Histopathological Evaluation of A/chicken/iran/339/02 (H9N2), an Iranian Field Isolate of Influenza Virus, on Japanese Quail (Coturnix coturnix japonica)

The respiratory and gastro intestinal systems of the quail could be a major source of avian influenza virus shedding and transmitting viral particles to other birds and species and endanger public health. So, in this study, the gross and histopathological findings of quail exprementally infected with field isolate of A/chicken/Iran/ 339/02 (H9N2) avian influenza virus were examined to evaluate the mortality and mobility rate of this isolate. A total of 50 quails at the age of 14 days were randomly divided in two groups: 25 quails in the trial group and 25 in the control group. The trial group was inoculated with 10 50% embryo infectious 6.3 dose/bird of avian influenza virus via nasal inoculation. Moderate clinical signs were observed 6 days post-infection (p.i.). No mortality was observed. Macroscopic and microscopic findings were observed mostly in the respiratory system, liver and intestine. No clinical and pathological signs were determined in the internal organs of the quails in the control groups. This study determined that the respiratory and gastro intestinal systems were affected more sever than other organs. However, the level of cell injuries has direct relation with accumulation of infection agents. Key word: Avian influenza H9N2 Quail Histopatology clinical signs INTRODUCTION influenza A virus infection in quail. So far the studies on There is increasing evidence that H9N2 avian virus are in deed limited. Therefore, in the present study, influenza viruses are endemic in chickens and other landthe gross and histopathological findings of quail based poultry, such as quail, pheasant, chukar and other exprementally infected with field isolate of minor domestic poultry in many Asian and European A/chicken/Iran/339/02 (H9N2) avian influenza virus were countries [1-3]. examined. More recently, quails have shown a high incidence of infection with influenza A viruses, particularly H9N2 MATERIALS AND METHODS viruses in Hong Kong where 16% of quails in the markets were found to be positive for H9N2 viruses Specimen Collection: Lungs, trachea and Liver were [4]. Besides, the H5N1 viruses were found to be collected from chicken showing clinical signs and cocirculating with H9N2 and H6N1 viruses in the Hong mortality of avian influenza virus (AIV) in Fars province Kong poultry markets. Sequence analysis suggested of Iran in 2002 and were stored in-70°C. that the 1997 H5N1 virus was a reassortant containing genes from an H6N1 and/or an H9N2 virus. Both viruses Birds: A total of fifty two-week-old Japanese quails are endemic in quail in Hong Kong [5-6]. Also, there has (Coturnix coturnix japanica) were obtained from been concern about the role of Japanese quail in the commercial quail industry in Yazd province of Iran and genesis of influenza viruses with pandemic potential were fed ad libitum by standard grower diet throughout [7].Thus, since 1997; there has been increased interest in our experiment according to the national research council characterizing the incidence and subtypes of avian requirements [8]. the pathological changes of the quails caused by the World Appl. Sci. J., 9 (2): 226-229, 2010 227 Virus Preparation: The virus isolation was performed by 56 (13 days post-infection) and 66 (23 days post-infection) injecting infected bird tissue (mucosal surface of trachea) days of age in order to monitor the macroscopic and homogenates into allantoic cavity of 10-day-old specificmicroscopic findings from birds of trial group in pathogen–free embryonated chicken eggs, according to comparison to control group, sequencely. Tissue samples standard procedures [9] and avian influenza (H9N2) virus such as: trachea, lung, air sac, liver, spleen, kidney, was isolated and named A/chicken/Iran/339/02. The titer pancreas, adren, cardiac system, skin, brain and intestine of the propagated virus was then calculated as a median were fixed in 10% neutral formalin solution and then embryo infectious dose (EID ) per milliliter. The Reed and subjected for histopatological examination. 50 Muench method was used for virus titration [10]. This virus stock was used in current experiment. RESULTS Birds and Avian Influenza Challenging: A total of No mortality was observed, clinical signs such as 50 quails at the age of 14 days were randomly divided into depression, ruffled feathers, diarrhea and nasal and eye two groups of 25birds/group. Each group was reared in discharges were observed on 49 and 56 days of age in separate room, with similar environmental conditions, some birds of trial group. Macroscopic findings were same availability of feeders and drinkers conditions, with observed between days 6 and 13 after inoculation, these separate ventilation systems and offered feed and findings were not appreciable different. No macroscopic drinking water ad libitum at the animal research unit of finding was determined in the quails slaughtered on day Shiraz university school. Before the experiment, rooms 23 after inoculation (not illustrated). were vigorously washed and fumigated using Macroscopic findings in trial group were mild in formaldehyde. Before inoculation, birds were bled to severity. Congestion and haemorrhagia were observed confirm negative avian influenza infectious status. Each mostly in the respiratory system, liver and intestine. birds in group 2 (trail group) received at 43 days of age a Trachea in some birds was moderately exudated, the similar challenge of H9N2-AIV field isolate. Briefly, each content of the intestines was dilute and petechial quails in trial group were intranasally inoculation with 10 haemorrhagia was observed in the intestines mucosal 6.3 embryo infectious dose/bird of A/chicken/Iran/ 339/02 surface. (H9N2) isolate. Birds in group 1 (control group) received Microscopic findings varied from sever to mild in the sterile allantoic fluid as a negative control. Birds were trial group on days 6, 13 and 23 after inoculation, monitored daily for clinical signs and death until the end respectively. The hyperemia, edema and infiltration of of study. Quails were anesthetized, then sacrificed on lymphocytes, macrophages and heterophils were mostly 42 (1 day before infection), 49 (6 days post-infection.), observed in the respiratory systems, liver, spleen and Fig. 1: Lung, inflammatory cells infiltration in the interstitial area with hemorrhage and edema of a quail infected with H9N2 of avian influenza virus subtype on day 6 post-infection, H&E;×360. World Appl. Sci. J., 9 (2): 226-229, 2010 228 Fig. 2: Duodenum, Hyperemia, edema, hyperplasia and degeneration in cells of lamina epithelia were observed in quails infected with H9N2 of avian influenza virus subtype on day 13 post-infection, H&E;×360. intestines, but these findings were determined to be In this study, the mentioned macroscopic and moderate in kidney and pancreas organs. However, no microscopic findings were determined mostly in the microscopic finding was observed in the skin, brain and respiratory system, liver, spleen and intestines of the cardiac system (not illustrated). Deciliation and sloughing quails to be sever more than other organ. Swayn et al. were observed in the tracheal epithelium and microscopic (1997) reported that in chickens the respiratory system, findings in lung was considerable (Fig. 1). Hydropic spleen, kidney and pancreas were affected in the low degeneration, apoptosis, focal necrosis and kupffer cell severity AIV infections; however, the skin, brain, cardiac activation were found in liver. Degeneration in cells of and the adren were affected in the sever cases [16]. It lamina epithelia, desquamation and hyperplasia were seems there is a relation between the severity of the AIV observed in the intestines (Fig. 2). infections and the tissue and organ tropism in avian No macroscopic and microscopic findings were species. It is reported that the occurrence of the disease determined in the internal and external organs of the syndrome or the degree of severity of the disease quails in the control groups (not illustrated). depends on multiple factors, including the pathogenicity DISCUSSION species, age of the host, route of infection and existence In this experimental study, the virus was isolated This study determined that the respiratory and gastro from the broiler farm which was showing obvious intestinal system were affected more server than other influenza clinical signs and mortality rated up to 30%, did organs and according to other study which indicated that not caused any mortality or disease in quails. It was the level of cell injuries has direct relation with reported that the severity of the disease depended accumulation of infection agents [19-20]. Therefore, these whether the infection was natural or experimental. systems could be a major source of AIV shedding and Although some avian influenza virus (AIV) strains caused transmitting viral particles to other birds and species. sever systemic infection and high mortality in natural The limitation of our study was that we did not conditions and this virus strain caused only a mild consider chicken as a positive control group to be infection without any mortality in the experimental study compared histopathologicaly with quails in negative in chicken [11-13]. In an outbreak of H9N2 in Iran, control and trial groups. We did not follow the level of mortality rate increased up to 65% in chickens [14], while viral shedding on separate days after inoculation. Thus, experimental studies using all H9N2 AI viruses from the further study will be needed to follow up the level of AIV Middle East shown to have low or no mortality in in internal organs of quail and viral shedding on different chickens [15]. days after inoculation. of the virus, care and feeding conditions, the host of secondary bacterial infection [17-18]. World Appl. Sci. J., 9 (2): 226-229, 2010 229 Our study with H9N2 virus isolated from a 7. Makarova, N.V., H. Ozaki, H. Kida, R.G. Webster and broiler farm which was showing influenza clinical D.R. Perez, 2003. Replication and transmission of signs and mortality could not produce disease and influenza viruses in Japanese Quail. Virol., 310: 8-15. mortality in quails. Hence, quails might have played role 8. National research council, 1984. 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