Abstract
Importance
Few studies have examined the interactions of the chicken anemia virus (CAV) with the immune system of embryos.
Objective
To study the pathogenicity of the CAV in chicken embryos with respect to its tissue tropism and viral load in different tissues.
Method
The pathogenicity of the CAV was examined in about-to-hatch embryos through cytology, histopathological examinations, molecular detection, immunocytochemistry (ICC), immunofluorescent test, and immunohistochemistry techniques.
Results
The experimental study using a polymerase chain reaction (PCR) assay revealed the presence of CAV in the liver, bone marrow, and thymus tissues of embryos where embryos were inoculated with CAV-positive bone marrow specimens obtained from chicken anemia-suspect cases. Intracytoplasmic and intranuclear inclusion bodies were detected cytologically in the bone marrow of the embryos, whereas no inclusion body was detected in the thymus. On the other hand, both kinds of inclusion bodies were observed in bone marrow and thymus in the histopathology. In addition, various organs of PCR-positive CAV embryos revealed the depletion of lymphocytes and hematopoietic cells in the thymus and bone marrow, respectively. The CAV antigen was confirmed in the bone marrow and thymus cytological smears using ICC and immunofluorescence techniques.
Conclusions and Relevance
Viral antigen exhibited more tropism towards the bone marrow of embryos than the thymus in ICC and immunofluorescence techniques. The presence of CD44, a marker for progenitor cells and an antigen in the serial sections of bone marrow, confirmed the relevance of viral tropism for the hemocytoblasts of the bone marrow as the main target cells in embryos and subsequently in chicks for viral replication.
Keywords: Chicken anemia virus; Antigens, CD44; embryo; immunosuppression; progenitor cells
INTRODUCTION
The chicken anemia virus (CAV) is a potent immunosuppressive virus affecting chickens [1]. The CAV, the causative agent of chicken infectious anemia (CIA), belongs to the family Anneloviridae, which is grouped under the genus Gyrovirus. The virus, which has tropism for T lymphocytes of the thymus, erythroid, and myeloid progenitor cells of bone marrow, is spread by horizontal and vertical transmission [2,3].
The persistence of CAV in the tissues of adults results in intermittent shedding of the virus through eggs. The virus may also be present in immune-privileged cells of the reproductive tract to avoid immune detection. Furthermore, once these organs are formed in the embryo, they remain quiescent until the onset of reproductive maturity and undergo remarkable growth and differentiation [4]. The presence of CAV in the reproductive tissues and viral transcription tied to cell regulation might allow this virus to expand at specific stages of embryo development and then remain dormant until the chicken reaches reproductive maturity [5].
The production performance is severely hampered when the immune system of the birds is affected by the CAV. The increased susceptibility to the secondary pathogens paves the way for severe outbreaks. The affected birds also develop profound immunosuppression against various vaccines, resulting in vaccination failure [6,7].
The selective depletion of erythroid and myeloid progenitors has consequent effects on the immune function, making CAV infections an ideal model for studying virus and immune system interactions [8]. On the other hand, there is a lack of information on the stage at which the precursor cells become susceptible to infection. Moreover, understanding the virus–host relationship in the developing embryo is essential. Hence, the present study examined the interaction of the virus with the immune system of the embryos, which can provide useful insight into the impairment of the immune system. This study examined the CAV and its impact on the immune organs of developing embryos for the first time to the best of the authors’ knowledge.
METHODS
Ethics statement
No ethical involvement was required because no chicks or adult birds were used in this research [9]. Only embryonated eggs were used for the study. The tissue samples were collected from the dead carcasses of adult birds affected with CAV in field cases.
Source of virus
The PCR-positive CAV samples from field cases were used to inoculate the embryonated chicken eggs.
Preparation of tissue homogenate for chicken embryo inoculation
The polymerase chain reaction (PCR)-positive tissue samples comprised of bone marrow, thymus, and liver obtained from CAV-positive cases were pooled and finely minced. Subsequently, a 10% tissue suspension was made in sterile phosphate-buffered saline (PBS). Subsequently, the suspension was centrifuged at 6,708 × g for 15 min. The supernatant was filtered through a 0.22 μm syringe filter (M/s Sartorius), treated with antibiotics (penicillin [1,000 IU/mL] and streptomycin [1 mg/mL]), and stored at −20°C until needed.
Chicken embryonated egg inoculation
A 0.2 mL sample of the inoculum was injected into the yolk sac of 10 eight-day-old chicken embryonated eggs of an SPF origin (M/s Venky’s Pvt. Ltd., India) and incubated for 12 days. Candling was conducted daily to check the viability of the embryos. The embryos that died within 24 h were discarded. After 12 days of incubation (20th day of embryonation), the eggs were chilled at 4°C for eight to 12 h before harvesting. The embryos and lymphoid organs were collected for molecular diagnosis, histopathology, and immunohistochemistry (IHC). In addition, bone marrow smears were prepared and subjected to cytology, immunocytochemistry (ICC), and immunofluorescence studies. Suitable control SPF eggs were kept by inoculating sterile saline onto a sufficient number of eggs and subjected to further assays.
Gross examination
A necropsy was conducted on the embryos, and alterations in relation to the appearance of embryos with visceral lesions were documented. The bone marrow, thymus, and liver were collected from embryos during necropsy.
Molecular detection of viruses in chicken embryos
DNA extraction and purification from the collected samples were carried out using a QIAamp DNA Mini Kit (M/s Qiagen, Germany). The VP2 gene of the CAV (419 bp) was detected using the primer sequence of 5’-CTAAGATCTGCAACTGCGGA-3’ and 5’-CCTTGGAAGCGGATAGTCAT-3’ [10]. The following conditions were used for thermal cycling: 95°C for 3 min (initial denaturation) and 35 cycles of denaturation (94°C for 1 min), annealing (60°C for 1 min), extension (72°C for 1 min), and final extension (72°C for 10 min).
Cytology
Bone marrow smears were prepared from the femur of embryos using the impression method. The cytological smears were air-dried and fixed in methanol, followed by Giemsa staining [11].
Histopathology
The organs collected from the embryos were fixed in 10% neutral buffered formalin. Ethylenediaminetetraacetic acid (EDTA) formalin was used to decalcify the bone. The paraffin-embedded sections were cut at 5 μm thickness and stained with hematoxylin and eosin (H&E) for the histopathological examination. Tissue sections of the thymus and bone marrow were stained with phloxine-tartrazine to reveal the viral inclusions [11].
IHC
The IHC staining technique for antigen demonstration was performed using the procedure described elsewhere with some modifications as per the guidelines given by the manufacturer (M/s PathnSitu, India; M/s Bioss, USA) [12,13]. The formalin-fixed, paraffin-embedded (FFPE) tissue sections were subjected to immunohistochemical staining. Three-micron-thick sections were prepared from the samples, followed by deparaffinization with xylene. Table 1 provides details of the antibodies used.
Table 1. Antibody and immunohistochemistry methodology.
| Sl. No. | Antibody | Organ | Type | Marker for | Antigen retrieval | Dilution | Source |
|---|---|---|---|---|---|---|---|
| 1 | VP2 | Thymus | Rabbit polyclonal | Viral antigen | 37°C, proteinase K, 20 min | 1:100 | In house |
| Bone marrow | |||||||
| 2 | CD3 | Thymus | Rabbit polyclonal | T cell | 120°C, 15 min, pH high | Ready-to-use | Pathnsitu |
| 3 | CD44 | Bone marrow | Rabbit polyclonal | Hemocytoblast | 120°C, 15 min, pH low | 1:300 | Bioss |
For CAV antigen demonstration, enzymatic antigen retrieval was achieved using proteinase K for 45 min. For CD44, heat-induced antigen retrieval was carried out at 120°C using a citrate buffer (pH 6) for 15 min. The tissues were treated with goat serum to prevent non-specific binding. The tissue sections were then incubated overnight with anti-CAV rabbit serum (for CAV antigen) and CD44 (BS-2507R) primary antibodies (for CD44) at 4°C. The polyexcel horseradish peroxidase (HRP)/3,3'-diaminobenzidine (DAB) detection system of M/s PathnSitu (PEH002), India, was used for staining according to the manufacturer’s protocol, and hematoxylin was used for counterstaining.
CD3 was confirmed using Tris EDTA buffer (pH 8.5–9.0) for antigen retrieval from the tissue sections for 15 min at 120°C and incubated with the CD3+ primary antibody for one hour, followed by H&E staining according to the manufacturer’s protocol.
ICC
In ICC, the bone marrow smears of embryos were fixed in acetone for 10 min, followed by incubation with primary antibodies of 1:10 diluted anti-CAV rabbit serum and 1:300 diluted CD44 (BS-2507R) for 2 h at 37°C in a moist chamber. The slides were washed with PBS, followed by a secondary antibody using a polyexcel HRP/DAB detection system (PEH002; M/s PathnSitu) according to the manufacturer’s protocol, and hematoxylin was used for counterstaining.
Immunofluorescence test (IFT)
An indirect immunofluorescent test was conducted to detect the viral-specific antigen as described elsewhere [14]. The bone marrow impression smears were fixed in chilled acetone for 10 minutes, washed with PBS, and 1:50 diluted anti-CAV rabbit serum was added. The slides were incubated for 1 h at 37°C in a moist chamber, washed with PBS, followed by treatment with 1:160 diluted anti-rabbit fluorescein isothiocyanate conjugate (F9887; Sigma Aldrich, USA) and incubated for 1 h at 37°C in a humidified chamber. The slides were rewashed with PBS, mounted on 50% glycerol saline, and examined by fluorescent microscopy.
RESULTS
Embryo studies
Out of ten embryonated eggs inoculated on the 8th day with CAV-positive tissue homogenates, three embryos were found dead within the first 24 h of inoculation and discarded. The remaining seven embryos were harvested after 12 days of inoculation, i.e., the 20th day of embryonation, to assess the infectivity of the virus on various tissues of the embryos. No changes occurred in the embryos inoculated with sterile saline.
Gross examination of embryos
The CAV-inoculated embryos did not exhibit any tangible difference in appearance except for one with stunted growth compared to normal control embryos (Fig. 1A). Similarly, yellowish liver (n = 1), paleness (n = 1), and hemorrhage in breast muscle (n = 1) were observed compared to control embryos (Fig. 1B). In the remaining embryos, there were no visible gross lesions in visceral organs (Fig. 1C), including the thymus (Fig. 1D) and bone marrow (Fig. 1E).
Fig. 1. Gross examination of CAV pV-positive embryos.
(A) PCR-positive CAV embryo showing stunted growth (4) as compared to the normal (C) control. (B) PCR-positive CAV (20-days-old) embryos showing paleness (3) and haemorrhage (5) in breast muscle as compared to normal (C) control. (C) The PCR-positive CAV (20-days-old) embryos showing no lesions in visceral lesions. (D) The PCR-positive CAV (20-days-old) embryos showing normal thymus without gross lesions. (E) PCR-positive CAV embryos showing normal bone marrow without gross lesions.
CAV, chicken anemia virus; PCR, polymerase chain reaction.
Confirmation of CAV in embryo
The VP2 gene of CAV was detected in the bone marrow, thymus, and liver of all seven chicken embryos (Fig. 2A) and negative for the control uninfected embryos.
Fig. 2. Molecular detection and examination of CAV pV-positive embryos.
(A) CAV-specific amplicons visualized by agarose gel electrophoresis (Lane 1: 100 bp molecular weight marker; Lanes 2–8: Samples showing CAV-specific 419 bp product of VP2 gene; Lane 9: Positive control; Lane 10: Negative control). (B) CAV: Bone marrow cytology showing intracytoplasmic inclusion (arrow) in the orthochromatic erythroblasts (Giemsa staining). (C) CAV: Bone marrow cytology showing intranuclear inclusion (arrows) in the orthochromatic erythroblasts (Giemsa staining). (D) CAV inoculated embryo: Thymus showing presence of intracytoplasmic inclusion bodies (arrow) in thymocytes (phloxine tartrazine staining). (E) CAV-inoculated embryo: Thymus showing the presence of intranuclear inclusion bodies (arrow) in the thymocytes (phloxine tartrazine staining). (F) CAV-inoculated embryo: Bone marrow showing intracytoplasmic inclusion bodies (arrow) in haehemocytoblasts (phloxine tartrazine staining). (G) CAV-inoculated embryo: Bone marrow showing intranuclear inclusion bodies (arrow) in haehemocytoblasts (phloxine tartrazine staining). (H) CAV-inoculated embryo: Bone marrow smear showing CD44 immunostaining in the membrane of the haematopoietic cells (ICC staining). (I) Bone marrow smear showing CAV viral antigen in the cytoplasm of the haematopoietic cells (ICC staining).
CAV, chicken anemia virus; ICC, immunocytochemistry.
Cytology
In the CAV-inoculated embryos, the bone marrow cytology showed low cellularity of the erythroid and myeloid series (Supplementary Fig. 1A) compared to normal uninfected control embryos, which showed densely packed erythroid and myeloid series (Supplementary Fig. 1B). The orthochromatic erythroblasts revealed the presence of intracytoplasmic (Fig. 2B) and intranuclear inclusion bodies (Fig. 2C).
Histopathology
Although no gross lesions in the thymus and bone marrow of embryos were observed, the histopathological sections revealed moderate to severe depletion of T lymphocytes and hemocytoblast, respectively. The thymus of the CAV-affected embryos showed the depletion of lymphocytes in the medullary region, the presence of intracytoplasmic (Fig. 2D), and the intranuclear (Fig. 2E) inclusion bodies in thymocytes. The bone marrow of the CAV-affected embryos revealed a severe reduction of the erythroid and myeloid cell population and the presence of adipose tissue (Supplementary Fig. 1C) compared to the normal control embryo (Supplementary Fig. 1D). In addition, intracytoplasmic (Fig. 2F) and intranuclear (Fig. 2G) inclusion bodies were also observed in the hematopoietic cells of the bone marrow according to the phloxine tartrazine staining technique. In addition, the liver also showed mild degeneration, necrosis, and thrombi.
ICC
The observation of CD44-positive cells in the bone marrow smear of the CAV-positive embryos through ICC revealed the mild expression and positive reaction was observed as dark golden-brown deposits in the cell membrane and the cytoplasm of hemocytoblast cells (Fig. 2H).
ICC of the bone marrow smear for CAV detection revealed strong expression of the viral antigen as dark golden-brown deposits were observed in the cytoplasm of hemocytoblast cells (Fig. 2I). The smears from the uninfected control embryos showed negative staining for the CAV antigen (Supplementary Fig. 1E).
Fluorescent antibody test
Molecular detection of the CAV antigen in tissues by PCR was confirmed by an indirect immunofluorescent test in the bone marrow smears of embryos. The smears exhibited apple green fluorescence, indicating the characteristic intranuclear immunofluorescent viral particles in hematopoietic cells, which showed a fluorescent dotting pattern specific to CAV (Fig. 3A). At higher magnification, such fluorescent dots appeared as doughnut-shaped apoptotic bodies, which are the characteristic features of CAV (Fig. 3B). The smears from uninfected control embryos were negative for specific immunofluorescence (Supplementary Fig. 1F).
Fig. 3. Immunofluorescence and immunohistochemical studies of CAV-inoculated embryos.
(A) CAV-inoculated embryo-FAT: Bone marrow smear showing intranuclear immunofluorescent antigen in haematopoietic cells (FA staining, scale bar = 100 µm). (B) CAV-inoculated embryo-FAT: Bone marrow smear showing intranuclear fluorescent antigen dot (doughnut-shaped apoptotic bodies) pattern (FA staining, scale bar = 40 µm). (C) CAV-inoculated embryo-IHC: Thymus showing more CD3+ lymphoid cells in cortex and medulla (IHC staining, scale bar = 100 µm). (D) CAV-inoculated embryo-IHC: Thymus showing mild antigen expression in the cytoplasm of thymocytes (IHC staining, scale bar = 100 µm). (E) CAV-inoculated embryo-IHC: Bone marrow showing mild focal immunostaining of CD44 haemocytoblasthemocytoblasts (IHC staining, scale bar = 100 µm). Inset: Bone marrow showing expression of CD44 in the membrane and cytoplasm of haemocytoblasthemocytoblasts (IHC staining, scale bar = 40 µm). (F) CAV-inoculated embryo-IHC: Bone marrow showing marked antigen expression in haemocytoblastthe hemocytoblasts (IHC staining, scale bar = 100 µm). Inset: Bone marrow showing brown granular deposits of antigen in the cytoplasm and nucleus of haemocytoblasthemocytoblasts (IHC staining, scale bar = 40 µm).
CAV, chicken anemia virus; FAT, fluorescent antibody test; FA, fluorescent antibody; IHC, immunohistochemistry.
IHC
Immunolabelling of the viral antigen and serial sections of CD3+ for the lymphoblasts of the thymus and CD44 hematopoietic progenitor cells of the bone marrow were carried out in the CAV-inoculated embryos. The IHC technique emphasized the relevance of tropism for the CAV.
Comparative immunolabelling of CD3 and CAV antigen in the thymus of embryo
Immunolabelling with CD3+ revealed marked expression in the thymus, with these cells distributed over the entire thymic parenchyma. The CD3+ positive cells exhibited brown granular deposits in the membrane and cytoplasm (Fig. 3C). On the other hand, the CAV antigen in lymphoblast cells of the thymus revealed mild expression in serial sections as granular brown deposits (Fig. 3D). The uninfected control embryos revealed negative staining of the CAV antigen in serial sections, whereas marked expression of CD3+ was observed in the membrane and cytoplasm (Supplementary Fig. 1G). Table 2 presents the results regarding the expression of markers.
Table 2. Expression of markers in the thymus and bone marrow of CAV-inoculated embryos.
| Sl. No. | Organ | Method | CD44 | CD3 | CAV |
|---|---|---|---|---|---|
| 1 | Thymus | IHC | NA | +++ | + |
| 2 | Bone marrow | IHC | ++ | NA | +++ |
| ICC | + | NA | ++ |
Expression of markers: +, mild expression; ++, moderate expression; +++, strong expression.
CAV, chicken anemia virus; IHC, immunohistochemistry; ICC, immunocytochemistry; NA, not applicable.
Comparative immunolabelling of CD44 and CAV antigen in the bone marrow of embryos
Immunolabelling with the anti-CAV antibody was pronounced in the bone marrow, whereas the presence of CD44 cells was found only in focal areas (Fig. 3E) in the bone marrow. The positive reaction was characterized by brown granular deposits in the membrane and the cytoplasm of CD44-positive cells. Furthermore, the CAV antigen could be disseminated throughout the bone marrow tissue in the serial sections of the same location where the CAV antigen exhibited granular brown deposits in the nucleus and cytoplasm of the hemocytoblasts in the bone marrow serial sections (Fig. 3F). The uninfected control embryos revealed the mild expression of CD44 in focal areas in serial sections (Supplementary Fig. 1H). In contrast, the negative staining of the CAV antigen was revealed in the bone marrow (Supplementary Fig. 1I).
DISCUSSION
The CAV-inoculated embryo did not exhibit any significant changes in appearance except for stunted growth in one embryo. A severe reduction in the size might be due to liver protein synthesis in that particular embryo. Similarly, yellowish liver, paleness, and hemorrhage were observed in the breast muscles. The paleness of the muscle changes might be attributed to the tropism of the CAV to hemocytoblasts, resulting in paleness of the embryo liver [15,16].
The CAV was detected in the organs of all seven embryos by molecular detection using the PCR technique. The tropism for bone marrow and thymus revealed the immunosuppressive capability of CAV [15]. The bone marrow cytology showed the low cellularity of the erythroid and myeloid series as well as the intracytoplasmic and intranuclear inclusion bodies. The low cellularity was well supported by the depletion of hemocytoblasts in histopathology. The low cellularity and the presence of inclusion bodies in the progenitor cells indicate the influence of the CAV on bone marrow. The hypoplasia of the hematopoietic cell lineages in this study agrees with previous studies [17].
No significant gross lesions were observed in the thymus and bone marrow of the embryos. In contrast, histopathological examination revealed the depletion of lymphocytes in the medullary region of the thymus and the depletion of the erythroid and myeloid series in the bone marrow. In addition, intracytoplasmic and intranuclear inclusion bodies were also observed. The liver exhibited mild degeneration and necrosis. These histopathological lesions revealed the effects of the virus on the immune organs of the CAV-affected embryos [15,18].
In this study, the imprints of bone marrow were taken from the embryos PCR positive for the CAV and subjected to ICC for immunoreactivity studies [19]. The CD44-positive cells showed mild expression compared to the strong expression of the viral CAV antigen. No earlier studies on the ICC of the CAV-affected organs have been published. Therefore, reports have not been quoted in support or against the current observation.
The presence of CAV particles in hematopoietic cells by the IFT in the bone marrow was indicated by the apple green fluorescence of the intranuclear viral particles, which showed a fluorescent dotting pattern specific for the CAV [14,20]. An antigen was observed in the bone marrow smear by recognizing the fluorescent antigen with an IFT, highlighting the test as a rapid diagnostic technique for diseases.
The significance of the thymus and bone marrow as the major target organs for viral replication was highlighted by viral antigen demonstration. Immunolabelling with CD3+ revealed marked expression over the entire thymic parenchyma, whereas the CAV antigen showed mild expression in the thymus. As per the current observation, the mild expression of viral antigen in the thymus suggests that the CAV load in the thymus of the embryo is comparatively lower than that of the bone marrow of the embryo [8,21]. Hence, the viral antigen might have more affinity towards bone marrow in CAV-affected embryos. No study on the immunolabelling of the thymus in the CAV-infected embryos has been published. The viral antigen was in CD3+ cells where mature T lymphocytes are responsible for disseminating the virus in the thymus [21].
Labeling of the CAV antigen and CD44 highlighted the relevance of the viral tropism for the hemocytoblast cells of the bone marrow as the main target organ for their replication. CAV-affected cells originating from bone marrow are responsible for the widespread distribution of the CAV throughout the body.
The mesenchymal stem cells of the bone marrow are multipotent cells that can be differentiated in vitro into many different types of cells [22]. They are also called stromal progenitor cells, which are self-renewable, expandable stem cells used for regenerative studies [23]. They express specific markers like CD44, which produce cytokines and growth factors for regulating hematopoiesis [24]. Mesenchymal stem cells regulate the differentiation and proliferation of hematopoietic cells in the bone marrow, which could be the target of viral infection. The viral infection may result in the depletion of mesenchymal stem cells from the bone marrow, leading to the substantial loss of cells and their ability to renew and differentiate into specialized cells and tissues. The CAV infection alters the cytokine milieu in the bone marrow, affecting hematopoiesis and changing the properties and functions of the immune progenitor cells [25].
In compliance with the above-mentioned studies on chickens, the viral antigen was present in CD44 stem cells of embryos inoculated with CAV-positive tissue homogenates in the present study. This event might negatively impact the proliferation and differentiation of hematopoietic cells in the bone marrow during embryonic development.
In conclusion, this study provides insight into the interaction of the virus with the immune organs of embryos in the face of CAV infection. Demonstration of the antigen by IHC and IFT revealed more expression in the bone marrow than the thymus, indicating that the virus shows more tropism towards the bone marrow of the embryos than the thymus. The expression of CD44 and the CAV antigen in serial sections of the bone marrow of the embryo affirms the tropism of the CAV for hemocytoblast cells for virus replication.
ACKNOWLEDGMENTS
The authors are thankful to the Tamil Nadu Veterinary and Animal Sciences University for providing the necessary facilities to carry out the research work. The authors also thank Dr A. Balasubramaniam for the critical corrections in the manuscript preparation.
Footnotes
Funding: The authors received funding for research from Tamil Nadu Veterinary and Animal Sciences University. The authors received no financial support for authorship, and/or publication of this article.
Conflict of Interest: The authors declare no conflicts of interest.
- Conceptualization: Suohu S, Raja A, Arulmozhi A, Balasubramaniam GA.
- Funding acquisition: Suohu S, Balasubramaniam GA.
- Investigation: Suohu S.
- Methodology: Suohu S, Arulmozhi A, Raja A, Gopalakrishnamurthy TR.
- Supervision: Balasubramaniam GA, Arulmozhi A, Gopalakrishnamurthy TR.
- Writing - original draft: Suohu S, Balasubramaniam GA, Arulmozhi A.
- Writing - review & editing: Suohu S, Balasubramaniam GA, Arulmozhi A.
SUPPLEMENTARY MATERIAL
Examination of normal control embryos, histopathological examination of CAV-inoculated and normal control embryo.
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Associated Data
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Supplementary Materials
Examination of normal control embryos, histopathological examination of CAV-inoculated and normal control embryo.