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ISDS 2016 Conference Abstracts

Immune response and distribution of antigen in 
chickens after infection LPAIV (H4N6)
Denys Muzyka*1, Hyun Lillehoj2, Olexandr Rula1 and Borys Stegniy1
1Laboratory of Avian Diseases Epizoothology, National Scientific Center Institute of Experimental and Clinical Veterinary Medicine, 
Kharkiv, Ukraine; 2Animal Parasitic Diseases Laboratory, Beltsville, MD, USA

Objective
To study the immune response in chicken on the administration of 

LPAIV isolated from the natural reservoir.

Introduction
Influenza is a serious problem for the health of people, animals and 

birds. Therefore, comprehensive study of influenza virus, its natural 
reservoir, pathogenesis and immune response will provide further 
opportunity to ensure protection for animals, birds and people from 
this infection.

Methods
Four-week-old commercial chickens were intranasally inoculated 

with a H4N6 LPAIV A/Garganey/Chervonooskilske/4-11/2009 
(H4N6), isolated from the cloacal swab of clinically healthy garganey 
in 2009 in Ukraine. Cecum, spleen, lung, and trachea samples were 
collected from infected chickens on 1 - 14 dpi and examined by 
immunohistochemical and virology techniques. On these days, 
we collected blood samples for serological analysis. Detection of 
antibodies to avian influenza virus subtype H4 was performed with 
chicken serum samples by HI test and ELISA. The studies were done 
according to IACUC.

Results
Upon intravenous and intranasal infection with this virus  

(A/Garganey/Chervonooskilske/4-11/2009), no clinical signs were 
observed in chickens and no pathological changes were found at 
necropsy. Infection of poultry with this virus provoked an antibody 
response at 10 days after intranasal inoculation which ranged from 
1:8 to 1:32 serum antibody titers. Only 2 of 5 chickens were positive 
by the HI test and 3 of 5 were positive by ELISA at intranasal 
inoculation. All 10 chickens were positive both by HI test and by 
ELISA after intravenous inoculation. Specific antibodies (HI test) to 
influenza virus H4 were detected in titer ranges of 1:128 to 1:1024.

In immunohistological studies, the respiratory tract organs (lungs 
and trachea) showed higher level of humoral immunity (IgM, IgG, 
IgA-expressing cells) in the lung compared to the trachea. Also, 
indicators of cell mediated immunity as measured by the CD4 and 
macrophage markers were higher in LPAIV-infected chickens in 
the lungs at 14 days post infection compared to uninfected chickens. 
Lymphocytes expressing CD8 were increased starting 7dpi.  
The chickens in the infected group showed 2 times higher levels of 
CD8 cells compared to the control chickens. IFN-γ transcripts were 
observed in the AI-infected chickens starting at 7dpi that coincides 
with the increasing level of

CD4 cells. The number of lymphocytes which secrete IL-2 and 
IL-15 in AI-infected chickens were in general 1.5 to 2 times higher 
compared to the uninfected chickens. In AIV-infected chickens, the 
level of cells expressing IFN-γ, IL-2, and IL-15 increased at 7-days 
after infection. The peak time coincided with a period of increasing 
CD8 cells. However, there was no significant difference in these 
cytokine levels between the AIV-infected and uninfected groups.

In the cecum, lower levels of CD4 cells were seen on 5 dpi but 
levels slowly increased from 7 dpi to 14 dpi following AIV infection. 

In the ceca, a significant increase in the number of cells expressing 
IgM and IgG was found.

LPAIV infection induced an increase in macrophages and 
lymphocytes expressing CD4 and CD8 in the spleen throughout the 
period examined in this study indicating their role in host response 
to viral infection. The levels of macrophages in chickens of AIV-
infected group were 2 times higher than the control after 1 dpi.

Conclusions
Although infection with a LPAIV did not cause obvious clinical 

disease, viral replication was detected in the trachea and spleen and 
both local and systemic cellular and humoral immune responses were 
elicited in these LPAIV-infected chickens. Our results indicate the 
potential possibility for infection of poultry with viruses isolated from 
wild birds. But currently it is not completely known why some viruses 
from wild birds can cause infection in poultry, while others can not. 
Further study of the immune response will enable us to determine the 
features of the pathogenesis of low pathogenic avian influenza.

Keywords
avian influenza; immune response; chicken

Acknowledgments
Our research was funded by USDA projects P382a and P444 through the 
Ukrainian Science and Technology Center. The authors express special 
gratitude Dr. Pavlo Shutchenko.

*Denys Muzyka
E-mail: dmuzyka77@gmail.com

Online Journal of Public Health Informatics * ISSN 1947-2579 * http://ojphi.org * 9(1):e107, 2017