Case report CONGENITAL HEPATIC FIBROSIS IN AN ABORTED HOLSTEIN-FRIESIAN FETUS

Congenital hepatic fibrosis (CHF) is a rare condition characterized by abnormal accumulation of fibrous tissue in the liver, leading to liver dysfunction. While CHF has been documented in various animal species, it has rarely been reported in cattle. This report presents a case of CHF in an aborted Holstein-Friesian fetus. During the necropsy, the most notable macroscopic findings included an enlarged liver with an irregular “cobblestone appearance” on the surface and firm parenchyma, as well as moderate ascites. Histopathological examination revealed extensive hepatic fibrosis in the portal areas, accompanied by bridging fibrosis extending between portal tracts and bile duct proliferation within the fibrous tissue. Screening for infectious agents as the cause of abortion or liver lesions was unremarkable. The necropsy and histopathological findings confirmed CHF, constituting the first case described in Holstein-Friesian calves in Serbia. Further research is required to determine whether this condition has a genetic basis or is influenced by yet to be identified factors in Holstein-Friesian cattle


INTRODUCTION
Congenital hepatic fi brosis (CHF), fi rst described by Kerr et al. [1] is a condition characterized by the aberrant accumulation of fi brous tissue within the liver, leading to hepatocellular dysfunction, portal hypertension, and compromised liver function.This disease is relatively rare in humans, and has been reported in some veterinary species such as horses [2], rat [3], cat [4], an equine fetus [5] and dogs [4].CHF is a rare and sporadic cause of perinatal mortality in cattle, however in literature there are few reports of occurrence of congenital hepatic fi brosis in calves [6][7][8][9][10].
In this report, we describe the macroscopic and histopathological aspects of hepatic fi brosis in an aborted Holstein-Friesian fetus.Additionally, we tested the serum from the aborting dam, parts of the placenta, and organs of the aborted fetus to identify infectious agents commonly associated with cattle abortions.We also examined the fetal liver tissue for the potential presence of hepatitis E virus (HEV).Our goal was to identify infectious agents that could have been the cause of the cow's abortion and liver lesions in the aborted fetus.

Case presentation
An abortion in a third parity Holstein-Friesian cow, in a village in Mladenovac municipality, Belgrade, Serbia, was reported to the local veterinarian.An aborted female fetus (at the 8th month of gestation) was sent for necropsy to the Department of Pathology of the Veterinary Institute, with no history of disease in the cow during pregnancy, as part of the active program of veterinary authorities for the detection of infectious abortive agents in domestic animals.Accordingly, a serum sample from the aborting cow, as well as parts of the placenta were also sent to the institute for analysis.According to the submission form, there were two family milk cows in the backyard, raised in an extensive system, both cows were subjected to artifi cial insemination at different times, and only this cow had an abortion.The cow had no previous history of disease, she aborted late at night, and in the morning the owner did not notice any clinical signs in the cow.
At the necropsy of the calf fetus, signifi cant changes were observed in the liver, which appeared enlarged with rounded edges, measuring approximately 20 x 15 x 7 cm.The liver's capsule surface exhibited irregularities and increased hardness.On the cut surface, extensive irregular white-gray fi rm structures were observed extending into the hepatic parenchyma (Fig. 1A).Additionally, a larger amount of red-tinged ascites fl uid had accumulated in the peritoneum, notably in the omentum and mesentery.The thoracic and pericardial sacs were also fi lled with a moderate amount of free reddish fl uid (Fig. 1A).
Furthermore, the face of the fetus appeared slightly shortened, and the mucous membranes of the eyes, mouth, vagina, and anus exhibited cyanosis.In the subcutaneous tissue, noticeable edema composed of a reddish liquid was observed.No gross lesions were noted in other organs.Tissue samples from the liver were fi xed in 10% neutral buffered formalin and processed routinely for histological examination, and embedded into paraffi n blocks.Sections of the liver tissue were cut to 5 μm and stained with haematoxylin and eosin (HE), Masson's trichrome (MT) and Van Gieson (VG), and examined histologically.
Microscopically, extensive hepatic fi brosis was observed in the portal areas (Fig. 1B), along with bridging fi brosis extending from one portal tract to another, as demonstrated by specialized MT staining (Fig. 1C).The hallmark of portal fi brosis was an increase in extracellular matrix (ECM) components, including collagen type I deposits, as revealed by the specialized VG staining (Fig. 1D).This resulted in the expansion of the portal tract, accompanied by the proliferation of bile ducts.Bridging fi brosis was characterized by hyperplastic proliferation of fi ne fi bers, forming fi brotic bands that connected the portal spaces (porto-portal bridging fi brosis).
Short cords or islands of vacuolated hepatocytes could be observed in the liver parenchyma and besides, we did not observe any notable infl ammatory reaction in the liver parenchyma.Additionally, we observed large cells with a dark, displaced nucleus at the periphery and cytoplasm containing droplets, which were localized in proximity to the collagen fi bers within fi brotic areas.These cells were assumed to be stellate cells (Fig. 1B).
The serum of the aborting cow was negative for antibodies against BVDV, BoHV-1, Brucella spp., C. burnetii, C. abortus, N. caninum, Leptospira spp., and HEV, while placenta and fetal tissues were negative by real-time PCR for all seven agents, including fetus liver tissue for HEV-RNA, and cultivation of Listeria spp.
The diagnosis of congenital hepatic fi brosis in this aborted calf fetus was established through macroscopic observations, consistent with previously described fi ndings of this condition in calves [6][7][8][9][10].Confi rmation was obtained through histopathological evaluation, as well as collagen fi ber staining using MT and VG techniques.These staining methods provided evidence of portal and bridging fi brosis, along with bile duct proliferation and the presence of cells resembling stellate cells in the liver parenchyma, as observed through HE staining.Furthermore, the absence of positive results for the most common infectious abortive agents in both the cow and fetal tissues, along with the absence of HEV RNA in the fetal liver tissue, supports the diagnosis of CHF in the aborted calf.

DISCUSSION
Congenital hepatic fi brosis is a rare developmental disorder pathologically based on ductal plate malformation, resulting from abnormal embryogenesis of the biliary ductal system [6].In humans, CHF is a hereditary, autosomal recessive disease, usually associated with autosomal recessive polycystic kidney disease childhood [28,29].In cattle, CHF has been diagnosed in fetuses, newborns [6][7][8], and in a ten-month-old calf [9].However, unlike CHF cases in horses, cats, dogs, and rats, this fi brocystic hepatic disease in calves is not associated with malformations in other organs, which is also a feature of this case.The sporadic occurrence and the absence of other etiologies indicate that congenital hepatic fi brosis in prenatal and postnatal calves is similar to the disease in humans and may result from a heritable or spontaneous genetic mutation, possibly related to biliary dysgenesis [6].
The pathological lesions described in our case are similar to those reported in cases of CHF in calves of the Holstein-Friesian [6,7], Nellore [10], and Hollando-Argentino [9] cattle breeds, as well as liver fi brosis described in cases of 'Paunch Calf Syndrome' (PCS) in Romagnola cattle [30].Subsequent investigations of PCS cases identifi ed a missense mutation in the KDM2B gene (c.2503G>A) on BTA17, which is associated with this condition [31].In our case, in addition to the defi ned liver fi brosis, we also observed moderate ascites and mild subcutaneous edema, likely resulting from postmortem changes or alterations in hydrostatic and osmotic blood pressure caused by liver disease.No pathological lesions were observed in other organs or systems.Therefore, even though we did not perform genetic testing for PCS genotyping, we ruled out PCS as the cause of fetal death in this case, which, along with hepatic fi brosis, is characterized by multiorgan developmental dysplasia.To the authors' knowledge, there have been fi ve reported cases of this condition in Holstein-Friesian calves, with one case described in a 1-day-old calf [7], two in aborted fetuses at 8 month of gestation [6,8] and two in aborted fetuses at 7 month of gestation [6].This suggests that CHF is a very rare condition in this breed of cattle.Therefore, additional research will be necessary to confi rm a genetic etiology for this condition with convincing evidence that the defect might be regulated by a single autosomal locus acting in a recessive manner, or to detect the other factors responsible for the occurrence of this liver disease in Holstein-Friesian calves.
Since an infectious etiology was absent in our case, and a toxic etiology, metabolic abnormalities, nutritional defi ciencies, or trauma were unlikely due to the lack of clinical signs in the dam, the characteristic macroscopic "cobblestone appearance" of the lesions on the surface of the fetal liver, associated with marked fi brosis in the liver parenchyma as observed in the histopathological evaluation, favored the diagnosis of CHF in the aborted fetus.
In conclusion, this is the fi rst report of liver disease with a description of fi brosis and proliferation of bile ducts in fi brous tissue in a Holstein-Friesian calf in Serbia, and the described lesions are characterized as congenital hepatic fi brosis.This disease may have a genetic basis in Holstein-Friesian cattle, or it may be caused by factors not identifi ed in our research.In both cases, it is necessary to continue the research on aborted and stillborn fetuses in this breed of cattle to detect the causes of this fatal liver disease.Given its potential genetic basis as well as unrecognized factors that cause its appearance, it is crucial to recognize and monitor this disease.This recognition will serve to make decisions regarding the use of breeding animals that carry genes for this condition and their exclusion from further breeding, or if other causes are responsible for its occurrence, they need to be addressed.

Figure 1 .
Figure 1.Liver; calf.Congenital hepatic fi brosis in a Holstein-Friesian calf.(A) Irregularities on the surface of the calf 's liver, showing a "cobblestone appearance".A larger amount of red-tinged ascites fl uid accumulated in the peritoneum and omentum (yellow arrow).The thoracic sac fi lled with free reddish fl uid (black arrow).Histopathological evaluation of CHF in a Holstein-Friesian calf.(B) Excessive deposition of connective tissue in the portal and periportal space (black arrow) with proliferation of bile ducts (yellow arrows).Large cells with a nucleus at the periphery and cytoplasm fi lled with droplets near the collagen fi bers within the fi brous area, which we suspect are stellate cells (green arrows), hematoxylin and eosin staining × 40 objective.(C) Increased fi brous tissue expanding the portal tract (portal fi brosis) (black arrows) and fi brotic bands connecting portal tract to portal tract (porto-portal bridging fi brosis) (yellow arrows), Masson Trichrome staining × 10 objective.(D) Type I collagen deposits in the portal area (black arrows), Van-Gieson staining × 40 objective.