ABSTRACT
In the present investigation, the thermostability of a live attenuated buffalopox vaccine prepared with an indigenous baffalopox virus isolate (BPXV Vij/96) and freeze-dried under conventional lyophilizing conditions is described. Three different stabilizer combinations like LS (lactalbumin hydralysate + sucrose), LHT (lactalbumin hydralysate + Trehalose dihydrate) and TAA (Trehalose dihydrate + l- Alanine + l-Histidine) were used to prepare the vaccine. The study indicated that the LS stabilizer was found to be the stabilizer of choice followed by LHT and TAA for buffalopox vaccine at all temperatures studied. The presence of stabilizers has beneficial influence in preserving the keeping quality of the vaccine. Further, among the diluents used to reconstitute the freeze-dried buffalopox vaccine, double distilled water, 0.85% normal saline solution and phosphate buffer saline were the choice of diluents in that order. However, 1M MgSO4 did not perform well at higher temperatures. Investigation suggests for using LS as a stabilizer for freeze-drying and any of the three diluents except 1MgSO4 for reconstitution of buffalopox vaccine.
Subject(s)
Excipients/chemistry , Vaccinia virus/chemistry , Viral Vaccines/chemistry , Animals , Chlorocebus aethiops , Freeze Drying , Vero CellsABSTRACT
In the present study, SYBR Green and TaqMan real time PCRs (rt-PCR) based on the C18L gene (encodes ankyrin repeat protein) of camelpox (CMLV) and buffalopox viruses (BPXV) were, respectively employed for potency evaluation of live attenuated camelpox and buffalopox vaccines. Cells infected with the respective vaccine viruses were harvested at critical time points and subjected to respective PCRs. The critical time points of harvests for CMLV and BPXV respectively, were 36 and 30 h post infection and were respectively determined based on maximum slopes of (-3.324) and (-3.321) standard curves. On evaluation of eight batches of camelpox and seven batches of buffalopox vaccines, the results indicated that the titres estimated by respective rt-PCRs were well comparable to the conventional TCID(50) method. The rt-PCR assays were found relatively more sensitive, specific and rapid than end point dilution assay. Thus, they could be used as additional tools for estimation of live CMLV and BPXV particles in camelpox and buffalopox vaccines.
Subject(s)
Genes, Viral , Orthopoxvirus/genetics , Real-Time Polymerase Chain Reaction/methods , Vaccinia virus/genetics , Vaccinia/genetics , Viral Vaccines/genetics , Animals , Chlorocebus aethiops , Orthopoxvirus/immunology , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccinia/immunology , Vaccinia virus/immunology , Vero Cells , Viral Vaccines/immunologyABSTRACT
Both conventional and real time PCR (rt-PCR) assays based on the amplification of a 103bp fragment from the DNA polymerase (DNA pol) gene (conserved, non-structural) of Orf virus (ORFV) were developed for detection and semi-quantitation of ORFV DNA from infected cell culture and clinical samples. The latter technique was based on TaqMan chemistry. The rt-PCR assay was specific and sensitive as it could detect as low as 3.5fg or 15 copies of ORFV genomic DNA. Both intra- (0.38-1.0%) and inter-assay (0.53-2.87%) variabilities of rt-PCR were within the acceptable range meaning the high efficiency and reproducibility of the assay. The rt-PCR was applied successfully to detect ORFV DNA from suspected clinical samples. Further, the assay has shown a relative diagnostic sensitivity and specificity of 100% and 93.5%, respectively, when compared to B2L gene based semi-nested PCR implying a wide potential of this rt-PCR for rapid field diagnosis of Orf in sheep and goats.