Testing of a dengue 2 live-attenuated vaccine (strain 16681 PDK 53) in ten American volunteers.

A live-attenuated dengue 2 vaccine (strain 16681 PDK 53) developed at Mahidol University, Thailand was evaluated for safety and immunogenicity by administering 10(4) p.f.u. subcutaneously to ten flavivirus non-immune American volunteers. The vaccine was safe; there were no serious adverse reactions. Eight recipients experienced no or mild side effects. One recipient reported headaches on 7 separate days. One volunteer, who had a fracture of the humerus 1 day after vaccination requiring surgical repair, experienced generalized malaise with fever (maximum temperature = 38.9 degrees C), headache, eye pain and myalgia lasting less than 24 h. The vaccine was highly immunogenic; all recipients developed neutralizing antibody that persisted for two years.

Attempted transmission of Ehrlichia risticii, causative agent of Potomac horse fever, by the ticks, Dermacentor variabilis, Rhipicephalus sanguineus, Ixodes scapularis and Amblyomma americanum.

Dermacentor variabilis, Rhipicephalus sanguineus, Amblyomma americanum, and Ixodes scapularis ticks were investigated for their ability to transmit Potomac horse fever. Larval and nymphal ticks were exposed to Ehrlichia risticii by feeding on mice inoculated with the organism. Molted exposed ticks were then allowed to feed on susceptible ponies or mice. No evidence of transmission, either clinically or by detection of antibodies to E. risticii in mice or ponies, was observed for any tick species examined.

Antigenic and genetic relatedness of eight Rickettsia tsutsugamushi antigens.

The genetic and antigenic relatedness of eight antigens in three strains of Rickettsia tsutsugamushi has been studied by using recombinant organisms expressing epitopes of the 150-, 110-, 72-, 58-, 56-, 49-, 47-, and 20-kilodalton (kDa) polypeptide antigens of the Karp strain. Southern blot analysis of Karp, Kato, and Gilliam strain genomic DNA by using probes specific for each antigen class indicated that while strong homology exists between each of the corresponding antigen genes in these three strains, some restriction fragment length polymorphism exists. Antibodies affinity purified against each recombinant antigen class reacted with a comparably sized polypeptide in the Karp, Kato, and Gilliam strains in Western blots (immunoblots). Against more recent human isolates of R. tsutsugamushi, the affinity-purified antibodies against the 58-kDa recombinant antigen (anti-58-kDa) reacted with all nine isolates, anti-56-kDa reacted with eight of nine isolates, anti-47-kDa reacted with eight of nine isolates, anti-72-kDa reacted with eight of nine isolates, and anti-110-kDa reacted with four of nine isolates. Additional analysis indicated that the 110-kDa antigen may contain strain-specific epitopes similar to those previously reported for the 56-kDa polypeptide. Evidently, the strain heterogeneity among scrub typhus rickettsiae is a result of multiple components that exhibit variability in a background of strong homology.

Epidemiology of Potomac horse fever: an investigation into the possible role of non-equine mammals.

A serological study of antibodies to Ehrlichia risticii was carried out on 10 species of wild and domestic mammals found on or near 21 horse farms in an area of the USA in which Potomac horse fever is endemic. No antibodies were found in 133 peridomestic rodents (Norway rats and house mice), nor in 108 wild rodents (white-footed mice and meadow voles) captured on farms. Three of the six domestic animal species examined, cats, pigs and a goat, showed serological evidence of exposure to E risticii. Seropositive animals were detected on three of the 21 premises. The eight seropositive cats (of 48 cats tested) were on two farms, and the three seropositive pigs (of 14 tested) were all on one farm which lay some 3 km from where the one seropositive goat (of three tested) was found. None of the 79 dogs, 75 cattle and seven sheep tested had antibodies to E risticii. The significance of these findings is discussed in the light of current understanding of the transmission of Potomac horse fever and of the epidemiology of other related ehrlichial diseases.

White-footed mice: tick burdens and role in the epizootiology of Potomac horse fever in Maryland.

One hundred ten white-footed mice (Peromyscus leucopus) were captured on horse farms in south-central Maryland, examined for ticks, and tested for specific antibodies to Ehrlichia risticii, the causative agent of Potomac horse fever. Peromyscus leucopus were consistently infested with immature American dog ticks (Dermacentor variabilis), with monthly prevalences as high as 80%. Sera from all 97 P. leucopus tested for antibodies to E. risticii were negative. This indicates that P. leucopus is not a reservoir of E. risticii, and suggests that immature D. variabilis do not acquire E. risticii in feeding upon white-footed mice.

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.

Molecular cloning and expression of Rickettsia tsutsugamushi genes for two major protein antigens in Escherichia coli.

Several polypeptide antigens of Rickettsia tsutsugamushi are recognized by human or primate convalescent sera and may be important protective immunogens. Molecular cloning and expression of the genes encoding the 110K (110 kilodalton) and 56K polypeptide antigens of R. tsutsugamushi Karp were accomplished in the lambda gt11 expression vector system. Southern blot analysis with the cloned fragments for the 56K polypeptide antigen (0.7 kilobases) and the 110K polypeptide antigen (5.4 kilobases) confirmed that the insert DNA was rickettsial and not host cell in origin. Expression of a complete 110K polypeptide was shown to be independent of isopropyl-beta-D-thiogalactopyranoside induction, suggesting that an intact rickettsial promoter was operational. Epitopes of the 56K polypeptide were expressed as lac promoter-dependent beta-galactosidase fusion proteins. Polyclonal antibody, affinity purified against the recombinant 110K and 56K polypeptides, reacted with polypeptides of similar size in the Kato and Gilliam strains of R. tsutsugamushi. Group-reactive, but not strain-specific, monoclonal antibodies against the 56K polypeptide reacted with the cloned portion of the 56K polypeptide. Western blot analysis demonstrated that the cloned 56K Karp antigen gene product is recognized by human convalescent serum.

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.

Synthesis and bioevaluation of a rapidly biodegradable tissue adhesive: 1,2-isopropylidene glyceryl 2-cyanoacrylate.

A rapidly biodegradable tissue adhesive, 1,2-isopropylidene glyceryl 2-cyanoacrylate, was synthesized and characterized by NMR. Bond strength was determined. Three types of corresponding polymers were prepared and evaluated for in vitro and in vivo toxicity. The polymers were generally found to be in the moderately toxic and toxic groups similar to the lower alkyl 2-cyanoacrylates.

Development of antigen-specific cell-mediated immune responses after infection of cynomolgus monkeys (Macaca fascicularis) with Rickettsia tsutsugamushi.

Cynomolgus monkeys were evaluated for cellular immune responses after infection with the Karp strain of Rickettsia tsutsugamushi. Antibody and clinical signs of localized and systemic infection were also evaluated. Animals challenged with homologous or heterologous strains at various times after a primary infection were also followed up. Naive monkeys developed eschars, lymphadenopathy, rickettsemia, and elevated body temperatures. Antibody in these animals was IgM followed by IgG. Lymphocyte proliferation and production of gamma-interferon by peripheral blood mononuclear leukocytes also were demonstrated. If challenged six years after the initial infection, clinical signs and cellular responses were indistinguishable from naive animals but an anamnestic IgG antibody response was noted. If challenged eight months after the initial infection, complete resistance was noted, but if challenged at one year, a localized cutaneous lesion developed. The majority of animals infected previously had preexisting lymphocyte activity, a characteristic suggesting long-term immunologic memory that was not protective against rechallenge.

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.

Biocompatibility testing of polymers: in vivo implantation studies.

An in vivo method is described for screening polymeric materials for biocompatibility. The test is based on grading acute and subacute tissue reactions at 7 and 28 days, respectively, following implantation in rats. The methods is reproducible and reliable. It is designed to provide uniform test criteria for biocompatibility assessment in the early phases of the development of surgical implant materials.

Biocompatibility testing of polymers: in vitro studies with in vivo correlation.

An in vitro method has been developed for screening of candidate biomaterials in an early phase of their development. The test is based on L-929 mouse fibroblast cultures and their response to powdered polymer samples. It applies microscopic observation for the detection of morphological changes, uses dye exclusion testing for cell viability determination, and utilizes estimation of population doublings as an end point. The test is shown to be reliable and reproducible and is compared to in vivo implantation studies in rats, previously reported.

Specimen holders for simultaneous critical point drying of multiple biological specimens.

Specimen holders were developed for simultaneous critical point drying of multiple specimens of monolayer cell cultures grown on Leighton tube glass cover slips or for specimens of cells collected on silver or cellulose membrane filters. The use of these multiple specimen holders makes it possible to process several specimens in parallel and thus significantly reduce the time required for handling large numbers of samples and also eliminate the possible variations that occur in processing individual samples one at a time in series.

Physical characteristics and performance of synthetic wound dressings.

From these studies it is concluded that: 1) Physical characteristics of wound dressings can be quantified and evaluated for their contribution to in vivo performance. 2) A total performance scoring system can be reliably used to quantitatively separate series of wound dressings by their in vivo performances. 3) The physical characteristics most determinate of good perfprmance in these 5 series of dressings were undersurface texture and water vapor transmission.