Validation of polymerase chain reaction assay as an alternative method for detection of chicken anemia virus as a vaccine contaminant

Viral safety and potency are considered the two cornerstones for production of satisfactory vaccines. The three main aspects of viral safety of vaccines are the residual pathogenicity of vaccine virus, the presence of extraneous agents, and the inadequate inactivation of vaccine virus (Dodetetal. 2010). Vaccine safety mostly have the priorityover the potency; as the problems originating from the use of low-potent vaccine are still confined to the recipient flocks, while the use of contaminated vaccine will explore a series of crises in vaccinated flocks, and in most cases, the contaminant pathogens finds their way to the surrounding environment causing disastrous losses. The main source of vaccine contamination is the use of contaminated avian substrates for propagation of vaccine virus. Most vaccines are currently produced on chicken embryo-derived materials such as tissue cultures and whole embryos. Use of such biological materials in vaccine production and development may lead to potential contamination of vaccines with various extraneous viruses, especially the vertically transmitting viruses. Therefore, these vertically transmitting viruses must have the priority in vaccine screening practice. Chicken anemia virus (CAV) is one of chicken viruses that can transmitted vertically, and its role as vaccine contaminant is well established since the first isolation of the virus from chicken flocks that have been vaccinated with contaminated MDV vaccines (Yuasa 1983, Yuasa et al. 1979). The use of specific pathogen free (SPF) eggs in vaccine preparation did not eliminate the possibility of vaccines contamination with CAV. Different studies reported the persistence of CAV in gonads of SPF chickens and its vertical transmission to eggs and embryos (Cardonaet al.2000, Milleretal. 2003, Schat . and Woods 2013). Until now, thedetection of CAV contamination in avian live vaccines is currently performed by conventional methods such as tissue culture inoculation and /or chicken inoculation (CFR 2012, Pharmacopoeia2009 -b). Although these methods are establishedand routinely used, it is time consuming and laborious. In recent years the European Pharmacopoeia (Ph.Eur) advised the application of the three R’S rule that are directed to reduce, refine and replace the use of animals in the manufacturer and testing of veterinary vaccines, and replacement of in-vivo methods with the more advanced biotechnology-based techniques (Dodetet al.2010,Van der Kamp 1996). The polymerase chainreaction( PCR) proved to be a valuable tool for the screening of avian vaccines for extraneous virus pathogens such as New-castle disease virus (Stäuber et al. 1995), avian leukosis virus (Häuptli et al. 1997); infectious laryngotracheitis virus(Vögtlin et al. 1999), infectious bursal diseasevirus(Timgren-Cizinauskas 2001), avian Reovirus (Bruhn et al. 2005), Marek´s disease virus (Lang 2005), avian encephalomyelitis virus (Xie et al. 2005) and CAV (Amer et al. 2011, Hermann et al. 2012). The aim of this study is the application ofPCR assay as a routine test for detection of CAV contamination in avian live vaccines as an alternative to the in vivo and in vitro assays.

A commercial CAV live vaccine (Nobilis® CAV P4, Intervet /Schering-Plough) containing 106 TCID50 of strain 26p4a / vial, was used as the source of CAV. The virus was served as positive control in PCR assay, and for spiking of various types of vaccines (experimental contamination) to evaluate the efficiency of the PCR in detection of CAV in contaminated vaccines. A stock virus was prepared by rehydration of the original vaccine vial in 5 ml nuclease-free water. This stock was divided into 0.5 ml aliquots and stored at-70° C.

Different types of commercial avian live viral vaccines were used for several purposes; firstly, to check the PCR specificity, by testing templates from different viruses against CAV DNA; secondly to utilize these vaccines as models to evaluate the efficacy of the PCR in detection and recovery of CAV from experimentally contaminated vaccines. Another aims were the application of the developed assay to screen a large number of vaccines for detection of CAV contamination in one working area at the same time (to simulate the routine testing of vaccines); and the last objective was to evaluate the effect of vaccine matrix or formula on the assay sensitivity. Twenty-six batches of different avian live viral vaccines were utilized. Types and number of tested vaccines are as follows: three batches of fowl pox virus( FPV) vaccine, two batches of Marek´s disease virus (MDV) vaccine, 14 batches of Newcastle disease virus (NDV) vaccines, two batches of infectious bronchitis virus (IBV) vaccines, two batches of infectious bursal disease virus (IBDV) vaccines, two batches of avian encephalomyelitis virus (AEV) vaccine, and one batch of avian Reovirus (ARV) vaccine). Prior to put in test, each vaccine sample was rehydrated in 5.0 ml PCR- grade water, and each of these stocks was used to prepare further dilutions required to conduct PCR assay and for verification of assay specificity and sensitivity as described under each heading.

Viral nucleic acid was extracted using GENEAID viral nucleic acid extraction kit II, Lot no.JN07607 – P, Cat. No. VR100. The procedures were carried out using 200 µl / sample according to the manufacturer’s protocol. The purified nucleic acids was recovered in 50-µl elution buffer and stored at -20° C until used.

Two sets of CAV specific primers were used. Primer “A” was designed to amplify the overlapping region of VP2-VP3 and VP1-VP2 (Hermann et al. 2012, Ottiger 2010) with anexpected product size of 419 bp Primer “B” was designed to amplify the highly conserved sequence within the CAV VP1gene (Ameret al.2011, vanSanten et al. 2001) with an expected product sizeof 676 bp; The two primers were synthesized by BIOMATIK™. Data of Oligonucleotide primers are shown in table-1

The PCR reaction was carried out in 25-µl reaction volume in 0.2-ml PCR tubes with 5.0 µl Ultra-Pure Taq PCR Master Mix, 1.5 µl (0.6-µM final concentration) of CAV specific primer (Biomatik), 5.0 µl of nucleic acid template, and the reaction volume was completed to 25 µl by addition of nuclease-free water. Thermocycling process was performed in TECHNETC- PLUS thermocycler as follows; initial cycle at 95°C for 5 min. followed by 40 cycles of 95°C for 30 sec , 55°C for 35 sec, and 72°C for 60 sec, and finally one cycle of 72°C for 7 min. After amplification, 8-µl of the PCR product was electrophoresed through 1.5% agarose gel in TAE buffer and visualized under UV trans-illuminator.

Assay specificity was verified by testing of various avian viral nucleic acids against templates containing CAV DNA. Viral nucleic acid was extracted from each of seven avian viral vaccines including DNA viruses (FPV, and MDV), and RNA viruses (NDV, IBV, IBDV, AEV, and ARV).The extracted nucleic acids was tested by the same procedures. Each vaccine was represented by one 200-µl sample taken from the vaccine stock.

To determine the detection limit, serial 10- fold dilutions of CAV stock were prepared in PCR-grade water (from 10^-1 – 10^-6), and were used for DNA extraction and PCR amplification as described above. Assay repeatability was evaluated by application of this PCR assay in three different days - using fresh preparations each test day- and results of the three occasions were compared in between.

Twenty-six batches of avian live viral vaccines were screened by the described CAV PCR assay to assure freedom of CAV contamination. Prior to put in test, each vaccine was rehydrated with PCR grade water at the rate of 5 ml / 1000 doses, and 200µl samples were used for DNA extraction.

Three different types of live avian vaccines, namely NDV, FPV, and ARV, were utilized as models for experimental contamination with CAV. Serial 10-fold dilutions (from 10^-1- 10^-6) of CAV stock were prepared in each of these three vaccines (the vaccine matrix was used as diluents), as well as in PCR-grade water. DNA was extracted from all dilutions and tested by PCR to verify the assay efficiency in detection of CAV in experimentally contaminated vaccines, as well as to evaluate the adverse effect – if any – of vaccine matrix or formula on the efficiency of the assay.

Different parameters such as primer concentrations, DNA template volume, and thermal cycling protocols, were trialed in occasions to assess the optimal conditions for performing PCR assay. Two sets of Oligonucleotide primers (A and B) were used and tested to assess their specificity for CAV. As shown in figure-1, both primers were efficient in amplifying its own specific sequence (419 bp for primer A, and 676 bp for primer B). A slightly higher performance was recorded by using primer B; therefore, it was selected to complete the PCR assay in all tests downward.

The specificity of CAV PCR assay was verified by testing different templates extracted from seven different avian viruses including DNA viruses ( FPV, and MDV), and RNA viruses ( NDV, IB , IBDV, AEV, and ARV) in parallel to template of CAV. As shown in fig. (2), a specific sequence was detected only in template containing CAV DNA (lane 3). No amplification was detected in samples containing templates of NDV, IBV, IBDV, ARV, FPV, or MDV.

The assay repeatability was verified by detection of specific bands in all test occasions without significant difference between the three test repeats.

To determine test sensitivity or detection limit, PCR was performed on templates extracted from serial 10-fold dilutions (from 10^-1 to 10^-6) of CAV stock containing 10⁶ TCID50/5 ml. As shown in fig. (3), CAV specific band of 676 bp size was detected up to dilution 10^-3. This dilution is equivalent to 200 TCID50 /ml, and it means that the sensitivity of the assay is 40 TCID50/reaction.

To verify the effect of vaccine formula on assay efficacy in detection of vaccine contamination with CAV, three different vaccine matrices were used as vehicles and the developed PCR assay was conducted on these vaccines. Namely, NDV vaccine, ARV vaccine, and FPV vaccine were used for preparation of serial 10- fold dilutions of CAV from 10^-1 to 10^-6 and the assay^’s sensitivity was determined and compared with that obtained with CAV diluted in PCR grade water. The assay could detect CAV specific bands of 676 bp in up to the 10^-3 dilution in NDV and ARV vaccines matrices (figure-4 and 5); while in FPV vaccine the reaction could detect a specific band up to 10^-2 dilution, compared to 10^-3 in CAV samples diluted in PCR-grade water (figure-6).

 

Twenty-six batches of different avian live vaccines were screened by using the used PCR assay to detect CAV contamination. Tested vaccines included DNA viruses (5 batches) and RNA viruses (21 batches). Results indicated that all tested vaccines were free from CAV contamination as no product was detected in any vaccine, while a specific band of 676 bp size was detected only in samples containing CAV template.

Samples of extracted CAV DNA were used as additional external control to verify the performance of master mix and thermal cycling. Results indicated a successful amplification of target sequences in all samples spiked with external CAV-DNA controls (data not shown).

The role of CAV as an extraneous pathogen in avian viral vaccines is well established since the first isolation of the virus from contaminated Marek's disease vaccine (Yuasa 1983, Yuasaet al.1979).

CAV can be transmitted vertically from dams to offspring, and consequently embryos or cell cultures prepared from infected embryos could serve as a source of CAV contamination in vaccines prepared from such substrates. The use of specific pathogen free (SPF) eggs in vaccine preparation did not eliminate the possibility of vaccine contamination with CAV (Cardonaet al.2000). Detection of CAV contamination in vaccines is currently performed by cell culture inoculation (in -vitro) and/ or chicken inoculation (in - vivo)(CFR2012, Pharmacopoeia 2009 -b). Each one of thesetechniques has its own obstacles to be performed on a large-scale pattern or as a routine work destined for screening large number of vaccine samples in one working area at the same time. The detection of CAV in cell culture (in-vitro) is laborious and time consuming and require multiple passage in MDCC-MSB1 lymphoblastoid cells and specific antiserum to confirm virus detection by immunofluorescence (Hermann et al. 2012). The other choice (in-vivo)is the more difficult, as the environmental stability and ubiquity of CAV poses the maintaining of seronegative source flocks is out of hands. Due to the above-mentioned considerations, and others, there is much support for the use of alternative methods that could replace the current conventional tests (Ottiger 2010). The Council of Europe legislates an overall movement towards the replacement, reduction, and refinement of animal experimentation, or the ^“3 RS” rule (Bruckneret al.2000, Pharmacopoeia 2009 -a). The use ofalternative methods of analysis is accepted under the European Pharmacopoeia chapters 2.6.24 and 2.6.25, which stated that nucleic acid amplification test (NAT) give specific detection for many agents and can be used after validation for sensitivity and specificity (Pharmacopoeia 2009 -a, Van derKamp 1996). PCR based approach proved to bevaluable tool for the detection of vaccine contamination with extraneous viruses such as NDV (Stäuberet al.1995), ALV (Häuptliet al.1997), ILTV (Vögtlin et al. 1999), IBDV((Timgren-Cizinauskas 2001), avian Reovirus (Bruhn et al. 2005), MDV(Lang 2005), AEV (Xie et al. 2005), and CAV(Amer et al. 2011, Hermann et al. 2012). In this study, we described theapplication of PCR assay for successful detection of CAV in deliberately contaminated avian vaccines. The specificity of the assay was verified by detection of CAV- specific product only in samples containing CAV template , but not in other samples containing no templates (negative control) nor in samples contained templates of other avian viruses including DNA viruses (MDV& FPV ) and RNA viruses (NDV, IBV, IBDV, AEV and avian Reovirus), indicating assay specificity to CAV. The sensitivity or detection limit was evaluated by testing serial 10-fold dilutions of CAV. The assay enabled detection of specific bands up to 10^-3 dilution; this dilution is the equivalent to 200 TCID50 / ml; or 40 TCID50 / reaction. This limit of detection, although it is slightly lower than that obtained in some developed PCR assays (Imaietal. 1998, Soine et al. 1993), it still comparable withother reports (Ameret al.2011, Hermannet al.2012) who reported detection limits of 5 and 90TCID50 per reaction, respectively. The higher sensitivity of the former reports may be due to the use of nested PCR. Amer et al. 2011 stated that although the use of nested PCR showed improved detection sensitivity by10-100 folds, it increases the cost and time required for vaccine evaluation with no extra positive impact on the result obtained (Amer et al. 2011). Two sets of Oligonucleotideprimers were used in this study; the first one (primer A)  was designed to amplify the overlapping regions of VP2-VP3 and VP1- VP2 (Hermannet al.2012,Ottiger 2010), while the second primer (B) wasdesigned to amplify the highly conserved sequence of viral protein 1; VP1 (Ameret al.2011, vanSanten et al. 2001). Although one thermal cyclingpattern was used, the specificity of both primers was confirmed, and a CAV sequence specified to each primer was obtained at the expected size related to each one. This means that each of both primers could be used in application of CAV PCR with the same efficacy. The effect of vaccine matrix on assay efficiency was evaluated. Fowl poxvirus vaccine matrix reduced the assay sensitivity by one log, as the detection limit in fowl pox vaccine was 400 TCID50 / reaction, compared to 40TCID50/reaction in NDV and in avian Reovirus vaccines. It is not the first report for this adverse effect of fowl pox vaccine matrix in detection of extraneous viruses. This effect was reported in previous studies (Badawi 1997, Fadly . and Witter1997) who suggested that this effect might beresulted from special additives in vaccine formula. The substrate used in fowl pox vaccine preparation may play a role in this interference. Fowl pox vaccines are currently prepared from homogenates of chorioallantoic membranes from inoculated embryos, while matrices in other vaccines are mainly composed of cell culture fluid or from the allantoic fluid of inoculated chicken embryos, this may make the difference. The use of extracted CAV DNA as an external quality control widened the assay benefit. This external control can be used in each reaction to verify that master mix contains appropriate and functioning reagents, as well as the thermal cycling is suitable for amplification of target sequences.

In conclusion, this study describes the application of sensitive and specific PCR assay for the detection of CAV as a contaminant in avian live vaccines. This assay could be used as an alternative to the time- consuming, tedious, and laborious conventional in- vivo and in- vitro assays. Application of this technique can facilitate routine testing of vaccines for extraneous virus contamination.

Ahmed Badawi, Magda Sayed, Hytham Ibrahim, Manar Seioudy, Wael Elfeil and Abd El- Hakim Ali conceived and designed the experiments. Ahmed Badawi, Magda Sayed, Hytham Ibrahim, Manar Seioudy performed the experiments. Ahmed Badawi, Magda Sayed, Hytham Ibrahim, Manar Seioudy, Wael Elfeil and Abd El- Hakim Ali analyzed the data. Ahmed Badawi, Magda Sayed, Hytham Ibrahim, Manar Seioudy, Wael Elfeil and Abd El- Hakim Ali contributed reagents/materials/analysis tools. Ahmed Badawi, Magda Sayed, Wael Elfeil wrote the paper.

All authors show no conflict of interest.