Edited by: Kuldeep Dhama, Indian Veterinary Research Institute, India
Reviewed by: Hari Mohan Saxena, Guru Angad Dev Veterinary and Animal Sciences University, India; Mithilesh Kumar Singh, Indian Veterinary Research Institute, India
Specialty section: This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Immunoglobulin Y (IgY) is the predominant antibody found in hen’s (
Over recent years, there has been an increase in the use of oral passive immunotherapy. One novel protective strategy to achieve public health in humans and domestic animals is the production of specific immunoglobulin Y (IgY) in laying hens. This IgY is produced after the immunization of laying hens, when the humoral immune response (IR) is activated and produces specific IgY in the blood. After approximately a week, this IgY is transported to the yolk to confer natural passive immunity to the embryo and offspring (
Different research results have been reported concerning IgY levels, both in total and those specifically present in the serum or yolk, after laying hens have been immunized with different enteric pathogens employed as antigens (Ags) (
In poultry, GALT encompasses esophageal tonsils, pyloric tonsils, Meckel’s diverticulum, Peyer’s patches, and two cecal tonsils. Cecal tonsils are functionally and anatomically the most important organs of the GALT (
Although there are studies regarding the immunization schedule for IgY production (i.e., different methods of purification and quantification from eggs), research on humoral immune mechanisms after laying hen immunization with enteric bacterin is limited. For example, the activation of GALT has not been studied.
Therefore, the aim of the present study was to evaluate the relationship between the humoral IR and the activation of GALT in laying hens intramuscularly immunized with
The immunogen was made using an enterotoxigenic
To extend the immunogen, ETEC was grown in Minca broth medium for 72 h at 37°C to overexpress fimbria Ags. After growth, the bacteria were disabled by incubating in 0.5% formaldehyde overnight. The disabled ETEC’s strain was pelleted by centrifugation (4,000 ×
Forty-four Shaver hens (1-day-old) were obtained from a poultry farm (Monte Buey Avícola, Monte Buey, Córdoba, Argentina). The animals were kept in individual cages with food and water
The hens were fed a balanced ration for laying hens (Nutriarte, Córdoba-Argentina). A light/dark cycle of 16/8 h was used. Room temperature was 20 ± 2°C, and the relative humidity was between 55 and 60%.
All experiments were performed according to the institutional guidelines for animal care and experimentation. The protocol and procedures employed were reviewed and approved by the Ethics Committee of the Universidad Nacional de Rio Cuarto (
At 22 weeks of age, laying hens were immunized with 1 mL of bacterin (F4 group) or sterile PBS with 7% adjuvant (aluminum hydroxide; Control group); this was injected intramuscularly and distributed in both breast muscles. Booster immunizations were given at 2, 4, 6, and 8 weeks after the initial immunization. Eggs were manually collected every day from 23 to 42 weeks; egg yolks were separated from egg whites and stored at 4°C until needed for further analysis. Blood samples were taken from hens on the first day of immunization and every subsequent 15 days until the end of the test (57 days after the first immunization). Sera samples were preserved at −20°C for the determination of IgY and immunoglobulin A (IgA).
Approximately 2 mL of blood was collected from the brachial wing vein of each hen using heparinized syringes and then transferred into heparinized tubes. Blood samples were processed with 1 h between each drawing. Mononuclear cells were separated from the whole blood by using density gradient separation with Histopaque-1077 (Sigma-Aldrich, St. Louis, USA) according to a previously established protocol (
Immediately following death, half of the spleen of each laying hen was placed into a Petri dish with frost tamponed saline solution without Ca2+ and Mg2+ (CMF). The cells from the spleen were collected by mashing the tissue through a 60-µm nylon mesh into 5 mL of frost Roswell Park memory institute (RPMI)-1640. A further 2 mL of media was used to rinse any remaining cells through the mesh. The cells were separated by density gradient separation using Histopaque-1077 according to a previously established protocol (
The monoclonal anti-chicken antibody used in this research was Bu-1 a/b (AV20) coupled to fluorescein isothiocyanate (FITC; Santa Cruz Biotech). Bu-1 is expressed on avian B lymphocytes and used for single staining at 1:10 dilution of primary Ab (0.5 mg/mL) in PBS (pH 7.4). For blood samples, 50 µL of this dilution was mixed with 106 B lymphocytes and kept at 4°C in the dark for 15 min. After this incubation period, cells were washed with 100 µL of PBS and centrifuged at 400 ×
Fixation of blood and spleen B lymphocytes was performed according to a previously published procedure (
All samples underwent cytometry within 4 h of staining. Flow cytometry was performed on a BD FACS Canto II flow cytometer (San Jose, CA, USA). Green fluorescence (from FITC) was detected on the FL1 channel (530/30 nm), cells were analyzed at up to 10,000 events in the lymphocyte gate, based on forward and side scatters (
After necropsy, samples from esophageal, pyloric, and cecal tonsils and Peyer’s patches, spleen, and intestine (2 cm segment of the jejunal region before Meckel’s diverticulum) were fixed in buffered formalin (pH 7.4), dehydrated through graded alcohol to xylene, and paraffin embedded at 56–58°C for at least 3 h (1172601; Cicarelli). Subsequently, 3–4 µm sections were cut (Reichert-Jung ultramicrotome Leica RM 206) and placed on slides. Hematoxylin/eosin (H/E) staining was performed; sections were observed (Axiostar Plus Carl Zeiss microscope); and microphotographs were taken using a Canon PowerShot G6, 7.1 megapixels.
AV20-FITC was used to stain GALT and spleen sections of both the F4 and Control groups; these were used to measure germinal center (GC) that displays abundant fluorescent B lymphocytes using Image J software. Briefly, counting started from a point, moved through all the GC, and returned to the starting point. For this study, a digital camera (PowerShot G6, 7.1 megapixels, Canon Inc., Japan) was attached to a microscope (Axiophot, Carl Zeiss). In each spleen slide, 12 measurements of the GC were taken from each laying hen.
Hematoxylin–eosin stained intestinal sections from the F4, and Control groups were used to measure height, area, and perimeter of villi using a light microscope (Axiophot, Carl Zeiss). The data were processed using computer software (Axio Vision release). The height was the distance between the villous–crypt axis and the top of the villus. Villi area and perimeter were measured starting from the base thereof, crossing the edges with the cursor hairs and returning to the starting point. For each one of the variables, at least 15 measures of complete villi were taken from each slide of each hen.
The total concentration of IgA was evaluated by IgA Enzyme-Linked Immunosorbent Assay (ELISA) kit (ab157691 ABCAM). Sera samples were tested according to the manufacturer’s instructions. To measure secretory IgA, 2-cm segments of the jejunal region near to Meckel’s diverticulum were excised and then exposed. Mucus was collected by scraping the mucosal surface of the intestine, which was then weighed to equal quantities. Thereafter, the mucus from each gut sample was suspended in a 4-fold volume of PBS (wt/wt), vortexed thoroughly, and centrifuged at 5,000 ×
Serum IgY levels were evaluated by ELISA test, as previously described (
Anti-ETEC F4 serum levels were determined using the ELISA method developed by our research group, using the same procedure as for IgY serum levels, except that initially, the plates were coated with 50 µL of a suspension of concentrated and inactivated ETEC F4 strain (1 × 109 UFC/mL) in carbonate–bicarbonate buffer (0.05 M, pH 9.6) and incubated overnight at 4°C (
A water-soluble fraction (WSF) containing IgY from egg yolk was prepared using the water dilution method (
Avian immunoglobulin (Ig) was precipitated by sodium sulfate as described previously for the purification of IgY (
The total concentration of serum IgY was evaluated by ELISA test as previously described (
Anti-ETEC F4-IgY levels were determined using the ELISA method developed by our research group; it was similar to that for specific IgY serum levels, except that the wells of the microtiter plates were coated with 50 µL of a suspension of concentrated and formaldehyde-inactivated ETEC F4 strain (>1 × 109 UFC/mL) in carbonate–bicarbonate buffer (0.05 M, pH 9.6) and incubated overnight at 4°C. Serial dilutions of anti-ETEC F4-IgY and Control IgY (1/101 to 1/1,015) in PBS-Tween with 2% non-fat milk were incubated at 37°C for 1 h in a humidity chamber. Subsequently, an ELISA test was performed as mentioned for total IgY serum levels (
Statistical analyses were performed using Infostat software (
GC in GALT and in the spleen were greater in the F4 group than in the Control group (Table
Group | Peyer’s patches | Esophagic tonsils | Pyloric tonsils | Cecal tonsils | Spleen |
---|---|---|---|---|---|
Control ( |
78,532 ± 890*** | 9,786 ± 1,389*** | 47,316 ± 144 |
4,731,651 ± 6,789 |
499,318.8 ± 26,340*** |
F4 ( |
354,899 ± 1,245*** | 18,897 ± 2,567*** | 51,027 ± 1,877 |
5,102,797 ± 8,976 |
5,568,072.6 ± 11,380*** |
Peyer’s patches microphotography, with marked (abundant) lymphocyte production in intestinal histological sections in the F4 group and control group.
In addition, immunization triggered cecal and pyloric tonsil activation (increased 10-fold;
In ileum tissue sections, increased villi height (
Duodenum |
Ileum |
||||||
---|---|---|---|---|---|---|---|
Group | High (μm) | Area (μm2) | Perimeter (μm) | High (μm) | Area (μm2) | Perimeter (μm) | |
Control ( |
628.08 ± 296.22 | 10,347.20 ± 270.81 | 674.84 ± 50.2 | 731.38 ± 193.45* | 12,145.80 ± 1,198.70* | 957.90 ± 150.00 * | |
F4 ( |
1,257.86 ± 353.75 | 14,205.46 ± 365.02 | 901.99 ± 139.42 | 1,379.37 ± 245.70* | 17,456.40 ± 820.50* | 15,850.60 ± 135.40* |
In concordance with the numerous plasmatic cells located near to the epithelial cells in the villi of F4 group samples, the levels of secreted IgA were also significantly higher (1,624 ± 131 ng/mL,
In concordance with the expanded GC found in GALT and in the spleen, higher percentages of B lymphocytes were found in the F4 group in the blood (11.8 vs. 7.8%; Figure
Moreover, histological observations on the spleen corresponded well with the flow cytometry results.
In the F4 group, total IgY serum levels were high for all assays (
In the yolk, total IgY serum levels were already high after the first immunization in the F4 group, reached a peak by 3 weeks (
Anti
In the yolk, anti-
This work showed that intramuscular immunization of laying hens with
Despite the importance of other organs in the GALT, such as esophageal and pyloric tonsils, they were not studied in other researches. Interestingly, we found that both types of tonsils have been activated by the
The intestine is the major site of development, residence, and a route of entrance for pathogenic microorganisms into the body. Any perturbation of intestinal physiology can result in substantial clinical consequences (
Contrary to our data, in other research, no IgA serum was detected by ELISA test, after immunization of hens with an
According to the increased mucosal IgA levels registered into the bacterin-immunized hens, we detected an increment in intestinal protection, produced by an increase in both goblet cell number and their mucus production.
This work revealed that activated B lymphocytes entered the general circulation and caused a 50% increase in the number of circulating B lymphocytes, as recorded in the flow cytometry study. This is the response of the activation in GALT and the spleen, as we registered in the histopathologic and immunofluorescence study and in flow cytometry in the spleen. These data are consistent with GC activation in both the GALT and the spleen (
The spleen is the second most important secondary lymphatic organ after the MIS, controlling the blood and generating an IR to Ags through specific Ig secretion and clonal expansion of B lymphocytes in the GC (
With reference to IgY production, B lymphocyte activation in the spleen GC of
Immunoglobulin Y levels in our research were double the levels found in other research, where hens were immunized with
In previous research, specific IgY levels in hens immunized with
Laying hens, intramuscularly immunized with
Subsequently, a portion of B lymphocytes, after their clonal expansion, migrated from the blood to the spleen, where they activated other B lymphocytes in the spleen GC, resulting in systemic protection by high specific IgY serum levels. After one week, this IgY was present in the egg yolk.
These results suggest that GALT is a key immunologic tissue inside the MIS, working as a “command center” of humoral reaction.
This study received formal approval from the ethics committee of the Universidad Nacional de Río Cuarto. In order to guarantee a safe, correct and careful use and handling of experimental animals, the investigators proceeded according to the ethical guidelines of animal welfare committee (59) (see
Conceived and designed the experiments: MP, FA, AN, RM, AV. Performed the experiments: MP, AM, AN, FA, AV. Analyzed the data: MP, AV. Wrote the paper: MP, AV, AM, FA.
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, MS, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.
We thank Nelsy Scheleef for histological slides and Pilar Crespo for flow cytometry support.
Ab, antibody; Ag, antigen; BALT, bronchial-associated lymphoid tissue; CMF, tamponed Saline solution without Ca and Mg; CFU, colony forming unity; DCs, dendritic cells; ELISA, enzyme-linked immunosorbent assay;