PATHOLOGICAL STUDIES ON LIVER AND BILE DUCT TUMORS IN SUDANESE CATTLE

Due to their rare occurrence in bovine species, neoplasms of the liver and bile ducts were reported in few available researches. In Egypt, their prevalence in domestic animals was documented in a few records. An abattoir-based survey was conducted at Abu-Simbel slaughterhouse, Aswan Governorate, from December 2020 to October 2021. The study aimed to report the incidence rate of these tumors in imported Sudanese cattle. The percentage was 4 % and affected specimens revealed lesions ranging from single compact to multiple, nodular, metastatic masses with umbilicated appearance and necrotic centers. Diagnosis was confirmed histologically and tumorous lesions were identified as cholangioma, in which bile ducts showed marked proliferation and formed tubular structures lined with hyperplastic cuboidal cells with fibrous tissue proliferation and inflammatory cellular reaction, cholangiocarcinoma with its acinar pattern, mucin production and the fibrous stroma and mixed hepatocholangiocarcinoma that was characterized by simultaneous presence of hepatocellular and cholangiocellular carcinomas’ features. In conclusion, neoplasms are prevalent in Sudanese cattle, which could be exposed to carcinogenic chemicals or aflatoxins that cause tumor induction. Careful veterinary inspection at slaughterhouses and more studies with larger sample sizes are recommended.


INTRODUCTION
The most recorded neoplasms in bovine species are lymphoma and squamous cell carcinoma, which can occur in different parts of the body, including the alimentary tract (Lucena et al., 2011).However, tumors of hepatocellular and cholangiocellular origin are rarely reported, and represent less than 10% of all diagnosed neoplasms (Cullen and Stalker, 2016).
Primary hepatic neoplasms may appear in the form of focal tumors in which a single nodule or mass is present in the liver, multifocal that is characterized by a random distribution of multiple nodules in the liver parenchyma, or diffuse neoplasms that can affect the majority of the liver and accompanied by changes in color and consistency (Vielmo et al., 2020) Tumors of hepatocytic origin may be classified as adenomas, hepatocellular carcinomas, and hepatoblastomas.However, those possessing biliary epithelial cell origin include biliary cystadenomas (cholangioma) and cholangiocarcinomas (Head et al., 2003).Cholangioma is a benign tumor of the bile duct that appears as well-circumscribed, multiple nodular lesions with cavernous aspect and small amounts of bile, and sometimes it is represented as compact nodules (Baba and Câtoi, 2007).It has not been mentioned as a predisposing cause of cholangiocellular carcinoma without complete exclusion of this possibility (Lendavi et al., 2020).Cholangiocellular carcinoma is a malignant tumor that originates from bile duct epithelial cells.Dogs and cats are more frequently affected than cattle and other ruminants (Cullen and Popp, 2002).Combined hepatocellular cholangio-cellular carcinoma is sporadically reported in cattle and has neoplastic characteristics of both hepatocytic and biliary epithelial cell origins (Meuten, 2016).
These tumors have a high metastatic tendency, especially towards regional lymph nodes and lungs.Recognition of tumor emboli in pulmonary blood vessels indicates that hematogenous spread plays an important role in metastasis (MacLachlan and Cullen, 1995).Spontaneous cases may arise with an unknown cause (Kato et al., 2004).Whereas, concerning etiological agents, chronic parasitic infestation, different strains of bacteria and viruses, and ingestion of various carcinogenic chemicals such as nitrosamines, aflatoxins, and pyrrolizidines constitute an important role in tumor development (Gholami et al., 2006).
Diagnosis of liver neoplasms, as well as distinguishing benign from malignant types, have relied on both gross examination and histological features of lesions (Meuten, 2016).Histological evaluation can be performed according to the following criteria: cellular arrangement, stromal pattern, cell pleomorphism, vascular or lymphatic invasion, necrosis, hemorrhage, intratumoral inflammatory infiltrate, intratumoral vascular spaces, and the degree of mitoses (Vielmo et al., 2020).The objective of this study is to record the incidence and prevalence rates of hepatic and bile duct tumors in imported Sudanese beef cattle, which are supposed to be a major source of animal protein for Egyptians, in addition to characterization of gross and histopathological picture of the detected tumors.

Ethical approval:
The study protocol was approved by the Animal Ethical Committee for Veterinary Research of the Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt.Under the No. 06/2023/0098.Used samples were collected postmortem and no animals were killed for the study.

Animals and study area:
Imported Sudanese cattle were quarantined for up to 21 days under the supervision of Egyptian Quarantine Veterinarians in Wadi Halfa, a Sudanese city bordering Egypt.Then they were transported to Abu Simbel city, Aswan Governorate, Egypt, where they were slaughtered at Abu Simbel abattoir.All animals were males and aged from two to three years.The period of study extended from December 2020 to October 2021.

Sampling and visual examination:
1575 slaughtered cattle were inspected and examined with particular attention to liver affections.Of these, 172 (10.9%) cases showed gross lesions.Specimens suspected to have tumors were discarded, collected, and photographed by a digital camera.Small sections of cubic shapes were taken from lesions and fixed in 10% neutral buffer formalin for preservation.

Histopathological examination:
Fixed specimens were processed routinely, according to the procedure of Bancroft et al. (1996).Tissue sections of 3-5 µm thickness were stained with Hematoxylin and Eosin for microscopic examination by light microscope, and were photographed using a Sc30 Olympus camera.
Following histopathological examination, 57.1% of liver neoplasms were identified as benign, and 42.9% showed features of malignancy.Cholangioma, as a benign form, was detected in 4 cases with an incidence of 2.3%.Intrahepatic cholangiocarcinoma was present in 2 cases with an incidence of 1.1%, and mixed hepatocellular cholangiocellular carcinoma was diagnosed in one case with an incidence of 0.5%, which were categorized as malignant forms (Table 1 & Fig. 2).

Gross morphology:
Affected livers were enlarged and slightly congested, with rounded borders and irregular surfaces.An intrahepatic pale whitish-colored compact mass with approximately 5 cm diameter was detected and protruded from the liver surface.This mass was pedunculated and attached to hepatic tissue through dense pale yellowish fibrous tissue.In addition, varying degrees of biliary fibrosis and necrosis were noticed in affected cases (Fig. 3a).

Histopathology
Normal hepatic parenchyma was replaced with the tumorous mass, which was characterized by large numbers of variably sized tubular to papillary structures lined by a single layer, occasionally hyperplastic, low cuboidal or columnar cells with basally arranged round to oval nuclei and pale eosinophilic cytoplasm.There is no evidence of cellular pleomorphism, mitosis, necrosis, desmoplasia, or vascular invasion.These glandular structures contained minimal amounts of eosinophilic mucinous secretions and were infiltrated with inflammatory cells, especially lymphocytes and neutrophils (Fig. 3b).Massive fibrous connective tissue proliferation was detected, separating papillary structures from each other, as well as surrounding the benign tumorous lesion.The occasional presence of epithelial cell nests in the stroma suggests progression toward malignancy (Figs.3c,  d).

Gross morphology:
The liver was paler than normal, and friable with an irregular surface.It exhibited multiple large nodular lesions with firm consistency, pale yellow color, and approximately 5 cm diameter distributed randomly throughout the parenchyma and attached to the surface.These lesions had central raised necrotic areas with grayish coloration, giving them somewhat an umbilicated appearance and on cut section, spreading to the biliary tree was observed (Fig. 4a).In association with large nodules, multiple smaller ones were present and scattered within affected hepatic tissues (Fig. 4b).Excision of nodular lesions had resulted in depressed areas or cavitation with dark red necrotic patches (Fig. 4c).

Histopathology:
The neoplasm was formed of anaplastic, large, cuboidal to rounded cells that were highly pleomorphic and disorganized with moderate to minimal amounts of cytoplasm, large nuclei with varying shapes, and prominent one or more nucleoli.These cells formed isolated gland-like structures.Cellular atypia was an important feature and occurred in the form of cytoplasmic basophilia, nuclear hyperchromasia, high nuclear to cytoplasmic ratio, loss of nuclear polarity, and abundant mitotic figures.However, many cells were binucleated (Fig. 5a).Cholangiocarcinoma was characterized by a poorly differentiated acinar histological pattern in which variably sized acini or tubules that were lined by a single layer of neoplastic biliary epithelial cells were detected.Also, they were filled with acidophilic mucin and distributed in the fibrous stroma (Fig. 5b).Adjacent non-neoplastic hepatic cells showed variable degrees of hemorrhage, degenerative and necrotic changes as a result of compression (Fig. 5c).

Gross morphology:
The liver was congested with dark coloration and sharp edges.Its surface was irregular and showed a diffuse large yellowish-white circumscribed lesion with a firm texture, measuring approximately 15 cm and replacing most of the hepatic parenchyma.At the site of the junction, minute hemorrhagic zones with yellowish necrotic patches were detected.A tumorous mass was attached to the liver surface by dense whitish collagenous fibers.Generally, the lesion was unencapsulated with evidence of hematogenous invasion and the possibility of metastasis (Fig. 6a).

Histopathology:
Cellular features of both hepatocellular and cholangiocellular carcinomas were detected.In areas of hepatocellular carcinoma, hepatocytes showed abnormal arrangement in nearly parallel cords, with the formation of thick irregular trabeculae in stromal fibrosis.Neoplastic hepatocytes were large, rounded, or polygonal in shape with finely granular, acidophilic cytoplasm.Also, they contained large vesicular nuclei with obvious nucleoli.However, mitotic figures were visible (Fig. 6b).Along with neoplastic hepatocytes, hemorrhages, and the characteristic neoplastic clear cells with clear cytoplasm that contained variable amounts of glycogen, fat vesicles and non-uniformly round to oval nuclei were observed.Sinusoidal capillaries suffered from compression, which was caused by large neoplastic cells (Figs. 6b,  c).In Cholangiocarcinoma and combined areas, neoplastic bile duct epithelial cells were cuboidal to columnar with pale basophilic cytoplasm, round to oval one or more nuclei whose chromatin was condensed and nucleoli were prominent.These cells formed tubules distributed in a desmoplastic scirrhous stroma (Fig. 6d).

Figure (1):
The overall incidence of tumors among the diagnosed liver lesions.

DISCUSSION
In Egypt, few researchers report the incidence rate of liver neoplasms in cattle.
So the present study aimed to record their incidence and prevalence in slaughtered cattle imported from Sudan, as well as describe their detailed gross and histopathological picture.The incidence rate was 4% among the affected specimens.
A lower incidence was recorded by Mohammed et al. (2012) et al. (2021) mentioned that lesions of cholangiocarcinoma in cattle were similar to those caused by tuberculosis and occurred in the form of numerous coalescing pale yellowish nodules that were not only present in the liver, but also in the omentum and ruminal serosa.
The most characteristic histological features of cholangiocarcinoma are mucin production and the fibrous stroma, which were regarded as valuable findings for tumor recognition (Head et al., 2003).Meuten (2016) stated that cholangiocarcinomas might be either well differentiated with an acinar or tubular histopathological pattern or undifferentiated with a solid pattern.However, in agreement with our observed acinar form, Vielmo et al. (2020) reported that it was formed of undifferentiated cells that displayed high mitotic index, marked cellular pleomorphism, and vascular invasion.In their study, the solid pattern was the most frequent, in which large neoplastic cells with prominent, multiple nuclei mostly replaced the hepatic parenchyma.Jubb et al. (2007) and Ohfuj (2012) mentioned that cholangiocellular carcinomas form acini, ductules, or papillary projections within the lumen of neoplastic ducts.Cells were cuboidal or columnar with minimal amounts of clear cytoplasm.Mitotic figures were abundant.Tubules were filled with mucinous secretions and didn't contain bile.In contrast, Azizi et al. (2016) detected mucinfree acinar structures or solitary islands located mostly in periportal areas and were embedded in excessive fibrous tissue stroma.Cuboidal to columnar neoplastic cells appeared with variable-sized, roundto-oval nuclei and basophilic cytoplasm.According to Elbert et al. (2021), neoplastic epithelial cells formed ductules surrounded by dense or edematous areas of desmoplasia, necrosis, and dark basophilic clumps of calcium.Honda et al. (2020) recorded a case of hepatocholangiocarcinoma in an aged cow.During the post-mortem examination, the liver showed some degrees of adhesion with various internal organs, its external surface was irregular and nodular.Some hepatic lobes were mostly replaced by whitish connective tissue.The cut section revealed a pus-like fluid filling distended bile ducts and orange to yellow-colored liver tissue.Also, they observed a severely enlarged gall bladder that contained bile with blackish stones.However, a nearly similar gross picture was reported in our study, with the presence of a large diffuse yellowish-white lesion measuring about (15) and replacing most of the hepatic parenchyma.This was associated with a narrow hemorrhagic zone and necrotic areas at the site of its attachment.Aishma et al. (2010) and Yeh, (2010) mentioned that the characteristic histopathological feature of hepatocellular carcinoma was trabecular or pseudo glandular arrangement of neoplastic hepatic cells that appeared to resemble hepatocytes with fat, while cholangiocellular carcinoma was characterized histologically by glandular structure, production of mucin and the desmoplastic stroma.Honda et al. (2020) observed the collision of both features of hepatocellular and cholangiocellular carcinomas, which resulted in the development of combined hepatocellular cholangiocellular carcinoma.In hepatocellular carcinoma-affected areas, neoplastic hepatocytes were polygonal in shape, with round to oval nuclei, abundant pale acidophilic cytoplasm, and were arranged in solid nests or trabeculae, while in areas of cholangiocellular carcinoma, neoplastic cholangiocytes were cuboidal to columnar, with round to oval nuclei and mucin on its apical surface or in the cytoplasm.Neoplastic cholangiocytes formed tubular-like structures embedded in stromal fibrosis.This picture was in complete agreement with our histopathological findings.

CONCLUSION AND RECOMMENDATION
Liver and bile duct tumors are prevalent in beef cattle imported from Sudan.These animals might be exposed to certain hepatocarcinogens as aflatoxins or certain drugs administered for treatment purposes.Strict veterinary inspection at slaughterhouses should be applied to these animals, as they are considered a major source of meat in our country.In addition, a larger sample size and further studies concerning lesions in other organs are required to be conducted.
Figure (2): Percentage of each type of liver tumor in relation to their overall incidence

Table 1 :
Numbers and incidence rates of different types of liver neoplasms.
Histological classification of tumors of the alimentary system of domestic animals.Vol 5 2nd series.Armed Forces Institute of Pathology Washington 257p.Papillary adenocarcinoma of the extrahepatic bile duct in a Holstein cow.Vet.Pathol., 42: 74-77.