.A modelof a simple inactivation study could comprise evaluation of the titre of livevaccine before and after inactivation and measuring the log10 fallin titre during the inactivation process. This would give an indication of theefficacy of the inactivation process. There is evidence that virus titrationtests may not have satisfactory sensitivity to ensure complete inactivation. Inthese circumstances, a specific innocuity test would need to be developed andvalidated to be fit for increased sensitivity.
To increase sensitivity morethan one passage would be essential depending on the virus (OIE Terrestrialmanual tests of sterility, 2017).Asample representing at least 200 doses of vaccine (final formulated product)should go through innocuity test to show absence of infectious virus by inoculation of sensitive cell culture monolayers. Thefollowing instructions should be considered for innocuity test:(1)The test method should be highly sensitive to the virus.(2) Alarge enough number of vaccine doses to satisfy statistical requirements shouldbe tested.(3)Under the selected conditions inactivated virus should not interfere with thereplication of non-inactivated virus (Barteling, 1983).
Inthe final product, antigen must be extracted from adjuvant following anappropriate validated method (OIE Terrestrial manual FMD, 2017). Antigen recoveryfrom oil-adjuvanted vaccines using n-butyl alcohol as nine volumes of anoil-emulsion vaccine was mixed thoroughly with one volume of n-butyl alcoholand centrifuged for 5 min at 4?, 5000g. Collection of the lower aqueous phasesto be tested (Feng et.al, 2016).Concentrationof antigen may be necessary in which case it must be shown that theconcentrated does not affect with the sensitivity or reading of the assay. Inspectionof The cell sheets is done daily for 2–3 days, then the consumed medium istransferred to fresh monolayers and the original monolayers are replenishedwith fresh medium.
Using this method, traces of live virus can be propagated bythe passage technique and detected on the basis of CPEobserved. Three passages of the original virus preparation are usually used. Avariant on this method is to freeze–thaw the old monolayers to releaseintracellular virus, which can be detected by additional passage (OIE Terrestrialmanual FMD, 2017).Aslong as the 146S antigen concentration was below 1 ?g per 106 cells,suspension cultures of baby hamster kidney cells is proved to be the mostsensitive detection system for traces of infective virus. Above this levelinterference may cover the presence of non-inactivated virus (Barteling, 1983).
2.Sterility testØ Definition:Sterilityis defined as the absence of viable microorganisms. Freedomfrom contamination is defined as the absence of specified viable microorganisms(OIE Terrestrial manual tests of sterility, 2017).Ø Country regulations:Testsfor sterility should be applied on every batch of vaccine.
Based on theiranimal health status, each country or regions will have specific requirements towhat microorganisms are necessary to eliminate and what appropriate testingprotocols are acceptable. As well as applying general testing procedures recognizedin national or regional standards, it may be necessary to apply rigorousexclusion testing for specific agents that are exotic to the precise country orregion (OIE Terrestrial manual tests of sterility, 2017).Ø Tests for sterility Testsfor sterility shall be convenient to prove that the vaccine is free fromextraneous viruses, bacteria, fungi and protozoa.
· Traditional techniques AmplificationAmplification of viable extraneousagents arises by using of cell culture that is susceptible to particularviruses of concern, bacterial, and fungal culturing techniques and, wherenecessary and possible, tests including animal inoculation. Some techniqueshave been accurately validated and found to be “fit for purpose”, whereasothers may have undertaken only partial validation studies. For instance, methods for bacterial and fungal sterility have notbeen officially validated although they have been used for many years.
Inparticular, the in-vivo and cell culture methods have basically unknownsensitivity and specificity (Sheets et al., 2012) though there is a believedtheoretical sensitivity of 1 colony-forming unit (CFU). It is thereforeimportant to interpret results in the light of specific circumstances ofcultures employed and bearing in mind sensitivity and specificity of detectionsystems (OIE Terrestrial manual tests of sterility, 2017). DetectionFluorescenceantibody test (FAT), presence of colonies or cytopathic effects (CPE) andenzyme-linked immunosorbent assay (ELISA) will be used for detection purposesafter amplification using culturing techniques. Care must be taken when using ELISAtechnique for detection as such tests do not distinguish viable from non-viableagent detection (OIE Terrestrial manual tests of sterility, 2017).· Recent TechniquesMoresensitive techniques such as molecular assays may have the ability to detect agents,which may not be fruitfully amplified in traditional culturing methods.
Thedetection range can be widened using family specific primers and probes whendesigned properly. Yet, such new tests are mostly able to detect evidence fornon-infectious contaminants, such as remnants of nucleic acid frominactivated contaminants. Additional tests would be required to determine thenature of the contaminant such as non-infectious nucleic acid or infectiousvirus. Trails at virus isolation or sequencing may remedy this. Molecularassays if not designed as fit for purpose may miss detection of contaminatingagents or lack sensitivity to do so (Hodinka, 2013).
Likewise,recent enhancements in protein and peptide separation proficiencies and highly precisemass spectrometry have endorsed the identification and quantification ofproteins in a given sample. However, most of these new technologies cannot discriminatebetween viable and non-viable organisms. Therefore, targeted assays, e.
g.amplification in cell culture followed by polymerase chain reaction (PCR) maybe superior (Wang et al., 2014).· Extraneous agents require specific methodsfor detection General procedures are useful as screening tests to detect extraneousagents present in biological material as they will not necessarily detect all contaminants.
Some agents may involve specific methods for detection (Table 1). Remarkably, some subtypes of anagent type may be detectable by general methods, and some may require specifiedtesting for detection. Such as, bovine adenovirus subgroup 1 (serotypes 1, 2, 3and 9) can be readily isolated using general methods (Vero cells) howeverbovine adenovirus subgroup 2 (serotypes 4, 5, 6, 7, 8 and 10) are not readilyisolated and required specialized methods for isolation (OIE sterility test).