Real time RT-PCR assay for detection of different serotypes of FMDV in Egypt

Aim: The present study indicated that rRT-PCR could be provided for the detection of FMDV in infected, contact and carrier cattle and also provide a rapid sensitive tool aiming to aid in rapid disease detection and control. Foot and Mouth disease virus serotypes O and A still existing in Egypt. In January 2012, sever outbreaks struck the animal population in most Egyptian 1 governorates. The causative virus was identified as FMDV SAT2. 
 
Material and Methods: Five samples of tongue epithelium (ET) and five oesophageal-pharyngeal (OP) fluid samples were collected from FMD suspected cattle in infected farm at El-Fayoum and 20 OP samples from in-contact cattle at the same farm in addition to 30 OP samples from apparently healthy cattle at three different localities in El-Fayoum governorate (12 from Fayoum; 9 from Sinoras and 9 from Edsa) in order to detect carrier cattle. All of these samples were collected during November and December 2011 and January 2012. 
 
Results: All the ET and OP samples were inoculated on BHK cell culture and baby mice. The obtained results were identified using complement fixation test in addition to real-time reverse transcriptase polymerase chain reaction (rRT-PCR). In the infected farm at El-Fayoum FMDV type SAT2 was detected in cattle which are considered as the first introduction of this type while FMDV type O and SAT2 were detected in the in-contact cattle in the same farm. The sensitivity of rRT-PCR was cleared in the in-contact cattle as 13 out of 20 OP samples were positive to FMDV by rRT-PCR while 11 out of 20 OP samples were positive to FMDV by CFT. The OP samples collected from apparently healthy cattle from Fayoum, Sinoras and Edsa localities in Fayoum governorate demonstrate the circulation of the FMDV type A, O and the recent SAT2 in carrier cattle which threaten cattle population in Fayoum governorate. Also the sensitivity of real time RT-PCR over the CFT in detection of FMDV carrier cattle was clearly noticed in these localities as 19 out of 30 OP samples were positive by rRT-PCR while in contrast there were only 16 out of 30 samples positive by CFT. 
 
Conclusion: In conclusion, this study demonstrates that real-time RT-PCR currently used at the WRL for FMD provides an extremely sensitive and rapid additional procedure for improved laboratory diagnosis of FMD especially in-contact and carrier cases. The rRT-PCR generated results in less than one day from test commencement, in contrast to up to four days to define some positive and all negative samples by combined use of CFT and virus isolation. This is an important feature when definitive diagnostic results are required in a short timescale during emergencies. Also this study demonstrates the current situation of FMDV circulating in EL-Fayoum governorate and the introduction of new SAT2 serotype beside type A and O.


Introduction
the virus [2,3].Asymptomatic persistent infection is a common after-effect following infection of ruminants Foot and mouth disease causes serious economic with FMD virus [4,5].A carrier is defined as an animal losses due to reduction in productivity and mortality of from which live viruses can be recovered for longer young animals.Control of outbreaks is depend upon a than 28 days after exposure [2, 6,7].These carrier system of monitoring and early detection, which animals can precipitate new outbreaks of disease [3, requires basic familiarity with clinical signs and the 8,9].ability to characterize the strain of virus responsible by Identification of FMD carrier ruminants mainly laboratory tests [1].
depends on isolation of the virus from oesophageal / A critical issue in this respect is the occurrence of pharyngeal fluid samples (probang samples) on primary carrier animals and the risk that pose in transmitting calf thyroid cells (BTY) cells [10] and definitive determined.The quantification arises by measuring diagnosis of FMD requires the detection of virus, the amount of amplified product at each stage during antigen or genome in clinical material.For almost the PCR cycles.Quantification of amplified product is twenty years, the WRL for FMD has used an indirect, obtained using fluorescent SYBR green.SYBR green sandwich enzyme-linked immunosorbent assay is a dye bind to double stranded DNA.The intensity of (ELISA) [11,12] to identify FMDV, and however the fluorescent emissions increase as more double ELISA is not 100% sensitive.Consequently, suspensions stranded amplicon is produced with the dye signal of each specimen are also propagated in sensitive cell increase.The dye will bind to any double strand DNA cultures [11] and the specificity of any isolated virus molecule, while the 5' nuclease probe assay is specific confirmed by the ELISA.Whilst such virus isolation to a pre-determined target [22].(VI) methods are highly sensitive, they require four The present study was planned to investigate the days before a negative result can be concluded and efficacy of a fully automated real-time RT-PCR with reported as 'no virus detected' (NVD).
virus isolation (VI) and CFT for the detection of A number of reverse transcription polymerase FMDV in infected and apparently healthy cattle chain reaction (RT-PCR) assays have been developed (carrier) to provide a rapid sensitive tool aiming to aid as an alternative to viral culture [13-18], but these in disease control.assays require other laboratory techniques to detect

Materials and Methods
the amplified product, which is associated with considerable hands-on time, poses a serious hazard for Samples: Epithelial tongue (ET) and Oesophageal/ amplification product carryover, and limits the number pharyngeal fluid samples (OP) were collected of specimens that can be processed simultaneously.
according to [6] from El-Fayoum governorate during Recently, the development of a real-time reverse November and December 2011 and January 2012.transcription polymerase chain reaction (rRT-PCR) Five ET and five OP samples were collected procedure has provided an additional tool which can from 10 suspected infected cattle and 20 OP samples be used for FMD diagnosis [19].Furthermore, this were collected from in-contact cattle from the same real-time RT-PCR method can be automated allowing farm.In addition to 12, 9 and 9 OP samples were increased through put samples with fewer userreceived from Fayom, Seniores and Edsa localities dependent steps [20].
respectively in El-Fayoum governorate (fig- 1).These Also Real-time RT-PCR has several advantages samples were examined by tissue culture and baby over conventional RT-PCR where it is more rapid and mice, then typed using complement fixation test sensitive and performed in a closed one-tube system (CFT) and SYBR green real time RT-PCR.and avoids potential cross contamination during

Real-time RT-PCR or quantitative PCR is a
A-On Tissue culture: The infectivity of ET and OP variation of the standard PCR technique used to samples were determined by inoculation in monolayers quantify DNA or messenger RNA in sample using of baby hamster kidney cells (BHK), as described by sequence specific primers, the relative number of [10] through three successive passages.The cells were copies of a particular DNA or RNA sequence can be examined daily for the presence of viral cytopathic complications to disease control.So the detection of effect (CPE) for 3 days according to [23].FMD virus in persistently infected carriers among B-In Baby mice inoculation: Suckling Swiss Albino exposed cattle is of great importance that needs a mice of 2-3 days old were used to detect FMD virus in suitable sensitive and rapid tool and accordingly the ET and OP samples inoculated in 4-baby mice I/P in a present study was planned to evaluate rRT-PCR assay dose of 0.1ml / mice and deaths were recorded from 48 for detection of FMD virus either in naturally infected; th hours to the 7 day post inoculation.
in contact or apparently health cattle.

VI has been the recommended laboratory procedures
Typing of obtained suspected isolates using for FMD diagnosis, based on their suitability to detect CFT: CFT was carried out according to [24] for typing the presence of FMDV antigen in tissue samples.If of FMDV 7 serotypes using known hyperimmune sera one considers that VI procedures actually measure against 7 serotypes.
then it is evident that its effectiveness for diagnostic

Real time RT-PCR technique:
use is inherently compromised.Virus isolation depends A-RNA extraction: RNA extraction was carried out on the presence of infectious virus in sample submissions.using the QIAamp® Viral RNA kit (Qiagen, Germany) It depends upon the antigen being present in sufficient according to the manufacturer's protocol to all samples concentration to work [25].Complement fixation test in a final volume of 50 ml according to the manufacturer's and rRT-PCR were used for detection of FMDV.CFT o instruction and stored at -80 C until used.had a reasonably high specificity but the sensitivity B-Primers: Primer pair (PoR/PoF) for real time RTwas moderate and for this reason, the technique would PCR was synthesized by BioBasic, Canada.PoF (5´not be used for quantitative measurement of FMDV CCT ATG AGA ACAAGC GCA TC -3´) and PoR (5´-[26] while Real time RT-PCR can detect a small CAA CTT CTCCTG TAT GGT CC -3´) were derived fragment of FMDV genome RNA, not just live virus.from FMDV 3D polymerase for detection of FMDV Real-time RT-PCR provides an extremely sensitive and have no cross reaction with swine vesicular and rapid procedure that contributes to improve disease (Universal primer for FMDV).laboratory diagnosis of FMD [23,25].

C-Real-time RT-PCR (rRT-PCR): rRT-PCR was
The obtained results showed that all of ET and performed using QuantiTect® SYBR® Green RT-OP obtained from suspected cattle induced charac-PCR Kit (Qiagen, Germany) as manufacturer's teristic cytopathic effect of FMD virus on BHK cell instructions.The cycling parameters were 50 °C for 30 culture and specific signs of FMD in baby mice min and 95 °C for 15min; then 30 cycles consisting of indicating the presence of FMD virus as shown in table 94 °C for 15 s, 55°C for 30 s and 72 °C for 30 s. (1).Such methods for detection of FMD virus were Negative control specimen was involved.Each sample recommended by [10,25] they showed that FMD virus was tested in duplicate.PCR amplification was carried isolation depend on the presence of infectious virus in out in the Thermocycler Rotor-Gene Q (Qiagen, Germany) sample submission.Regarding the identification of the detected FMD virus, CFT and rRT-PCR; using

Results and Discussion
specific primers; confirmed that the obtained virus The occurrence of persistently infected (carrier) isolate is SAT2 (Table -1).These findings indicates that of FMD virus in ruminants represents further the results of CFT and rRT-PCR came in a parallel  while in Edsa types A, O and SAT2 were detected in 1; Table-1 clarified that OP samples obtained from 5 and 2 out of 9 samples respectively.These findings the same in-contact cattle induced positive results in showed that the three types of FMDV are circulating in both of BHK cell culture and baby mice inoculation Fayoum Governorate threaten cattle population.Also tests (8 out of 20 and 11 out of 20 respectively) with the sensitivity of real time RT-PCR over the CFT in positive CFT and rRT-PCR which identified the same detection of FMDV carrier cattle was clearly noticed FMD virus strain (SAT2) which was detected in 5 out in Sinoras and Edsa as in Sinoras where 2 OP samples of 20 OP samples indicating that these in contact cattle were positive by rRT-PCR and in contrast they were are carriers to the same virus strain isolated from negative by CFT while in Edsa one OP sample was infected cattle and clarify the idea about FMD carrier positive by rRT-PCR while it was negative by CFT.cattle.However; 6 OP samples; from in-contact cattle These findings agree with those of [22,27] who stated were found to be positive to FMDV type O.The that the real-time RT-PCR has proven to be highly presence of carrier cattle having the same FMDV type sensitive and specific under laboratory condition.SAT2 in contact cattle to infected ones appears to be logic while the presence of type O could be attributed Conclusion to a mixed infection or a recent sub-clinical infection.
This study demonstrates that real-time RT-PCR Such findings come in agreement with [4,5] they currently used at the WRL for FMD provides an stated that asymptomatic persistent infection is a extremely sensitive and rapid additional procedure for common after-effect following infection of ruminants improved laboratory diagnosis of FMD especially inwith FMD virus and [2,6,7] they identified animal contact and carrier cases.The rRT-PCR generated carrier as an animal from which live virus can be results in less than one day from test commencement recovered for several days after exposure.In addition in contrast to up to four days to define some positive the sensitivity of rRT-PCR assay for detection of and all negative samples by combined use of CFT and carrier cattle was 100% compared with virus isolation virus isolation.This is an important feature when suggesting its suitability for screening FMDV carrier definitive diagnostic results are required in a short animals as reported by [28] who also stated that cell timescale during emergencies.Also this study demonculture technique detects virus infection while the

Figure- 1 .
Figure-1.Schedule of collected samples for detection of FMDV in cattle

*
TC= Tissue Culture, **BM= Baby Mice, ***CFT= Complement Fixation Test, T= Total no. of positive typed FMDV serotypes Table-1 that 2 OP samples in the in-contact cattle were positive in rRT-PCR while sensitive technique suitable for detection and negative in CFT.identification of FMD virus.Also these observations Table-2 demonstrated that by using the same support the suggestion that rRT-PCR has a sensitivity techniques; 6 samples out of 12 at Fayoum and 2 out of of 100% compared with the virus isolation test as 9 samples at Sinoras were positive to FMDV type O demonstrated by [27,28].

Table - 2
. Detection of FMD virus in OP samples in apparently healthy cattle at 3 localities in El-Fayoum governorate