TNF-a and FGF immunohistochemical expressions and pathological evaluation of the duck's liver lesions in Mosul city

Introduction

Liver ailments pose a health concern for poultry. They have implications for their overall well-being, productivity, and a range of vital bodily functions the liver governs, including metabolism and immunity regulation (1). Any harm or inflammation to this organ can disrupt the birds' health and their ability to fight illnesses due to its functions in energy storage and detoxification (2). Changes in liver tissue due to pathogens, infections, and inflammation play a role in evaluating damage and disease progression within the organ system (3). These alterations involve disturbances in liver cells' normal functioning and inflamed blood vessels and dead tissues, which collectively affect the liver's functioning (4). Research indicates that such alterations are frequently triggered by acute or long-term reactions prompted by bacteria, viruses, and external factors like pollution and inadequate nutrition (5). Cytokines, such as TNF alpha, play a role in understanding how the liver ΄s immune system responds to injury (6). Tumor necrosis factor alpha (TNF -α) is a cytokine that triggers tissue inflammation and is released by blood cells and damaged liver cells when an injury occurs (7). This substance increases the permeability of blood vessels to help more immune cells reach the affected area and combat infections or injuries (8). However, although this reaction offers some protection, heightened levels of TNF alpha can result in inflammation and permanent harm to tissues, adversely impacting liver functions (9).  Keeping track of TNF alpha through methods may prove beneficial in gauging the level of inflammation and the severity of the response (10). An important protein known as the fibroblast growth factor (FGG) promotes cell growth and aids tissue healing after an injury. Increased levels of FGG in the liver are believed to be the body ΄s way of responding to damage and assisting in the repair of tissues (11).  In liver cells harmed or injured, other hepatic cells grow and multiply to compensate for the loss, promoting this process through FGG (12). It also helps to create blood vessels that deliver the necessary oxygen and nutrients to that area of the body, which is crucial for its recovery following injuries or illnesses, in the liver (13). Studying liver diseases in ducks in Mosul is important due to the factors that can harm birds, such as pollution and unsustainable farming practices, which are prevalent in the region and negatively affect their health. Research on liver diseases in duck' areas is limited locally; hence, this study is crucial for understanding disease occurrence within the region (14,15).

Immunohistochemical staining techniques provide in-depth insight into the changes linked to liver damage by allowing the investigation of specific proteins involved in tissue inflammation and cell renewal processes. The analysis of TNF alpha and FGF using Immunohistochemistry is a method for obtaining information that aids in assessing liver health conditions. This helps researchers identify strategies to effectively prevent and manage disease prevalence in ducks and enhance their productivity. This study aims to examine the frequency of liver abnormalities in ducks using histopathology and analyze TNF alpha and FGF proteins with Immunohistochemistry to understand disease severity and its consequences.

Materials and methods

Ethical approval

Ethical approval was granted by the College of Veterinary Medicine at the University of Mosul, with the approval number UM.VET.2022.093 dated 01/11/2022.

Duck samples

The research involved 25 ducks collected from various areas of Mosul City, which were examined for signs of liver disease. Those displaying symptoms were selected and taken to the Mosul University, College of Veterinary Medicine research lab for testing. From November 2022 to November 2023, 25 adult ducks of different genders weighing between 2 to and 3 kilograms and aged between 1 and 1.5 and a half years were chosen from various areas in Mosul city. Veterinarians from the University of Mosul carefully assessed the ducks for signs of liver disease by observing symptoms such as skin tone changes and abdomen swelling, which may indicate health issues related to the liver.

Liver sample collection

After gently putting down the ducks to end their suffering and prevent the further spread of infection in the flock, we gathered samples of their livers and carefully transported them to the Poultry Disease Laboratory at the Veterinary Teaching Hospital at the University of Mosul for further analysis. The samples are processed according to established procedures to maintain their integrity. Then, it is prepared for detailed examination using histological and immunohistochemical techniques. The infection rate was calculated using the following formula; Infection rate = (Number of infected ducks / Total number of ducks examined)*100.

Histopathological examination and staining preparation

The liver tissue samples were prepared for assessment using fixation techniques and staining processes with hematoxylin and eosin (15). This enabled the observation of histopathological changes in liver tissues (16).

Scoring system of the pathological changes

The data presented in table 1 reflect the histopathological changes categorized into five main parameters: cell injury, circulatory disturbances, cell adaptation, inflammation, and an increase in fibrous connective tissue. These changes were scored based on their severity (17).

Table 1: Ordinal descriptive scoring system for severity of histopathological changes severity in duck liver

Immunohistochemistry IHC of TNF alpha and FGF

The immunohistochemistry test was performed to identify the expression of TNF alpha and FGF expression in liver tissues using antibodies located in these proteins according to the staining protocol by the Dako^® EnVision™ FLEX system. This study's immunohistochemical detection of TNF alpha and FGF was conducted using a FLEX Polyclonal Mouse Anti-avian antibody (Dako^® EnVision™ FLEX system). Tissue sections were prepared on positively charged slides, baked overnight at 60ºC, and deparaffinized in xylene, followed by rehydration in graded ethanol. The sections were then treated with hydrogen peroxide to inhibit endogenous peroxidase activity and washed with PBS. A protein block was applied to prevent non-specific binding, followed by the primary antibody Anti-TNF alpha and FGF, diluted 1:200), incubated for 90 minutes. After washing, a biotinylated secondary antibody was added and incubated for 20 minutes, then streptavidin peroxidase and further PBS washes. The chromogen DAB, diluted with substrate, was added to visualize the antibody-antigen complex. Finally, slides were counterstained with Harris Hematoxylin, washed, and cover slipped using an aqueous mounting medium.

Results

Infection rate

The study revealed that 52% of ducks in the area were affected by liver diseases, indicating the presence of these health issues within the duck community.

Liver histopathology

Upon examination of the liver samples under a microscope and analysis of the tissue structure, signs of damage and inflammation over time in the liver tissue were revealed, such as obvious foci of coagulative necrosis of the hepatocytes, focal infiltration of inflammatory cells, and severe fatty degeneration of the hepatocytes (Figure 1). The results also demonstrated severe massive hydropic degeneration of the hepatocytes, necrosis of other tissue, infiltration of inflammatory cells surrounding the portal area, proliferation of Kuepfer cells, and expansion of sinusoids (Figure 2 and Table 2).

Liver histopathological changes

The histopathological severity scores for liver changes in ducks were assessed and summarized in table 2. The table provides scores as the median and interquartile range (IQR) for various pathological changes and these significance levels. Degeneration was rated moderate with a median of 2 (IQR: 1.5), indicating consistent tissue damage throughout the samples. Fatty Change and Necrosis: Both were scored as mild with a median of 1 (IQR: 2.5). However, the high IQR variability suggests some severity variability across samples. Edema scored as zero with a median of 0 (IQR: 1), indicating minimal or no observable tissue swelling. Congestion of blood vessels rated as moderate with a median of 2 (IQR: 1.5), indicating significant vascular changes in the liver. Hypertrophy was rated null with a median of 0 (IQR: 1), suggesting no hypertrophic changes in the liver tissues. Hyperplasia scored mild with a median of 1 (IQR: 1.5), showing slight cellular adaptation through proliferation. Acute inflammation rated as mild with a median of 1 (IQR: 2), indicative of mild yet consistent acute inflammatory changes. Chronic inflammation was rated as null with a median of 0 (IQR: 1), showing no chronic inflammatory patterns. The increase in fibrous tissue scored mild with a median of 1 (IQR: 1.5), indicating mild fibrotic changes in the liver.

Figure 1: Photomicrograph of duck's liver. (A&C): Congestion and expansion in the sinusoids, with hemorrhage in the liver tissue, obvious focus (tow heads arrow) of coagulative necrosis of the hepatocytes (black arrow), focal infiltration of inflammatory cells (yellow arrow), and fatty degeneration of the hepatocytes (red arrow). (B&D): severe fatty change of the hepatocytes (black arrow), necrosis of the hepatocytes (red arrow), and infiltration of inflammatory cells (yellow arrow). (A&B: 100X), (C&D: 400X). H&E stain.

Figure 2: Photomicrograph of duck's liver. (A&B): the inflammatory cells infiltrate around the central vein and not the portal area, severe massive hydropic degeneration of the hepatocytes (black arrow), necrosis of others (red arrow), and infiltration of inflammatory cells surrounding the portal area (yellow arrow). (C&D): the blue arrow indicates the presence of necrosis and not expansion of the sinusoid, severe hydropic degeneration (black arrow) and severe necrosis of the hepatocytes (red arrow), proliferation of Kupfer cells (yellow arrow), and expansion of sinusoids (blue arrow). (A&C: 100X), (B&D: 400X). H&E stain.

Table 2: Scores of ordinal descriptors for the severity of histopathological changes in the duck's liver

Data expressed as Median and Inter-Quartile-Range. N= 25. Different letters mean there is a significant difference at P≤0.05.

Immunohistochemical changes for TNF-alpha and FGF

The immunohistochemical staining revealed TNF alpha levels in the inflamed regions, with a presence in the affected areas signifying a robust inflammatory reaction. On the contrary, FGF exhibited weak expression levels in regions demonstrating cell regeneration and fibrous tissue growth (Figures 3 and 4).

Figure 3: Photomicrograph of duck's liver. [A&C]: immunohistochemical expression of the TNF reveals intense expression (score 3+). [B&D]: immunohistochemical expression of the FGF reveals weak expression (score 1+).  (dark brown color is positive reaction). [A&B: 100X], [ C&D: 400X]. Hematoxylin.

Figure 4: Photomicrograph of duck's liver. (A&C): immunohistochemical expression of the TNF reveals moderate expression (score 2+). (B&D): immunohistochemical expression of the FGF reveals weak expression (score 1+). (Dark brown is a positive reaction). (A&B: 100X), (C&D: 400X). IHC TNF.

Discussion

This research indicates that ducks residing in Mosul city, Iraq, have a liver disease occurrence rate of about 52%. This percentage signifies the presence of liver abnormalities within the duck population and highlights the necessity of investigating potential causes and environmental factors that could further exacerbate this issue (18). The results indicate that the duck liver experienced significant histopathological changes, with degeneration and vascular congestion being the most prevalent. Conversely, edema and chronic inflammation were either absent or mild. This suggests the presence of environmental or pathological stress on the liver, which can greatly affect its vital functions. These findings underscore the necessity of monitoring histopathological changes to identify underlying causes and formulate appropriate therapeutic strategies (19,20).

Upon examination, it was observed that ducks experiencing health issues displayed abnormalities in their liver tissues, such as persistent inflammation and extensive damage to hepatocytes. These changes indicate that the liver is under strain, likely caused by an infection that has triggered a prolonged response or repeated episodes of inflammation over time (20,21). The microscopic findings suggest that the liver is facing challenges, possibly stemming from infections or exposure to environmental toxins, resulting in gradual deterioration and harm to the tissue over an extended period (22).

Furthermore, hypertrophy, congestion of liver blood vessels, and blockage were signs of blood circulation in the liver. This vascular congestion often results from stress on the liver due to efforts to combat pathogens or toxins, resulting in the widening of hepatic vessels and damage to their walls (23). Gathering cells near blood vessels suggests that the system's reaction to liver inflammation is an ongoing response to infection or long-term inflammation that may further harm liver tissues (23-25).

The immunostaining results revealed TNF alpha protein levels in areas affected by inflammation, indicating inflammatory activity in those regions. In response to injury and infection, TNF alpha, a cytokine, is secreted by immune cells (26). Its primary function involves stimulating cells and enhancing blood flow to the affected site, allowing defensive cells to reach it. However, the prolonged increase in TNF alpha levels observed in the samples can lead to persistent inflammation, damaging liver tissue and hindering the liver's recovery capacity (27). The results underscore the function of TNF alpha in this process; while it serves as an initial barrier against infections when present at normal levels, the body's defense mechanisms may be compromised if it remains elevated for an extended period. This can result in prolonged inflammation that negatively impacts liver health (28). The higher TNF alpha levels could also suggest that the liver is having difficulty combating threats from pathogens, leading to persistent inflammation and gradual deterioration of tissue quality (29).

In contrast to that finding, researchers discovered levels of the growth factor FGF in certain liver zones where cell growth was active. This observation signifies the body's efforts to mend injured tissues by triggering cellular renewal and promoting the formation of fibrous tissue and new blood vessels, crucial for tissue repair and nourishment (30,31). Elevated levels of FGF found in damaged areas of the liver indicate that the liver is attempting to heal and renew the affected tissue as a response to inflammation-induced damage (32).

However, this regeneration process might be inadequate or restricted due to inflammation, as shown by increased TNF alpha levels, suggesting that the repair attempts may not be sufficient to combat the damage (33). The significant occurrence of infection, 52%, provides evidence of the prevalence of liver ailments in ducks, suggesting that these birds encounter various environmental factors or infections that negatively affect their liver health. Potential contributors may include nutrition, pollution, the environment, or the existence of agents leading to liver inflammation) 34).

The high infection rate may result from a combination of factors such as diet and contaminated water, which can directly impact health. Pollution levels in the environment are closely linked to increased occurrences of liver diseases alongside inadequate healthcare and poor hygiene practices that elevate birds' exposure to diseases, ultimately leading to long-term liver inflammation issues (35,36). The results underscore the importance of taking steps to control the transmission of liver ailments in ducks (37). The suggestions include improving animal healthcare practices by maintaining cleanliness standards and providing nutrition to reduce pathogen exposure and counteract the impact of detrimental environmental elements (38).

Conclusion

This research offers information about liver issues in ducks in Mosul city, which helps us better comprehend the reasons for and signs of the illness. The TNF alpha and FGF immunohistochemical expression findings provide insight into the disease's progression mechanisms, which might lead to treatment and prevention strategies.

Acknowledgment

The authors express their gratitude to the Veterinary Medicine College at the University of Mosul for helping to raise the caliber of this study.

Conflicted interest

The author does not have any financial or personal information about individuals or organizations that would improperly influence the article's content.