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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This article presents a simplified method for preparing 4 hemagglutination units antigen for Newcastle disease virus serological testing. By accurately determining the hemagglutination titer of the antigen, combined with a more rigorous back-titration method, and a well-defined adjustment process, it enhances test efficiency, reduces false positives, and improves disease surveillance in poultry.

Abstract

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article introduces an optimized method for preparing 4 hemagglutination units (4-HAU) antigen solution, a key component of the hemagglutination inhibition assay (HI) used in NDV serological detection. Unlike conventional methods, which involve time-consuming and undefined back-titration and adjustment steps, this approach streamlines this process by accurately measuring the HA titer using an initial series of dilutions (1:3, 1:5, 1:7, and 1:9). We also provide a specific method for adjusting or reformulating based on the back-titration results, reducing the need for repeated back-titrations. In addition, we evaluated the effect of the accuracy of the 4-HAU antigen solution on serum HI titer and found that when the titer of the 4-HAU antigen was lower than 3, it resulted in the appearance of false-positive HI samples. By providing an accurate method and minimizing computational tasks, this approach increases test efficiency and reliability, contributing to improved disease surveillance and control in poultry populations.

Introduction

Newcastle Disease (ND) is a widespread and severe poultry affliction recognized globally1-3. It manifests through various symptoms such as high fever, respiratory distress, dysentery, nervous disorders, and mucosal hemorrhage4. The causative agent, Newcastle Disease Virus (NDV), has endured for almost a century, afflicting more than 200 avian species, including chickens, ducks, geese, and pigeons5. Transmission primarily occurs through direct or indirect contact with infected birds, with poultry, pigeons, and free-ranging birds serving as potential reservoirs6. Despite NDV's single serotype, its genetic diversity poses significant challenges to disease management and control efforts3,7.Vaccination serves as the primary strategy for controlling ND, complemented by stringent biosecurity measures5,8. Various commercial vaccines are globally available for poultry, provoking robust serum antibody responses following immunization9. These antibodies play a critical role in mitigating symptom severity upon exposure to virulent strains and in curbing interflock transmission10. Standard revaccination protocols, typically involving live attenuated vaccines administered every 6-12 weeks, are standard practice in regions endemic to ND9. Routine monitoring of postvaccination antibody titers in commercial poultry flocks is essential for assessing vaccine effectiveness11-13. Low antibody titers post vaccination may indicate vaccine failure, prompting timely corrective measures such as supplementary vaccination or investigation into potential immunosuppressive factors affecting immune response14.Multiple techniques are utilized for detecting serum antibodies against NDV, including enzyme-linked immunosorbent assay, hemagglutination inhibition (HI), and neutralization assay9,13,15,16. Each method presents unique advantages and limitations concerning sensitivity, specificity, and cost-effectiveness.This article delineates a step-by-step protocol based on the World Organization for Animal Health (OIE)'s protocol for conducting HI to quantify serum-specific antibody titers against NDV11. Hemagglutination (HA), a phenomenon induced by certain enveloped viruses like NDV, involves the clumping of red blood cells (RBCs)9. The hemagglutinin-neuraminidase (HN) protein on the NDV surface interacts with RBCs, resulting in cell clumping and lattice formation2. HI assay is preferred as a serological method due to its ability to assess serum antibody specificity towards the HN protein of NDV8,9. Furthermore, its cost-effectiveness and independence from specialized instrumentation render it accessible and practical for routine use.To improve assay efficiency, we refined the workflow of the OIE protocol11, with a focus on attaining more accurate antigen HA titers and offering detailed adjustments for the 4 hemagglutination units (4-HAU) antigen solution. Additionally, through comparative analysis, we assessed the impact of 4-HAU accuracy on HI results, providing valuable insights for field practitioners. This approach is not limited to NDV antibody testing but extends to the detection of viral subtypes and other hemagglutinating viruses, including measles, polyomavirus, mumps, and rubella.

Protocol

  1. Approval for the protocol was granted by the local institutional animal care and use committee. All procedures involving live virus antigens and clinical serum samples were performed in a Biosafety Level 2 laboratory, in compliance with established safety protocols.
  1. 1. Preparation of 1% chicken RBC suspension
  1. 1.1. Add 3 mL of Alserver's solution (anticoagulant) into a sterile 15 mL conical centrifuge tube.1.2. Collect 1 mL of blood from the wing vein of each of the three non-immune chickens (without NDV antibodies). Immediately transfer the blood to the tube containing the anticoagulant and gently mix. 1.3. Fill the tube with sterile 1x phosphate-buffered saline solution (PBS, pH 7.2-7.4) to a total volume of 12 mL, and mix gently. Centrifuge at 500 × g for 10 min using a horizontal rotor.1.4. Carefully remove the supernatant and the white blood cell layer. Repeat steps 1.3 and 1.4 2x to obtain the RBC pellet.1.5. Reverse pipette 1 mL of the RBC pellet into 99 mL of sterile PBS, to prepare a 1% RBC suspension.NOTE: The required volume of the 1% chicken RBC suspension is estimated at 3 mL/plate (25 µL/well × 96 wells × 1.25 = 3.0 mL; where 1.25 accounts for pipetting wastage to ensure sufficient volume).
  1. 2. Reconstitution of lyophilized antigen or serum
  1. 2.1. Add 2 mL of sterile PBS to the ampoule containing the lyophilized antigen or serum. Gently shake the ampoule and allow it to stand for 2-3 min to minimize bubbles and ensure complete dissolution.NOTE: Follow the specific manufacturer's instructions, as different types of lyophilized materials were not compared in this study.2.2. Aliquot the reconstituted solution into 1.5 mL centrifuge tubes and freeze at -20 °C until use. NOTE: Re-determine the HA titer each time the antigen is thawed and used, or when a new RBC suspension is prepared.
  1. 3. HA titration of antigens
  1. 3.1. Dispense 40, 80, 120, and 160 µL of PBS, respectively, into four adjacent wells of a disposable 96-well V-bottom microtiter plate, then add 20 µL of antigen to each well, and mix by pipetting up and down 10x to achieve dilutions of 1:3, 1:5, 1:7 and 1:9, respectively. A schematic diagram of the dilution operation is shown in Figure 1.[Place Figure 1 here]3.2. Label a new microtiter plate. Add 25 µL of PBS to each well in rows 1-5.3.3. Place 25 µL of the diluted antigen solutions into the first wells of rows 1-5 using a multichannel pipette, and mix by pipetting up and down at least 5x.3.4. Transfer 25 µL from the first well of each row to the second well, mixing thoroughly. Continue transferring from one well to the next, through to the 11th column, to create two-fold serial dilutions (1:2 to 1:2048). Discard 25 µL after the 11th column. 3.5. Add 25 µL of PBS to each well, starting with the wells containing the lowest antigen concentration.3.6. Add 25 µL of 1% chicken RBC suspension to each well, again starting with the wells containing the lowest antigen concentration.3.7. Shake the plate on a microplate shaker for ~20 s to mix thoroughly. 3.8. Leave it undisturbed on benchtop at room temperature (20-25 °C) for ~30 min until the RBC of the 12th column completely settled. For the plate layout refer to Figure 2.[Place Figure 2 here]3.9. Read and record the results. HA is determined by tilting the plate by 90° for ~25 s and observing the presence or absence of tear-shaped streaming of the RBCs. The highest dilution showing complete hemagglutination (no streaming), represents 1 HA Unit. Wells in the 12th column serve as RBC (negative) controls.3.10. Calculate the HA titer of each row using Table 1, the maximum of which is used for the preparation of the 4-HAU antigen solution. For example, if a, b, c, d, and e are equal to 9, 8, 7, 6, 6, respectively, then 3×28=768 is the maximum value, and is the HA titer of the antigen stock.[Place Table 1 here]
  1. 4. Preparation of the 4-HAU antigen solution
  1. 4.1. Calculate the dilution factor for the preparation of the 4-HAU antigen solution: D=HA Titer4 . Determine the total 4-HAU volume needed: V=3 mL×Number of microplates. Then, calculate the required volumes of antigen stock solution: Va=VD , and PBS: Vp=V-Va.NOTE: Volumes are measured in milliliters. Estimate 3 mL per microtiter plate of the 4-HAU (25 µL/well × 96 wells × 1.25 = 3 mL).4.2. Pipette the calculated volume of PBS into a container, then add the corresponding volume of antigen stock solution and mix well to obtain the 4-HAU antigen solution.NOTE: The 4-HAU antigen solution should be used as soon as possible.
  1. 5. Back-titration of the 4-HAU antigen solution
  1. 5.1. Dispense 25, 50, 75, 100, 125, and 150 µL of PBS into six adjacent wells of a microtiter plate, then add 25 µL of the 4-HAU antigen solution to each well, and mix by pipetting up and down 10x. NOTE: Dilute the 4-HAU antigen solution with PBS at dilutions of 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7. A schematic diagram of the dilution operation is shown in Figure 3.[Place Figure 3 here]5.2. Transfer 25 µL of each diluted antigen solution to the wells of another row using a multichannel pipette. Add 25 µL of PBS to an extra well as a negative control.5.3. Add 25 µL of PBS to each well, then add 25 µL of 1% chicken RBC to each well.5.4. Shake the plate on a microplate shaker for ~20 s to mix. 5.5. Leave itundisturbed on the benchtop at room temperature (20-25 °C) for ~30 min until the RBCs of the negative control are completely settled.5.6. Read and record the results. NOTE: The negative control well should show no HA. Ideally, the 1:4 dilution is exactly the highest dilution with complete HA. If the titer of the 4-HAU solution is more than 4, the highest dilution may be 1:5, 1:6, or 1:7; if less, it may be 1:2 or 1:3. A schematic layout of the back-titration and representative results are shown in Figure 4.[Place Figure 4 here]5.7. Adjust or reformulate the 4-HAU antigen solution if necessary.NOTE: If the volume of the 4-HAU antigen solution used in back-titration is ≤1% of the total volume (the volume of 4-HAU antigen solution ≥???? 15 mL), refer to Table 2 for specific adjustments. For example, if the highest dilution with complete HA is 1:2, the titer of the 4-HAU antigen solution is now 2, and an equal volume of antigen should be added as in the previous preparation of the 4-HAU antigen solution. Conversely, if the highest dilution of complete HA is 1:6, the titer is now 6, and half the volume of PBS should be replenished. [Place Table 2 here]
  1. If the volume used in the back-titration exceeds 1% of the total (the volume of 4-HAU antigen solution
  1. 6. Preparation of serum
  1. 6.1. Collect ~1 mL of blood from chicken wings without using an anticoagulant. 6.2. Transfer the blood to a 1.5 mL centrifuge tube and incubate at 37 °C for ~2 h. Centrifuge at 3,000 × g for 10 min, and carefully aspirate the supernatant into a new tube.
  1. 7. HI assay
    7.1. Label microtiter plates according to the assay layout. Add 25 µL of PBS to each well in columns 1-11, and 50 µL to the wells in column 12, using a multichannel pipette.7.2. Add 25 µL of serum to the first well of each row, including positive and negative serum control rows. Mix thoroughly by pipetting up and down at least 5x.7.3. Transfer 25 µL from the first well of each row to the second well and mix thoroughly. Continue transferring and mixing from the second well to the third, and so on, until the 10th column. Discard 25 µL of liquid from the well in the 10th column after mixing.7.4. Add 25 µL of 4-HAU antigen to each well in columns 1-11 in the direction of low to high serum concentration.7.5. Shake the plate on a microplate shaker for ~20 s. Leave it undisturbed on the benchtop for ~30 min.7.6. Add 25 µL of 1% chicken RBCs to each well in the direction of low to high serum concentration.7.7. Shake the plate on a microplate shaker for ~20 s to mix thoroughly. Leave it undisturbed on the benchtop for ~30 min until RBCs of the PBS control wells are completely settled.7.8. Read and record the results. NOTE: The HI titer is the highest serum dilution that completely inhibits hemagglutination of the 4-HAU antigen. Tear-shaped streaming of RBCs can be observed in the last column of each row, by tilting the plate 90° for 25 s. Complete HA (no streaming) can be observed in the second last well from the end of each row. Valid test results require the HI titer of the positive serum control to be within one dilution of the known HI titer; the HI titer of the negative serum control to be ≤ 2 log2; and the absence of self-HA of the RBC control. For a schematic diagram of the HA assay workflow, see Figure 5.[Place Figure 5 here]

Representative Results

Validation of the titer of 4-HAU antigen solution formulated using the optimized methodThe study employed various dilutions of the antigen stock solution to accurately determine the HA titer, facilitating the calculation of the dilution factor for preparing the 4-HAU antigen solution. The results reveal that the optimized method is both efficient and precise, reducing the number of repetitive back-titration and adjustment procedures. In the first scenario, an initial HA titer of ~512 yielded a final titer of 768, aligning with the requirements for the 4-HAU antigen solution (Figure 6). Similarly, in the second instance, an initial titer of ~256 remained unchanged, ensuring the prepared 4-HAU antigen solution met experimental criteria (Figure 7). Lastly, with an initial titer of ~128, the determined exact titer of 144 maintained compatibility with experimental specifications (Figure 8). This streamlined approach significantly diminishes the time investment, taking merely 1-2 h compared to conventional methods. Overall, these findings underscore the efficacy and expediency of the optimized approach for formulating the 4-HAU antigen solution. [Place Figure 6 here][Place Figure 7 here][Place Figure 8 here]Effect of the accuracy of the 4-HAU antigen on serum HI titerTo assess the impact of the 4-HAU antigen accuracy on serum HI titers, serum samples with known HI titers were selected and prepared across a titer range of 2log2 to 9log2 via 2-fold dilutions. Antigen solutions ranging from 1-HAU to 8-HAU were then prepared using the optimized method. In the HI assay, it was observed that when the 1 HAU antigen solution was used, the measured antibody titer was 1log2 higher than the correct value. Similarly, for the 2-HAU antigen solution, the measured antibody titer was 1log2 higher when the target antibody titer ranged from 4log2 to 9log2. Conversely, using the 3- or 5-HAU solutions resulted in the measured antibody titers that matched those of the 4-HAU. Finally, with the 6- or 7-HAU antigen solutions, the measured titer values were 1log2 lower when the actual antibody titer ranged from 2log2 to 3log2. The results are summarized in Table 4 and the experimental results are shown in Figure 9. These findings highlight the nuanced relationship between the 4-HAU antigen accuracy and measured antibody titers in HI assays, providing valuable insights for assay interpretation and optimization.[Place Table 4 here][Place Figure 9 here]Figure 1: Schematic diagram illustrating the procedure for diluting of the antigen solution in the hemagglutination assay. The antigen solution is diluted in PBS at ratios of 1:3, 1:5, 1:7, and 1:9, respectively. PBS in wells or centrifuge tubes is represented in blue, while antigens are shown in green.Figure 2: Schematic layout of a microplate used in the hemagglutination assay. The first column of rows 1-5 contains undiluted and various initial dilutions of the antigen solution, followed by a two-fold serial dilution from left to right until column 11. PBS control is placed in the last column. The gradient from dark to light indicates decreasing antigen concentration.Figure 3: Schematic diagram of dilution procedure of 4 hemagglutination units' solution in back-titration. The 4-HAU working solution is diluted with PBS at ratios of 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7. PBS is depicted in blue, and antigens in green.Figure 4: Schematic layout of the wells for back-titration and representative result. The diluted 4-HAU working solution is transferred to a new row, with an additional PBS control added. The gradient from dark to light green indicates antigen concentration from high to low. Representative results demonstrate that the 1:4 dilution is the highest dilution showing complete hemagglutination.Figure 5: Schematic diagram of hemagglutination assay workflow. PBS is represented in blue, serum in yellow, the 4-HAU antigen solution in green, and the 1% chicken RBC suspension in red. Arrows indicate the order of liquid dispensation.Figure 6: HA titer determination of the antigen and the potency confirmation of the prepared 4-HAU antigen solution. The upper panel (A) shows the results of the HA assay (HA titer ~512). The middle panel (B) shows the HA titer calculation, with the maximum value in bold (768) used to calculate the dilution factor of the 4-HAU antigen solution. The lower panel (C) confirms the potency of the 4-HAU antigen.Figure 7: HA titer determination of the antigen and the potency confirmation of the prepared 4-HAU antigen solution. The upper panel (A) shows the results of the HA assay (HA titer ~256). The middle panel (B) shows the HA titer calculation, selecting the maximum value in bold (256) to calculate the dilution factor of the 4-HAU antigen solution. The lower (C) shows the 4-HAU potency confirmation.Figure 8: HA titer determination of the antigen and the potency confirmation of the prepared 4-HAU antigen solution. The upper (A) shows the results of the HA assay (HA titer in this example is ~128). The middle (B) shows the calculation of HA titer, selecting the maximum value in bold (144) to calculate the dilution factor of the 4-HAU antigen solution. The lower (C) shows the 4-HAU potency confirmation.Figure 9: HI titer measurement of serum samples through two-fold serial dilutions (undiluted to 128 times) corresponding to the potency of the 4-HAU solution, ranging from 1 to 7 HA units.Rows 1-8 of A-G utilized serum with hemagglutination inhibition titers from 9 log2 to 2 log2, diluted to column 10 (2-1,024x) by a two-fold serial dilution. The last column serves as the PBS control, while the penultimate column contains the 4-unit antigen control. The antigen solutions in A-G contained 1-7 HA units, respectively. The back-titration of the 1-7 HA unit antigen is shown in H.Table 1: HA assay results and calculation of HA titer of stock solution.Table 2: Reference table for adjusting 4-HAU concentration based on back-titration results. Va and Vp represent the volumes of antigen and PBS used in the formulation of 4-HAU antigen solution, respectively.Table 3: Reference table of dilution factor correction for re-preparation of 4-HAU antigen. D represents the dilution factor previously used in 4-HAU antigen preparation.Table 4: HI titer readings for 2-fold serial dilutions of serum when 4-HAU solution contained 1 to 7 HA units.

Discussion

The optimized method proposed in this article presents an approach for accurately preparing the 4-HAU antigen solution. Although the OIE and EU guidelines suggest initial dilutions for HA titration, they do not provide precise details regarding the dilution ratios to be used or offer specific methodologies11,12. Furthermore, although the FAO and OIE recommend back-titration as a means of enhancing the accuracy of 4-HAU, they lack a clear protocol11,13. This article addresses these shortcomings by delineating a protocol using precise dilution ratios (1:3, 1:5, 1:7, 1:9) in HA titration of antigens, selecting the maximum values for 4-HAU preparation. Moreover, the protocol incorporates dilution ratios of 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7 in the back-titration process. Additionally, a comprehensive method of adjustment or reformulation is provided, to enhance the feasibility of the assay.Rationale of initial dilution in HA titrationSignificance of initial dilution The HA titer of the antigen stock solution is denoted as x，where 2n≤x<2n+1 (for n=1, 2, ..., 11), the theoretical experimental value in the HA assay is 2x. Consequently, the dilution factor for the 4-HAU preparation is 2x22, and the HA titer of the 4-HAU is 4x2x. It is evident that the 4-HAU titer is precisely 4 only when x=2n. In all other scenarios, the 4-HAU titer resides between 4 and 8.Reasons for excluding dilution factors of 2, 4, 6, and 8The dilution factor is denoted as D (where D=1, 2,..., 9); the theoretical value of the HA titer of the antigen stock solution is D×2xD. It is observed that when D=2, 4, 8, the value remains equivalent to that of D=1. Additionally, whenD=6, the value matches that of D=3. Therefore, utilizing dilution factors of 2, 4, 6, and 8 only increases the workload without enhancing the accuracy of the results.Benefits of initial dilution factors of 3, 5, 7, and 9The HA titer of the antigen stock solution is D×2xD. The HA titer of the 4-HAU is 4xD×2xD.When x=2n, the maximum HA titer of antigen stock solution occurs at D=1; when x=2n-2×5, it occurs at D=5; for x=2n-2×6, it is at D=3; and for x=2n-2×7, it is at D=7. Using these maximum values for 4-HAU preparation ensures that the titer is exactly 4, enhancing the accuracy of the experimental results.Significance of the method in relation to existing methods The World Health Organization (WHO) has specified the use of standardized RBCs, standardized antigen solutions, and test controls for quality control in animal influenza virus-associated HA/HI testing17. For example, the concentration of the RBC suspensions must be consistently confirmed and adjusted with a hemacytometer counting; antigen dilutions must be back-titrated on each test day; red blood cell controls must be set up in HA tests to determine results based on them; negative and positive serum controls must be set up in HI tests to determine the presence of variability.In the FAO Newcastle Disease HA/HI test protocol13, a 10% chicken RBC stock suspension is firstly prepared without leukocyte removal, in which the pellet of packed blood cells was measured using one of three methods: a micro-hematocrit centrifuge, a micropipette, or a graduated centrifuge tube. The stock solution is then diluted to the desired concentration. Other similar measures have also been requested, but there is a lack of specific details on the back-titration, as prementioned.In this study, for feasibility reasons, instead of using a hemacytometer or micro-hematocrit centrifuge for RBC suspension preparation, we measure the pellet of packed blood cells by reverse pipetting to reduce the dependence on test equipment. In the antigen standardization section, we improved the resolution of the HA test results with the initial dilution, gave a more stringent dilution method of back-titration, and supplied the 4-HAU-related adjustment and reconstitution steps to facilitate a step-by-step start. Therefore, our approach complements the existing methods.Limitations of the methodOur method incorporates additional dilution and pipetting steps to improve the theoretical accuracy of the results, but manual addition of liquid and dilution using a pipette can introduce inaccuracies due to variability in technique among different personnel. In addition, chicken RBC suspension tends to settle at the bottom of the container during the experiment, and if they are not mixed adequately before use, they can introduce variations due to the differences in the concentration of RBCs dispensed. Therefore, we recommend choosing a technician who is skilled in pipetting to perform the test and avoid changing personnel during the test to minimize variability. Of course, if the equipment conditions allow and the batch size is large, it is best to use an automated pipetting workstation. Lack of standardization of RBC suspension may contribute to batch-to-batch inconsistencies. RBC suspension has a limited shelf life and is typically freshly prepared for each experiment, usually daily. Factors such as centrifugal force and duration during RBC pellet preparation can influence their volume. Additionally, RBCs may adhere to the tip surface during reverse pipetting, complicating accurate measurements. Incorporating WHO- or FAO-related instrument-based concentration validation would help reduce the variability.Current HA/HI results still need to be visually assessed. Variability in results is inevitable due to subjective factors in human judgment despite relevant comparative controls, so differences in interpretation criteria at different time points or persons can affect the accuracy of 4-HAU titers. In the future, if the results can be judged numerically, it will be beneficial to reduce variability.Importance and potential applications of the method in specific research areasThis article offers clear guidelines and rationale for dilution ratios and numerical treatment, thereby standardizing and optimizing HA assays for improved accuracy and efficiency of 4-HAU preparation. By elucidating a precise methodology and simplifying theoretical requirements for technicians, it streamlines testing tasks. Furthermore, by providing methodologies for back-titration and adjustment of antigen solutions, it contributes to standardizing serological testing protocols, thereby bolstering the reliability of test results in veterinary diagnostics.Serologic antibody level testing plays a crucial role in assessing the immune status of birds affected by diseases like ND. The OIE defines a positive sample as one with an HI titer greater than or equal to 4log211. This article confirms that false-positive samples were more likely to be detected at 4-HAU antigen titer below three. However, within the 3 to 7 HA units, minimal impact on sample positivity is observed. It is worth noting that the OIE permits individual laboratories to use an 8-HAU antigen solution in HI assay11. Overall, our findings stress the need to consider the 4-HAU antigen titer in serologic testing for improved disease diagnosis and surveillance.

Acknowledgements

1.Ayllon, J., García-Sastre, A., Martínez-Sobrido, L. Rescue of recombinant Newcastle disease virus from cDNA. J Vis Exp. (80), e50830 (2013).2.Chen, Y. et al. The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization. PLoS Pathog. 20 (2), e1011981 (2024).3.Miller, P. J., Decanini, E. L., Afonso, C. L. Newcastle disease: evolution of genotypes and the related diagnostic challenges. Infect Genet Evol. 10 (1), 26-35 (2010).4.Terregino, C., Capua, I. Clinical traits and pathology of Newcastle disease infection and guidelines for farm visit and differential diagnosis. Avian Influenza and Newcastle Disease: A Field and Laboratory Manual. 113-122 (2009).5.Dimitrov, K. M., Afonso, C. L., Yu, Q., Miller, P. J. Newcastle disease vaccines-A solved problem or a continuous challenge? Vet Microbiol. 206, 126-136 (2017).6.Sheng, W. et al. Molecular characteristics and phylogenetic analysis of pigeon paramyxovirus type 1 isolates from pigeon meat farms in Shanghai (2009-2012). Sci Rep. 14 (1), 10741 (2024).7.Yates, J. G. E. et al. Production of high-titer recombinant Newcastle disease virus from allantoic fluid. J of Vis Exp. (183) 63817 (2022).8.van Boven, M. et al. Herd immunity to Newcastle disease virus in poultry by vaccination. Avian Pathol. 37 (1), 1-5 (2008).9.Oberländer, B. et al. Evaluation of Newcastle disease antibody titers in backyard poultry in Germany with a vaccination interval of twelve weeks. PloS One. 15 (8), e0238068 (2020).10.Bhattacharya, S. et al. Spillover of Newcastle disease virus to Himalayan Griffon vulture: a possible food-based transmission. Virus Genes. 60 (4), 385-392 (2024).11.World Organisation for Animal Health (OIE). Chapter 3.3.10. Newcastle disease (Infection with Newcastle disease virus). Manual of diagnostic tests and vaccines for terrestrial animals. (2024).12.Council of European Union. Council Directive 92/66/EEC of 14 July 1992 Introducing Community measures for the control of Newcastle disease. Document 31992L0066. L260, 1-20 (1992).13.Grimes, S. E. A basic laboratory manual for the small-scale production and testing of I-2 Newcastle disease vaccine. RAP Publication. 2002/22, 1-129 (2002).14.Dortmans, J. C. F. M., Peeters, B. P

Materials

NameCompanyCatalog NumberComments
(3-Glycidyloxypropyl)trimethoxysilaneSigma440167GOPS
0.25% Trypsin-EDTA (1X)Gibco25200-056
4-Dodecylbenzenesulfonic acidSigma44198DBSA
96-well plateFalcon353075
AcetoneTechnic530
Acrylic resinFischer scientificNC1455685
agaroseSigmaA9539
autoclaveTuttnauer3150 EL
AZ 10XTMicrochemicalsPositive photoresist
AZ 826 MIF DeveloperMerck10056124960Metal-ion-free developer for the negative photoresist
AZ DeveloperMerck10054224960Metal-ion-free developer for the positive photoresist
AZ nLof 2070MicrochemicalsNegative photoresist
BuprenorphineAxience
CarprofenRimadyl
Centrifuge Sorvall Legend X1RThermo Scientific75004260
CMOS camera Prime 95BPhotometrics
CO2 incubator HERAcell 150iThermo scientific
DAC boardNational InstrumentsUSB 6259
Déco spray PébéoCultura3167860937307Black acrylic paint
Dextran Texas Red 70.000ThermofisherD1830
Die bonding paste "Epinal"HitachiEN-4900GCSilver paste
Dimethyl sulfoxideSigmaD2438
Dispensing machineTianhaoTH-2004C
Dulbecco’s Modified Eagle’s Medium + GlutaMAX™-IGibco10567-014
Dulbecco's Modified Eagle's MediumSigmaD6429
Egg incubator COUVAD'OR 160lafermedemanon.com
Ethylene glycolCarl Roth6881.1
Fertilized eggs of Japanese quailJapocaille
Fetal Bovine SerumVWRS181BH
FlaskGreiner658170
Fluorescence macroscopeLeica MZFLIII
Gl261DSMZACC 802
Gold pellets - Dia 3 mm x 6 mm thNeyco
Handheld automated cell counterMilliporePHCC00000
Heating and drying ovenMemmertUF110
Hexadimethrine Bromide Sequa-breneSigmaS2667
hot plate Delta 6 HP 350Süss Microtec
Illumination system pE-4000CoolLed
Infrared tunable femtosecond laser (Maï-Taï)Spectra Physics (USA)
Ionomycin calcium saltSigmaI3909
Kapton tape SCOTCH 92 33x193MPolyimide protection tape
Lab made pulse generator
Labcoter 2 Parylene Deposition system PDS 2010SCS
Lenti-X 293 T cell lineTakara Bio63218HEK 293T-derived cell line optimized for lentivirus production
Lenti-X GoStix PlusTakara Bio631280Quantitative lentiviral titer test
Mask aligner MJB4Süss Microtec
Micro-90 Concentrated cleaning solutionInternational ProductsM9050-12
Microscope slides 76 x 52 x 1 mmMarienfeld1100420
Needles 30GBD Microlance 3304000
PalmSens4 potentiostatPalmSens
parylene-c : dichloro-p-cyclophaneSCS300073
PCB Processing TanksMega ElectronicsPA104
PEDOT:PSS Clevios PH 1000Heraeus
penicillin / streptomycinGibco15140-122
Petri dishFalcon351029
pGP-CMV-GCaMP6fAddgene40755plasmid
Phosphate Buffer Saline solutionThermofisherD8537
Plasma treatment system PE-100Plasma Etch
PlasmaLab 80 Reactive Ion EtcherOxford Instruments
Plastic maskSelba
Plastic weigh boat 64 x 51 x 19 mmVWR10770-454
Poly-dimethylsiloxane: SYLGARD 184 Silicone Elastomer KitDow chemicals1673921
Polyimide copper film 60 µm (Kapton)GoodfellowIM301522
Propan-2-olTechnic574
Protolaser SLPKF
puromycinGibcoA11103
Round cover glass 5 mm diameterFischer scientific50-949-439
Scepter Sensors - 60 µmMilliporePHCC60050
Silicone adhesive Kwik-SilWorld Precision Instruments
spin coaterSüss Microtec
Spin CoaterLaurellWS-650
Super glueOffice depot
tetracycline-free fœtal bovine SerumTakara Bio631105
Thermal evaporator Auto 500Boc Edwards
Two-photon microscopeZeiss LSM 7MP
U87-MGATCCHTB-14Human glioblastoma cells
Ultrasonic cleanerVWR
Vortex VTX-3000LLMSVTX100323410
Xfect single shots reagentTakara Bio631447Transfection reagent

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