The inflexibility of existing serological techniques for detection of rabies in surveillance constrains the benefit to be gained from many current control strategies. We analysed 304 serum samples from Tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using lentiviral vectors bearing the CVS-11 envelope glycoprotein. Compared with the widely used gold standard fluorescent antibody virus neutralisation assay, a specificity of 100% and sensitivity of 94.4% with a strong correlation of antibody titres (r = 0.915) were observed with the pseudotype assay. To increase the assay's surveillance specificity in Africa we incorporated the envelope glycoprotein of local viruses, Lagos bat virus, Duvenhage virus or Mokola virus and also cloned the lacZ gene to provide a reporter element. Neutralisation assays using pseudotypes bearing these glycoproteins reveal that they provide a greater sensitivity compared to similar live virus assays and will therefore allow a more accurate determination of the distribution of these highly pathogenic infections and the threat they pose to human health. Importantly, the CVS-11 pseudotypes were highly stable during freeze–thaw cycles and storage at room temperature. These results suggest the proposed pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections.

The major Pneumocystis carinii antigens inducing antibody responses in infected hosts were identified by Western immunoblotting techniques. The biochemical nature of these antigens was also elucidated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by protein staining revealed a major component with a molecular weight (MW) of greater than 205,000. This major component disappeared and a new major protein staining component of approximately 110,00 to 116,000 MW appeared when electrophoresis was done in the presence of ,3-mercaptoethanol. Periodic acid-Schiff staining revealed that this major component contains carbohydrate moieties. A major component in the 55,000to 60,000-MW region was visible with periodic acid-Schiff stain, but not with a protein stain, after electrophoresis in the presence of 1mercaptoethanol. The majority of sera tested from humans with diagnosed pneumocystosis and from rats allowed to recover from steroid-induced pneumocystosis reacted strongly with 110,000to 116,000-, and 55,000to 60,000-MW components. These sera often, but not always, detected antigens with MWs of approximately 170,000, 125,000, and 30,000 to 32,000. The data suggest that the antigenic composition of P. carinii is relatively complex and that rat and human P. carinii probably share antigenic determinants. Competitive studies between infection-derived human and rat antisera for the major rat P. carinii components revealed competition; rat antisera appeared to recognize a greater range of antigenic epitopes than did human antisera. Protease treatment of the antigenic components that had been immobilized on nitrocellulose paper destroyed their antigenic reactivity with rat antibody. Treatment with sodium periodate decreased reactivity of this 110,000to 116,000-MW component and completely destroyed the reactivity of the 55,000to 60,000-MW component with rat antibody. Pneumocystis carinii is a ubiquitous organism infecting many mammalian species, including man (14). Serological surveys indicate that detectable antibodies to P. carinii occur in 40 to 75% of the healthy adult population (9, 12). These data suggest that P. carinlii often occurs as a subclinical infection (14). Severe P. car-inii pneumonitis (PCP) similar to that seen in humans can be induced in rats by giving corticosteroids and a low-protein diet (17). In rats and humans, antibody titers against P. carinii usually rise upon

In rabies endemic regions, a proportionally higher incidence of rabies is often reported in dogs younger than 12 months of age, which includes puppies less than 3 months of age; this presents a serious risk to public health. The higher incidence of rabies in young dogs may be the effect of low vaccination coverage in this age class, partly as a result of the perception that immature immune systems and maternal antibodies inhibit seroconversion to rabies vaccine in puppies less than three months of age. Therefore, to test this perception, the authors report the virus neutralising antibody titres from 27 dogs that were vaccinated with high quality, inactivated rabies vaccine aged three months of age and under as part of larger serological studies undertaken in Gauteng Province, South Africa, and the Serengeti District, Tanzania. All of these dogs seroconverted to a single dose of vaccine with no adverse reactions reported and with postvaccinal peak titres ranging from 2.0 IU/ml to 90.5 IU/ml. In light of these results, and the risk of human beings contracting rabies from close contact with puppies, the authors recommend that all dogs in rabies endemic regions, including those less than three months of age, are vaccinated with high quality, inactivated vaccine.

Cryptosporidiosis, previously seen mostly among immunocompromised patients, is now recognized among immunocompetent patients. During a large outbreak of cryptosporidiosis in two day-care centers, we compared two procedures for the demonstration of the organism in preserved stool specimens. Of 703 stool specimens tested by both techniques, Sheather sucrose flotation (SSF) identified 127 (18.1%) as positive for Cryptosporidium sp. oocysts. Ritchie Formalin-ethyl acetate sedimentation (F/EA) plus a modified cold Kinyoun acid-fast stain (MCK) of the sediment identified 129 (18.4%) as positive for Cryptosporidium sp. oocysts. The degree of agreement between the two tests was statistically highly significant (P < 0.0001). A total of 161 (22.9%) were positive by one technique or the other; 95 (13.5%) were positive by both techniques. A total of 32 specimens were positive by SSF but negative by F/EA plus MCK, and 34 specimens were positive by F/EA plus MCK but negative by SSF. The discrepancies between the two techniques occurred in stool specimens that contained rare to a few oocysts. Other parasitic forms were found by both techniques. F/EA plus trichrome staining recovered 126 (17.9%) specimens with Giardia lamblia, whereas SSF recovered only 42 (6.0%) specimens with G. lamblia. No association (X2 = 0.02, P = 0.89) was observed between the presence of G. lamblia and Cryptosporidium sp. in these stool specimens. We concluded that F/EA plus MCK of the sediment was as effective in the concentration and identification of Cryptosporidium sp. oocysts as SSF. F/EA plus MCK may be advantageous as a single concentration method for general parasitology when Cryptosporidium sp. is also being sought.

Hybridoma-producing monoclonal antibodies against Pneumocystis carinii were produced by the fusion of nonsecreting mouse myeloma cells (P3X63-Ag8.653) with splenocytes from BALB/c mice that had been immunized with partially purified preparations of P. carinii. Of 227 hybridoma clones producing antibodies against P. carinii, as measured by an enzyme-linked immunosorbent assay, 12 monoclonal antibodies showing the highest reactivity in the enzyme-linked immunosorbent assay were further characterized. The majority (11 of 12) of the monoclonal antibodies did not cross-react with Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, or Mycobacterium avium as determined by absorption experiments. By using the indirect immunofluorescence assay, serological reactivity was shown for these antibodies with titers ranging from 1:40 to 1:10,240. By using a competitive binding assay, these 12 monoclonal antibodies could be divided into seven groups, each group reacting with a different antigenic determinant of P. carinii. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of P. carinii, followed by Western immunoblot analysis, allowed the identification of one major antigen with an apparent molecular weight of 110,000 by all 12 monoclonal antibodies. Other minor bands with molecular weights of approximately 116,000, 90,000, 55,000, and 35,000 were recognized by several of the monoclonal antibodies.

Necropsy revealed pulmonary infarction, subsequent to heart failure from dilated cardiomyopathy.

No abstract

We developed a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that detected relatively low concentrations of known Pneumocystis carinii antigen added to buffer or rat sera. Artificial immunization-derived polyclonal rabbit anti-P. carinii antibody was used on the solid phase to capture the antigen. Infection-derived (after P. carinii pneumonia) polyclonal rat anti-P. carinii antibody or a mixture of five murine monoclonal antibodies was used as the antigen detector antibody. Rabbit anti-rat immunoglobulin G antibody or goat anti-mouse immunoglobulin G antibody conjugated to alkaline phosphatase was used as the final antibody. After standardization and optimization of the various reactants in this ELISA system, approximately 53 ng of known P. carinii antigen per ml suspended in phosphate-buffered saline-Tween 20 buffer or 210 ng of antigen per ml suspended in normal rat serum diluted 1:4 could be detected. In addition, an indirect ELISA for P. carinii antibody measurement was developed, using as the antigen a soluble supernatant from a sonicated preparation of Percoll-purified whole cysts and trophozoites to coat the solid phase. Limited studies with sera from a small number of caesarian-obtained, barrier-sustained rats from Charles River Breeding Laboratories, Inc., and the National Institutes of Health and sera from normal and heavily infected rats indicated that the caesarian-obtained, barrier-sustained rats had negligible levels of antibody. The normal and heavily infected rats had variable antibody titers. A significantly high level of P. carinii antigenemia was detected in only 2 (11%) of 18 heavily infected rats. Extensive studies of the P. carinii pneumonia rat model with the ELISA did not reveal significant serum P. carinii antigenemia during the acute stage of infection. However, soluble P. carinii antigen was detected by the ELISA and Western blot assays in the supernatant of lavage fluid after centrifugation to sediment intact organisms. As expected, P. carinii antigens were detected by these assays in the lavage pellet recovered after centrifugation. In conclusion, the antigen assay used in this study detected P. carinii antigen in lung lavage but failed to detect P. carinii antigen in rat serum during the acute phase of infection.