Avian influenza virus (AIV) infects different types of birds, including chickens, causing respiratory or systemic diseases that vary in severity and cause heavy losses to the poultry industry in the world (Goudrazi et al., 2013). This disease is caused by type A influenza virus that belong to family Orthomyxoviridae. To date, 18 H subtypes have been recognized (H1 to H18) and 11 N subtypes (N1 to N11) (Tong et al., 2013). Possibly the most significant problem facing the poultry industry in the world at this time is the spread of AIV serotype H5N1. Another underestimated challenge is the extensive spread of AIV serotype H9N2 in Asia. Lately, many countries in the Middle East were reporting a low pathogenic AIV H9N2 in their commercial flocks especially in Saudi Arabia, Jordan, Israel and Egypt (Monne et al., 2007; Golender et al., 2008; Afifi et al., 2012). H9N2 viral infection in domestic poultry may lead to diverse clinical manifestation of varying severity depending on the strain of virus, route of entry, host species, age of host, immune status, presence of secondary pathogens and management (Dash et al., 2015). Clinical sign in H9N2 infected chickens include nasal discharge, gasping, conjunctivitis, facial edema, reduced feed intake and mortality (Nili & Asasi, 2003; Lee et al., 2011). Based on biological methodologies, real-time RT-PCR (RRT-PCR) were developed in the last decade for rapid detection of influenza viral RNA in clinical and laboratory specimens (Rahman et al., 2015). At the same time, previous phylogenetic studies of H9N2 hemagglutinin, have focused on sequences from a single location especially on surface glycoprotein hemagglutinin (HA) (Fusaro et al., 2011; Dalby & Iqbal, 2014). In Egypt, the recent emergence of H9N2 virus from clinically healthy commercial bobwhite quail flock was reported in May 2011 (ElZoghby et al., 2012). Then, the virus became endemic in Egypt and continuously isolated from breeders, layers with drop in egg production and broilers (Abdel-Moneim et al., 2012; ELbayoumi et al., 2013; El Miniawy et al., 2014; Dabbour, 2015). In addition, sero-surveillance data indicated occupational exposure of humans to H9N2 in Egypt (Gomaa et al., 2015). In January 2015, the first human H9N2 case was reported from Egypt (EMPRES, 2015). According to phylogenic analysis, all H9N2 AIV viruses isolated from broilers farms science its emerging till today, grouped in the G1/97-like lineage in one group with Israeli strains and other related strains that circulating in the Middle East (Ahmed et al., 2013; Naguib et al., 2015). So, the present study was conducted to ascertain the pathogenicity and infectivity of the new Egyptian isolates of AIV H9N2 isolated from broiler chickens under experimental conditions.
2. Materials and methods
Samples and virus isolation. Oropharyngeal swabs and organ samples collected from 100 broiler flocks in the Egyptian governorates of Alexandria, ElBheira, El-Gharbia, Kafr Elsheikh, El-Fayoum, ElMmenia and Assiut from the period from May 2014 till March 2015. The collected samples were placed into 3 mL of transport media consisting of 10% glycerol, 200 U of penicillin per mL, 200 mg of streptomycin per mL, 250 mg of gentamicin per mL, and 50 U of nystatin per mL (OIE Manual, 2014). At the time of sampling, affected birds showed respiratory signs and depression with daily mortality rate within the flocks ranging between 1% and 5%. A 0.2 mL aliquot of the sample suspension was inoculated into the allantoic cavity of 9 - 11 day-old embryonated chicken eggs. The eggs were incubated at 35 C for 2–3 days and subsequently the allantoic fluid was harvested. Allantoic fluids were tested for the presence of hemagglutinating viruses by the HA assay using 0.5% chicken erythrocyte suspension.
Viral RNA extraction and RT-PCR. The extraction of viral RNA was conducted from a virus containing allantoic fluid using a spin column purification kit (Qiagen, Valencia, Calif., USA). All allantoic fluids were examined for AIV H9N2 subtype by real-time reverse transcription-PCR (rRT-PCR) using Quantitect probe RT-PCR kit (Qiagen, Inc. Valencia CA) on a real-time PCR machine Stratagen MX3005P machine (Stratagene, USA).
Molecular detection and characterization. HA gene Partial sequencing for 7 positive AIV H9N2 isolates was performed by purification of PCR products using QIAquick PCR Product extraction kit (Qiagen, Valencia). The sequence reaction was done using Bigdye Terminator V3.1 cycle sequencing kit (Perkin-Elmer) and the sequence reaction was purified using Centrisep spin column (ABI, USA). DNA sequences were obtained using Applied Biosystems 3130 genetic analyzer (ABI, USA). A phylogenetic tree was created by the MegAlign module of Lasergene DNAStar. Nucleotide sequences were aligned and phylogenetic analysis was performed using the neighbor-joining method in the Molecular Evolutionary Genetics Analysis (MEGA5) program.
Virus used in the experimental challenge. H9N2 LPAIV Egyptian isolate (A/Chicken/Alex/2016) that used in this study was randomly selected from the Egyptian H9N2 viruses that isolated in this study. Virus was propagated in the allantoic cavities of 9- 11 days old fertile specific-antibody-negatives (SAN) chicken eggs. The allantoic fluid inoculum used in this study had a titer of 106 embryo infective dose (EID50)/mL, and it was tested by reverse transcription polymerase chain reaction to make sure it was negative for other common avian pathogens, such as Newcastle disease virus, infectious bronchitis virus and AIV H5N1 virus.
Source of chickens. Broiler chickens (cobb type) that carrying a significant level of MDA to H9N2 AIV, representative of the common situation in Egypt, were obtained from a commercial hatchery at 1 day of age.
Experimental design. Twenty broiler chicks were divided in two equal groups (10 chicks/group) at one day of age and chicks were reared in controlled environment conditions. At 21 days of age one group of broilers was infected by dropping of 50 ul of AIV H9N2 inoculum (106 EID50/ml) in both nostrils and both eyes and the other group served as negative control group.
Clinical signs and gross lesions. All the chickens were observed daily for signs of disease and mortality for 14 days' post challenge. Birds and feed in all groups were weighted at the age of 21st, 28th and 35th day old (end of the experiment) to determine the body weight and food conversion ratio "FCR". Necropsy was performed on each dead chicken, and lesions were recorded.
Virus shedding. Oropharyngeal and cloacal swab samples from experimental chickens were collected at 4 th, 7th, 11th and 15th days post challenge in sterile phosphate buffered saline (PBS) with 2% antibiotic solution of penicillin streptomycin-neomycin and immediately frozen and kept at -80°C till examined by qRT-PCR for detection and titration of challenged AIV H9N2 virus.
3.9. Viral RNA copy number quantification in oropharyngeal (OR) and cloacal (CL) swabs. RNA extraction from swabs was carried out by QIAamp viral RNA extraction kit (Qiagen, Valencia, Calif., USA) as per manufacturer’s protocol. Viral RNA copy number quantification was done by qRTPCR using 1μl of eluted RNA.
3.10. Serology. At 8th, 21st, 28th and 35th day of age, 1.5 cm blood sample from the wing vein was collected from 5 birds/group in tubes, serum was separated and haemagglutination inhibition (HI) test was performed as per standard protocol according to OIE Manual, (2014). In this test 1% (v/v) chicken red blood cells and 4 haemagglutination units (HAU) of H9N2 virus were used. Known positive and negative sera were used as controls.
3.11. Histopathological examination. One chicken from both challenged and non-challenged groups was sacrificed and subjected to throughout necropsy at 4th, 7th and 11th day post challenge. Trachea, kidneys, bursa of Fabricius and thymus were collected and kept in 10% buffered formalin for 24 hr. Tissues were processed, embedded in paraffin, and stained with hematoxylin and eosin according to routine histologic techniques (Bancroft et al., 2012).
In the current study, the positive commercial broiler flocks with AIV H9N2 infection were 32% from 100 examined flocks. LPAI H9N2 was isolated in six out of seven governorates. Upper Egypt governorates recorded the highest incidence (35.70%) while Lower Egypt recorded 30.60% from the total examined flocks. Alexandria has the highest rate among the incidence of H9N2 virus infection (47.80%) then Assuit (36.64%), El-Menia (35.70%) and finally El-Fayoum (33.33%), While Lower Egyptian governorates showed low incidence of LPAI H9N2 infection in comparison to Upper Egypt, in which, El-Behira recorded 25.80% and El-Gharbia recorded 21.40% (Table 1).
Table (1): Results of AIV H9N2 prevalence among tested broiler flocks from different governorates in Egypt:
Table (2): Seasonal incidence of H9N2 flocks during 2014 – 2015:
Partial hemagglutinin (HA) gene (segment 4) sequencing was conducted in the Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP) to investigate the nucleotide sequence of the H9 gene in order to identify the pathogenicity and genetic characterization of the isolated H9N2 AIV strains (Table 3).
After the challenge, tracheal rales started to be evident in the H9N2 AIV-infected broiler group 3 days post infection and reached to its peak at 7th day (89% from challenged birds) and continued till the end of observation period associated with depression and ruffling feather (89% & 78%, respectively) from the challenged birds. there was greenish and whitish diarrhea in 30% from the challenged birds (at 5th & 6th DPI) and continued to the end of observation period (14th DPI). Mortality started at 6th days and continued till the 8th DPI (20%) (Table 4). While the negative control group had no remarkable signs or gross lesions.
Table (3): Total number of selected H9 isolates for sequencing in this study:
Phylogenetic analysis of the HA gene showed that the seven Egyptian isolates of H9N2 were grouped in the Qa/HK/G1/97 lineage, which is similar to the viruses circulating in the Middle East, with close phylogeny to the Israeli viruses (Fig. 1).
Fig. (1): Nucleotide phylogenetic tree of H9 protein of the seven H9N2 AIV isolates analyzed in this study (Marked) and other H9N2 strains published in GenBank.
Table (4): Results of daily observation for clinical signs and mortality for challenged group:
Average body weight (g). It's clear that H9N2 infection has pathogenic effect on the average body weight of broiler chickens, at 28 days of age (7 days post challenge). The difference in average body weight between infected group and negative control group was about 160 g (14%) and 290 g (17%) at the end of the experiment (35 days) (Table 5).
Feed conversion ratio (FCR). There was great difference in the FCR between the infected and none infected group after the challenge with the LPAI H9N2 isolate. Seven days post challenge, the FCR was increased in the infected group with percentage 84 % when compared with negative control group. While, FCR in the infected group was 46 % higher than negative control group during the whole experiment (Table 6).
Fecal shedding. Challenged group started to shed the H9N2 virus in the feces beginning from the 4th day post challenge (33.3 % from challenged individuals). At 7th day post challenge, the infected group (66.6 % from its individuals) shed the virus in the feces (1.114 x103 copies / 200 µl dilution). At 11th post challenge, the viral genome was detected in the fecal samples of infected group in 33.3 % from challenged birds (5.8 × 102 copies / 200 µl dilution) (Table 7).
Tracheal shedding. The H9N2 virus genome was detected in the trachea of all challenged individuals at the 4th day post challenge (1.152 x 104 copies / 200 µl dilutions). At 7th day post challenge, the amount of the shed virus reached to its highest amount (2.589 x 104 copies / 200 µl dilutions). At 11th and 15th day post challenge, the amount of the virus was 7.89 x 103 & 5.2 x 102 copies / 200 µl dilutions, respectively, (Table 7).
Table (5): Average body weight (g) of challenged and none challenged groups at 21, 28 and 35 days of age
Table (6): Feed conversion rate (FCR %) of the challenged and none challenged groups.
Table (7): Detection and molecular quantitation of H9N2 AIV in tracheal and cloacal swabs from challenged birds in different days post challenge
Both groups of chickens were negative, and they had GMTs of < 21 day before infection. But at 7 days and 14 days post challenge, the infected birds had a GMT 24.2 and 24.5 , respectively, continued to raise till 14 days post challenge, whereas the negative control group remained negative (GMT < 21 ) (Table 8).
Histologically, at 4th day post challenge, tracheal epithelium in the birds from infected group showed degeneration (A1) which extended to desquamations and sloughing of the tracheal mucosa at 7th day (A2) then leukocytes infiltration at the 11th day post challenge (A3) (Fig. 2). At the same time, vacular degeneration, necrosis in the tubular epithelium was seen in the kidneys of infected birds that ended with interstitial edema at days 4th, 7th and 11th day post challenge (C1), respectively (B1, B2 & B3, respectively) (Fig. 2). The bursa of Fabricius showed slight necrosis at 4 th day post challenge, degeneration in the follicles at the 7th day post challenge (C2) and ended with depletion and necrosis in the lymphoid cells at 11th day post challenge (C3) (Fig. 2). While thymus of infected birds showed hemorrhage, necrosis at 7th day post challenge (D1) and the pathological changes continued with necrosis in the lymphoid cells in the thymic lobule at 11th day post challenge (D2) (Fig. 2).
Table (8): Results of geometric mean of HI titers for challenged and none challenged group with H9N2 virus:
Fig. 2: Histologic changes in chickens 15 days after infection with AIV H9N2 (H&E× 400). (A1) trachea from infected birds at 4th day PC: deciliation and degeneration of the tracheal epithelium. (A2): trachea from infected birds at 7th day PC: desquamation and sloughing of epithelial cells lining the trachea. (A3): trachea from infected birds at 11th day PC: widely dilated blood mucosal blood vessels with leukocytes infiltration. Necrotic debris is evident in the tracheal mucosa. (B1): kidney from infected birds at 4th day PC: beginning of vacular degeneration of the tubular epithelium. (B2): kidney from infected birds at 7th day PC: degeneration and necrosis of the tubular epithelium. (B3): kidney from infected birds at 11th day PC: necrobiotic changes of the renal tubules. (C1): bursa from infected birds at 4th day PC: depletion and necrosis of the lymphoid cells in the bursal follicles. (C2): bursa from infected birds at 7th day PC: multifocal vaculation of cortex & medullary zone with lymphocytes depletion and degeneration. (C3): bursa from infected birds at 11th day PC: depletion and necrosis of the lymphoid cells beside multiple cortical and medullary vacculation. (D1): Thymus of infected birds at 7th day PC: hemorrhage in the interstitial tissue beside necrosis of the lymphocytes. (D2): Thymus of infected birds at 11th day PC: necrosis of lymphoid cells in both cortical & medullary zone of the thymic lobule.
It seems that H9N2 has been circulating in an undetectable manner in Egypt since a serological evidence of H9 spreading has been recorded in 2009 - 2010 (Afifi et al., 2012). Since then, Avian Influenza H9N2 outbreaks in commercial poultry industry were reported by several researchers' from Egyptian poultry flocks (Soliman, 2014; Dabbour, 2015). So, the present investigation determined the pathogenicity and infectivity of an Egyptian H9N2 virus isolated during a disease outbreak in 2015. Firstly, from the current study, by application of RRT-PCR, it was noticed that the pattern of H9N2 incidence among commercial broiler chickens was dramatically increased in Egypt in which, out of 100 tested flocks, there were 32 positive cases (32%) during the period from June 2014 till February 2015. Some recent studies in Egypt indicated that there was an increase in the incidence of H9N2 infection among broiler flocks in which the isolation rate varied from 10 % (Shalaby et al., 2014), 15 % (Soliman, 2014) and up to 55 % (Dabbour, 2015). Also, some of these positive H9N2 cases were isolated from flocks suffering from severe respiratory manifestations with mortality which approached 5% in some tested flocks at the time of sampling. Although H9N2 viruses are characterized as LPAI viruses, they may cause high morbidity and mortality (Arafa et al., 2012). Low biosecurity levels in the farms, insufficient measures and facilities to restrict the movement of poultry from infected areas to none infected ones, unorganized backyards and rooftop rearing as well as live bird markets, byproducts, manure from infected flocks to other susceptible birds, allow H9N2 AIV disease to be distributed all over Egypt among broiler chickens (Ahmed et al., 2013). The obtained results showed that circulation of the H9N2 virus were higher in cooler temperatures in the winter season of 2014 & 2015. Previous studies have shown the high prevalence of H9N2 AIVs infection in winter and spring rather than in summer in chicken farms (Chen et al., 2012; Zhao et al., 2015).
Our analysis confirmed that the H9N2 virus population circulating in the countries bordering Egypt may be considered also the main source for the viruses detected in the country in which, the topology of the phylogenetic tree showed that the HA sequences of the seven H9N2 Egyptian tested samples showed identity with a close relationship between Egyptian and neighboring Middle Eastern as well as Israel H9N2 isolates and identified an ancestor relationship to low pathogenic H9N2 Quail/HK/G1/1997 prototype. These results also resemble the results obtained by (Arafa et al., 2012). According to Kandeil et al. (2014) the emergence of H9N2 in Egypt might have been due to the importation of this virus through wild birds, legal or illegal trade of poultry, or another unidentified mechanism.
Previously, it was widely believed that H9N2 viruses can cause infection in chickens without any overt clinical signs of the disease, but instead farm conditions together with bacterial or viral co-infections, age and breed of chickens, were important contributing factors for higher morbidity and mortality that associated with field H9N2 infection (Nilli & Asasi, 2002; Kishida et al., 2004). In our study, the AIV H9N2 was more pathogenic to broilers, and it produced more severe gross and histologic changes in the respiratory, urinary and lymphoid organs. There was respiratory manifestations in the form of rales, nasal discharge, sneezing, conjunctivitis, facial edema and mouth breathing beside depression and ruffling feather began from 3rd day and continued to the 12th day PC. The high frequency of challenged birds showing respiratory manifestations could be a result of the efficient viral dissemination in an infection site that is rich in trypsin-like proteases. These enzymes enable the virus to replicate easily in the epithelial tracheal cells as well as upper respiratory tracts (Shaib et al., 2011). Also, the cumulative mortality in the challenged group with A/chicken/Alex/2015/H9N2 Egyptian isolate was 20% when compared with negative control group which confirmed the low pathogenic nature of H9N2 AIV virus. Mild tracheal rales, respiratory distress, nasal and ocular discharge, coughing and sneezing in broiler chickens were reported by many workers (Seifi et al., 2012; Iqbal et al., 2013). In some other studies mortality was not reported in experimental chickens with LPAI H9N2 infection (Bijanzad et al., 2013). This variation in the severity of the symptoms as well as level of mortalities either in experimental of field infection possibly because of difference in the amount or the virus in the inoculation dose, genetic variation in the virus isolate or could be due to either these previously experiments were done on SPF or commercial broiler chickens, the presence or absence of co-infection with other pathogens either bacteria or viral infection.
In the present investigation, it was noticed that the challenged birds with LPAI H9N2 isolate began to shed the virus at 4th day post challenge. Virus quantity in the cloacal shedding reached to its peak level at 7th day post challenge, and then the copy number of the virus decreased at the 11th day PC till it stopped completely at 15th day post challenge. Broiler and SPF chickens shed the H9N2 virus in cloacal swabs up to 9 day post inoculation (Gharaibeh, 2008). The dissemination and adaptation of H9N2 to the digestive tract cells which represented by the number of challenged birds showed diarrhea or presence of H9N2 virus with high level in the feces of challenged birds could be an indication that the virus acquired new tissue tropism towards enteric cells that have a mixture of furin-like and trypsin-like proteases (Swayne et al., 2013). Interestingly, it was noticed that tracheal shedding of the H9N2 virus strongly correlated with the severity of clinical symptoms that appeared on the challenged birds as well as the lesions found in the upper respiratory tract, lesions which were mostly seen in the nasal cavity (rhinitis) and tracheal sections (tracheitis) (Morales et al., 2009). Earlier oral shedding as compared to cloacal shedding was also observed in experimentally infected chickens (Dash et al., 2015). Due to the high shedding of H9N2 viruses in oral swabs, Guo et al. (2000) assumed that these viruses are transmitted mainly by aerosol and, to a lesser extent, in feaces.
The most important pathogenic effect of H9N2 in this study was the significant decrease (290 g) in the average body weight of the infected broiler group compared to the control group. These results were probably because of the effect of viral infection on pancreatic tissue which resulted in impairment in pancreatic enzymes production essential for efficient digestion which affected the food digestion and nutrients utilization (Subtain et al., 2011).
Histopathological results revealed that the Egyptian H9N2 isolate is epitheliotropic especially toward respiratory, urinary and lymphoid organs. The trachea showed hyperemia and edema, followed by deciliation, necrosis, and sloughing of the epithelial cells at 7th day post challenge which associated with the peak of the disease. But as noticed, there was an improvement in the severity of the histopathological changes in trachea at 11th day post challenge which associated with an improvement in the clinical manifestations. El Miniawy et al. (2014) noticed that the tracheal lesions in SPF chicken group challenged with LPAI H9N2 at 28th day of age were most prominent on the 9 th day and subsided by 14th day of experiment. The most predominant histological lesions in the kidney of challenged bird with AIV H9N2 were severe inflammation and focal lytic necrosis which associated with infiltration of lymphocytes in the renal tissues as well as tubule-interstitial nephritis, especially at 7th and 11th day post challenge. These results were close to the results of (El Miniawy et al., 2014) who said that renal lesions observed in infected group with Egyptian isolate of LPAI H9N2 were in the form of tubule-interstitial nephritis.
In the present study, the predominantly histologic changes in the bursa of Fabricius were congestion, edema and apparent hemorrhages, vacculation, lymphocytes depletion and degradation in the immune organs were seen in the thymus and bursa in the challenged birds with the Egyptian isolate of H9N2 at 7th and 11th day post challenge which showed that damage and dysplasia of immune organs are possible after H9N2 infection. Furthermore, thymic lobular dysplasia was seen, as was thinning of the cortex, fewer lymphocytes in the medulla, increased size of epithelial reticular cells, and invasion of fat cells of the interlobular connective tissue into the thymus substance, all of which lead to degradation of the thymus. These pathological changes in the immune organs could be an indication of the involvement of immune system as well as the immunosuppressive effect of H9N2 subtype on broiler chickens in Egypt. Our results were similar to the lesions observed in the thymus and bursa of Fabricious in chickens inoculated with 106 EID50 intra-nasally per bird of H9N2 avian influenza virus at 20 days of age (Hadipour et al., 2011b). Also, thymic lobular dysplasia, as well as atrophy in the bursa was reported after the inoculation of H9N2 in broiler chickens at 8th day of age (Qiang & Youxiang, 2011). According to Pazani et al. (2008), presence of the mentioned histologic changes in the thymus and bursa of Fabricious confirmed that the H9N2 is viremic in nature, or possibly secondary to stress or as a result of activation of commensal bacterial infection. Conversely, Doustar et al. (2012) suggested that the H9N2 LPAI virus might be disseminated to the lymphoid organs of the infected chicken through the blood or lymph vessels. But of course, further studies are needed to show such effect of H9N2 subtype on immune system of infected chickens in Egypt.
This study demonstrated the pathogenic nature of the local Egyptian H9N2 isolate. This disease should be taken seriously by broiler producers in Egypt and the region, and the chicken industry should vaccinate broilers against this disease. In addition, serious steps should be taken to start an eradication program for this disease not only because of its pathogenicity for chickens but also for its human pandemic potential.
This article was originally published in Alexandria Journal of Veterinary Sciences, AJVS. Vol. 51(2): 90-100. November 2016 DOI: 10.5455/ajvs.236275. This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
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