Vet Pathol32:635-640 (1995)

The Lesions of Hepatic Fatty Cirrhosis in Sheep

R. G. HELMAN,L. G. ADAMS,
AND C. H. BRIDGES

Department of Pathobiology, Texas Veterinary Medical Center, Texas A&M University,

College Station, TX 77843-4467

Abstract. During a natural outbreak of hepatic fatty cirrhosis (HFC) in western Texas, 500 2-6-year-old
Rambouillet ewe sheep were sequentially studied to determine the pattern of lesion development. All sheep
developed lesions of HFC. Grossly, changes first began in the subcapsular hepatic parenchyma along the porta
hepatis and spread peripherally until, in the final stages of the disease, approximately 80% of the liver was
affected. Ascites, hydropericardium, and acquired hepatic vascular shunts were present in sheep with severe
HFC. Light microscopic lesions initially appeared as accumulations of fine lipid droplets in the cytoplasm of
periacinar hepatocytes but, with time, involved all hepatocytes of the lobule. The fat vacuoles in the periacinar
hepatocytes coalesced to form larger vacuoles; and after rupture of adjacent fat-laden hepatocytes, fatty cysts
appeared. Fibrosis began in the periacinar zone associated with the ruptured fatty cysts and continued until
there was widespread bridgingperiacinar fibrosis. Islands of regeneratinghepatocyteswere frequently sequestered
within the bands of fibrous tissue. Characteristically,the hepatic and posterior mediastinal lymph nodes, lung,
and spleen contain ceroid. No lesions of hepatic encephalopathy were found in any animal. HFC is a progressive,
chronic disease of sheep, and the morphology of the hepatic lesions is similar to lipotrope-deficientforms of
nutritional cirrhosis. These findings are discussed in relation to similar nutritional deficiencies and toxicoses in
sheep.

Key words: Ceroid; cirrhosis; fatty change; liver; ruminant; sheep.

The earliest descriptions of hepatic fatty cirrhosis
(HFC) demonstrated it to be a disease responsible for
serious economic losses in livestock in a five-county
area in western Texas4Typically, morbidity varies from
85 to loo%, and mortality approaches 85% over an
extended 3-year period. The disease primarily affects
domestic and sylvatic ruminants-chiefly, sheep, goats,
cattle, deer, and pronghorned antelope.'^^ Animals ini-
tially show signs of failure to gain and maintain body
weight. Signs typically associated with hepatic enceph-
alopathy, including head pressing, depression, abnor-
mal head positioning, and coma, are displayed in an-
imals in the final stages of the disease. Necropsies of
affected sheep having clinical signs of terminal hepatic
disease reveal livers with varying degrees of pale yellow
discoloration and firmness believed to represent fatty
degeneration and cirrhosis, respectively. Regional
lymph nodes frequently had a similar yellow discol-
~ration.~
HFC has appeared in years when there was above-
average winter rains supporting a bloom of spring veg-
etation.’ Furthermore, in years of HFC occurrence, the
wet winters have been consistently followed by drought
conditions through the summer months. Lambs born
in the spring following wet winters have lesions of HFC
when slaughtered 4-5 months later. Therefore, it has
been suggested that a hepatotoxin is consumed by an-
635
imals during the late winter and spring, thus causing
the initial hepatic damage. Because of drought con-
ditions, the reduced range of vegetation through late
summer and fall creates a nutritional stress on an al-
ready diseased liver, and fat mobilization with hepatic
fat accumulation continues. This is aggravated in ewes
due to most animals being in the last trimester of ges-
tation.
Since the original description of HFC in 1932, the
bulk of investigative work has concentrated on iden-
tifying an etiologic agent and epizootilogic factors as-
sociated with the disease. Cases of HFC have been
diagnosed in southern Texas, northeastern Mexico, and
eastern New Mexico and in Texas counties surround-
ing the five-county area where HFC was originally de-
As an economically important and unique
disease of ruminants, it is important for veterinary
pathologists to recognize the lesions of HFC and their
progression in order to make an accurate diagnosis.
The purpose of this paper is to characterize the gross
and microscopic lesions of HFC in sheep.
Materials and Methods
All procedures, examinations, and determinations were
conducted on spontaneously affected sheep. Two groups of
animals used in this study were Rambouillet ewe sheep, 2-
6 years old, purchased off one ranch located in an HFC-

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636 Helman, Adams, and Bridges Vet Path01 3 2 6 , 1995

Fig. 1. Liver; sheep. The earliest macroscopic lesions are

multifocal, pale areas of fatty change (arrows) around the Fig. 3. Liver; sheep. The visceral capsular surface is al-
large blood vessels in the porta hepatis. most diffusely affected by fatty change that is represented as
homogeneous to lacy pale regions.
affected area of western Texas in Reagan County. One group
of 400 sheep was maintained on range at the ranch of pur- Tissues were fixed by immersion in 10% neutral-buffered
chase for a 7-month period from February through August. formalin, embedded in paraffin, sectionedat 6 pm, and stained
This ranch had had consistent problems with HFC over the with hematoxylin and eosin. Frozen sections of hepatic lymph
previous 20 years, such that in years of HFC occurrence 99% nodes, kidneys, lung, and liver were stained with the Oil Red
of the sheep on the ranch developed the disease. 0 method to demonstrate cellular lipids. Paraffin-embedded
The second group of 100 animals included 80 sheep with sections of liver were stained by Wilder’s silver impregnation
various stages of HFC and 20 unaffected sheep. These ani- method for reticulin and by Van Gieson’s picrofuchsin tech-
mals were brought to Texas A&M University, College Sta- nique for collagen fibers. Ceroid was demonstrated with the
tion, Texas, for study over a 1-year period beginning in March. acid-fast stain of Ziehl-Nielsen and with ultraviolet fluores-
The latter sheep were maintained on native pasture grasses cent microscopy in unstained sections. Microscopic lesions
in the spring, summer, and fall and were dewormed at 1-3- in samples taken from multiple sites in affected livers were
month intervals. During the winter, the sheep were supple- coordinated with the macroscopic lesions.
mented with prairie grass hay and grain, mostly milo maize.
The characteristics of the macroscopic lesions are based
Results
on the examination of 38 1 livers obtained by necropsy after All sheep in both groups of the study developed
HFC. The earliest gross lesions were focal to multifocal
natural death or slaughter of sheep in the first group of sheep
during the months of May, June, and August (months 4,5 , and consisted of pale, yellow, and firm subcapsular
and 7). Results were tabulated and sketches were made of areas around the porta hepatis on the visceral surface
the distribution, size, and extent of the macroscopic lesions.
of the liver (Fig. 1). The papillary process and the
Data on changes in other organs were gathered while per-
ventral border of the left lobe sometimes were involved
forming complete necropsies on 65 additional sheep, the sec-
ond group, affected with various degrees of HFC. Sheep in
to a minor extent. The lobular pattern of the liver was
the second group were necropsied at 1-month intervals accentuated throughout the organ and characterized
throughout the year-long period of study at Texas A&M. by a yellow, 1-mm central focus surrounded by nor-
mal-appearing tan to red-brown parenchyma.
Later, livers of affected sheep had moderately ex-
tensive lesions extending from the subcapsular areas
deeper into the parenchyma of the liver. The areas of
discoloration were firmer and completely surrounded
the porta hepatis extending along the boundaries of the
caudal vena cava and the attachment of the lesser
omentum (Fig. 2). The lesions located along the um-
bilical fissure and the ventral border of the left lobe
were more extensive also, having expanded toward the
porta hepatis and appearing on the parietal surface of
the liver.
In animals with more advanced HFC, there were
Fig. 2. Liver; sheep. Pale areas of fatty change (arrows) extensive lesions involving the entire parenchyma be-
have extended around the entire porta hepatis and into the tween the parietal and visceral surfaces of the left lobe
surrounding right and left lobes. (Fig. 3). The lesions were confined mostly to the retic-

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Vet Pathol326, 1995 Ovine HFC 631

Fig. 5. Liver; sheep. Earliest microscopic change in HFC

is the presence of finely vacuolated cytoplasm of periacinar
hepatocytescharacteristicof lipid accumulation.Central vein
is just out of the field of view. Hematoxylin and eosin. Bar
= 25 Fm.

vascular anastomoses and fibrous adhesions between

Fig. 4. Liver; sheep. There is prominent hypertrophy of
the margins of the liver accompanying the extensive fatty
change and atrophy of the more central regions of the liver
(a). A cross-section adjacent to the hilar region of the liver
showing sharp demarcation between fatty, atrophic, and hy-
pertrophied liver (b).
the hepatic capsule and the diaphragm. The afferent
lymphatics of the hepatic and caudal mediastinal lymph
nodes frequently were distended and tortuous. The per-
icardial sac commonly contained between 30 and 100
ml of clear, yellow, thin fluid. The thoracic and ab-
dominal cavities of these animals contained from 50
to 200 and 500 to 2,000 ml, respectively, of a similar
fluid. The mesentery commonly was very edematous.
The hepatic lymph nodes were enlarged and measured

ular impression of the left lobe and to the omasal im-

pression of the right lobe. The caudate process was
occasionally involved, and the left lobe was atrophic
in some sheep.
In sheep with the terminal stages of the clinical dis-
ease, the surface of the liver was yellow and uniformly
pitted with small depressions. The more central regions
of both major lobes were firm and small while the right
lateral borders of the left and right lobes typically had
undergone massive hypertrophy (Fig. 4). The capsule
of the hypertrophied regions of liver was thickened and
had a lacy, gray to tan pattern. In some animals, the
entire liver was yellow and markedly atrophic. In the
atrophic central regions of the major lobes, there fre-
quently were bulging, 0.5-2.0 cm, green biliary cysts
Fig. 6. Liver; sheep. Hepatocytes with more advanced
adjacent to the caudal vena cava and porta hepatis. fatty change adjacent to the periacinar region (central vein
In more severely affected sheep, the gall bladder usu- out of view). Individual hepatocytes have one to numerous
ally was distended with dark green, viscous bile. The cytoplasmic fat vacuoles. Sinusoids are inconspicuous be-
parietal capsule of the liver often was thickened and cause of hepatocellular enlargement with fat. Hematoxylin
edematous, and in severely damaged livers there were and eosin. Bar = 25 wm.

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638 Helman, AdamIS, and Bridges
Fig. 7. Liver; sheep. Multiple fatty cysts in the periacinar
region (central vein out of field). There is early fibrosis ad-
jacent to the fatty cysts with minimal numbers of mono-
nuclear inflammatory cells. Ceroid-laden mononuclear phag-
ocytes (see inset) are adjacent to hepatocytes and fatty cysts.
Hematoxylin and eosin. Bar = 50 pm.
up to 3.5 cm. The cortex had many small, 1-2 mm,
yellow-orange foci throughout. The caudal mediastinal
lymph node had a similar but less pronounced ap-
pearance. The thoracic and abdominal fat frequently
displayed serious atrophy.
Microscopically, the initial lesion was an accumu-
lation of many fine, intracellular lipid droplets in the
hepatocytes located in the periacinar region of the lob-
ule (Fig. 5). The lipids stained intensely with Oil Red
0. With progression of the disease, the lipid accu-
mulation in hepatocytes spread toward the portal areas
of the hepatic lobule and eventually affected practically
all of the hepatocytes. Often, mitotic figures, one to
three per 400x field, were prominent in hepatocytes
throughout the lobule.
Coalescence of smaller lipid droplets to form larger
lipid vacuoles was obvious within hepatocytes located
in the periacinar region, spreading peripherally in the
hepatic lobules until a majority of hepatocytes were
laden with one or more large lipid vacuoles. Over time,
the larger intracellular vacuoles continued to increase
in size, particularly in those hepatocytes of the peria-
cinar region of the hepatic lobule (Fig. 6 ) . Eventually,
adjacent, enlarged hepatocytes ruptured, giving rise to
fatty cysts with multiple hepatocyte nuclei compressed
at the periphery. Such fatty cysts were most frequently
located eccentrically to or surrounding the central veins
(Fig. 7).
The first appearance of fibrosis was in the sites around
the central veins where fatty cysts were most numerous
(Fig. 7). Often, the larger fatty cysts had ruptured, re-
sulting in the release of lipids into the sinusoids. De-
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Vet Pathol326, 1995
Fig. 8. Liver; sheep. Portal region with hyperplastic bile
ducts, bands of fibrosis, and a regenerative hepatocytic nod-
ule. Hematoxylin and eosin. Bar = 100 pm.
position of collagen occurred initially on the collapsed
fatty cysts and condensed hepatic reticulin. Fibroplasia
continued along a line linking adjacent central veins.
Increased numbers of Kupffer cells were prominent in
the periacinar regions where fibrosis was greatest. In
the later stages of the disease, bands of fibrous con-
nective tissue with entrapped fatty cysts extended be-
tween central veins and portal areas, resulting in pseu-
dolobulation. When fibrosis was advanced, there were
often secondary hypoxic changes of the residual he-
patic tissue in the periacinar and periportal regions
characterized by coagulation necrosis. Secondary bil-
iary hyperplasia occurred frequently in the portal areas
and often extended along the usually indistinct bile
ductules adjacent to the terminal branches ofthe portal
veins (Fig. 8). Nodular hyperplasia of the hepatocytes
with lobular hypertrophy was noted in the right lateral
borders of the right and left lobes of the liver.
Kupffer cells in and around the fibrotic areas con-
taining fatty cysts commonly had abundant intracy-
toplasmic pigment interpreted to be ceroid based on
resistance to organic solvents and yellow-brown au-
tofluorescence under ultraviolet light (see also Fig. 7).
Similar collections of pigmented mononuclear phag-
ocytes were present in lymphatics in portal triads and
the hepatic lymph nodes. In addition, there were in-
creased numbers of mononuclear phagocytes in the
cortical and medullary sinuses and lymphoid follicular
hyperplasia throughout the cortex.
In the final stages of the disease, the fibrous tissue
became the predominant element with few normal,
discernible landmarks remaining. An occasional slump
of surviving, regenerating hepatocytes was present ad-
jacent to or surrounding a branch of a portal vein in
Vet Path01 325, 1995
proximity to a fibroticportal triad. The number of fatty
cysts was reduced as the fibrosis became the predom-
inant feature.
The caudal mediastinal lymph nodes, spleens, and
alveolar capillaries of severely affected sheep also con-
tained ceroid-laden macrophages. In the spleen, pig-
mented macrophages initially appeared in the central
portions of the Malpighian corpuscle adjacent to the
central artery but, after time, were increased in and
around the corpuscles. In advanced HFC, there was
lymphoid depletion of the spleen. No lesions of hepatic
encephalopathy were found in any animal in the ter-
minal stages of HFC.
Discussion
HFC in sheep is a distinct and characteristic hepatic
disease in which there is a progressive fatty change of
the liver leading to cirrhosis. The development of he-
patic lesions consistently follows a pattern with the
parenchyma adjacent to the major vessels and capsule
along the portal and umbilical fissures being affected
first with gradual extension into the right and left lobes
of the liver. The development of hypertrophy in the
least affected regions of liver occurs in the late stages
of the disease and is a presumed, compensatory re-
sponse to increase liver mass to meet bodily needs.
All morphological hepatic lesions of HFC can be
related to the abnormal accumulation of intrahepa-
tocytic lipid. The gradual distension of hepatocytes
with lipid is followed by rupture of neighboring he-
patocytes and formation of fatty cysts. It has been shown
in experiments on other forms of fatty cirrhosis that
fibrosis will OCCUT only if the accumulation of lipid is
sufficient to rupture hepatocytes and form fatty cysts.2,6
As fatty cysts undergo dissolution, fibrosis develops by
condensation of reticulin and production of collagen
by fibroblasts, and possibly myofibroblasts, stimulated
by the presence of extracellular lipids, especially cho-
lesterol-containing lipids.
The appearance of ceroid in hepatic lesions, regional
lymph nodes, spleen, and lung follows the rupture of
fatty cysts and the onset of fibrosis. Ceroid is com-
monly seen in adipose tissue of animals with vitamin
E deficiency and is believed to represent an abnormal
lipid product formed by the polymerization of oxi-
dized, unsaturated fatty acids.5 The amount of ceroid
correlated directly with the severity and extent of fatty
cyst formation and rupture in sheep with HFC. For
the most part, ceroid is believed to be formed locally
in the liver in the regions of the fatty cysts and appears
in distant lymphoid organs and the lungs as a result of
lymphatic and/or venous transport of ceroid-laden
mononuclear phagocytes.
The earlier and more extensive involvement of the
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Ovine HFC 639
ventral lobe of the liver may represent a streamlining
effect, which has been defined in dogs and cats.3It has
been suggested that portal blood from the caudal mes-
entenc and splenic veins is distributed to the dorsal
lobe while blood from the cranial mesenteric vein flows
into the ventral lobe. Although the exact percentage of
distribution is not known in ruminants, it may help
explain the differences in involvement between the
dorsal and ventral lobes of the liver in sheep with HFC.
Lesions of portal hypertension are a common se-
quela to the severe liver cirrhosis in sheep in the later
stages of HFC. This is evident by the development of
vascular anastomoses on and between the parietal he-
patic capsule and diaphragm. Distension of the afferent
lymphaticsto the hepatic and caudal mediastinal lymph
nodes plus ascites are also commonly associated with
portal hypertension.*However, the ascites may also be
exacerbated by hypoalbuminemia, which has been de-
scribed in sheep with HFC and would be expected in
animals with chronic liver failure.'J4
HFC in sheep closely resembles experimental nu-
tritional cirrhosis in laboratory r o d e n t ~ . ~The
, ~ J dis-
~
ease in laboratory animals has typically been induced
by feeding a lipotrope-deficient diet in which the feed
has low levels of methionine, folic acid, and/or vitamin
BIZ.As in HFC, the development of fatty change in
nutritional cirrhosis due to lipotrope deficiency begins
in the periacinar region and extends to involve the
entire lobule. Similarly, fatty cysts form around the
central veins followed by rupture and the development
of fibrosis eventually progressing to cirrhosis with cer-
oid formation.
Lipotropic agents are essential for the processing of
intracellular lipids destined for export from the liver.12
It is suggested that the synthesis and/or secretion of
triglycerides in the form of very low-density lipopro-
tein (VLDL)is blocked with lipotrope deficiency. Stud-
ies have shown that lipotrope-deficient diets result in
a decrease in intracellular phosphatidyl choline (leci-
thin), which is an essential component in VLDL syn-
thesis.
The closest situation to lipotrope deficiency in ru-
minants has been described in ovine white liver disease
(WLD)I6J7In this condition, it is speculated that a soil
cobalt deficiency results in lowered levels of vitamin
BIZin the animal. Sheep with WLD have a diffuse and
progressive periacinar hepatocellularfatty change lead-
ing to formation of fatty cysts and accumulation of
ceroid. Fibrosis along the tracts of fatty change with
portal biliary hyperplasia also is described. Grossly,
the liver of animals with WLD is diffusely pale tan to
gray and becomes firm but does not have the charac-
teristic pattern of macroscopic lesion development as
seen in sheep with HFC.
In WLD, sheep typically develop lesions and clinical
640 Helman, Adams, and Bridges
signs of fatty liver in the spring immediately following
a winter season of above-average rains. A similar sea-
sonal pattern of disease occurrence is seen in HFC. In
WLD, it is thought that the resulting bloom of cobalt-
deficient vegetation is responsible for the development
of a vitamin B,, deficiency in the animal. Fatty liver
results from two factors: reduced lipoprotein formation
and export from the liver due to lowered B,, levels and
increased supply of proprionates from the lush pasture
vegetation. 16,17
There are, however, other potential mechanisms for
the development of fatty cirrhosis. Lipoprotein syn-
thesis and secretion by the liver is a complex and mul-
tistep process utilizing diverse metabolic pathways plus
coordinated intracellular movements through the cy-
tosolic filament and microtubule network.lo Xeno-
biotics like ethionine and carbon tetrachloride also
produce fatty liver but do so through a metabolically
different mechanism than lipotrope deficiency, al-
though generically there is an interference with lipo-
protein, specifically VLDL, synthesis. The mycotoxins
aflatoxin and phomopsin A also result in, among other
things, fatty but, aflatoxins result in decreased
protein synthesis, which reduces lipoprotein synthesis,
whereas phomopsin A targets microtubular function
and, thereby, interferes with lipoprotein secretion.
Phomopsin A-induced damage to microtubules also
results in a second, consistently demonstrated signifi-
cant effect-an increase in the number of mitotic fig-
ures in hepatocytes. I 3 Although the latter lesion has
not been quantified in sheep with HFC, the promi-
nence of mitotic figures in the early lesions could be a
consequence of exposure to an antimicrotubular com-
pound similar to what is seen in sheep with lupinosis
due to phomopsin toxicosis.
Admittedly, the exact cause of HFC is unknown at
this time. Results of extensive plant feeding trials since
1932 (over 120 species of plants) have consistently
failed to produce HFC in sheep.' The seasonal and
climatic pattern of occurrence of HFC (6 to 9 months
following a winter with above-average rainfall) could
support either a mycotoxin etiology or nutritional de-
ficiency as is seen in lupinosis or WLD, respectively.
Certainly, the similarities in the morphology of lesions
between HFC and experimental and naturally occur-
ring nutritional cirrhosis suggest the possibility of a
common or, at least, partially shared pathophysiologic
mechanism.
Request reprints from Dr. R. G. Helman, Department of Pathobiology, Texas Veterinary Medical Center, Texas A&M
University, College Station, TX 778434467 (USA).
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Vet Pathol 32:6, 1995
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