Volume 5 Supplement 4
Different immune responses to three different vaccines following H6N1 low pathogenic avian influenza virus challenge in Taiwanese local chicken breeds
© Chang et al; licensee BioMed Central Ltd. 2011
Published: 3 June 2011
H6N1 low pathogenic avian influenza virus (LPAIV) are frequently isolated in Taiwan and lead to significant economic losses, either directly or indirectly through association with other infectious diseases. This study investigates immune responses to three different vaccines following a H6N1 challenge in different local breeds.
Experimental animals were sampled from six local chicken breeds maintained at the National Chung-Hsing University, namely Hsin-Yi, Ju-Chi, Hua-Tung (Taiwan), Quemoy (Quemoy Island), Shek-Ki (China), Nagoya (Japan) and a specific pathogen free (SPF) White Leghorn line. A total number of 338 chickens have been distributed between a control and a challenge group, H6N1 challenge was performed at 7 weeks of age; vaccination against Newcastle Disease (ND), Infectious Bursal Disease (IBD) and Infectious Bronchitis (IB) was performed at 11 weeks. The anti-H6N1 LPAIV antibody titers were measured by ELISA at days 0, 7, 14 and 21 after challenge, and the anti-ND, anti-IBD and anti-IB antibody titers were measured by inhibition of hemagglutination test and ELISA at days 0, 14, 28 after vaccination.
There was no effect of the H6N1 LPAIV challenge at 7 weeks of age on the subsequent responses to ND and IBD vaccine at 11 weeks of age, but, surprisingly, the H6N1 LPAIV challenge significantly affected antibody levels to IB vaccine in some breeds, since IB0 and IB14 antibody titers were lower in the challenge groups. However, there was no significant difference in IB28 antibody titers among the experimental groups.
Local breeds have different immune response to H6N1 LPAIV challenge and subsequent vaccines. Differences dealt mainly with kinetics of response and with peak values. Quemoy exhibited higher antibody levels to H6N1, ND and IBD. The negative effect of the H6N1 LPAIV challenge on IB vaccine response may be related to the fact that both viruses target the lung tissues, and the type of local immune response induced by LPAIV challenge may not be favourable for birds to make optimum IB-specific antibody response.
Since 1982, National Chung-Hsing University is maintaining six local chicken breeds: Hsin-Yi, Ju-Chi and Hua-Tung were collected from small villages in Taiwan, Quemoy was collected from Quemoy Island near China, Shek-Ki was from China, and Nagoya was from Japan . In previous studies, Quemoy had significantly higher antibody titers against Newcastle Disease (ND) after vaccination than other local breeds, Shek-Ki, Hua-Tung and Ju-Chi had lower anti-ND antibody titers . Thus, immune response was shown to differ within this set of local chicken breeds.
H6N1 Low pathogenic Avian Influenza Virus (LPAIV) is frequently isolated in Taiwan and lead to significant economic losses, either directly or indirectly through association with other infectious diseases . This study investigated immune responses to ND, Infectious Bronchitis (IB) and Infectious Bursal Disease (IBD) vaccines following a H6N1 LPAIV challenge on six local chicken breeds and SPF chicken.
In this study we used the six local breeds and added an SPF White Leghorn as a control genotype.
A total of 314 chicks were hatched from 23 sires and 91 dams with full pedigree in six local breeds. Twenty-five SPF chicks were purchased from Animal Health Research Institute (Council of Agriculture, Executive Yuan R.O.C.). Day-old chicks were wing-banded and raised in floor pens until 5 weeks of age, and they were transferred to experimental cages after 6 weeks of age. Sire families were distributed between the control and the challenge group. Individual body weights were recorded weekly from hatch to 16 weeks of age.
Vaccination program and challenge
Day-old chicks were all vaccinated against Marek’s disease and ND. At two weeks of age, chicks were vaccinated against ND, IB, IBD, Fowl Pox and Avian Reovirus infection. At four weeks of age, chickens were vaccinated against ND, IB, IBD and Infectious Laryngotracheitis. The H6N1 LPAIV (A/chicken/Taiwan/0825/2006) challenge was performed at 7 weeks of age, birds from the challenge group received a drop with 107 EID50 of viruses into eye and nose. Blood samples were collected at days 0, 7, 14 and 21 post-challenge. Chickens health condition and mortality were recorded and monitored. At 11 weeks of age, all chickens, from both challenge and control groups, were vaccinated again against ND, IB and IBD, blood samples were collected at days 0, 14 and 28 post-inoculation. Sera from blood samples were collected and stored in -20 °C refrigerator.
Immune response measurement
The antibody responses to H6N1 LPAIV, IBD and IB were measured by enzyme-linked immunosorbent assay (ELISA) with commercial test kit (IDEXX Laboratories, Inc., Westbrook, ME), the antibody titer calculation was performed according to IDEXX’s formula. The antibody responses to ND were measured by hemagglutination inhibition test (HIT), the antibody titer was expressed as the log2 of the reciprocal of the highest dilution. Each measure was defined by the virus name (AI, ND, IBD or IB) and the day of sampling after inoculation (either days 0, 7, 14, 21 or 28). In addition, response to IB vaccine was calculated by the difference in antibody titers between stages, i.e. day 0 to day 14, day 14 to day 28 and day 0 to day 28.
where Y ijkl is the antibody titer of the lth animal of the ith breed, the kth sex after the jth challenge treatment, i=1, 2,…,7, j=1,2, k=1,2, l=1,2,…,338, μ is the mean, τ i is the fixed effect of the ith breed, Στ i =0, α j is the fixed effect of the jth challenge treatment, Σα j =0, (τα) ij is the fixed interaction effect between the ith breed and the jth challenge treatment , ΣΣ(τα) ij =0, and e ijkl is the residual random error, .
All statistical analyses were conducted by using SAS software (SAS Institute).
H6N1 LPAIV challenge
Sample size and mortality per breed in H6N1 challenge experiment
Mortality rate (%)
ND vaccine response
IBD vaccine response
IB vaccine response
Effects of H6N1 challenge on antibody titers in different periods across breeds.
6253 ± 499
5106 ± 499
8119 ± 609
8769 ± 609
1852 ± 726
3722 ± 738
4598 ± 553
3374 ± 541
3474 ± 682b
5720 ± 608a
-946 ± 564b
2408 ± 564a
4818 ± 877
3175 ± 784
7149 ± 604
7461 ± 540
2331 ± 766
4303 ± 699
3633 ± 508
3424 ± 469
5261 ± 447
6362 ± 413
1663 ± 502
2935 ± 463
2050 ± 687
1235 ± 615
9464 ± 623
9809 ± 623
7148 ± 938
8516 ± 884
6117 ± 548a
3165 ± 437b
7038 ± 748
8718 ± 642
580 ± 844b
5433 ± 695a
3205 ± 679
3063 ± 679
3186 ± 962b
6118 ± 962a
-19 ± 1107
3055 ± 1107
Breed comparison showed that Quemoy had the highest antibody titers for IB0. Quemoy, Hsin-Yi and Nagoya had the highest values for IB14. These three breeds had still high values for IB28, but Shek-Ki had also high values for IB28, although it exhibited low values for IB0 and IB14. Thus, this breed was characterized by a late and strong response to IB vaccine, without any significant effect of the previous H6N1 challenge.
Discussion and conclusions
H6N1 LPAIV challenge effect
Mortality data and the increased antibody titers of challenge group at day 14 post-challenge showed that the challenge test had been effective. Estimation of breed effects may be affected by limited sample size, particularly for Shek-Ki, SPF and Nagoya. However, Quemoy had the largest sample size, and can be identified as the most resistant breed since it did not show any mortality after the challenge, and exhibited the most rapid immune response at day 7 post-challenge. At the opposite, Hsin-Yi can be identified as the most susceptible breed, with a rather high mortality (7 birds out of 26) in spite of the use of a low pathogenic virus.
Breeds effect, vaccine efficacy and duration of immunity
Antibodies were detected for ND, IB and IBD at day 0 post-inoculation because all chickens had been vaccinated for ND, IB and IBD at earlier ages. Thus, the immune response following the inoculation at 11 weeks of age may be considered as a secondary immune response. Antibody titers at day 14 post-inoculation were significantly higher than at day 0, showing vaccine efficiency, except in the case of Nagoya and SPF for IBD vaccine where no change in antibody levels was observed.
Breed significantly affected immune response. Quemoy had high and rapid responses to the three vaccines and to H6N1 LPAVI challenge test, in contrast to Ju-Chi which had low immune response to vaccines and challenge test. Breed’s effect on the antibody titers at day 0 revealed differences in the duration of immunity to previous vaccines. Quemoy appeared to have a better immunity than other breeds, Shek-Ki had a slow response to ND and IB, and Ju-Chi presented lowest response to IBD. The better immune response of Quemoy is consistent with previous results .
H6N1 LPAIV challenge effect on IB immune response
H6N1 LPAIV challenge had a negative effect on antibody levels to IB even before the vaccination at 11 weeks of age. Immune response to IB vaccine took place in H6N1 challenge groups with some delay (after 14 days). The negative relationship of H6N1 LPAIV challenge and IB vaccine response could be related to the fact that both viruses target the lung tissues. Recently, Haghighat-Jahromi et al.  showed that coinfection of H9N2 AI virus with IB live virus enhanced the virulence of H9N2 and increased the rate of mortality. In addition, Karimi-Madab et al.  showed that IB live vaccine could be an important risk factor resulting in enhanced virulence of H9N2 LPAIV in field conditions. Although these studies were focusing on broilers and H9N2 LPAIV, the present study shows also an interaction between IB and H6N1 LPAIV infection in some local chicken breeds. One could speculate that the type of local immune response induced by H6N1 LPAIV infection was not favourable for birds to make optimum IB-specific antibody response.
In conclusion, local breeds have different immune response to H6N1 LPAIV challenge and subsequent vaccines. The H6N1 LPAIV challenge influenced the response to subsequent vaccination against IB, but had no effect on ND and IBD subsequent vaccines.
We would like to thank many graduate and undergraduate students of National Chung-Hsing University for their assistance in data collection.
This article has been published as part of BMC Proceedings Volume 5 Supplement 4, 2011: Proceedings of the International Symposium on Animal Genomics for Animal Health (AGAH 2010). The full contents of the supplement are available online at http://www.biomedcentral.com/1753-6561/5?issue=S4.
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