Equine infectious anemia

In studying Equine Infectious Anemia (EIA), a specific viral disease of
Equidae, characterized by intermittent fever, anemia and progressive weakness,
questions have arisen as to the existence of a viral hemagglutinin. Raised
hemagglutinin levels are present in the infected sera, but this is further complicated
by the fact that some horse serums contain relatively high levels of
hemagglutinins which are believed to be non-specific natural agglutinins. This
investigation, which parallels research currently in progress on the EIA virus
proper, is designed to characterize those agglutinins found in non-infected sera.
Two principal types of equine hemagglutinins were studied; (1) the natural
occurring non-specific blood agglutinin, and (2) hyperimmune isoagglutinins produced
in a primary immune response following injection of heterogenic equine
erythrocytes. Both species of agglutinins were obtained from whole serum.
Standard immunological techniques such as reduction and alkylation, heat
inactivation, ion-exchange chromotography, density gradient ultracentrifugation,
and electrophoresis were employed in the investigation of structural and biological
characteristics of the hemagglutinins.
The results have shown that natural equine hemagglutinins are heterospecific.
They are sensitive to heat (56°C) and reducing agents such as mercaptoethanol.
Natural agglutinins have a sedimentation coefficient of 18.65 and electrophoretically
migrate as alpha2 globulins. The hyperimmune isohemagglutinin is a specific antibody reactive with a
well defined antigen. It is relatively heat stabile and hemagglutinating activity
is reduced with mercaptoethanol. Similarly, the isoagglutinin has the some
sedimentation coefficient and electrophoretic mobility as the natural antibody.
The isoagglutinin is unique in that it represents a new class of antibody not previously
reported.

The disease of equine infectious anemia
(EIA), since the first records in 1847, has been one
of the most serious and deadly virus diseases of the
Equidae. The most difficult factor concerning this
disease was adequate diagnosis. The clinical diagnosis
has proven itself undependable and until 1966 with the
Immune-adherence test the laboratory diagnosis was
inconclusive. The indirect passive hemagglutination
test was used as a possible test for the disease due
to its high sensitivity. The indirect passive hemagglutination as
the diagnostic test for EIA was based on the use of
antibody as the titrating agent instead of the virus.
The antibody was removed from the sera of infected
horses. The seperation of the antibody was
accomplished by use of a DEAE-cellulose column and an
increasing salt gradient. The various proteins were
sepRrated and measured in a spectrophotometer. The
antibody portion was collected and concentrated by
polyethylene gycol. The more specific type of antibody
(19S) was used in all tests. The indirect passive hemagglutination test
was run on a total of 92 sera of which 58 were
negative and 34 sera were positive. The test proved
reliable in all the sera tested. In support of the
IPHA the IA test was run as a comparison.

Liver, kidney and spleen cells from healthy horses
and horses with equine infectious anemia were cultured
in vitro. Primary culture of organs from healthy horses
yielded typical elongate fibroblast cells. Primary culture
of organs from diseased horses yielded a variety of cell
types; pleomorphic giant cells being most common. The giant
cells did not divide, but either underwent degeneration and
death or became transformed into squamous-like cells lacking
contact inhibition resulting in the production of foci.
Inoculation of normal equine fibroblasts with either serum,
plasma, or liver, kidney or spleen extracts from diseased
horses resulted in a proliferation of the fibroblasts. Normal liver or kidney fibroblasts cocultivated with equine
leucocytes and inoculated with serum or plasma from a diseased
horse became transformed. This research supports the
proposal that equine infectious anemia virus is an oncornavirus.

It has been shown that sideroleukocyte formation in
equine infectious anemia is related to the presence of
detectable amounts of ferritin in the serum of the infected
animal. The presence of ferritin has been associated with
tissue breakdown as a result of the infectious process.
A relatively simple agar diffusion technique has been reported
which is of value in the screening of horses for the
acute phase of equine infectious anemia.
It has further been shown that infected leukocytes can
be induced to form sideroleukocytes earlier and in greater
number by the addition of ferritin or calcium. Non-infected
horses can not be induced to form sideroleukocytes by the
addition of only ferritin or calcium or infected sera.
However, addition of both ferritin and calcium does cause
induction of sideroleukocytes in normal leukocytes.
Levels of serum calcium in infected horses were shown
to be inversely proportional to both temperature of the
animal and the formation of sideroleukocytes.
The data indicated the important role of serum levels
of ferritin and calcium to the mechanism of sideroleukocyte
formation both in vivo and in vitro.

Newborn, albino Swiss mice were inoculated with either
infectious EIA material or a normal, negative control
sample o At weekly intervals, one mouse from each group was
sacrificed and its liver assayed for xanthine oxidase
activity, nitrogen content, and its relative weight.
By the third week of age, mice which had received an
infected inoculum displayed no outward signs of illness;
yet, on examination, their livers were higher in xanthine
oxidase activity, had a greater concentration of nitrogen,
and were smaller in size to those of litter mates which had
been injected with non-infected material o
These effects were accomplished by either infected
horse serum or a suspension of white blood cells from an
infected horse. In addition, the same effects were demonstrable
with either chronic serum from a febrile period,
or serum from a true asymptomatic carrier state. White blood
cells from an inapparent carrier were unable to induce elevated
responses in young mice.