Detection of intranuclear inclusion bodies in hepatocytes of the poultry infected with fowl adenoviruses (Adenoviridae: Aviadenovirus)

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Introduction

Avian adenoviruses (Adenoviridae: Aviadenovirus: Fowl adenovirus, FAdV) are viruses that infect domestic and synanthropic birds (chickens, turkeys, pheasants, and others). FAdVs are important infectious pathogens responsible for significant economic losses in the poultry industry worldwide [1]. FAdVs are associated with highly contagious diseases such as inclusion body hepatitis and chicken hydropericarditis syndrome, which cause significant economic damage to the poultry industry. Inclusion body hepatitis is caused by several FAdV serotypes (FAdV-11, FAdV-8a, and FAdV-8b), while the etiological agent of adenoviral hydropic pericarditis syndrome is FAdV-4 [2].

Previously, serotype FAdV-4 (type C) was the dominant pathogen causing adenovirus infections. For example, in South Korea, until 2019, FAdV-C accounted for 63% of detected cases, FAdV-E for 24%, and other serotypes for 13%. Since 2019, the situation has changed—serotype FAdV-E has come to prevail over serotype FAdV-C (18.5% and 77.8%, respectively) [1].

Hepatitis with inclusion bodies primarily affects young broiler chickens older than 2 weeks [3]. Between 2011 and 2021, broiler chickens accounted for 92% of all cases of hepatitis with inclusion bodies in Spain. The virus spreads primarily through horizontal transmission, but vertical transmission has also been observed [4]. The virus has a latent phase and reactivates at the onset of egg production, causing a subclinical course of the disease, which facilitates its vertical transmission [3]. Group 1 adenoviruses can infect not only chickens but also other bird species, including pigeons, ducks, quail, and ostriches [4]. Clinical signs are nonspecific, typically last 4–5 days, and include depression, ruffled feathers, reduced mobility, and a crouched posture. Necropsy reveals extensive or focal necrosis in the liver, which becomes soft, pale, and enlarged [3]. Cases of pancreatic involvement–including necrosis and atrophy–as well as kidney [5] and spleen [6] lesions have been reported. Histological examination reveals lymphoid infiltration, foci of necrosis and dystrophy [5], and intranuclear basophilic bodies or inclusions in hepatocytes [3].

Thus, FAdV causes various clinical forms of the disease, leading to significant economic losses [6], which result from high mortality, reduced productivity, and the need for forced antibiotic therapy against secondary bacterial infections arising against the backdrop of adenoviral immunosuppression. The ability of adenoviruses to persist for long periods, reinfection, their ability to infect not only farm poultry but also synanthropic and wild birds– and to spread through them–as well as their capacity to cause infection without visible clinical signs and to overcome interspecies barriers (the SSY-76 virus) makes this issue particularly relevant for the commercial poultry industry [7].

In the laboratory diagnosis of diseases caused by FAdV, molecular, pathological-anatomical, and pathomorphological studies are widely used [8–11]. The clinical picture and pathological-anatomical signs are not specific enough to establish a diagnosis, unlike histological examination. Histological analysis reveals intranuclear basophilic bodies–inclusions that are a pathognomonic sign [12] of FAdV Group 1 infection in birds. Despite the informative value of histological examination, it has a significant drawback: the lengthy time required to perform it, which ranges from 5 to 10 days and, in some cases, several weeks¹ [13].

The research findings presented in this article are consistent with the results of a study conducted by a group of scientists (McCullough K. et al.) in 2025 [14].

To reduce the time required to detect intranuclear inclusions, it is advisable to find an alternative to histological examination. The cytological diagnostic method will not only reduce the time required for analysis but, due to its simplicity and convenience, can also be effectively used at animal disease control stations and poultry farms without the need to fix the specimen and send it to a specialized laboratory. The cytological method for detecting intranuclear inclusions can be used for rapid preliminary diagnosis and differential diagnosis.

The aim of the study is to develop a cytological method based on the preparation and staining of imprint smears to detect, under microscopy, specific intranuclear inclusions in hepatocytes–a pathognomonic sign of avian adenoviruses–which does not require special, expensive equipment and is more rapid than histological examination.

Materials and methods

Preparation of histological slides

Pathological specimens (liver) from 13-day-old SPF (specific-pathogen-free) chicken embryos were fixed in a 10% solution of neutral formalin. To prepare overview sections, the material was processed in a TLP-720 histoprocessor (Mt Point, Russia), and paraffin embedding was performed on an ESD-2800 embedding station (Mt Point, Russia); 5–8 µm-thick sections were prepared on an RMD-3000 semi-automatic rotary microtome (Mt Point, Russia), flattened on the HWB-75 Water Bath histological section flattening device, dried on the HWB-75 Thermostolik histological section drying device, and stained with hematoxylin and eosin in the ALS-96 linear automatic steiner (Mt Point, Russia). The slides were examined using a Mikmed-6 microscope (Lomo, Russia) at magnifications ranging from ×100 to ×1000.

Preparation of smear samples

Pathological material–liver (from 13-day-old SPF chicken embryos, both intact and those previously infected at 6 days of age with field isolates of FAdV-E having an infectious titer of 5.2 log₁₀ EID₅₀/cm³) was excised at random to obtain organ fragments measuring up to 2 × 2 × 1 cm; the resulting fragments were immersed in a container of distilled water and thoroughly rinsed twice, with the water changed each time. The washed organ fragments were then lightly pressed with their cut surfaces against microscope slides (previously degreased with alcohol), and the slides with the resulting organ smears were placed in a tray, where, after the specimens had completely dried, they were fixed with 96% ethyl alcohol, ensuring that the alcohol completely covered the smears. After 15 minutes, the alcohol was removed, and the smears were left to dry completely. Once dry, the smears were considered fixed and ready for staining.

Preparation of azure and eosin: Azure-eosin was diluted in distilled water in a 1 : 10 ratio–1 part dye to nine parts distilled water. The required volume of the prepared stain was calculated based on the number of smears–1 milliliter of stain per 1–2 smears.

Preparation of Ziehl’s fuchsin: Pfeiffer’s fuchsin was diluted in a ratio of 1 part Pfeiffer’s fuchsin to 9 parts distilled water; the mixture was stirred by shaking the vial several times. The required volume of dye was calculated based on the number of smear-imprints–1 cm³ of Ziehl’s fuchsin per 2–4 smear-imprints.

Azure and Eosin Staining (Method 1): The fixed smears were treated with the stain prepared as described above; the exposure time was 60 minutes. The smears were then rinsed in distilled water (gently, using a fine stream directed along the edge of the microscope slide) and dried.

Once dry, the smears were considered ready for microscopy.

Gram staining (Method 2): Staining was performed using the standard method in the following sequence:

1) A solution of gentian violet was applied to the smears for 2 minutes; 2) A solution of Lugol’s iodine was applied to the smears for 2 minutes, until the smears turned black; 3) The smears were immersed in 96% ethyl alcohol for 20 seconds; 4) the smear prints were rinsed in distilled water; 5) Ziehl’s fuchsin was applied to the smear prints for 1 minute; 6) the smear prints were rinsed with distilled water; 7) the smear prints were dried.

Once dry, the smears were considered ready for microscopy.

Microscopy of the smears was performed at magnifications ranging from ×80 to ×1000 in transmitted light.

Ethics approval. The authors confirm compliance with institutional and national standards for the use of laboratory animals in accordance with the Consensus Author Guidelines for Animal Use (IAVES, July 23, 2010). The study protocol was approved by the Ethics Committee of the Federal State Budgetary Institution ARRIAH (Protocol No. 4, dated October 10, 24).

Results

Results of Phase 1 of the study

In the first stage of the study, no significant histological changes were observed in the livers of intact embryos. Externally, the organ was covered by a thin connective-tissue septum. No signs of the formation of interlobular connective-tissue septa were detected. Connective tissue elements were detected only in the region of the hepatic triads. The triads contained an artery, a vein, and a bile duct. The parenchyma of the lobules consisted of a system of hepatic beams (Fig. 1), which comprised 2 or 3 consecutively arranged rows of hepatocytes.

 

Fig. 1. Micrograph. Normal liver of an intact 13-day-old chicken embryo. Hematoxylin-eosin staining. Magnification ×200. пб – hepatic plates; ск – sinusoidal capillaries; ко – blood islands.

Рис. 1. Микрофото. Печень 13-дневного интактного куриного эмбриона в состоянии морфологической нормы. Окраска гематоксилином и эозином. Ув. ×200. пб – печеночные балки; ск – синусоидные капилляры; ко – кроветворные островки.

 

The liver cells were cuboidal in shape, with moderately vacuolated cytoplasm and a rounded nucleus (Fig. 2).

 

Fig. 2. Micrograph. Hepatocytes of an intact 13-day-old chicken embryo. Hematoxylin-eosin staining. Magnification ×1000. вц – vacuolized cytoplasm; эх – euchromatin in the nuclei.

Рис. 2. Микрофото. Гепатоциты печени 13-дневного интактного куриного эмбриона. Окраска гематоксилин и эозином. Ув. ×1000. вц – вакуолизиованная цитоплазма; эх – эухроматин в ядрах.

 

Euchromatin (active chromatin) predominated in the hepatocyte nuclei. The hepatic beams branched and anastomosed with one another, forming a network of beams. The bile capillaries consisted of a barely discernible canal whose wall was formed by adjacent hepatocytes. Sinusoidal capillaries filled with blood were located between the beams. Multiple, relatively large foci of embryonic hematopoiesis were identified (Figs. 3, 4), consisting of groups of 10–15 cells (lymphocytic, granulocytic, erythrocytic, and thrombocytic precursors). Signs of mild edema (widening of the Dissé spaces) were noted; in some areas, granules of hemoglobinogenic pigments were present.

 

Fig. 3. Micrograph. Foci of embryonic hematopoiesis in the liver of an intact 13-day-old chicken embryo. Hematoxylin-eosin staining. Magnification ×80.

Рис. 3. Микрофото. Очаги эмбрионального кроветворения в печени 13-дневного интактного куриного эмбриона. Окраска гематоксилином и эозином. Ув. ×80.

 

Fig. 4. Micrograph. Hepatic plates (пб); sinusoidal capillaries (ск); embryonic hematopoiesis foci (оэк) in liver of intact 13-day-old chicken embryo. Hematoxylin-eosin staining. Magnification ×200.

Рис. 4. Микрофото. Печеночные балки (пб), синусоидные капилляры (ск) и очаги эмбрионального кроветворения (оэк) в печени 13-дневного куриного эмбриона контрольной группы. Окраска гематоксилином и эозином. Ув. ×200.

 

Histological examination of the livers of 13-day-old embryos experimentally infected with a field isolate of FAdV-E revealed processes of granular, hyaline-droplet, and large-droplet fatty degeneration, occurring in different regions either independently or in combination (Fig. 5).

 

Fig. 5. Micrograph. Structural abnormalities in the liver of the 13-day-old chicken embryo infected with FAdV-E. Hematoxylin-eosin staining. Magnification ×600. ккжд – large-droplet fatty degeneration of hepatocytes; лг – hepatocyte lysis; кх – chromatin condensation; л – lymphocytes; э – eosinophils.

Рис. 5. Микрофото. Структурные нарушения в печени 13-дневного куриного эмбриона, зараженного полевым изолятом аденовируса FAdV-E. Окраска гематоксилином и эозином. Ув. ×600. ккжд – крупнокапельная жировая дистрофия гепатоцитов; лг – лизис гепатоцитов; кх – конденсация хроматина; л – лимфоциты; э – эозинофилы.

 

Oxophilic areas of parenchymal necrosis and necrobiosis were observed. Some hepatocytes had undergone lysis, exposing a network of intertwined reticulocytes with processes and Kupffer cells. Isolated hemorrhages were noted. The hepatocyte nuclei appeared swollen. In some cells, chromatin condensation occurred, resulting in the formation of sharply basophilic clumps (Figs. 6–8). This phenomenon may be associated either with the early stages of karyorrhexis or with the development of virus-induced apoptosis. The formation of basophilic intranuclear bodies—inclusions characteristic of adenoviral infections–was also noted (Figs. 6–8). These bodies were relatively large, blue-violet in color, and round or round-oval in shape (mirroring the shape of the hepatocyte nuclei). The morphology of these affected hepatocyte nuclei was preserved even against a background of cytoplasmic lysis.

 

Fig. 6. Micrograph. Initial stages of inclusion body (тв) formation, chromatin condensation (кх) in nuclei of hepatocytes of the 13-day-old chicken embryo infected with FAdV-E. Hematoxylin-eosin staining. Magnification ×1,000.

Рис. 6. Микрофото. Начальные стадии формирования телец-включений (тв), конденсация хроматина (кх) в ядрах гепатоцитов печени 13-дневного куриного эмбриона, зараженного полевым изолятом аденовируса FAdV-E. Окраска гематоксилином и эозином. Ув. ×1000.

 

Fig. 7. Micrograph. Formation of basophilic intranuclear inclusion bodies in hepatocytes of the 13-day-old chicken embryo infected with FAdV-E. Hematoxylin-eosin staining. Magnification ×400.

Рис. 7. Микрофото. Формирование внутриядерных базофильных телец-включений в гепатоцитах печени 13-дневного куриного эмбриона, зараженного полевым изолятом аденовируса FAdV-E. Окраска гематоксилином и эозином. Ув. ×400.

 

Fig. 8. Micrograph. Histological lesions in liver of the 13-day-old chicken embryo infected with FAdV-E. Hematoxylin-eosin staining. Magnification ×1,000. кх – chromatin condensation; тв – inclusion bodies; э – eosinophils.

Рис. 8. Микрофото. Гистологические изменения в печени 13-дневного куриного эмбриона, зараженного полевым изолятом аденовируса FAdV-E. Окраска гематоксилином и эозином. Ув. ×1000. кх – конденсация хроматина; тв – тельца-включения; э – эозинофилы.

 

All of the processes described occurred against a background of serous edema and moderate lymphoid and eosinophilic infiltration (Figs. 5, 8).

A reduction in the number and size of embryonic hematopoietic islets was observed.

Results of Phase 2 of the Study

In the second phase of the study, the possibility of making a morphological diagnosis of adenovirus infection was investigated by examining liver smears stained with azure and eosin using the Romanowsky–Giemsa method.

The following structural elements were identified in liver smear-imprints from 13-day-old intact chicken embryos (Fig. 9):

– hepatocyte nuclei of a pale violet color. The cytoplasm was in a state of lysis. It is extremely difficult to prevent mechanical destruction of the parenchymal epithelial cells when preparing smear preparations of any organ. Intact hepatocytes with complete nuclei and cytoplasm were present only as isolated specimens;

– cells of the erythrocyte lineage at various stages of maturity (normocytes, erythrocytes) with basophilic nuclei and cytoplasm that was weakly to moderately oxophilic. Lysis of individual red blood cells was noted; in their place, only swollen, rounded nuclei with weakly basophilic staining were identified. The red blood cells themselves had either a characteristic oval shape or a rounded shape, which was the result of mechanical stress during the preparation of the smear. There were also isolated erythroblasts–large oval cells with a large, eccentrically located nucleus and markedly basophilic cytoplasm;

– lymphocytes with a characteristic morphology– a round or round-oval, distinctly basophilic nucleus and a narrow, barely discernible cytoplasmic rim. It was easy to distinguish the lymphocytes from the nuclei of lysed red blood cells, given the shape, size, and staining pattern of these structures;

– vacuoles in place of fat droplets, which appeared at the sites of lysed hepatocytes. The fat was washed out during the fixation of the smear imprints after their preparation. The vacuoles were round in shape, measuring 0.5–1.0 µm, and could merge to form larger structures–up to 5 µm.

 

Fig. 9. Micrograph. Structural elements identified in the imprint smear of liver from intact 13-day-old chicken embryo. Romanowsky–Giemsa staining. Magnification ×200. г – hepatocytes; яг – hepatocyte nuclei; л – lymphocytes; э – red blood cells.

Рис. 9. Микрофото. Идентифицированные структурные элементы в мазке-отпечатке печени 13-дневного интактного куриного эмбриона. Окраска по Романовскому–Гимзе. Ув. ×200. г – гепатоциты; яг – ядра гепатоцитов; л – лимфоциты; э – эритроциты.

 

Similar structural elements were identified in liver smears from 13-day-old chicken embryos experimentally infected with the field isolate of adenovirus FAdV-E. At the same time, certain morphological features were identified (Figs. 10–12):

– absence of hepatocytes with characteristic morphology;

– a relatively smaller number of hepatocyte nuclei, but with more pronounced signs of karyolysis;

– a relatively larger number of lymphocytes compared to liver smears from embryos in the control group;

– the presence of characteristic basophilic inclusions at the sites of lysed hepatocytes (1–3 per field of view at a total magnification of ×400).

These changes indicated intense processes of cytolysis, inflammatory cellular infiltration, and structural changes in hepatocyte nuclei specific to adenoviral infection.

 

Fig. 10. Micrograph. Structural elements identified in the imprint smear of liver from FAdV-E-infected 13-day-old chicken embryo. Romanowsky–Giemsa staining. Magnification ×200. яг – hepatocyte nuclei; тв – inclusion bodies; э – red blood cells; л – lymphocytes.

Рис. 10. Микрофото. Идентифицированные структурные элементы в мазке-отпечатке печени 13-дневного куриного эмбриона, зараженного полевым изолятом аденовируса FAdV-E. Окраска по Романовскому–Гимзе. Ув. ×200. яг – ядра гепатоцитов; тв – тельца-включения; э – эритроциты; л – лимфоциты.

 

Fig. 11. Micrograph. Morphology of the imprint smear of liver from FAdV-E-infected 13-day-old chicken embryo. Romanowsky–Giemsa staining. Magnification ×200. Тв – inclusion bodies; яг – hepatocyte nuclei; л – lymphocytes; э – red blood cells.

Рис. 11. Микрофото. Морфология мазка-отпечатка печени 13-дневного куриного эмбриона, зараженного полевым изолятом аденовируса FAdV-E. Окраска по Романовскому–Гимзе. Ув. ×200. тв – тельца-включения; яг – ядра гепатоцитов; л – лимфоциты; э – эритроциты.

 

Fig. 12. Micrograph. Light microscopy of the imprint smear of liver from FAdV-E-infected 13-day-old chicken embryo. Romanowsky–Giemsa staining. Magnification ×200. яг – hepatocyte nuclei; тв – inclusion bodies; э – red blood cells; л – lymphocytes.

Рис. 12. Микрофото. Результаты световой микроскопии мазка-отпечатка печени 13-дневного куриного эмбриона, зараженного полевым изолятом аденовируса FAdV-E. Окраска по Романовскому–Гимзе. Ув. ×200. яг – ядра гепатоцитов; тв – тельца-включения; э – эритроциты; л – лимфоциты.

 

Results of Phase 3 of the Study

In the third stage, we investigated the comparative effectiveness of Romanowsky–Giemsa and Gram staining of liver smears from embryos for establishing a morphological diagnosis of adenoviral hepatitis. The results of the examination of smears stained using the Romanowsky–Giemsa method were obtained in the second stage.

An examination of Gram-stained liver smears from 13-day-old intact and FAdV-E field isolate-infected chicken embryos yielded the following results (Figs. 13–16):

– the contours of hepatocyte nuclei were less distinct than in smears stained with Romanowsky–Giemsa;

– the relatively low intensity of erythrocyte nuclei made it difficult to determine their structure, the pathological process, and artifacts arising during smear preparation;

– the cytoplasm of erythrocyte-lineage cells remained either unstained or weakly oxophilic;

– lymphocyte nuclei and intranuclear inclusions stained strongly basophilic, which made it difficult to identify them and to describe and analyze the results obtained.

 

Fig. 13. Micrograph. Morphology of the imprint smear of liver from intact 13-day-old chicken embryo. Gram staining. At low magnification of light microscope ×50.

Рис. 13. Микрофото. Морфология мазка-отпечатка печени 13-дневного интактного куриного эмбриона при малом увеличении светового микроскопа. Окраска по Граму. Ув. ×50.

 

Fig. 14. Micrograph. Structural elements identified in the imprint smear of liver from intact 13-day-old chicken embryo. Gram staining. Magnification ×200. яг – hepatocyte nuclei; э – red blood cells; л – lymphocytes.

Рис. 14. Микрофото. Идентифицированные структурные элементы в мазке-отпечатке печени 13-дневного интактного куриного эмбриона. Окраска по Граму. Ув. ×200. яг – ядра гепатоцитов; э – эритроциты; л – лимфоциты.

 

Fig. 15. Micrograph. Morphology of the imprint smear of liver from FAdV-E-infected 13-day-old chicken embryo. Gram staining. Magnification ×200. тв – inclusion bodies; л – lymphocytes; э – red blood cells.

Рис. 15. Микрофото. Морфология мазка-отпечатка печени 13-дневного куриного эмбриона, зараженного полевым изолятом аденовируса FAdV-E. Окраска по Граму. Ув. ×200. тв – тельца-включения; л – лимфоциты; э – эритроциты.

 

Fig. 16. Micrograph. Light microscopy of the imprint smear of liver from FAdV-E-infected 13-day-old chicken embryo. Gram staining. Magnification ×200. яг – hepatocyte nuclei; тв – inclusion bodies; э – red blood cells; л – lymphocytes.

Рис. 16. Микрофото. Результаты световой микроскопии мазка-отпечатка печени 13-дневного куриного эмбриона, зараженного полевым изолятом аденовируса FAdV-E. Окраска по Граму. Ув. ×200. яг – ядра гепатоцитов; тв – тельца-включения; э – эритроциты; л – лимфоциты.

 

Conclusions

1. The microarchitecture of the liver in intact 13-day-old chicken embryos corresponds to the stage of incubation; no pathological processes are detected.
2. In the livers of chicken embryos experimentally infected with a field isolate of FAdV-E, histological changes characteristic of adenoviral hepatitis develop the following: granular, hyaline-droplet, and large-droplet fatty degeneration, necrosis and lysis of hepatocytes, extensive areas of necrobiosis and parenchymal necrosis, serous edema, lymphoid and eosinophilic infiltration, formation of intranuclear basophilic bodies, and atrophy of embryonic hematopoietic islets.
3. In liver smears from 13-day-old embryos infected with the FAdV-E field isolate, stained with azure and eosin according to the Romanowsky–Giemsa method, pathological processes are sufficiently identifiable to establish a morphological diagnosis of adenoviral hepatitis: total hepatocyte cytolysis, a lymphocytic reaction, and intranuclear basophilic bodies or inclusions.
4. The method of morphological diagnosis of adenoviral hepatitis, based on microscopy of liver smears stained using the Romanowsky–Giemsa method, is quite promising, given its simplicity, significant time savings, and reduced costs for expensive reagents and specialized equipment. The results of these studies also allow for differential diagnosis from acute toxic hepatitis.
5. Gram staining of liver smears is less effective for detecting morphological features of adenoviral hepatitis than Romanowsky–Giemsa staining.

 

¹ Bykov V.L., Yushkantseva S.I. Histology, Cytology, and Embryology. Atlas: A Study Guide. Moscow: GEOTAR-Media, 2013. Electronic text. Available at: https://www.rosmedlib.ru/book/ISBN9785970424377.html (accessed: 16.01.2026).

About the authors

Yury R. Zelensky

Federal Centre for Animal Health

Author for correspondence.
Email: zelenskiy@arriah.ru
ORCID iD: 0009-0007-0467-0182

Post-Graduate Student, Veterinarian, Laboratory for Avian Disease Prevention

Russian Federation, 600901, Vladimir

Igor N. Gromov

Vitebsk Order of the Badge of Honor State Academy of Veterinary Medicine

Email: gromov_igor@list.ru
ORCID iD: 0000-0001-8065-5661

Doct. Sci (Veterinary Medicine), Professor, Head of the Pathological Anatomy and Histology Chair

Belarus, 210026, Vitebsk

Olga A. Chupina

Federal Centre for Animal Health

Email: chupina@arriah.ru
ORCID iD: 0000-0002-6832-0348

Cand. Sci. (Biology), Head of the Preclinical Study Centre

Russian Federation, 600901, Vladimir

Mikhail S. Volkov

Federal Centre for Animal Health

Email: volkov_ms@arriah.ru
ORCID iD: 0000-0002-8819-0720

Doct. Sci (Veterinary Medicine), Associate Professor, Head of the Laboratory for Epizootiology and Monitoring

Russian Federation, 600901, Vladimir

Natalia V. Moroz

Federal Centre for Animal Health

Email: moroz@arriah.ru
ORCID iD: 0000-0002-9672-8594

Cand. Sci. (Veterinary Medicine), Head of the Laboratory for Avian Disease Prevention

Russian Federation, 600901, Vladimir

References

Supplementary files