Prestorage leucoreduction improves several in vitro red cell storage parameters following gamma irradiation.

Gamma irradiation (GI) can prevent transfusion-associated graft-vs.-host disease, result in a small decrement in 24-h in vivo red blood cell (RBC) recovery and in a minor loss of maintenance of several in vitro parameters during routine storage. Results from studies by Davey et al. (Transfusion 1995, 35, 55S) demonstrated that the decrement in 24-h recovery was not observed if units were first leucoreduced (LR), suggesting that RBC damage on leucocytes from GI was secondary to the effects of irradiation . In this study, we further investigated how leucoreduction affected eight in vitro storage parameters of gamma-irradiated units by using a matched sample study design. Leucoreduction significantly reduced (P < 0.05) the deleterious effect of GI on adenosine triphosphate glucose levels, haemolysis and mean corpuscular volume (MCV) for AS-3 units and significantly decreased (P < 0.05) the deleterious effect of GI on glucose levels, haemolysis and MCV for AS-1 units. Leucoreduction did not influence (P >> 0.5) the enhanced potassium release associated with GI. Some, but not all, the observed RBC damage caused by GI appears to be secondary to the effect of irradiation on leucocytes. Based on the in vivo survival studies of Davey et al. and the results of this study, there is justification to consider performing additional survival studies to support extended storage of LR gamma-irradiated RBC.

Disease features in horses with induced equine monocytic ehrlichiosis (Potomac horse fever).

Fifty-five horses were inoculated IV and/or SC with materials containing Ehrlichia risticii, ie, infected whole blood, buffy coat cells, or cell culture, to study clinical and hematologic features of equine monocytic ehrlichiosis (Potomac horse fever). Major clinical and hematologic features of induced E risticii infection were biphasic increase in rectal temperature with peak increases of 38.9 C and 39.3 C on postinoculation days (PID) 5 and 12, respectively; depression; anorexia; decreased WBC count (maximal decrease of 47% on PID 12); and diarrhea from PID 14 to PID 18. Increased WBC count was an inconsistent feature, with a maximal increase of 51.5% on PID 20. During times of decreased and increased WBC counts, lymphocyte/neutrophil ratios remained fairly constant. However, not all horses had all clinical and hematologic features, and these features were present in different degrees among horses. Increased rectal temperature, depression, anorexia, and decreased WBC count were more consistent features, whereas diarrhea developed in 73% of the horses. Of 55 horses, 39 (71%) had all clinical and hematologic features of the disease (classic disease), whereas 16 (29%) horses did not have greater than or equal to 1 of these features (nonclassic disease). The E risticii titer in the blood (ehrlichemia) was maximum during the peak increase in rectal temperature. In 55 horses, mortality was 9%. Significant differences (P greater than 0.5) in clinical and hematologic features were not detected between horses that survived and those that died of E risticii infection.

Detection of serum antibodies against Ehrlichia risticii in Potomac horse fever by enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed which was specific and sensitive in detecting antibodies to Ehrlichia risticii in Potomac horse fever (PHF). The ELISA antibody titers were correlated with the indirect fluorescent antibody (IFA) titers. E. risticii propagated in human histiocyte culture was purified on renografin gradient and the band of the organisms at a density of 1.182 g/ml was used as antigen. ELISA antibody titers were determined through computer assisted analysis, the observed antibody titers were derived by serial serum dilutions and using a resultant standard curve the predicted antibody titers were obtained from a single serum dilution. The standard curve had a correlation coefficient of 0.8975. The observed and predicted antibody titers were in good agreement, as the respective titers fell within a two-fold range. There was a good correlation between ELISA and IFA test results, but the ELISA titers were several times higher. In experimental infections of horses produced with the infected equine whole blood and the Ehrlichia infected macrophage culture, the antibodies were first detected in two weeks and one week postinoculation (PI), respectively. In both cases the titers reached a peak in about 4 weeks PI with a mean titer of 1:16558 and 1:4030, respectively. The antibody titers of the convalescent sera of field cases of PHF were comparatively lower than the experimentally infected horses.

In vitro interference between equine herpesvirus types 1 and 2.

Interference between equine herpesvirus types 1 (EHV-1) and 2 (EHV-2) was studied in equine dermis (ED) monolayer cell cultures and equine lymphocyte cultures. Cell cultures were infected with EHV-2, and after a short incubation period, the cultures were superinfected with EHV-1. At various intervals, different measurements of EHV-1 expression in dually infected cultures, compared with those in cultures infected with EHV-1 alone, were studied. In dually infected ED cell cultures, the EHV-1 cytopathic effect, EHV-1 titer, and EHV-1 enzyme-linked immunosorbent assay antigen titer were maximally reduced to values of 40%, 58.5%, and 54.9%, respectively, at postsuperinfection hour (PSIH) 36. Values of these EHV-1 expressions were subsequently increased at PSIH 48. However, thymidine kinase activity was reduced to a maximum of 67.3% reduction at PSIH 48. In dually infected lymphocyte cultures, the EHV-1 titer, EHV-1 infective centers, EHV-1 enzyme-linked immunosorbent assay antigen titer, and thymidine kinase activity were maximally reduced to values of 77.4%, 78.7%, 98.3%, and 72.9%, respectively, at PSIH 24. These reductions of EHV-1 expressions were completely abrogated at PSIH 48 to 72. In both cell culture systems, a marked interference of EHV-1 by EHV-2 was observed; this was transient in the lymphocyte cultures, but was more prolonged in ED cell cultures. This interference appeared not to be interferon mediated. The multiplication of EHV-2 in the dually infected ED cell cultures appeared unaffected.

Experimental reproduction of Potomac horse fever in horses with a newly isolated Ehrlichia organism.

Potomac horse fever, a recently recognized disease of equines, characterized by high fever, leukopenia, and a profuse diarrhea, was studied for its etiology. An Ehrlichia organism was isolated in equine macrophage-fibroblast cell cultures and mouse macrophage cell cultures from the mononuclear cells of blood of infected horses. The agent was continuously propagated in mouse macrophage cell cultures. The organism multiplied in the cytoplasm of mouse macrophage cells and was identified by Giemsa staining, acridine orange staining, and by indirect immunofluorescence with convalescent sera from infected horses. The disease was experimentally reproduced in horses inoculated with Ehrlichia-infected cell culture material. The Ehrlichia organism was reisolated from the blood of these infected horses during the course of the disease. Antibody against the organism was detected in the sera of experimentally infected horses. This study confirmed that the new Ehrlichia organism is the etiological agent of Potomac horse fever.

Detection of equine herpesvirus-1 antigen and the specific antibody by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assays (ELISA) were developed to detect equine herpesvirus-1 (EHV-1) antigens and specific antibodies. Detection of EHV-1 antigens was done by a 4-layer ELISA. In inoculated cell cultures, EHV-1 cell antigen was detected after postinoculation (PI) hour 4, reached approximately twice the value of EHV-1 viral antigen (extra-cellular virus) in PI hour 18, and peaked in PI hour 24, whereas EHV-1 viral antigen appeared after PI hour 12, increased steadily, and peaked higher than EHV-1 cell antigen in 24 hours. The ELISA titer and infectivity titer of 24-hour PI cultures were 200 and 9.2 X 10(5) plaque-forming units/0.1 ml, respectively. Equine anti-EHV-1 antibody (EAEHV-1) from experimentally inoculated pony foals and rabbit anti-EHV-1 antibody (RAEHV-1) were detected by indirect ELISA and direct ELISA, respectively. The RAEHV-1 had ELISA titers of 665,000 and 102,000 when rabbits were immunized with EHV-1 infected cell culture lysate and with sucrose-purified virus, respectively. The corresponding plaque-reduction virus-neutralization titers were 270 and 150 in the absence of complement and were 6,200 and 3,200 in the presence of complement. The EAEHV-1 had a mean ELISA titer of 60,000, and the corresponding mean virus-neutralization titer in the absence of complement was 34 and that in the presence of complement was 2,142.

Infectious center assay of intracellular virus and infective virus titer for equine mononuclear cells infected in vivo and in vitro with equine herpesviruses.

A novel, simple method of infectious center assay was developed to detect and quantitate the intracellular existence of equine herpesvirus 1 and equine herpesvirus 2 in peripheral blood mononuclear cells infected in vivo and in vitro with the viruses by cocultivation of these cells with a permissive equine cell culture. The infectious center titers were correlated with the infectious virus titers. In vivo equine herpesvirus 1-infected mononuclear cells obtained from ponies experimentally infected with the virus and equine herpesvirus 2-infected mononuclear cells obtained from selected naturally infected ponies with the virus gave by infectious center assay a mean value of 67 infectious center/2 x 10(6) cells as a peak titer on day 4 postinfection and 26 infectious center/2 x 10(6) cells for equine herpesvirus 1 and equine herpesvirus 2 respectively. The mononuclear cells, in both cases, did not contain detectable infectious virus, but the infectious virus was detected from the respective cells when they were cultured in the presence of mitogen. The equine herpesvirus 1 infected mononuclear cells in culture gave a mean count of 8.05 x 10(2) infectious center/2 x 10(6) cells/mL and contained 1.08 x 10(4) plague assay/mL of infectious virus. Similarly the equine herpesvirus 2 infected mononuclear cells in culture gave a mean count of 7.1 x 10(1) infectious center/2 x 10(6) cells/mL and contained <10(1) tissue culture infective dose(50)/mL of infectious virus. Mononuclear cells infected in vitro with equine herpesvirus 1 gave a mean count of 9.3 x 10(4) infectious center/2 x 10(6) cells/mL and contained 5.75 x 10(3) plaque assay/mL of infectius virus. Culturing these cells in the presence of mitogen gave a mean count of 5.5 x 10(3) infectious center/2 x 10(6) cells/mL and contained 9 x 10(3) plague assay/mL of infectious virus. A correlation between infectious center assay and infectious virus assay is discussed.

Lymphocytes from ponies experimentally infected with equine herpesvirus 1: subpopulation dynamics and their response to mitogens.

Six pony foals, free of detectable serum neutralization (SN) antibody against equine herpesvirus type 1 by the standard virus-neutralization (VN) test, were inoculated with equine herpesvirus type 1. The ponies showed typical clinical signs of respiratory tract disease and developed a transient leukopenia, involving lymphocytes as well as neutrophils. The leukopenia reached its lowest point on postinoculation days (PID) 3 to 5 and then returned to base-line values by PID 8 to 10. On quantitation of lymphocyte subpopulations, T and B lymphocytes were decreased during the onset of leukopenia and then recouped during the recovery from leukopenia. However, the proportions of the T and B lymphocytes remained constant during the lymphopenia, ranging from 70% to 80% and 20% to 30%, respectively. The lymphocyte blastogenic response to mitogens increased to peak by PID 2 to 5 and then decreased to base line values or below by PID 7. Mitogen responses of T lymphocyte and mixed lymphocyte preparations were nearly similar. However, the responses of 2 ponies wee somewhat different from the responses of others in that there was an increase in the B lymphocytes in the range of 40% to 50% during the recovery phase of lymphopenia. Also, the 2 ponies' mixed lymphocyte response to mitogens was considerably higher and the T lymphocyte response to mitogen was lower as compared with that of mixed lymphocyte preparation. The importance of these 2 types of responses is discussed.

Separation and identification of equine leukocyte populations and subpopulations.

Various methods of separation and identification of major equine leukocyte populations and subpopulations were used. The purity of T and B lymphocytes separated in Sephadex anti-equine F(ab')2 columns was 87% to 99% and 83% of 97%, respectively. The purity of T lymphocytes separated in nylon-wool columns was 89% to 98%. Preparations of B lymphocytes separated in glass-bead columns were 68% to 79% pure. The presence (or absence) of surface immunoglobulin by immunofluorescence was the most consistent and reliable method for the identification of B or T lymphocytes, respectively. However, the erythrocyte-antibody-complement-rosette method for the identification of B cells and the erythrocyte-rosette method for the identification of T cells were not suitable. Monocytes were separated by the adherence method, and the purity, as identified by the latex particle ingestion procedure, was 70% to 78%. Electron microscopy of monocytes stained by peroxidase activity did not identify these cells. The purity of neutrophils obtained by the Ficoll-Hypaque separation method was 95% to 97%. The merits and usefulness of these methods were discussed.

Complement requirement for virus neutralization by antibody and reduced serum complement levels associated with experimental equine herpesvirus 1 infection.

Pony foals, negative for detectable serum-neutralizing antibody to equine herpesvirus 1 by the standard tube-culture virus neutralization test, were experimentally infected with equine herpesvirus 1. Complement-requiring (CR) and non-complement-requiring (NCR) serum-neutralizing antibodies were evaluated in preinfection and postinfection sera by means of a complement-enhanced plaque reduction assay. Low levels of CR antibodies were found in the preinfection sera of only group II ponies. Upon infection, CR antibodies were detected by day 2 postinfection and reached peak titers between 7 and 14 days postinfection in the antisera of all ponies. NCR antibodies were detected later than CR antibodies and at levels approximately 40 to 150 times lower than the latter. CR/NCR ratios indicated that complement requirement was greatest early in the acute stages of disease and that this requirement decreased during the convalescent phase. Fractionation of 1-week and 2-week postinfection antisera of group I ponies indicated the CR antibody activity resided in both the 7S and 19S fractions. Total serum complement levels of the ponies were quantified throughout the infection with an equine anti-goat erythrocyte hemolytic system. In vivo, complement levels were depressed for all ponies during the first 2 weeks of infection. A decline in complement levels was seen as early as day 2, and they decreased to an average of 35% of preinfection levels on day 10 postinfection for all ponies.

Isolation and characterization of adeno-associated viruses from bovine adenovirus types 1 and 2.

Hemagglutinating DNA viruses of 20 nm diameter were isolated from bovine adenovirus types 1 and 2. The isolates were heat stable, chloroform resistant, and defective. Their densities were 1.38 to 1.39 g/cm3, and they were found to be serologically identical to the bovine adeno-associated virus strain X7. A partial antigenic relationship was found between these and the canine adeno-associated virus.