije). Inaktivirani virus bio je stabilan na karticama tijekom 30 dana pri sobnoj temperaturi i 4 ºC. Virus je bio dokazan RT-PCR-om u svima Flinders tehnologijom pripravljenim otiscima cekalnih tonzila, bubrega, voljke, slezene, dušnika, obriscima fecesa i crijevnih lezija ptica sumnjivih na newcastlesku bolest. Virus je bio inaktiviran nakon dodira s FT fifi ltrirnim papirom što je bilo potvrđeno činjenicom da se nije više mogao uzgojiti u kokošjim embrijima ni na kulturi fibroblasta podrijetlom od kokošjih embrija. Zaključuje se da su FT papirići prikladni za uzimanje i prijenos uzoraka koji sadrže virus newcastleske bolesti bez potrebe pohranjivanja u hladnom prostoru. Virus inaktiviran na FT papiriću (kartici) odgovarajući je izvor virusne RNA za njegov molekularni dokaz i identifikaciju

Procjena osjetljivosti lančane reakcije polimerazom za dokaz virusa anemije u pilića uporabom različitih početnica za tri gena.

Chicken Anaemia Virus (CAV) genes cloned plasmid DNA templates (pCR4-CAV-VP1 and pCR4-CAVVP2& 3) were used in this study to develop a sensitive polymerase chain reaction (PCR) for CAV gene detection. A total of nine sets of primers, which include one set of published primers and two sets of designed primers for each gene of CAV, viz. VP1, VP2 and VP3, were used to assess the sensitivity of PCR. The PCR cycle conditions were standardized with the designed primers to get a single, specific sized amplicon for each gene separately. PCR sensitivity assessment was done by making serial 10 fold dilutions of the cloned CAV plasmid templates and subjected to PCR with each set of primers for each gene of CAV. The highest dilution of the CAV plasmid DNA showing a visible PCR amplicon was taken as the detection limit. The results showed that the designed primer VP1.2 was found to be more sensitive for the VP1 gene and the concentration of the plasmid DNA was 0.05 fg/μL or 8.6 ×103 molecules/mL and VP 2.2 was found to be more sensitive for the VP2 gene and the concentration of the plasmid was 5 ag/μL or 9.9 ×102 molecules/mL. In the case of VP3, the published primer VP 3.1 was found to be more sensitive for the VP3 gene and the concentration of the plasmid was 5 ×10-4 ag/μL or 10.5 ×10-2 molecules/mL. The findings of this study may be very useful for diagnostic, sequencing, cloning and expression purposes.Matrice gena virusa anemije pilića klonirane u plazmidnoj DNA (pCR4 CAV-VP1 te pCR4-CAV-VP2 i 3) bile su rabljene radi razvijanja osjetljive lančane reakcije polimerazom (PCR) za njihovo dokazivanje. Za procjenu osjetljivosti PCR-a bilo je rabljeno devet kompleta početnica među kojima je bio jedan komplet već objavljenih i dva kompleta sintetiziranih početnica za svaki gen virusa anemije pilića, odnosno za VP1, VP2 i VP3. Uvjeti PCR-a za umnožavanje bili su standardizirani sa sintetiziranim početnicama kako bi se dobio pojedinačni specifični umnožak za svaki gen. Osjetljivost PCR-a bila je procijenjena na osnovi deseterostrukih serijskih razrjeđenja kloniranih plazmidnih matrica u reakciji rabljenih u svakom kompletu početnica za svaki gen virusa. Najveće razrjeđenje plazmidne DNA virusa koje je pokazivalo jasno vidljiv umnožak bilo je uzeto kao krajnja mogućnost dokaza. Rezultati su pokazali da je proizvedena početnica VP1.2 bila osjetljivija za gen VP1, a koncentracija plazmidne DNA iznosila je 0,05 fg/μL ili 8,6 ×103 molekula/mL. Početnica VP2.2 bila je osjetljivija za gen VP2, a koncentracija plazmida bila je 5 ag/μL ili 9,9 ×102 molekula/mL. U slučaju VP3, objavljena početnica VP3.1 pokazala se osjetljivijom za gen VP3, a koncentracija plazmida iznosila je 5 ×10-4 ag/μL ili 10,5 ×10-2 molekula/mL. Nalazi ovog istraživanja mogu biti korisni u dijagnostici, sekvencioniranju, kloniranju i ekspresiji

Brzi lateks aglutinacijski test za serološki dokaz serotipa 4 ptičjeg adenovirusa upotrebom rekombinantnog antigena

Hydropericardium hepatitis syndrome (HPS) is a newly emerging disease of poultry, which is caused by fowl adenovirus serotype 4. The virus was propagated in a primary chicken embryo liver culture. The cytopathic effect was observed from the third passage onwards. DNA was isolated from the infected culture and used as a template for amplification of a partial hexon gene (700 bp) using hexon gene specific primers. The amplified product was cloned into pProEX HT b vector and the ligated product was transformed into DH5α cells. The recombinant clones were analyzed by colony PCR and plasmid isolation, followed by restriction digestion to check the insert release. The positive clones were induced by IPTG. The induced culture fractions were checked at different hours and the induction was high at the 4th hour onwards. The expressed proteins were purified and confirmed by using hyperimmune serum against FAV4 by western blot analysis and the protein size of 50kda was obtained. The purified recombinant FAV4 protein was used as a serodiagnostic agent using enzyme linked immunosorbent assay and latex agglutination test.Sindrom hidroperikarda i hepatitisa nova je bolest peradi uzrokovana serotipom 4 ptičjeg adenovirusa. Virus je bio umnožen u primarnoj staničnoj kulturi podrijetlom od jetrenoga tkiva pilećega zametka. Citopatski učinak javio se nakon treće pasaže. DNK je bila izdvojena iz zaražene kulture i rabljena kao kalup za umnožavanje dijela gena heksona (700 bp) uz upotrebu specifičnih početnica. Umnoženi odsječak bio je kloniran u vektoru pProEX HT b, a proizašli proizvod prebačen u DH5α stanice. Rekombinantni klonovi bili su analizirani lančanom reakcijom polimerazom i izdvajanjem plazmida nakon čega je pomoću restrikcijske digestije provjerena uspješnost postupka. Pozitivni klonovi bili su inducirani pomoću IPTG-a. Frakcije induciranih stanica bile su provjeravane svakog sata, a indukcija je bila velika nakon četiri sata. Proizvedene bjelančevine bile su pročišćene i identificirane uporabom hiperimunoga seruma za FAV4 Western blot analizom te se pokazalo da je proizvedena bjelančevina veličine 50 kda. Pročišćena rekombinantna bjelančevina FAV4 bila je rabljena kao antigen u imunoenzimnom testu i lateks aglutinacijskom testu

A Defined Antigen Skin Test That Enables Implementation of BCG Vaccination for Control of Bovine Tuberculosis:Proof of Concept

In most low- and middle-income countries (LMICs), bovine tuberculosis (bTB) remains endemic due to the absence of control programs. This is because successful bTB control and eradication programs have relied on test-and-slaughter strategies that are socioeconomically unfeasible in LMICs. While Bacillus Calmette–Guérin (BCG) vaccine-induced protection for cattle has long been documented in experimental and field trials, its use in control programs has been precluded by the inability to differentiate BCG-vaccinated from naturally infected animals using the OIE-prescribed purified protein derivative (PPD)-based tuberculin skin tests. In the current study, the diagnostic specificity and capability for differentiating infected from vaccinated animals (DIVA) of a novel defined antigen skin test (DST) in BCG-vaccinated (Bos taurus ssp. taurus x B. t. ssp. indicus) calves were compared with the performance of traditional PPD-tuberculin in both the skin test and in vitro interferon-gamma release assay (IGRA). The IFN-γ production from whole blood cells stimulated with both PPDs increased significantly from the 0 week baseline levels, while DST induced no measurable IFN-γ production in BCG-vaccinated calves. None of the 15 BCG-vaccinated calves were reactive with the DST skin test (100% specificity; one-tailed lower 95% CI: 82). In contrast, 10 of 15 BCG-vaccinated calves were classified as reactors with the PPD-based single intradermal test (SIT) (specificity in vaccinated animals = 33%; 95% CI: 12, 62). Taken together, the results provide strong evidence that the DST is highly specific and enables DIVA capability in both skin and IGRA assay format, thereby enabling the implementation of BCG vaccine-based bTB control, particularly in settings where test and slaughter remain unfeasible

Comparative transcriptome analysis of spleen of Newcastle Disease Virus (NDV) infected chicken and Japanese quail: a potential role of NF-κβ pathway activation in NDV resistance

Newcastle disease (ND) affects a few hundred avian species including chicken and several species of domestic and wild birds. The clinical outcome of Newcastle disease virus (NDV) infection ranges from mild to severe fatal disease depending on the NDV pathotype and the host species involved. Japanese quails serve as natural reservoirs of NDV and play important role in NDV epidemiology. While infection of chicken with velogenic NDV results in severe often fatal illness, the same infection in Japanese quails results in inapparent infection. The molecular basis of this contrasting clinical outcomes of NDV infection is not yet clearly known. We compared global gene expression in spleen of chicken and Japanese quails infected with lentogenic and velogenic NDVs. We found contrasting regulation of key genes associated with NF-κB pathway and T-cell activation between chicken and Japanese quails. Our data suggests association of NDV resistance in Japanese quails to activation of NF-κB pathway and T cell proliferation