RESULTS

Clinical Findings
Daily monitoring did not show any changes in clinical behaviour of the birds in control group. Infected chickens showed clinical signs such as depression, puffing, oedema of face and head, conjunctivitis and ruffled feathers on days 3 or 4 PI.

Gross Necropsy Findings
Control chickens did not show any gross lesions. However, the most frequent gross lesions in infected birds were turbidity of the thoracic and abdominal air sacs and mild congestion of the trachea and lung and mild accumulation of fibrinous exudate on the tracheal mucosa.

Fig. 1:	(a) Indirect immunofluorescence technique shows the clear localization of AIV antigen in the trachea frozen section (arrows) on day 4 PI (IIF x100), (b) distribution of AIV antigen 6 days PI in the lung frozen section can be seen (arrows), using indirect immunofluorescence technique (IIF x250) and (c) distribution of AIV antigen 8 days PI in the kidney tubules can be seen (arrows), using indirect immunofluorescence technique (IIF x250)

Serological Findings
The specific antibody titer against H9N2 avian influenza virus at days 1, 2, 4, 6, 8 and 11 post-inoculation in treatment group was 3, 2.4, 1.7, 3.3, 4.2 and 5.6 and in control group was 3, 2.7, 2.34, 1.8, 1.24 and 0, respectively.

Viral Antigen Distribution
In the control chickens (18 birds), there was no detectable H9N2 virus antigen in all of the examined organs.

In inoculated chickens, using indirect immunofluorescence technique, influenza virus nucleoprotein was demonstrated in the trachea on days 2, 4, 6, 8 (Fig. 1a) and 11 PI, in the lung on days 2, 4, 6, 8 (Fig. 1b) and 11 PI and in the kidney on days 6, 8 and 11 PI (Fig. 1c).

DISCUSSION

Although experimental study of low pathogenic AI viruses in SPF chicken cause no or low mortality, frequent high mortality rates have been reported in the field cases (Bano et al., 2003; Naeem et al., 1999, 2003; Nili and Asasi, 2002, 2003).

Intranasal inoculation of chickens with Iranian AIV H9N2 subtype revealed that the virus is epitheliotropic in chicken and tracheitis, pneumonia and tubulointerstitial nephritis were the most frequent histologic changes.

In some researches (Slemons and Swayne, 1990; Slemons et al., 1990; Swayne and Slemons, 1992, 1994, 1995; Swayne et al., 1994; Swayne and Pantin-Jackwood, 2006), inoculation of chickens by intranasal (IN) and intratracheal (IT) route with low virulence chicken- or duck-origin influenza virus isolates produced mortality and kidney lesions in 1-day-old chickens and adult hens. However, in other studies absence of mortality has been reported by Shalaby et al. (1994) and Swayne et al. (1994). In the current study, presence of necrosis and inflammation in the trachea, lung and kidney, along with demonstration of influenza nucleoprotein using IFA in the trachea, lung and kidney indicate that the Iranian AIV H9N2 subtype has tissue tropism for respiratory system and kidney of chickens.

In this experiment, tracheitis, pneumonia and tubulointerstitial nephritis were the most frequent histologic lesions. The lesions were obvious on days 2-8 PI but on day 11 PI the severity of lesions was reduced. Clinically this was associated with improvement in general condition of inoculated birds.

Inoculation of an isolate of H9N2 avian influenza virus to chickens using different routes and subsequently challenged with other infectious agents. The AIV antigen was detected in the trachea, lung, kidney and cloacal bursa among infected birds (Bano et al., 2003).

In chickens intratracheally inoculated with avian influenza virus isolates of either low or high pathogenicity the low pathogenicity isolates produce histologic lesions most frequently in the trachea and lung or lacked histologic lesions. Viral antigen was present only and infrequently in the trachea and lung of inoculated chickens (Mo et al., 1997). In another study after intravenous inoculation of avirulent H4N4, H6N2 and H3N8 viruses into chickens, specific lesions and immunoperoxidase staining were noted in the kidney only (Hooper et al., 1995).

Inoculation of two low pathogenicity and two high pathogenicity avian influenza viruses into chickens by intranasal route showed that the LPAI viruses caused localized virus infections in the respiratory and gastrointestinal tracts (Swayne and Beck, 2005). Inoculation of three strains of AIV with chicken/duck origins to 5-week-old chickens by intravenous (IV), intranasal and IT routes revealed that the Chickens inoculated by IT and IN routes had mild to severe tracheitis, bronchitis and pneumonia associated with secondary bronchi but lacked renal tubule necrosis and nephritis (Swayne and Slemons, 1994). As abdominal air sacs are next to kidneys, presumably, presence of nephritis foci in the kidneys during 6-11 days PI could be resulted from infection of the respiratory tract (air sacs). Regarding kidney lesions, the result obtained from the present study is in agreement with finding by Slemons and Swayne (1990) and Slemons et al. (1990).

This finding indicate that renal failure resulted from kidney lesion could be encountered in H9N2 AIV infection in chicken. In current experiment common non-specific changes were lymphoid and reticuloendothelial hyperplasia in duodenum and spleen. These changes could be an immune response of B and T-lymphocytes to foreign antigens. Lymphoid atrophy in thymus and bursa of Fabricius was most compatible with non-specific endogenous glucocorticoid response and mild infiltration of lymphocytes in pancreas was similar to mild non-specific immunologic reaction (Swayne and Slemons, 1995). Absence of lymphocyte necrosis and viral antigens in primary and secondary lymphatic organs show that lymphoid tissues are not target of AIV H9N2.

CONCLUSION

Presence of viral antigen in the trachea, lung and kidney of chickens, concurrent with histologic lesions indicate that the H9N2 avian influenza virus is epitheliotropic, which in IN route of inoculation has tissue tropism and pathogenicity for the trachea and lung (pneumotropic) and kidney (nephrotropic).