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Full Scientific Reports |
Correspondence: 1Corresponding Author: Meilin Jin, Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 1 Shizishan Street, Wuhan 430070, e-mail: chenjianfeng2003{at}yahoo.com.cn
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Abstract |
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 TOPSources and manufacturers Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: Avian influenza virus • diagnosis • H5N1 • latex agglutination
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Introduction |
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The recent emergence and re-emergence of influenza viruses with pandemic potential is of great concern to both the veterinary and public health communities. Early diagnosis of influenza virus infection is therefore essential, especially for the highly pathogenic avian influenza (HPAI) virus H5N1. A rapid, sensitive and inexpensive diagnostic test is also required for H5N1 surveillance.
Presently, the identification of the H5N1 can be accomplished using various diagnostic assays, such as virus isolation (VI) in embryonated chicken eggs, direct antigen detection using enzymatic, fluorescent, optical, or chromatographic immunoassays,4 and nucleic acid detection assays (reverse transcription-PCR [RT-PCR],8 real-time RT-PCR,9 and nucleic acid sequence-based amplification3). The objectives of this study were: 1) to develop a rapid latex agglutination test (LAT) for detecting H5N1 in chicken tracheal swabs and tissues; and 2) to compare sensitivity and specificity of the newly developed LAT in relation to VI, immunochromatographic assay, and real-time RT-PCR.
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Materials and Methods |
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TOPSources and manufacturersAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monoclonal Antibody Preparation
Six-week-old female BALB/c mice were immunized with A/Chicken/HuBei/327/2004(H5N1). The spleen of immunized mice was harvested and the lymphocytes isolated were used in the generation of hybridomas as previously described.5 Hybridoma culture supernatants were screened for reactivity against H5N1 by hemagglutination inhibition (HI) assay, and hybridoma cells from wells containing HI titers of 1 : 32 were expanded and cloned by limiting dilution. To ensure that the monoclonal antibodies (MAbs) secreted by the hybridomas were specific for AIV H5N1, the hybridoma culture supernatants were analyzed by HI assay for reactivity against H9N2, H4N6, H3N2, and H1N1 viruses. Seven hybridoma cell lines secreting MAbs specific for the HA protein of H5N1 were established. The IgG antibodies in ascites were purified using protein A affinity chromatography.
Preparation of Sensitized Latex Particles (SLPS)
Affinity-purified antibody was coupled to carboxylated latex particles of 0.75 µm in diameter by modification of procedures recommended by the manufacturerb. Briefly, 500 µl of 2.5% (w/v) latex particles were washed 3 times in 0.1 M carbonate buffer (pH 9.6) and 3 times in 0.02 M phosphate buffer (pH 4.7) in 1.5-ml microcentrifuge tubes. All washes were done at 14,000 x g for 6 min at 25°C unless otherwise stated. Following the final wash, the particles were re-suspended in 625 µl of the 0.05 M 2-[N-morpholino] ethane sulfonic (MES) acid, pH 4.7 and transferred into 5-ml centrifuge tubes. Afterwards, 625 µl of freshly prepared 2% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide · HCl (EDC) was added drop by drop while the solution was slowly vortexed. The tubes were further incubated at 25°C for 4 hr while being rotated slowly end-to-end. The sensitized latex particles (SLPs) were then transferred into microcentrifuge tubes, washed 3 times in 0.02 M phosphate buffer (pH 4.7), and re-suspended in 3 ml of 0.01 M borate buffer (pH 8.0). Concentrations of IgG ranging from 200 to 2,400 µg/500 µl of particles were added to the SLPs and the tubes were rotated slowly end-to-end for 8 hr at 25°C. To block nonspecific binding, 50 µl of 0.1 M ethanolamine was added to the SLPs and rotated for additional 30 min. The SLPs were then transferred into 1.5-ml microcentrifuge tubes and centrifuged at 14,000 x g for 10 min. The supernatant was saved for protein determination and the SLPs were re-suspended in 1 ml 0.01 M borate buffer (pH 8.0, containing 1% bovine serum albumin) and rotated at 25°C for 30 min. Finally, the SLPs were washed one more time in BSA in 0.01 M borate buffer (pH 8.0) and re-suspended in 0.5 ml of latex storage buffer (0.1% BSA, 5% glycerol, and 0.1% NaN3 in 0.01 M PBS, pH 7.4).
Latex Agglutination Assays
Latex agglutination tests were performed by mixing 10 µl of SLPs with 10 µl of sample on a black-coated glass slide. The slide was then rotated manually for 30 sec. Test results were scored as follows: 4+, rapid agglutination of 100% SLPs, with formation of a ring; 3+, agglutination of >75% of SLPs, with some ring formation; 2+, agglutination of 
50% of SLPs, with no ring formation; 1+, agglutination of 
25% of SLP ; and -, no visible agglutination.
Sample Preparation for Testing by LAT
Tracheal swabs from birds were collected in 1.0 ml of 0.3 M borate buffer (pH 8.4, 0.85% NaCl, 0.5% N-acetyl-cysteine, 0.1% NP-40, 5,000 IU/ml penicillin G, 5 mg/ml streptomycin), and the suspensions were frozen and thawed 3 times and centrifuged at 14,000 x g for 10 min before testing by LAT. All tissue specimens were homogenized to give a 50% suspension (w/v) in sample buffer (PBS, pH 7.4, containing penicillin G 5,000 IU/ml and streptomycin 5 mg/ml). The suspensions were centrifuged at 14,000 x g for 10 min before testing by LAT.
Virus Isolation (VI) in Embryonated Chicken Eggs
Isolation of influenza virus was performed by inoculating 9-day-embryonated chicken eggs with 0.2 ml tissue suspensions or swab suspensions via the allantoic cavity. The eggs were incubated for 4 days and candled daily for viability; embryos that died within 24 hr of inoculation were discarded as nonspecific. Allantoic fluid from dead and surviving embryos was tested for Hemagglutinin (HA) activity. Samples that yielded no hemagglutination were re-inoculated for a second passage.
Hemagglutinin (HA) and Hemagglutination Inhibition (HI) Assays
HA and HI assays were performed in V-bottomed microtiter plates that contained 1% chicken erythrocytes solution following standard protocols recommended by International Animal Health Organization (OIE).13
Animal Infection Experiments
An experiment was designed to determine whether the LAT could be used in direct analysis of tracheal swabs and bird tissues. Seventy-two 8-wk-old White Stone chickens were experimentally infected with H5N1; these birds were divided into 3 groups of 24 chickens based on pre-existing H5N1 HI titers resulting from vaccination. Group I chickens had HI titers of 1 : 64, group II HI1 : 32, and group III HI = 0. Each group of 24 chickens was further divided into 6 subgroups of 4. Sub-groups 1, 2, 3, 4, and 5 received inoculation doses of 1,000, 100, 10, 1, and 0.1 50% egg infectious doses (EID50), respectively. Subgroup 6 was used as control and inoculated with virus-free PBS (0.01 M, pH 7.4). All birds were inoculated intramuscularly through the brisket muscle, housed in ventilated stainless steel isolation cabinets in a room with high efficiency particulate air under negative-pressure, and illuminated under continuous light for the duration of the experiment. Feed and water were provided ad libitum. All experiments were conducted in a biosafety level 3-plus laboratory. Tracheal swabs were collected daily after inoculation and treated as described above. Internal organs (heart, liver, spleen, lung, kidney, brain, pancreas, muscle, abd cloaca) of chickens that died were collected. The remaining chickens were euthanized 30 days postinoculation and internal organs were collected. All specimens were frozen at –70°C until tested.
Comparison of Lat with Other Diagnostic Tests
Two commercial kits were compared with LAT: a colloidal gold-based immunochromatographic test (H5 AIV Ag Test Kitc) and a real-time RT-PCR test (H5 AIV real-time RT-PCR Test Kitd). Ninety field specimens (23 livers, 41 lungs, 22 kidneys, 10 brains, and 4 tracheal swabs) were collected from birds during an H5N1 outbreak in Hubei Province, China in 2004. All specimens were tested by LAT, colloidal gold-based immunochromatographic test, real-time RT-PCR test, and VI. This work was conducted with the assistance of the Veterinary Diagnostic Center of Hunan Province, P. R. China and Shenzhen CIQ, P. R. China.
Field Evaluation of Lat Using Outbreak Specimens
A total of 828 tissue specimens (89 hearts, 102 livers, 102 spleens, 102 lungs, 102 kidneys, 23 pancreas, 27 tracheas, 77 cloacas, 102 brains, and 102 bone marrows) were obtained from chickens in the 2004 H5N1 outbreak in Hubei Province, China. These specimens were previously tested by VI for H5N1, and were used in this study for field evaluation of the LAT using procedures described previously.
Field Evaluation of Lat Using Routine Clinical Specimens
Six hundred and forty chicken specimens (160 tracheal swabs and 480 lung specimens) and 750 duck specimens (210 tracheal swabs and 540 lung specimens) were collected from slaughterhouses that processed animals from large commercial poultries. In addition, 260 lung specimens were collected from ducks on small family farms. All the specimens were tested for H5N1 using the LAT.
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Results |
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Analytical Sensitivity and Specificity of Lat
When H5N1 isolate Chicken/HuBei/327/2004 with a HA titer of 1 : 256 was serially diluted 2-fold (from 1 : 2 to 1 : 512), the highest dilution to produce agglutination (2+) was the 1 : 128 dilution. The specificity of LAT was evaluated using AIV subtypes H9, H4, H3, and H1 and other avian respiratory pathogens such as NDV, IBDV, IBV, EDSV-76, and PMV-2. No agglutination was observed with any viruses with the exception of AIV strain H5N1. Thus, LAT was highly specific for AIV H5N1.
Detection of H5n1 in Experimentally Infected Chickens by Lat
All chickens inoculated with 1,000 and 100 EID50 of A/Chicken/HuBei/327/2004 died within 36 hours of inoculation and those in the 5 subgroups of group III (HI = 0) died within 96 hours (Table 1). Members of group I (HI1:64) inoculated with 1 EID50 and 0.1 EID50 survived the infection and were euthanized 30 days postinoculation. Internal organ specimens were collected from all animals and tested using the LAT. Results of the LAT tests are described in the following 2 sections.
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Field Evaluations of Lat Using Outbreak Specimens
The sensitivity and specificity of the LAT were compared to those of the current gold standard for AIV detection, VI, using 828 chicken specimens obtained during the 2004 H5N1 outbreak. The performance characteristics of the LAT compared to the VI assay are shown in Table 5. A total of 477 were positive by VI, of which 439 were also positive by the LAT. In addition, 4 specimens were negative by VI but positive by the LAT. Thus, LAT had a sensitivity of 92.0%, specificity of 98.9%, positive predictive value of 99.1%, and negative predictive value of 90.1% for H5N1. The correlation between VI and LAT was 94.9%, and there was no significant difference in H5N1 diagnosis between the two techniques (P > 0.05).
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Discussion |
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In this study, IgG concentration affected the sensitivity of SLP. The best sensitivity was obtained when the largest percentage of the starting amount of IgG was bound to the latex beads, which was achieved using 1,000 µg of IgG per 0.5 ml of 2.5% latex particles. Although the total amount of IgG bounded to latex particles increased when more IgG was added, the rate of agglutination was significantly reduced when 1,800 µg of IgG or more per 0.5 ml of latex particles was used in conjugation.
To increase the specificity of the LAT, MAbs were used instead of polyclonal antibodies. Seven hybridoma cell lines were established that secreted MAbs specific for different regions of the HA protein of H5N1 virus. The reactivity of MAbs in culture supernatants was assessed by HI and the HI titer of culture supernatants was generally 32, indicating that high affinity MAbs were produced.
In the animal infection experiments, the chickens were divided into 3 groups based on the pre-inoculation HI titer and inoculated with varying virus doses (ranging from 1,000 EID50 to 0.1 EID50) in order to mimic field conditions in the laboratory but control other variables that might occur in the field, such as: medication, stress, and co infection with multiple bacterial and viral agents. Data obtained suggested that the LAT could detect H5N1 in tracheal swabs (Table 2). However tracheal swabs taken from the chickens inoculated with H5N1at 100 EID50 that died within 36 hours without obvious clinical signs, had a low positive rate (52.8%). It is suggested that this low positive rate might be due to the likelihood that the high dose of the inoculum might have attacked the brain and lung directly and killed the chickens before systemic replication, proliferation, and subsequent discharge of viruses through the trachea. In contrast, chickens inoculated with 1 EID50 and 0.1 EID50 lived longer and had higher positive rates (average 77.9% and 71.4%). The difference in the detection rates from various internal organs might be due to differential tropism. As expected, the detection rate was much higher in lung tissue than that in the heart, cloaca, and muscle. The low detection of H5N1 in the cloaca suggests that the current practice of using fecal droppings of waterfowls for AIV detection may seriously under-estimate the prevalence of H5N1 in these reservoir animals.
The evaluation results of the LAT when applied to 828 specimens of chickens collected during an outbreak (Table 5) and 90 specimens from naturally infected animals show excellent performance characteristics (sensitivity, specificity, and correlation) in comparison to VI, immunochromatographic test, and real-time RT-PCR test. Like immunochromatographic test, LAT results are read visually. However, unlike the former, the latter is less affected by the presence of blood in the specimens. Compared to real-time RT-PCR, LAT is much less expensive, less technically demanding, and can be performed in the field.
When LAT was evaluated in the field using specimens from large commercial operations that mandatorily immunize their birds and specimens from small family poultries that did not immunize, all specimens from large commercial operations were negative whereas those from family farms had a relatively high positive rate. This apparent discrepancy could be due to the following 3 reasons. First, these farms were far away from cities and widely scattered, which made governmental supervision very difficult. Second, veterinary services in China are relatively less developed in rural areas where the small family farms abound. Third, current poultry production systems in China vary tremendously in scale and technology, from large-scale intensively managed mega farms to backyard flocks with a small number of chickens, ducks, geese, and pigs housed in close contact. In the latter situation, no obvious severe losses would be felt when birds are infected with AIV and there are no significant incentives to the farmers for vaccination. Therefore, the occurrence of H5N1 in birds in small operations in the absence of apparent outbreaks demonstrates that the prevention and control of AIV in China is a very formidable task. It also suggests that AIV infections can only be controlled by wide-scale immunization and testing.
From 2003 to date, H5N1 has caused great losses to the poultry industry in China. As demonstrated in the study, the newly developed LAT test can accurately detect AIV subtype H5 without cross reactivities to the other viruses that were evaluated in the study. This test is applicable to internal organs and tracheal swabs. It is rapid, simple, and easy to perform and does not require expensive equipment or skilled personnel. Thus, this test is appropriate for use in field investigations and in rural animal clinics for the rapid detection of H5N1.
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Acknowledgments |
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a. Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, P.R. China.
b. Polysciences, Inc. Corporate Headquarters, Warrington, PA.
c. Animal Genetics, Inc. Jangan-gu, Suwon, Kyonggi-do, Korea.
d. Shenzhen Taitai Genomics, Inc. Shenzhen 518057, P.R. China.
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References |
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