Bull Vet Inst Pulawy 58, 517-520, 2014
DOI: 10.2478/bvip-2014-0079
Microscopic changes in the kidneys of cows infected
with Leptospira sp.
Vitalii Ukhovskyi1, Boris Borisevich2, Vlada Kulykova1,
Jacek Żmudzki3, Artur Jabłoński3
1Institute
of Veterinary Medicine of National Academy of Agrarian Sciences,
03151, Kiev, Ukraine,
[email protected],
2National University of Life and Environmental Sciences of Ukraine, 03041, Kiev, Ukraine
3
Department of Swine Diseases, National Veterinary Research Institute, 24-100 Pulawy, Poland
Received: March 10, 2014
Accepted: November 3, 2014
Abstract
The article presents data on histopathological studies of the kidneys of cows, which either recovered or died from
leptospirosis. Fragments of seven kidneys from slaughtered cows, positive for Leptospira antibodies in the microscopic
agglutination test (MAT) (titres of 50 and higher) were used in the study. The MAT was conducted with eight serological groups
of Leptospira: Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Pomona, Sejroe, Tarassovi, and Australis.
Microscopic changes in all morphological structures of the kidneys were presented. Micromorphological criteria, which can be
used for post-mortem diagnosis of leptospirosis were established. They included: serous glomerulonephritis with granular
dystrophy of podocytes, necrosis and collapse of the inner layer of Bowman's capsule, partial destruction of capsule and vascular
glomeruli, granular and vacuolar degeneration and destruction of tubular epithelial cells, foci of interstitial oedema, and
infiltrations predominantly with monocytes and isolated neutrophils. Microscopic changes in the kidneys suggest that the cows
died from leptospirosis as a result of toxic shock syndrome.
Keywords: cattle, leptospirosis, kidneys, microscopic changes, toxic shock syndrome.
Introduction
Leptospirosis, caused by pathogenic Leptospira
species, is one of the most widespread and best-known
zoonoses. Although there is considerable genetic
diversity among pathogenic Leptospira, the clinical
manifestation of the disease is constant for all species
and ranges from a mild, largely asymptomatic chronic
infection, to an acute, potentially lethal one (8). At least
0.5 million human cases occur each year, with
mortality rates ranging from 5% to 15% (14). They
infect a wide spectrum of hosts, including mammals,
reptiles, birds, and amphibians. A variety of clinical
symptoms may be seen in cows infected for the first
time: abortion, mastitis, loss of milk, stillbirth, or
giving birth to weak or infected but clinically healthy
calves. Infertility associated with persistent infection is
the most important economic consequence. Infection is
usually transmitted by direct contact with contaminated
urine, run-off water, or abortion fluids from infected
animals.
Leptospires usually survive in the renal tubules of
many domestic and wild animals (1, 9). In acute
infections, all organs may become infected. Bacterial
passage from infected kidneys to the bladder and
release of the bacteria during urination can expose new
hosts to leptospires (8).
The disease is characterised by the development of
vasculitis, endothelial damage, and inflammatory
infiltrates composed of monocytic cells, plasma cells,
histiocytes, and neutrophils. During gross examination,
petechial or even extensive haemorrhages are common
(2), and organs are often discoloured due to icterus
(19). Histopathological lesions are the most visible in
the liver, kidneys, heart, and lungs (6, 12, 16), but other
organs may also be affected according to the severity of
the individual infection. The overall structure of the
liver is not significantly disrupted, but there may be
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V. Ukhovskyi et al./Bull Vet Inst Pulawy/58 (2014) 517-520
intrahepatic cholestasis. Hypertrophy and hyperplasia
of Kupffer cells are evident, and erythrophagocytosis
has been reported. In the kidneys, interstitial nephritis
is the major finding, accompanied by an intense
cellular infiltration composed of neutrophils and
monocytes (20).
Pathological findings in the heart include
interstitial
myocarditis
with
infiltration
of
predominantly lymphocytes and plasma cells, petechial
haemorrhages (particularly in the epicardium),
mononuclear infiltrations in the epicardium, pericardial
effusions, and coronary arteritis (5, 7, 13). In the lungs,
pulmonary congestion and haemorrhage are common
(21, 23), and infiltrations of alveolar spaces by
monocytes and neutrophils occur (14). In the brain,
perivascular cuffings are observed (24).
The aim of the study was to investigate the
microscopic lesions in the kidneys of cows infected
with leptospirosis.
significant microscopic changes in all morphological
structures was observed.
Material and Methods
All blood vessels were dilated and filled with
blood. Endothelial cells of arteries and veins were in
a state of granular degeneration, some of them
collapsed. In most cases, disruption of the walls of
blood vessels of various types was registered, resulting
in a haemorrhage which originated in the stroma
(Fig. 1).
In all renal corpuscles extracapillary serous
glomerulonephritis was observed. Its early stage was
characterised only by accumulation of serous fluid in
the Bowman’s capsule.
Antigens. Reference strains of serological groups
of Leptospira interrogans: Canicola, Grippotyphosa,
Hebdomadis, Icterohaemorrhagiae, Pomona, Sejroe,
Tarassovi, and Australis were cultured to prepare the
microscopic agglutination test (MAT). The reference
strains are kept in the Laboratory of Leptospirosis at
the Institute of Veterinary Medicine of the National
Academy of Agrarian Sciences in Kiev. Leptospires
were cultivated in Kortgof liquid medium at 28-30ºC
under aerobic conditions. The strains were subcultured
every 7-10 d.
Microscopic agglutination test. The test was
carried out according to the OIE Manual of Standards
for Diagnostic Tests and Vaccines (4). The serum
samples diluted 1:25 were mixed with an equal volume
of each of the Leptospira serovars. Including this added
antigen, the serum dilution used during the preliminary
examination was 1:50. For samples reacting in the
preliminary examination with one or more serovars,
a series of twofold dilutions were prepared to titre end
point: 50% agglutination. The samples with titres of 50
were recognised as positive.
Histological examination. Fragments of the
kidneys of seven cows displaying specific antibody
titres of 50 and higher in the MAT were fixed in 10%
buffered formalin, dehydrated in rising concentrations
of ethanol, and embedded in paraplast through
chloroform. Sections were stained with haematoxylin
and eosin (H.E.).
Fig. 1. The kidney of a cow which died from leptospirosis:
1–expanded capillar of the stroma filled with blood; 2–haemorrhage
in the stroma; 3-granular degeneration of epithelial tubules;
4–hydropic degeneration of epithelial tubules. H.E. 200×
Fig. 2. The kidney of a cow which died from leptospirosis:
1–expanded capillar of the glomerulus full of blood; 2–swelling of
the glomerulus; 3–granular degeneration of mesangiocytes;
4–necrosis and destruction of the cells of the inner layer of the
Bowman’s capsule; 5–granular degeneration of the external layer of
cells of the Bowman’s capsule; 6–cell disruption of the outer layer of
the Bowman's capsule. H.E., 200×
Results
In histological studies of the kidneys of cows
which died from leptospirosis, the presence of
With the development of this process in the liver,
the severity of microscopic changes increased
markedly. Capillaries of vascular glomeruli were
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V. Ukhovskyi et al./Bull Vet Inst Pulawy/58 (2014) 517-520
clearly expanded, and they became swollen (Fig. 2).
Granular degeneration and decomposition of podocytes
were observed. All or almost all cells of the inner layer
of the Bowman’s capsule were necrotic and collapsed.
Part of the cells of the outer layer of Bowman's capsule
were in a state of granular degeneration and some of
them were destroyed.
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and the destruction of the basal membrane of the
epithelium was also recorded.
Fig. 4. The kidney of a cow which died from leptospirosis:
1–vacuolar degeneration of epithelial of the convoluted tubules;
2–destruction of the epithelial cells of convoluted tubules;
3–destruction of the basement membrane of the convoluted tubule.
H.E., 200×
Fig. 3. The kidney of a cow which died from leptospirosis:
1–expanded capillar of the glomerulus full of blood; 2–swelling of
the glomerulus; 3–destruction of mesangiocytes; 4–margination of
chromatin in the nucleus of mesangiocyte; 5–partial destruction of
glomerular vessels; 6–absence of the Bowman's capsule; 7–serous
exudate; 8–granular degeneration and destruction of the epithelial
cells of the convoluted tubules. H.E., 200×
Later, accumulation of a large volume of serous
fluid was observed in the cavity of the Bowman's
capsule. This led to an oedema in the glomerular
vasculature (Fig. 3). A significant number of
mesangiocytes were in a state of granular degeneration
or destroyed. In some dystrophic cells of mesangium
there was a margination of chromatin, which is,
according to the modern view, the precursor of cell
death. The accumulation of serous fluid also resulted in
partial destruction of glomerular vessels and partial
destruction of the Bowman's capsule.
The nature of the microscopic changes in different
nephron tubules was clearly dependent on the degree of
microscopic changes in renal corpuscles - the more
pronounced were the changes in the renal corpuscles,
the more severe were the changes in the renal tubules.
In all parts of the nephron tubules in the renal
corpuscles, where there was the initial stage of serous
extracapillary glomerulonephritis, the majority of cells
of the epithelium displayed visible signs of progressing
granular degeneration (Fig. 1). In some areas of these
tubules, separation of epithelial cells from the basement
membrane and full decomposition of epithelial cells
were identified. In all parts of the nephron tubules in
the renal corpuscles in which significant microscopic
changes were registered, cells of the epithelium were
subjected to vacuolar degeneration and gradual
destruction (Fig. 4). In one part of the tubules,
epithelial cells were completely or partially destroyed
The renal interstitium was swollen in some places
and a large number of monocytes and isolated
neutrophils were found in these places (Fig. 5).
2
1
Fig. 5. The kidney of a cow which died from leptospirosis:
1–interstitial oedema; 2–monocytes. H.E., 200×
Discussion
Leptospirosis is a worldwide disease except
Antarctica (1). This disease affects humans, wildlife,
and domestic animals, including cattle. In the severe
forms of the disease renal impairment is frequent. In
some cases it leads to the incidence of acute renal
insufficiency (11). It occurs when the kidneys become
unable to filter waste products from the blood. When
the kidneys lose their filtering ability, dangerous levels
of metabolites may accumulate and the balance of the
blood's chemical composition may be destructed (15).
Examinations of the main necropsy material of
fatal cases of leptospirosis under light and electron
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microscopies in consort with determination of
infectious and immune complexes demonstrated that
leptospires are capable of causing toxic-infectious
shock (TIS) with typical changes in the
microcirculatory bed and organ parenchyma (3, 10).
The results of our histological studies indicate that
the kidneys of cows with leptospirosis displayed
significant changes in the microscopic structure. In part
of the renal glomeruli, a destruction of the Bowman's
capsule and necrosis of a part of their cells were
recorded. In the epithelium of the renal tubules
degenerative changes and destruction of epithelial cells
were registered. We believe these changes reflect the
effects of leptospira toxins in renal parenchymal
patterns. It is confirmed by the case report where the
presence of microscopic changes typical for necrosis,
but not for cell apoptosis, was observed (22). This leads
to condensation, nuclear fragmentation, and
externalisation of membrane phospholipids, giving
a signal for these cells to be engulfed by
macrophages (18).
Additionally, the dystrophic cells lose their
specific functions. Therefore, degenerative and necrotic
changes in the cells of different parts of the nephron
reflect the total or partial cessation of function of
affected nephrons, resulting in acute renal failure.
Defeat of the endothelial cells in blood vessels, in our
opinion, was also due to the influence of leptospira
toxins circulating in the blood. The presence of
leptospira toxins and renal insufficiency causes the
development of the general intoxication (17).
It is known that toxic shock syndrome (TSS) is the
potentially fatal illness caused by different bacterial
toxins. Different bacterial toxins may cause the
syndrome, depending on the situation. Microscopic
changes in the kidneys suggest that the death of cows
from leptospirosis might occur as a direct result of TSS.
5.
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