Assessment of pathogenicity and tissue distribution of infectious bronchitis virus strains (Italy 02 genotype) isolated from moroccan broiler chickens

Clinical signs and gross pathological findings

Nasal discharge and watery eyes were also observed but were transient in some of infected by IBV/RA and IBV/MN chicks. These clinical symptoms were persisted in all groups until 12dpi. The birds infected by IBV/MN and IBV/TU appeared lethargic, reluctant to move (Fig. 1), whereas, chicks infected with by IBV/RA were not as apathetic. At autopsy, all infected chicks that were killed at 5 dpi to prevent their suffering were examined for the macroscopic lesions in the trachea, lung and kidney. These gross lesions consisted in hemorrhagic tracheitis, mucosal congestion and catarrhal exudates that mainly progressed. For the three strains used, the signs were observed in the most infected chicks with dominance in the lungs that were hemorrhagic and sometimes cyanotic at one lobe. Therefore, samples were taken at 14 dpi just for a macroscopic examination of organs to confirm whether the absence of clinical signs is correlative with the gross lesions. Whereas, gross lesions of kidney, were not observed in all inoculated chicks. During the experiment, the non-infected control group stayed normally without clinical signs or gross lesions. The statistical analysis was not performed as the three tested strains are phylogentically related to each other and the difference in clinical and tissues scores is not very significant.

Histopathological examination

The microscopic tissues analysis was performed in the euthanized chicks at 5 dpi. This period correlates with the maximum intensity of clinical signs (which begins between 2 and 7dpi). These signs fade from the 7dpi with a total restoration at 12 dpi, that why we only conducted the macroscopic examination at this time. Histopathological lesions developed at 5dpi in the three groups of experimentally infected chickens, were compatible of those previously described for infectious bronchitis virus [16]. And their scoring was mainly correlating with the ranking and scoring described in previous experiments [17].

In the trachea, most detected lesions in all infected groups were characterized by mucosal thickening, hyperplasia of the surface epithelium, loss of cilia, and mononuclear inflammatory cell infiltrate (lymphocytes, plasmocytes, and macrophage) of lamina propria (Figs. 2a and b). Mean total score of severity was 3,7; 4,7 and 6,7 in birds infected by IBV/MN; IBV/RA and IBV/14/TU strains respectively. This indicates that IBV/TU strain seems to have a higher degree of pathogenicity than the other tested strains (Table 4).

	Lesions		Mean lesion scores in IBV infected groups of birds
			IBV/MN	IBV/RA	IBV/TU
Trachea	Epithelium	Thickening	1	1,3	2
		Hyperplasia (1-4 +)	1	1,3	2,6
		Loss of cilia	0	0	0,6
		Degenerescence and necrosis of epithelial cells	0	0	0
		Inflammatory exudate (degenerate epithelial cells, Lc, heterophyles) on the surface	0	0,3	0,6
	Lamina propria	Oedema,	0	0,6	1
		Diffuse infiltration by mononuclear inflammatory cells (Lc, Plc, Mph)	1,3	1,3	1,3
		Mean total Score of severity	3.666	4.666	7,666

IBV infectious bronchitis virus

SPF Specific pathogen free

IBV/TU infectious bronchitis virus sampled from Abda-Doukkala region

IBV/RA infectious bronchitis virus sampled from Rabat-Sale-Azzemour Zaire region

IBV/MN infectious bronchitis virus sampled from Meknes Tafilalet region

In the lungs, lesions in all infected groups were mainly confined to the primary and secondary bronchi and included epithelial hyperplasia, loss of cilia, and heterophils and mononuclear inflammatory cell infiltrate (lymphocytes, plasmocytes and macrophages) of the lamina propria and peribronchial space (Fig. 3a and b). Other changes were located in the atria and parabronchi and consisted of hypertrophy and/or hyperplasia of pneumocytes. The score of these changes were from mild to moderate degree of severity and there was no big differences in the mean total score of severity among tissues infected with the 3 tested IBV strains (7,3; 8,3 and 6), thus indicating no differences in the pathogenicity of the three strains for the lung (Table 5). However, no kidney lesions were observed in chicks infected with the three tested strains. Tracheas and lungs of control chicks samples throughout the experiment, showed normal histological features.

	Lésions		Mean lesion scores in IBV infected groups of birds
			IBV/MN	IBV/RA	IBV/TU
Lung	Primary & secondary Bronchi	Epithelial hyperplasia	2	1,7	1
		Loss of cilia	1	1	0,7
		peribronchial infiltration by mononuclear inflammatory cells	2	1,3	0,3
		Infiltration of lamina propria by heterophils & mononuclear inflammatory cells	2	1,3	0,3
	Atria & parabronchi	Hypertrophy of pneumocytes	0	0,7	1,3
		Epithelial hyperplasia	0	0,7	1,3
	Inter-capillarysepta	hyperemia	1	1	1
		Mean of total Score of severity	7.333	7.666	6

IBV infectious bronchitis virus

SPF Specific pathogen free

IBV/TU infectious bronchitis virus sampled from Abda-Doukkala region

IBV/RA infectious bronchitis virus sampled from Rabat-Sale-Azzemour Zaire region

IBV/MN infectious bronchitis virus sampled from Meknes Tafilalet region

Ciliostasis: tracheal ciliary activity

Ciliary activity was observed in all tracheas of infected birds at 5dpi. But the severity differed between the three strains tested; the ciliostasi caused by IBV/MN and IBV/RA strains was 25 % and 50 % for IBV/TU strain, compared to the control group (100 %). However, a partial recovery of ciliary activity in infected birds was observed at 12dpi.

Viral re- isolation

Results related to IBV distribution was checked in all tissues samples collected at 5dpi of all infected chicks. The virus was found in the trachea, lung of all strains, but in the kidney of one strain (IBV/MN) with low viral load. The effect of the virus after one passage in SPF 10 old embryonated eggs showed dwarfism and hemorrhagic spots in all over the body of the embryo, which are considered pathognomonic signs of IBV (Fig. 4).

Serological response

Antibody titers against Infectious Bronchitis (Anti-IBV) in the serum collected from chickens of different groups on days 5 and 14 dpi was measured by ELISA test. In the all infected group, serum samples were negative at day 0 and 5 dpi but antibodies titers was increased at 14dpi. Serum of the control chicks was free from specific antibodies against IBV on the entire dpi.

Viral strains

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 [15]. 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 [27]. 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 [28].

Chickens

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.

Experimental infections

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 10^3.5 (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.

Histopathological examination

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 [17], 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) [29]. The mean score of each strain was then compared to evaluate the respiratory pathogenicity.

Viral re-isolation

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 [30]. 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) [31].

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).

Serological response

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.