editorial board
past issues
contributors
feedback
contact us

 

printer friendly page    email page

2010, Vol. 5 No. 2, Article 64

 

A Comparative Study on Efficiency of
Enzyme Linked Immunosorbent Assay and
Hemagglutination Inhibition Test in Detecting
Serum Antibody Against Avian Influenza Viruses
in Commercial Broilers

Sarfraz Ali Fazlani, Muhammad Abubakar*1,2, Faraz Shahid,
Muhammad Irfan3 and Muhammad Javed Arshed1

 

Faculty of Veterinary and Animal Sciences,
Lesbela University of Agriculture, Water and Marine Sciences,
Uthal, Lesbela, Pakistan

1National Veterinary Laboratory, Park Road, Islamabad.
2National University of Agricultural Sciences, NARC, Park Road, Islamabad, Pakistan
3Department of Veterinary Pathology, Faculty of Veterinary Sciences,
University of Agriculture, Faisalabad, Pakistan

 

*Corresponding Author; e-mail address: hayee41@gmail.com

 


ABSTRACT

A comparative study was undertaken to assess the diagnostic performance of HI and ELISA tests using a set of serum samples obtained from broilers post Avian Influenza vaccination. The geometric mean titers of HI antibodies as log2 indicated post-vaccination titres after 14 days. ELISA test showed positive results up to 1:16 HI titre, and a linear between these two serological tests was also confirmed. It was concluded that there was a positive relationship between HI and ELISA, although ELISA was more sensitive because it detected low level of antibodies than HI. Also, it was proved that the tests can be used efficiently for the detection of antibodies in the broiler serum samples. However, HAI and titration of antibodies by geometric mean titre was convenient to measure the protective level of antibodies to determine the efficacy of commercially available vaccines.

KEY WORDS

Avian influenza, Geometric mean titre, Haemagglutination Inhibition Test and ELISA.

INTRODUCTION

Avian influenza viruse is subclassified on the basis of antigenic differences in surface glycoprotein, haemagglutinin and neuraminidase. Sixteen H subtypes and nine N subtypes have been recognized so far. The first outbreak of AI in Pakistan, caused by a highly pathogenic AIV of H7 subtype was reported by Naeem and Hussain (1995). Naeem et.al (1999) isolated Subtype H9N2 from a breeder flock that showed a sudden dip in egg production along with respiratory signs.
Immunoassays like Haem agglutination Inhibition test (HAI), serum neutralization test (SNT), Agar Gel Precipitation Test (AGPT) and enzyme linked immuno-sorbent assay (ELISA), mainly based on detection of antibodies are practiced in the field to record serological surveillance (Perdue and Swayne, 2005).Among these Haem agglutination Inhibition Test has been extensively used in diagnostic laboratories to detect avian influenza antibody and is considered as a standard test. HI assay is based on the ability of Hemagglutinin (HA) protein present on the viral surface to agglutinate erythrocytes. Problems associated with accurate use of HI assay are nonspecific serum inhibitors, differences in the ability of virus to agglutinate erythrocytes and frequent antigenic drift, all of which may lead to false-negative results. The SN assay is labor intensive, virus-specific, and the correlation to antibodies detected by the other assays is largely unknown (Leuwarki et al., 2008).
The HI Test is relatively inexpensive serological test. In general, the presence of HI antibody in flock is indicative of proactive level against the subtype used in the test, antibody titre appears to correlate with the level of protection. However, the major hindrance is its labor intensiveness to use it on a large scale for mass screening. During the recent years, commercial kits of ELISA have been developed for specific detection of antibody against AIV of H and N subtype (Yoon et al., 2004).
The present study was undertaken with objective to assess the diagnostic performance of HI in comparison with ELISA using a set of serum samples obtained from broilers post Avian Influenza vaccination.

MATERIALS AND METHODS

Collection of serum samples
Known positive serum samples used in this study were collected from a group of 3 week old broiler chicken (n= 100, divided into two groups, A and B) vaccinated with oil emulsion H9N2 vaccine used to obtain positive sera.). Blood (0.5 to 1 mL) was collected from the wing vein, samples were allowed to coagulate and sera were separated, collected, marked, centrifuged, inactivated at 56C and stored at -20C prior to use. Group A chicks were vaccinated with H9N2 oil emulsion vaccine, while group B chickens were kept as control.
Virus
Local virus isolate chicken/ Pakistan/23/99 (H9N2) used with ELD50 of 109.26/ 0.1 mL.
Antibodies Detection Assays
Haemagglutination and Haemagglutination inhibition test were performed by adopting the technique described in OIE Manual of Diagnostic Test using 4 HA unit of virus with 1% suspension of washed chicken RBCs (OIE, 2005). Samples of sera were heat inactivated at 56C for 30 min and treated with an equal amount of 10% washed chicken RBCs at ambient temperature before subjecting to HI test for detection of antibodies. Each sample was serially 2-fold diluted in 0.1 Mol phosphate buffered saline (PH 7.4) and tested for HI activity against 4HA unit. The presence of HI antibody in each sample against H9N2 strain was observed by adding 0.5% chick RBCs and incubating at ambient temperature for 45 Min. HI antibody titre of each sample was determined as the reciprocal of highest dilution in which no heamagglutination was observed.
ELISA was performed in the presence of antiviral IgG with the H9N2 strain of virus that was used as infectious allantoic fluid. The fluid was centrifuged at 30000 RPM for two hours SW 41 rotor and viral pellet was re-suspended in TNE buffer (Tris 0.01 M, Nacl 0.1M, EDTA 0.001 M, pH 7.4) and further purified by sucrose gradient centrifugation (25-40%) at 20000 RPM for two hours. The viral band was harvested and its protein concentration was determined. Titres were expressed as highest dilution that yielded an optical density greater than mean plus three standard deviations of diluted sera (Lewis, 1988).

RESULTS

The geometric mean titres (log 2) in the haemagglutination inhibition (HAI) test of chick sera after vaccination with H9N2 oil emulsion vaccine was 5.7 0.29 (n=66) (Table-1). Results of control chicks were less than 1:2, calculated significantly lower than vaccinated chickens hence not presented in the table.
Positive anti sera showed 0.4 OD up to 1:256 dilutions which is considered positive for ELISA. Contrary to that negative control sera showed no absorbance even at 1:2 dilutions. The correlation between HI and ELISA indicated that serum samples showing HI titre log2 were positive up to 1:16 dilution (Table-2).

DISCUSSION

The percentage of chickens with subtype-specific antibodies was the highest (100 %) by the 21st day of infection followed by that of 14th (66.7 %) and the 28th (55.6 %) days, and was the lowest (44.4 %) by the 7th day. Serum titres ranged between 1:4 and 1:256 with predomination of 1:8 and 1:16 titres (29.2 % each). The mean arithmetic titre for the experiment was 1:38.2. The highest percentage of chickens with precipitins (55.6 %) was observed by the 14th day followed by that of 7th, 21st and the 28th days with 33.3 % each. The titres ranged from undiluted to 1:4 with prevailing of 1:2 titres. By means of ELISA, 55.6% sero positive birds were detected by the 14th day whereas the percentage on days 7, 21 and 28 was 33.3%. S/P values were the highest by the 14th day (up to 1.640) .These observations fall in line with the findings of Zarkov (2007).
There was a correlation between HI and ELISA test. Anti sera that showed high HI titres were also found positive in the ELISA. Similar results were reported by Meulemans et al. (1987) who carried out a comparative study between HI and ELISA for measuring antibodies against H7 serotype of avian influenza and did not observe any major difference between two tests In a Comparative study Meilin Jin et al (2004) also reported ELISA test run with nucleoprotein (N) a sensitive method for detection of antibodies and indicated that HI and ELISA tests had high agreement ratio and statistically non-significant difference.
A considerable variation in individual results and data obtained for comparative studies upon diagnostic test for detection of respective antibodies was reported by various workers (Zhou et al., 1998; Sala et al., 2003) obtained higher positive results in sera tested by means of HI compared to either IDT or ELISA .In another study ELISA was found to provide better results than HI or IDT (Adair et al., 1990 and Boer et al., 1990). However, no significant differences among tests was observed by Meulemans et al, (1987) and Beck and Swayne (1997).
Present study revealed that although ELISA is most sensitive to detect low antibody titre but comparatively expensive and difficult for standardization & purification of reagents.
It is concluded that HI and ELISA were of equal value for quick detection of the predictive level of antibodies to AI virus in a vaccinated or infected flock. However, HI is comparatively better in measuring the titre of antibodies by GMT because of being less expensive, feasible, convenient and commonly used test in diagnostic laboratories in Pakistan.

REFERENCES

  1. Adair, B.M., K. Burns, M.S. McNulty and D. Todd, 1990. A study of ELISA systems incorporating pooled viral and Mycoplasma antigen preparations for antibody screening of chicken sera. Avian Pathology, 19, 263−278.

  2. Beck, J.R. and D.E. Swayne, 1997. Evaluation of ELISA for avian influenza serologic and diagnostic programs: Comparison with agar gel precipitin and hemagglutination inhibition tests. In: Proceedings of the Fourth International Symposium on Avian Influenza, May 28−31, The University of Georgia, Athens, GA, USA, American Association of Avian Pathologists, Lib. Cong. Cat. Card No. 97-051243, 297−304.

  3. Boer, G.F., W. Back and A.D.M.E. Osterhaus, 1990. An ELISA for detection of antibodies against influenza nucleoprotein in human and various animal species. Archives of Virology, 115, 47−61.

  4. Lewis, G.D., 1988. Mete-calf TG Polyethylene glycol precipitation for recovery of pathogenic viruses. Applied Environ Microbial 54: 1983-1988.

  5. Meilin J.A.B., G. Wang, A.R. Zhang, S. Zhao, H. Li, Y. Tan, and H. Chen, 2004. Development of Enzyme-Linked Immunosorbent Assay with Nucleoprotein as Antigen Detection of antibodies to Avian Influenza Virus. Laboratory Animal Infectious Diseases, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070.

  6. Meulemans, M.C., M. Calier, M. Gonza and P. Petit, 1987. Comparison of haemagglutination Ininhibition, agar gel precipitation and enzyme-linked Immunosorbent assay for measuring antibodies against influenza virus. In: chicken. Avian Dis. 31: 560-563.

  7. Perdue M.L., and D.E. Swayne, 2005. Public health risk from Avian Influenza Viruses. Avian Dis., 49: 317-327.

  8. Naeem, K., and M. Hussain, 1995. An out break of avian influenza in poultry in Pakistan. Veterinary Rec. Pp. 137-439.

  9. Naeem, K., A. Ullah, R. J. Manvell and D. J. Alexander, 1999. Avian influenza subtype H9N2 in poultry in Pakistan, Vet. Rec. 145-560.

  10. Office International Des Epizootics (OIE), 2005. Manual of Diagnostic test and vaccines for terrestrial animals. Http/www/oieint/eng/ norms/manual A.2005.

  11. Sala, G., P. Cordioli, A. Moreno-Martin, M. Tolles, E. Brocchi, A. Piccirillo & A. Lavazza, 2003. ELISA test for the detection of influenza H7 antibodies in avian sera. Avian Diseases, 47, 1057−1059.

  12. Swayne, D.E., D.A. Senne and C.W. Beard, 1998. Avian influenza In: A laboratory manual for the isolation and identification of avian pathogens, 4th ed. Pp. 150-155.

  13. Yoon, K-J., B.H. Janke, R.W. Swalla and G. Erickson, 2004. Comparison of a commercial H1N1 enzyme-linked immunosorbent assay and hemagglutination inhibition test in detecting serum antibody against swine influenza viruses. J Vet Diagn Invest 16:197201.

  14. Zarkov, I.S., 2007. Diagnostic Potential of the Haemagglutination Inhibition Test, the Immunodiffusion Test and ELISA for Detection of Antibodies in Chickens, Intravenously Infected With A/Duck/Bulgaria/05 H6n2 Avian Influenza Virus Isolate. Bulgarian Journal of Veterinary Medicine, 10 (3): 169-178.

  15. Zhou, E.-M., M. Chan, R. A. Heckert, J. Riva & M.-F. Cantin, 1998. Evaluation of a competitive ELISA for detection of antibodies against avian influenza virus nucleoprotein. Avian Diseases, 42, 517−522.


Table-1 Mean Haemagglutination Inhibition Test (HI) antibody titre of chicks vaccinated with H9N2 AIV strain

Titre  Log2

No of samples
Mean titre

1

3

3

2

3

6

3

3

12

4

4

40

5

10

95

6

19

42

7

07

28

8

07

56

9

03

27

10

06

60

11

02

11

Mean S.E.M                          5.70.2 (n=66)

 

 

Table 2:  Optical density (405 nm wavelength) serial twofold dilution 

 

Dilution of sera showing the respective HAI titre (log2)

Positive

Control

Sera

Negative

Control

sera

HI

Titre

(Log2)

1:2

1:4

1:8

1:16

1:32

1:64

1:128

1:256

11

1.802

1.566

1.425

1.187

0.903

0.601

0.521

0.423

1.80

0.2

10

1.606

1.522

1.256

0.977

0.635

0.602

0.402

0.401

1.80

0.2

9

1.592

1.509

1.163

0.805

0.598

0.387

0.354

0.211

1.73

0.1

8

1.421

1.278

1.091

0.761

0.501

0.313

0,300

0.284

1.69

0.1

7

1.285

1.069

0.863

0.470

0.416

0.236

0.129

0.119

1.53

00

6

1.321

1.287

1.238

0.570

0.478

0.363

0.118

0.101

1.42

00

5

1.011

1.102

1.101

0.480

0.357

0.288

0.108

0.101

0.46

00

4

0.500

0.400

0.410

0.405

0.311

0.207

0.108

0.101

0.43

00

The samples with 0.4 OD and above were considered positive.

 


Copyright Vet Scan 2005-

All Right Reserved with VetScan
www.vetscan.co.in and www.kashvet.org
ISSN 0973-6980

 

Home | e-Learning |Resources | Alumni | Forum | Picture blog | Disclaimer

                   

 

 

powered by eMedia Services