Ultrastructural study of brain with experimental cowdria ruminantium infection (heartwater)
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Date
1991
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Abstract
Heartwater is an infectious, vector-borne, non-contagious disease of ruminants caused by
Cowdria ruminantium. The etiologic agent is an obligate, intracellular parasite infecting
phagocytic cells and, particularly, vascular endothelium. Transmission is effected by a three-
host tick of the genus Amblyomma. Clinically, the disease is characterized by fever and signs
of central nervous system disorder. There is general agreement on a deranged transport
mechanism across the vascular wall as the basis for the development of morphologic lesions
and clinical signs, but the actual mechanism of damage is poorly understood. Previous
ultrastructural studies are few and have mainly been concerned with the parasite itself. The
present morphologic study of the cerebral microvasculature is an attempt to elucidate the
pathogenesis of the central nervous system changes.
Five, clinically healthy, adult, Danish goats were used to establish the optimal procedure for
perfusion fixation of the brain and, at the same time, to serve as normal controls. The
animals were put under general anesthesia with pentobarbitone intravenously at 25 mg/kg and
placed in dorsal recumbency. Following dissection of the ventral neck region intubation was
performed and carotid arteries were isolated. Heparin (1250 i.u./kg) was injected and
perfusion done through one of the carotid arteries using 3% phosphate-buffered glutaraldehy
de solution at pH 7.4 and 500 mOsm. The best perfusion results were achieved with the tank
placed 1.5 m above the heart. The brain was removed and 1 mm3 samples were diced from
the best fixed areas of the cerebral and cerebellar cortices and further fixed i 3%
glutaraldehyde solution. Some of the samples were routinely processed and stained for
transmission electron microscopy. In an attempt to improve fixation and enhance demonstration of cellular membranes and other structures, other samples were subjected to
alternative processing and staining regimes including reduced osmium tetroxide, ruthenium
red, tannic acid, and uranyl block staining. On balance, however, these regimes did not
produce entirely satisfactory results and were therefore omitted in the experimental studies.
Corresponding pieces of brain tissue were post-fixed in 10% neutral, buffered formalin and
routinely processed for light microscopy. A complete necropsy was performed and various
tissues were taken and similarly processed for light microscopy.
Fourteen Tanzanian blended goats were experimentally infected by intravenous inoculation
with the Ball 3 strain of Cowdria ruminantium. Following an incubation period of about 2
weeks the animals developed temperatures up to 41.7°C. Other clinical signs included
bleating, loss of appetite, loss of body condition, listlessness, abdominal respiration, stupor,
head tremors and ataxia. In 2 goats, fits, opisthotonus, paddling movements and lateral
recumbency were also observed. When temperatures started to drop the animals were killed
and subjected to the same procedures as the controls.
Necropsy revealed mild hydropericardium (2-13 ml) and splenomegaly in all 14 goats. Other,
inconstant findings were urine retention in bladder, subepi- or subendocardial petechiae and
flabbiness of the heart. In areas of incomplete perfusion leptomeninges appeared hyperemic.
Light microscopically, Cowdria organisms were found in vascular endothelium of the brain
in 8 goats. Focal perivascular mononuclear cell infiltrations, sometimes extending into the
adjacent neuropil, were present in the brains of 12 animals. More extensive mononuclear cell
infiltrations, occasionally with a few eosinophils, were in the leptomeninges of 11 goats. Focal mononuclear cell infiltrations were also found, although variably, in pulmonary and
renal interstitium and in subepi- and subendocardial locations. All experimental animals ex
hibited moderate hyperplasia of splenic lymphoid tissue.
The only ultrastructural change of parasitized endothelial cells was compression and
displacement of cytoplasm and organelles by membrane-bound colonies of Cowdria
organisms. Perivascular accumulations of cells, consisting of macrophages, lymphocytes and
occasional plasma cells, were invariably associated with non-parasitized segments of
capillaries or venules.
Often intravascular mononuclear cells were seen adhering to the endothelium with portions
of their cytoplasm dipping into the endothelial cell membrane or interposed between the
endothelial cell and the basement membrane. Large, spherical, electron-dense inclusions,
often several in the same cell, were found in pericytes, both in control and experimental
animals but with a notably higher incidence in the latter. Similar inclusions as well as smaller
inclusions with the features of lipofuscin were seen in perivascular macrophages. An
additional and unique type of cytoplasmic inclusion was encountered in perivascular
macrophages. These inclusions presented as aggregations of irregularly round, membrane
bound particles, 0.25-0.4 /zm in diameter, in some cases with an internal structure remini
scent of partly degraded mitochondria. However, the aggregations were not convincingly
enclosed within membranes as would be expected in case of autophagocytosis. Another,
hypothetical, interpretation is that they represent abortive stages of Cowdria ruminantium
attempting to develop extravascularly and that possibly cell-mediated immunity, developed
during and after the incubation period, limits this second cycle within the host and results in the perivascular mononuclear cell infiltrations observed.
The findings in this study suggest the possibility of a direct involvement of circulating
monocytes in the pathogenesis of the brain lesions. Such cells may carry and shield the
antigen through the vessel wall into extravascular sites since no signs of vasculitis or
abnormal vascular permeability were found, making it unlikely that soluble antigens are
involved. The finding of a morphologically intact endothelium and vessel wall, even under
perfusion conditions, also casts doubt on a possible role of vasoactive substances in the
development of the lesions. If and when permeability changes of the brain microvasculature
occur, they apparently represent a later development of an inflammatory process rather than
a primary mechanism in the pathogenesis of cerebral heartwater.
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Keywords
Cowdria ruminantium infection, Goats microvasculature