Immunohistochemical Changes of Liver Fasciolosis in Cattle and Sheep in Jeddah, Saudi Arabia

Parasitic diseases are the major impediment in the growth and development of livestock health (Salmo, 2014). Fasciolosis is an important zoonotic disease widespread in animals and human beings, crucial to the economy and  health (Najib et al., 2020). The Fasciola hepatica and Fasciola gigantica cause fasciolosis. It is a neglected tropical disease and in more than 50 countries, most cattle and sheep are reared (Mehmood et al., 2017).
       
Fasciola hepatica and Fasciola gigantica are the two liver flukes commonly reported to cause Fascioliasis in ruminants. The disease is caused by the ingestion of encysted metacercaria of liver flukes (Ibrahim, 2017). Fasciola hepatica is found in sheep and cattle and is widely distributed in Europe, Africa, Asia, Oceania and North and South America  (Adarosy et al., 2013).
       
The liver is the most important organ for animal health and animal development since much of the body’s metabolism occurred in the liver. The liver parasite infections are one of the critical factors that decrease national income in animal production by either directly denouncing the pathologically-affected liver or by indirectly influencing animal growth and meat production (El-Hallawany and Abdel-Aziz, 2012).                
       
The adult Fasciola resides in the gall and bile ducts of infected animals leading to serious damage to animals. Sub-clinical and chronic disease usually results in decreased production of meat, milk and wool. Secondary bacterial infections, fertility problems and great expenses of anthelmintics should be considered (Degheidy and Al-Malki, 2012).
       
In rejecting the Fasciola hepatica infection and developing successful resistance to re-infection, there seems to be an essential part of the humoral and cellular immune response to the parasite. During their hepatic migration, the local immune response to young flukes may have a role in the resistance mechanisms. Lymphocytic infiltration of hepatic lesions has mainly consisted of CD4+ cells in the sheep during the early stages of infection, while CD8+ lymphocytes have been increasing in chronic infection and reinfection over CD4+ (Perez et al., 1998).
       
Therefore, this research was intended to analyze the histopathological and immunological reactions induced in the liver of cattle and sheep naturally infected with Fascioliasis.

Study area
 
The current research was a cross-sectional study carried out in one of the biggest abattoirs in Jeddah province in western region in Saudi Arabia. Examining of the slaughtered cattle and sheep to detect Fasciola spp in the liver was done periodically in the abattoir. A bout 150 cattle and 200 sheep were investigated. From these animals about 15 infected livers were collected from cattle and 25 infected livers were collected from sheep. The amount of worms in the gall bladder was ranged between 1-5 worms. 
Sampling
 
Examination of slaughtered cattle and sheep after skinning the slaughtered cow and sheep, the outer surface of the carcass was examined by visual observation for any abnormal lesion. Then the abdominal cavity was opened to check on internal organs. From each cow and sheep, the liver was inspected by making multiple deep incisions in the lobes and the gallbladder was opened using a knife (Fig 1). Observations were confirmed and the help of veterinary doctors was acquired throughout the inspection visits.
 

 
Histological examination
 
The liver samples of cattle and sheep were fixed in 10% neutral formalin for histopathological examinations. Then samples were dehydrated in ascending grades of alcohol. After that, the samples were embedded in paraffin wax. Sections of 5-micrometer thickness were prepared and subjected to staining with Hematoxylin and Eosin (H&E) (Bancroft and Gamble, 2008) for routine histological examination. The silver stain of Gordon and Sweet was used for displaying irregular reticular fibers, which may indicate cirrhosis or liver necrosis (Gordon and Sweets, 1936).
 
Immuno-histochemical examination
 
The paraffin liver sections were deparaffinized and hydrated. Incubation with 3% hydrogen peroxide for 5 minutes blocked endogenous peroxidase activity. Sections were incubated overnight with CD4+ and CD8+ monoclonal antibody (purchased from veterinary diagnostic test kits and reagents (VMRD Inc), Pullman, WA, USA). In a separated tissue sections, each primary antibody was added. The monoclonal antibody was then linked to the biotinylated goat anti mouse immunoglobulin serum (purchased from Dako Denmark A/S) for 30 minutes. A brown reaction was formed after 5 minutes of exposure to 3, 3-diaminobenzidine tetrahydrochloride solution (DAB) (purchased from Sigma_, St Louis, MO, USA), then washing with distilled water. Sections were countered-stained with hematoxylin (Behairy et al., 2018).

Fascioliasis is an endemic disease globally and causes severe economic conditions and affects large and small animals. Fascioliasis was the leading cause of liver damage and was responsible for full sheep (Degheidy and Al-Malki, 2013) and cattle liver condemnation (Okoye et al., 2015).        
       
In the current study, lesions were mostly around portal spaces with the extensive characteristic proliferation of fibrous connective tissue around the intra-hepatic bile ductules with peri-infiltration of mononuclear inflammatory cells as was reported by (Ashrafi et al., 2014; Labruna et al., 2018; Mendes et al., 2012). Besides, these findings were similar to the finding of Aljameel et al., (2017). They reported that the thickening of the bile duct is due to the chronic nature of Fasciola infection (Aljameel et al., 2017).
       
In the present study, the liver tissue’s histological structures appeared similar in both the cattle and sheep. The HandE liver sections of cattle and sheep exhibited dilated congested central vein with marked deeply acidophilic materials surrounding the central vein.  Most of the hepatocytes have a deep acidophilic cytoplasm with apoptotic nuclei. There was a pronounced inflammatory cellular infiltration across the central veins and close to the portal tracts in infected animals’ liver sections. Often this invasion has spread to the hepatocytes. There were no hepatocytes in areas of heavy cellular infiltration, only an acidophilic region (Fig 2). An extensive reticular fibers with hyperplasia of bile duct walls, which produced a thick wall picture, was marked in sheep and cows infected liver. These results in agreement with previous studies by (Okoye et al., 2015; Salmo, 2014), who proved that thickening of the bile duct is due to the chronic nature of Fasciola infection and the presence of mature flukes within the lumen of intra-hepatic bile ducts which brought a continuous irritation and led to hyperplastic proliferation (Okoye et al., 2015; Salmo, 2014).
 

       
An additional section showed major hepatocytes swelling that existed with clear cytoplasm. The nuclei relocated or disappeared from the cell’s usual location. The intra-sinusoidal cells, Kupffer cells, were hypertrophied (Fig 3). The portal area revealed a portal vein and a large proliferated, thick-walled bile duct surrounded with the nuclei of the cellular infiltrate (Fig 4).  Primary biliary cirrhosis showed extensive proliferation of fibrous connective tissue around the intrahepatic bile ductules with peri-infiltration of the bile duct’s mononuclear inflammatory cells. The thickening of bile ducts wall and surrounded by fibrous connective tissues and metaplasia of bile ducts with infiltration of inflammatory cells (Fig 5).
 

 

 

       
A marked hepatic infiltration with mononuclear inflammatory cells was also observed in this study. The formation of granuloma was also observed mainly near the portal area. These results were similar to the results detected by Okoye et al., (2015). Okoye et al., (2015) reported that the granulomatous reaction formed mainly of mononuclear infiltration in the form of eosinophils, macrophages and lymphocytes with overcrowded endothelial cells and fibroblast cells. They mentioned that these mononuclear infiltrations cells were considered the prominent features in the early stage and also the migratory phase of infection. Moreover, our results agree with the previous study done, which proved that immature liver flukes’ migration through the tissue causes hemorrhage and irritation and brought the cellular inflammatory reactions (Salmo, 2014). In addition, the present study revealed hypertrophied von-kupffer cells in the dilated blood sinusoids. This could be explained by (Lee and Friedman, 2011; Liu et al., 2010), who reported that Kupffer cells are associated with hepatocellular apoptosis, inflammation and fibrosis in liver fibrosis models.
       
In Gordon and Sweet reticulin stained sections, the reticular fibers in the stroma display a marked rise. Reticular fibers were observed as thin black structures appeared to rise in the area surroundings the central veins (Fig 6 and 7) and the structures in the portal area (Fig 8 and 9).
 

 

 

 

       
Immunohistochemically stained sections with CD 4 (Fig 10 and 11) and CD8 (Fig 12 and 13) showed a positive brownish reaction of CD4 and CD8 T- lymphocytes in-between the hepatocytes in the dilated blood sinusoids surrounding the central vein. In addition, positive brownish reactions are located mainly in the inflammatory cells infiltrations area of portal spaces.
 

 

 

 

       
The best-characterized function of intrahepatic lymphocytes is their high cytotoxic activity toward other cells. Several previous studies have investigated the immune response to Fasciola hepatica in sheep. These studies reported that sheep do not develop resistance to reinfection (Mehal et al., 2001; Moreau et al., 2002). 
       
The cellular immune response in sheep and cow to an infection with Fasciola hepatica were examined by immunohistochemistry of liver tissue. Interestingly, the presence of numerous activated CD4+ and CD8+ T-lymphocytes in the portal tract area, in-between the radiating hepatocytes and surrounding bile ducts, were marked observed in the present study. Similarly to studies done by Mendes et al., (2010); Molina and Skerratt, (2005) and Pérez et al., (2002). In addition, Zafra et al., (2010) reported that the heavy infiltration of CD2+, CD4+ and CD8+ T lymphocytes, both in hepatic lesions and hypertrophied  lymph nodes of infected goats suggested a strong local cellular immune response against flukes located within bile ducts.

The current study revealed that Fascioliasis’s most common lesions were biliary cirrhosis with marked mononuclear cellular infiltrations. Moreover, the histopathological changes in the livers of cattle and sheep infected with Fasciola hepatica represented cell damage, leading to severe economic losses in animals and could lead to massive health conditions in humans.