Saurino, Vincent R.

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.

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.

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 .