Three IBV field strains (coded, IBV/MN, IBV/RA and IBV/TU), were isolated from trachea, lungs and kidney of broiler chickens suffering from the specific clinical signs of IB, and were previously characterized to belong to Italy02 serotype at laboratory of Biopharma between 2010- 2014 . Phylogenetically, the three strains of Italy02 genotype branched and clustered with Spanish genotypes, are very closely related to Italy 497/02-1 (98.9 %); Spain/05/866 (98.9 %); and Spain/04/221 (97.4 %). The sequences of the three strains have a S1 gene nucleotide identity ranged between 96.9 % and 98.7 % when compared to each other.
These IBV strains were propagated by inoculation in 9- to 11-day-old SPF egg as described by Owen et al . For virus titration 6-fold dilutions were inoculated into the allantoic cavity of six SPF chickens embryos, and incubation during six days at temperature of 37 °C +/- 2 °C and relative humidity of 75 % +/- 10 %. The titer is calculated following the Reed and Muench method .
A total of 40 one day old SPF chickens, used for experimental infection were provided from the SPF chickens flock unit of Biopharma Laboratory, Rabat, Morocco.
In order to evaluate the pathogenesis of three Moroccan viral strains, an experimental infection study was performed in SPF chickens obtained from SPF chicken flock Unit of Biopharma, Rabat, Morocco. For this purpose, 40 one-day-old SPF chickens were housed in separate isolators with negative-pressure, the bright and thermal program was the same for all birds, and water and food were provided at will. Chickens were randomly divided into four groups of 10 birds (30 chicks in the experimental and ten chicks in the control group). The infection trials were approved by the local ethics committee of Biopharma (Biopharma Ethics and Scientific Committee) directed by its veterinarian in charge, with requirement of formal ethics approval referenced as N° 412/SB/2015, and were carried out following strict animal welfare guidelines.
Using a micropipette, chickens in the three groups were oculo-nasally administrated with 50 μl of inoculum containing 103.5 (EID50/ml) of virus strain, and control group was inoculated with phosphate-buffered saline (1XPBS). The specific clinical signs of IB were recorded at a rate of 2 times daily by the same observer during 14 days. Specific clinical signs of IB recorded by the same observer have included gasping, coughing, sneezing, depression, rales, and ruffled feather. Three chickens from each group were killed at 5dpi; one control animals and their tissues of trachea, lung and kidney were examined for gross lesions, and were collected for histopathology examination. Serum samples were tested for the presence of antibodies against IB using the ELISA test. Also the tracheal rings from each bird were obtained and placed in cell culture medium (D-MEM supplemented with 10 % of foetal calf serum) to evaluate tracheal ciliary activity at 5 and 12 dpi. Treatment organs of lung, trachea and kidney by the antibiotics and frozen at -80 °C were used for virus re-isolation and real time RT-PCR detection. At 14 dpi, all the birds were euthanized and necropsied for gross lesions examination before their elimination.
Sacrificed birds at 5 days of age were necropsied and gross lesions were recorded. Tissue samples from the trachea and lungs were collected and fixed in 10 % neutral-buffered formalin. They were processed for histopathological examination according to standard methods. They were dehydrated and embedded in paraffin wax. Five μm thick sections were stained with hematoxylin and eosin and examined under light microscope. Microscopic lesions in respiratory tissues were recorded and scored as described previously , from 0, 1, 2 and 3 (no, mild, moderate and severe, respectively). A mean of total severity index was then attributed to each IBV strains based on scores from 3 birds.
Ciliostasis: tracheal ciliary activity
Several parts of tracheal rings were prepared from each infected bird at 5dpi that coincides with the pic of the clinical signs. At 12 dpi, the ciliary activity was also evaluated from all the infected birds that have recovered. The prepared tracheal rings were immersed immediately in cell culture medium (D-MEM supplemented with 10 % of foetal calf serum), were microscopically analyzed for estimating the ciliary movement in tracheal infected and in tracheal control, and were scored on a scale from 0 (100 % activity) to 4 (no activity) . The mean score of each strain was then compared to evaluate the respiratory pathogenicity.
All individual samples of trachea, lung and kidney collected at 5 dpi from IBV-infected chickens, were immediately suspended in phosphate-buffered saline (1XPBS), treated by Gentamycin antibiotics (50 μg/g) and clarified by centrifugation at 2500 × g at 4 °C for 25 min. The homogenates samples were used and processed for IBV re-isolation as previously described . It’s was performed by inoculation in allontoic sac route of 9–11 day old eggs, with 100 μl of homogenates and incubation during 6 days (using five eggs by sample) at temperature of 37 °C +/- 2 °C and relative humidity of 75 % +/- 10 %. The embryos were examined for the presence of dwarfism and hemorrhagic traces (on the whole body of the embryo). The RT-PCR detection of viral RNA was performed in parallel with the homogenates tissues and the clarified allontoic fluid.
Extraction of viral RNA
Viral RNA was extracted from 200 μl of both organs samples and allantoic fluid (AF) using the Kits Macherey-Nagel according to the manufacturer’s instructions. Each RNA fraction was eluted in 50 μl of RNase-free water, and subjected to reverse transcriptase RT-PCR amplification.
Real time RT-PCR
A one step Real time RT-PCR was carried out using Invitrogen kit (SuperScript® III Platinum, Life Technologies, USA). The synthesis of the cDNA first strand was performed using 5 μl total viral RNA primed with a universal pair of primers a) downstream primer, AIBV-fr, targeting N gene nucleotide positions 811–832 (5′-ATGCTCAACCTTGTCCCTAGCA-3′); and b) upstream primer, AIBV-as, targeting N gene nucleotide positions 921–941 (5′-TCAA-ACTGCGGATCA-TCACGT-3′), and the probe TaqMan targeting N gene nucleotide positions 848-875 (5′FAM-TTGGAAGTAGAGTGACGCCCAAACTTCA-3′Tamra) .
The amplifications reactions (PCRs) were performed on a Smart Cycler. The following mix of each reaction was contained: 12.5 μl 2 × RT- PCR buffer mix, 0.5 μl MgSO4 (50 mM), 0.5 μl Rox (25 mM), 4.75 μl nuclease free water, 0.5 μl M-MULV reverse transcriptase enzyme (200U), 0.5 μl primers to a final concentration of 10 μM, 0.25 μl probe to a final concentration of 10 μM and 5 μl RNA template. The reaction was carried out in StepOneTM Plus real-time PCR system (Smart cycler Cepheid, USA) at 50 °C for 15 min, 95 °C for 5 min, and 40 cycles of 95 °C for 15 s and 60 °C for 45 s. All reactions amplifications were recorded, analyzed, and the threshold cycle (Ct) determined with the StepOne software (Smart Cycler).
An enzyme-linked immunosorbent assay (ELISA) commercial kit (FlockChek (IDEXX Laboratories, Inc., Westbrook, ME, USA), was carried out on serum samples collected from experimentally infected birds for antibodies detection at 5 and 14dpi. The test was performed according to the manufacturer’s instructions. Moreover, serology on SPF chicks of one day from the same rearing was also performed to ensure the negative status anti-IBV chicks antibodies used.