Description of the study areas
This research work was implemented in Aba'ala district (Erkudi and Hidmo kebeles), which is located in afar region, Ethiopia. This study district was purposively selected for the reason that the presence of active FMD outbreak case report in the course of the study period, January, 2019 to March, 2020. Afar regional state shares joint intercontinental borders with Eritrea in the north-east and Djibuti in the east part of the region. The region is described specifically through arid and semi-arid weather conditions with low and unpredictable rainfall. The altitude of the region ranges from 120m below sea level in Danakil depression to 1500 m above sea level. Majority of the pastoral community mainly depend on livestock production for their livelihood. According to APADB (2006), approximately there are 1.9 million cattle population in the region, and 90% of the study animals are managed under pastoral production and the rest 10% in agro-pastoral production system. The study area is situated in the North area of the region, Northeastern Ethiopia. It lies around between 13°15′ and 13°30′ 1atitude and 39°39′ and 39°55′ longitude. The high temperature of afar ranges as of 25°C in case of rainy period to 48°C during the dry season [42].
Study Population
The study populations were cattle that had experienced outbreak cases of FMDV and manifested typical FMD clinical signs in the Aba'ala district area during the study period of this research work. The study animals were cautiously inspected for the manifestation of distinguishing clinical signs of FMD such as vesicular lesions around the oral cavity, on the feet, salivation, lameness, anorexia and rise in temperature [43]. All ages and sexes of the study population reared by agro-pastoralists in the outbreak affected kebeles (subunits) of the study district were sampled.
Study Design
Prior to field level investigation and sample collection, district and kebele level animal health expertises were informed to report for regional veterinary laboratory centers when FMD outbreak occurred. Therefore, based on the occurrence of active FMD outbreak case report and active outbreak finding, a cross sectional study was used to collect tissue samples. Clinically, FMD suspected study populations were physically inspected for the manifestation of FMD with typical signs were sampled to collect biopsy samples that were intended for viral isolation, molecular detection and serotype identification purpose.
Sampling techniques and Sample Size Determination
Purposive sampling method was employed to select FMD affected study district, cattle herds, sampling animals as a result of the occurrence of FMD active case reports in the course of the study period, January, 2019 to March, 2020. Accordingly, within the study areas (subunits) animals with clear signs, symptoms and suspected to be infected with FMDV as indicated in (figure 3) were selected and sampled. From all outbreak affected kebeles, 27 swab, epithelial tissue and vesicular fluid samples were collected from clinically FMD suspected animals with active outbreak lesions for cell culture based virus isolation, molecular detection and identification of serotypes circulating in the study district.
Sample Collection and Transportation
Representative active bovine epithelial tissues, vesicular fluid and swab samples were aseptically collected with the help of tissue forceps from un-ruptured and freshly ruptured vesicles of clinically affected animals during the course of field outbreak to isolate the circulating viruses responsible for the occurrence of disease. These collected FMD suspected samples were kept in a sampling bottle containing virus transport medium that has equal volume of 0.04M phosphate buffer saline (PBS) with 50% glycerol enriched by antibiotics and antifungal according to the protocol recommended by OIE [18]. Collected clinical FMDV suspected specimens were transported to laboratory and stored at -20 °C and got transportation to National veterinary institute (NVI) using cold chain for virus isolation, molecular detection and serotype identification purpose.
FMD Virus Isolation
The samples collected were processed and cultured on BHK-21 cell monolayer with three subsequent passages as follows. About 1 gram of each tissue was taken and washed three times using sterile phosphate buffered saline containing antibiotics and antifungal (PBS) on petridish. Then, washed tissues were transferred to sterile mortar, cut into pieces using scissor and minced by scalpel blade. These minced tissues were then grounded and homogenized in sterile sand with a sterile pestle and mortal. Nine ml of PBS was added to the homogenized tissues and well mixed as well as small volume tissue culture made and small amount of five percent antibiotics (penicillin, streptomycin and Amphotericin B solution) containing medium were added so that the final volume was ten times that of the epithelial tissue, producing of ten percent suspension [18]. All procedures were conducted under the Biosafety cabinet level 2. About 1ml of filtered tissue suspension was inoculated on confluent cultured Baby hamster kidney (BHK-21) monolayer cells grown on 25cm2 tissue culture flasks and incubated at 37°C for 1hr for adsorption of the virus. Then, cell cultures were added 8ml of maintenance medium (2% MEM) and incubated at 37 °C and 5% CO2 in a humidified incubator. The appearance of virus induced cytopathic effect (CPE) was observed daily under the inverted microscope. The inoculated cell line was harvested when 85-100% of CPE was observed. These infected cells did not show CPE within 72hrs post infection on the third passage were supposed to be virus negative [18, 44]. Samples that showed typical CPE (positive cases), clinical tissue materials were used for serotype identification of the virus involved in the outbreak cases using antigen detection sandwich ELISA [45].
Serotyping of FMD Virus Isolates
FMD Serotyping was executed using both antigen detection sandwich ELISA and sets of serotype specific primers intended for testing of FMD virus and identifying the serotypes responsible for outbreaks cases. Sandwich‐ELISA was executed with particular combinations of anti‐FMDV monoclonal antibodies (MAb), used as coated and conjugated antibodies. The kit was developed for detection and serotyping of FMDV O, A, C, SAT1 and SAT2. A pan‐FMDV test, detecting any isolates of O, A, C, Asia1 and SAT serotypes, was also included in the kit to complement the specific serotyping of FMDV. The test was implemented based on the manufacturer's instruction and OIE [46]. A total of 13 positive sample suspensions that exhibited FMDV cytopathic effect (CPE) on BHK-21 cell were needed to be tested for detection of serotype identification using sandwich ELISA on a microplate containing 96 wells.
About 25μl of dilute buffer was dispensed into all wells of the test plate, then 25μl of previously diluted samples using ELISA buffer and ready‐to‐use controls was dispensed into the appropriate wells of the test plate pre‐coated with recombinant FMD viral antibody. One positive control for each FMD types O, A, SAT1 and SAT2 and negative controls were included in each plate. The plates were sealed using the enclosed plate sealer and incubated for 1hr at room temperature (20-25°C). After incubation, all fluids on the plates were discarded and the remaining residual fluids were removed. Then 200μl of washing solution were added and incubated for 3min at room temperature, subsequently wells were emptied and the washing repeated twice (three washing cycles in total). Then all residual fluids were removed by tapping on clean absorbent paper and 50μl of conjugate. A was added from columns 1 to 8 and the same volume of conjugate B was added from columns 9 to 12. Plates were covered and incubated at room temperature for 1hour. After incubation 50μl of substrate per well was added to all wells and plates were covered and left at room temperature for 20minutes in the dark. The reaction was stopped by adding 50μl of stop solution (sulfuric acid (H2SO4)). Immediately after stopping, reading the optical density (OD) of each well was done at 450 nm wavelength using micro plate reader.
Molecular Detection of FMD Virus
The presence of FMD viral genetic material in all 27 collected field samples was tested using conventional RT-PCR and specific primers that amplify Viral protein 1 (VP1) of FMDV using the RNeasy Mini Kit following the manufacturer‘s instruction (Qiagen, USA).
FMD Viral RNA Extraction
Total RNA was extracted from collected FMD suspected clinical samples suspension using Qiagen RNA extraction kit following manufacturer’s instructions as [47]. Briefly, 140 mirco-liters of sample suspension was added to 560μl buffer AVL carrier RNA in the mirco centrifuge and vortexed for 15second to mix and then incubated at room temperature (250c) for 10 min. The tubes were briefly centrifuged to remove drops from the inside of the lid. Then, 560μl of ethanol (70%) was added to the sample and mixed by pulse vortexing for 15 seconds followed by centrifuging to remove drops from the inside lid. Then, 630μl of the solution were applied to the QIAMP Mini-spin column in a 2ml collection tube and centrifuged 12,500rpm for 1min. The filtrate was discarded and the column was placed in a fresh 2ml collection tube. Then, 500μl of buffer AW2 were added and centrifuged at 12,500rpm for three min and the filtrate was discarded. Next, 65μl of Buffer AVE was added to the column equilibrated at room temperature for one min and centrifuged at 12,500 for 1min. Using reverse transcription polymerase chain reaction (RT-PCR) and specific primers set FMDV7-forward (FMDV7F) and FMDV7-reverse (FMDV7R) as depicted in (table 1), extracted RNA samples were detected for the presence of FMDV.
Table 1: The Universal primers and thermal cycle used for amplification of FMDV
Primer-FMDV7 universal-Forward 5pm/μl 5ʹ- GCCTGGTCTTTCCAGGTCT-3ʹ
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Primer-FMDV7universal-Reverse-5pm/μl 5ʹ-CCAGTCCCCTTCTCAGATC-3ʹ
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|
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Temperature
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Time
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Cycle
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Initial denaturation
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95
|
5min
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1 cycle
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1st denaturation
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94
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1 min
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35 cycles
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Annealing
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54
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1 min
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Elongation
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72
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1 min
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Final elongation
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72
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10min
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1 cycle
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Agarose Gel Electrophoresis
The PCR products were analyzed on the prepared 1.5% Agarose gel by adding 4μl gel red with loading dye and then the PCR product were loaded in the volume of 10μl in each well and 10μl molecular marker (ladder) was added started 100bp plus. Electrophoresis was run for one hour at 120V. Then, the DNA band was visualized by UV illumination, using desktop according to the base pair (bp), and then the size was determined and documented.
Data Management and Statistical Analysis
Data generated from laboratory investigations were recorded and coded using Microsoft Excel spreadsheet and analyzed using STATA version 14.2 for Windows. Cell culture results, CPE development and molecular characterization results were recorded and tabulated. Virus isolation and molecular detection of FMD virus were elaborated using descriptive statistics analysis. Moreover, regarding the molecular characterization, the banding patterns of individual sample were scored based on the presence or absence of the bands with the appropriate base pairs.