Horses--Diseases

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.

Normal and infected equine nasopharyngeal culture samples were investigated for their streptococcal species. Sampling and isolation were greatly facilitated with the use of disposable swabs and streptosel broth (B.B.L.). All nasopharyngeal culture samples contained streptococci.
With supplements of veal infusion or heart infusion added for the more fastidious organisms, phenol red broths were employed to determine the species of streptococci present. If the organisms were able to grow in Todd-Hewitt broth with 6.5 per cent saline added, they were subsequently tested for the ability to hydrolyze gelatin.
The absence of catalase was noted and the ability or inability to hydrolyze starch was tested.
Alpha, beta, and gamma hemolysis on blood agar plates was noted. Alpha-hemolytic colonies were isolated from the nasopharyngeal samples. These alpha-hemolytic streptococci were previously unreported in the literature. Optochin sensitivity tests were done on all alpha hemolytic colonies to confirm streptococcal growth by non-inhibition by ethyl hydrocuprein hydrochloride. Normal horses were found to possess beta-hemolytic streptococci, suggesting an immune carrier state. The presence of these beta-hemolytic streptococci in normal horses was also previously unreported in the literature. Since most of the infected horses sampled were also undergoing chemotherapy, antibiotic sensitivity tests were done on the streptococcal isolates. Penicillin, streptomycin, erythromycin, and bacitracin, antibiotics generally effective against Gram positive organisms were selected for the tests. All equine pathogenic streptococci were sensitive to the antibiotics used in chemotherapy. Streptococcus equi was sensitive to bacitracin where S. zooepidemicus was resistant to the same antibiotic. This may be new method to differentiate the two species. Antistreptolysin-O titers were done on 4 infected horses for more than one month following their upper respiratory symptoms. Only one horse showed a slight yet consistent, titer over the period tested. This particular horse did not possess S. equi in his nasopharynx. Thus the absence of S. equi in infected horses has been linked to the possession of an antistreptolysin-O titer in combination with antibiotic treatment. S. equi was cultured from horses receiving chemotherapy yet not possessing an antistreptolysin-O titer. Mouse protection tests using serum from horses infected with streptococci were inconclusive. Growth curves using stationary and agitated media showed better growth and more matt colonies in the richest stationary cultures. Rabbit immunization with bacterin made from disrupted pathogenic streptococci was begun. Using the Lancefield hot acid extraction method streptococcal cell extracts were prepared from the isolates. The extracts were then tested against known Group C antiserum in the small tube precipitin test. S. equi, S. zooepidemicus, S. bovis, S. fecalis, S. sanguis, and S. equisimilis were present in the normal equine nasopharynx. The presence of S. equi and S. zooepidemicus in the normal horses suggests an immune carrier state. The same organisms in the normal horses and S. equinus were present in the infected equine nasopharynx. S. equi and S. zooepidemicus were present more often in the infected horses than in the normals. S. zooepidemicus was present in infected horses not undergoing chemotherapy. The presence of this organism in the chronic equine suggests that protective antibody is neither present nor being produced.

An antigenic material was extracted from the
leukocytes of horses infected with Equine Infectious Anemia.
In unmodified form, this material was infectious, immunogenic,
and antigenic. After modification by anti-viral agents and cross-linkage
to rabbit gamma globulin, the material retained its
immunogenic and antigenic properties, but became non-infectious.
Inoculation of the non-infectious antigen into experimental
horses elicited antibody production. When these horses were
challenged with live virus, the classical symptoms of the disease
did not appear. The possibility exists that a protective vaccine
was developed from modified antigen preparations.

Leukocytes, harvested from EIA infected horse plasma, were
disrupted and partially purified by differential centrifugation.
This material was assayed for its capacity to inhibit migration
of leukocytes from infected horses. After 6 X 10^6 r of gamma
radiation the material was reassayed and 85% of the initial
activity remained, indicating that little antigenic destruction
had occurred. This material was coupled to rabbit gamma
globulin and used for horse inoculation. The above material,
uncoupled, and a partially purified irradiated normal horse
protein material were used as controls. Responses to
immunizations and challenges were monitored. Uncoupled irradiated
material produced the disease. Coupled irradiated
material did not produce the disease and the immunized horse
was resistant to challenge with 1 ml of a 10^-3
dilution of infectious serum for 30 days and, then, to 1 ml of undiluted
infectious serum for 30 more days .

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.