Indirect Fluorescent Antibody Assay Research Articles

Polymerase chain reaction evidence of Ehrlichia chaffeensis, an etiologic agent of human ehrlichiosis, in dogs from southeast Virginia

Abstract Objective To ascertain whether dogs are naturally infected with Ehrlichia chaffeensis. Animals 74 dogs from 5 animal shelters and 1 kennel in 3 cities and 3 counties in southeastern Virginia were tested during June 1991. Procedure Blood was drawn from 74 dogs; 73 were tested serologically for antibodies reactive to E chaffeensis and E canis, and 38 were tested for the presence of E chaffeensis, E canis, and E ewingii by polymerase chain reaction (PCR). Serologic testing by indirect fluorescent antibody assay. Nested PCR used Ehrlichia-wide outside primers to detect initial products, followed by use of species-specific primers for identification. Results 28 (38.4%) dogs had a positive test result (minimum titer, ≥ 1:64) for antibodies reactive to E chaffeensis, and 28 (38.4%) had a positive reaction to E canis. PCR analysis indicated that 8 (42.1 %) dogs were positive for E chaffeensis and 6 dogs (31.6%) were positive for E ewingii. All dogs had negative results of the PCR test for E canis. Conclusion Dogs are potential reservoirs of E chaffeensis. Clinical Relevance Canine E chaffeensis infection may be more prevalent than E canis or E ewingii infection in this region of the United States. (Am J Vet Res 1996;57:1175-1179)

A simple quantitative assay for the killing of in vitro culture cells by Ehrlichia canis infection

A simple colorimetric assay, on the basis of bioreduction of the substrate XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt) by dehydrogenases of actively metabolizing mammalian cells, was assessed for measurement of in vitro killing of canine macrophage-like cells by Ehrlichia canis infection. Formation of a highly colored, water soluble formazan which resulted from bioreduction by the test cells was significantly enhanced by phenazine methosulfate, an electron coupling agent. Normal cells markedly reduced XTT in the presence of phenazine methosulfate, yielding a post-/pre-bioreduction absorbance ratio of >4.0 under the assay conditions tested. E. canis-infected cells, however, markedly lost the capability for bioreduction, producing a post-/pre-bioreduction absorbance ratio as low as 2.0. Colorimetric assay results correlated with E. canis infections as tested by an indirect fluorescent antibody assay ( R = 0.95). This colorimetric assay involved a single step and results could be obtained in 1 h. Thus, the microtiter colorimetric assay was able to quantitate killing of the cells by E. canis infection with the advantages of reliability and simplicity. It will also prove valuable in facilitating the development of other in vitro assays in ehrlichiosis research.

Cat scratch disease. An overview for the ENT physician

Cat scratch disease (CSD) is a relatively common cause of chronic lymphadenopathy in the USA. In the present paper the authors describe recent advances in the understanding of this disorder focusing on etiology, clinical aspects, diagnostic management, and therapy. Rochalimaea henselae and Afipia felis, two gram-negative bacteria, have recently been isolated from lymph node tissue of patients suffering from CSD. The current literature reveals that Rochalimaea henselae seems to be the most probable agent responsible for CSD. Serum samples from CSD patients' cats have shown titers of 1:64 or higher for antibodies to Rochallmaea henselae. The incidence of CSD in the USA is between 0.77 and 9.3 per 100,000 per year. The incidence of CSD in Europe is unknown, but the prevalence of antibodies to Rochalimaea henselae among cats is comparable. Most common symptoms of CSD are regional lymphadenopathy, fever, and malaise. Other manifestations occur in about 5% of patients and include encephalitis, granulomatous hepatitis, and Parinaud's oculoglandular syndrome. Fatal complications and irreversible sequelae have not been reported. To establish the diagnosis of CSD, the presence of regional lymphadenopathy, cat contact, and papula are required. Additional procedures include indirect fluorescent antibody assays and PCR. Up to now there is no standard therapy for CSD. Antibiotic treatment, however, might be considered for CSD patients with severe symptoms. Rifampicin, ciprofloxacin, trimethoprim sulfamethoxazole, and gentamicin are known to be effective antibiotic agents.

Antigenic variability among North American and European strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein.

Two hybridoma cell lines producing monoclonal antibodies (Mabs) to the 19-kDa matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were obtained from BALB/c mice that were immunized with a reference Quebec tissue culture-adapted strain (strain IAF-Klop). The polypeptide specificities of the MAbs were determined by immunoblotting and immunoprecipitation tests with concentrated and purified preparations of the virus and by determining their reactivities with the Escherichia coli-expressed gene products of open reading frames 5 to 7. The two anti-M protein MAbs (MAbs IAFK3 and IAFK6) and another MAb (MAb IAFK8) directed to the 15-kDa nucleocapsid (N) protein were devoid of virus-neutralizing activity. A library of four anti-N protein MAbs (MAbs IAFK8, SDOW17, VO17, and EP147) and two anti-M protein MAbs (MAbs IAFK6 and IAFK3) was used to investigate, by an indirect fluorescent-antibody assay, the antigenic diversity of 15 Canadian PRRSV isolates, in comparison with those of the U.S. ATCC VR2332 attenuated vaccine strain and two reference European (Lelystad and Weybridge) PRRSV strains. The North American and European PRRSV isolates tested shared the epitopes recognized by anti-N protein MAbs IAFK8 and SDOW17, but three distinct patterns could be identified on the basis of their reactivities with the other anti-PRRSV MAbs. No reactivity to the anti-M protein MAbs was observed by either European PRRSV isolate or the attenuated U.S. vaccine strain.

Production and characterization of murine monoclonal antibodies to Blastocystis hominis

Several hybridomas producing antibodies detected by enzyme-linked immunosorbent assay (ELISA) were established by fusions of mouse myeloma P3.X63.Ag8.U1 with spleen cells from BALB c mice immunized against an isolate of Blastocystis hominis. Five strongly positive hybrids (6B6, 1D5, 1E7, 4F7 and 4G11) were cloned and all were found to secrete IgM monoclonal antibodies. Four MAbs (6B6, 1E7, 4F7 and 4G11) reacted in immunoblots with a number of B. hominis antigens (mol. wt ranging from 25,000 to 220,000) which were likely to be repeating oligosaccharide epitopes located on glycoproteins, as indicated by pronase and periodate treatment. Another MAb (1D5) reacted with a single antigenic band (mol. wt 30,5000). Similar results were obtained in immunoblots using 4 other B. hominis isolates. Indirect fluorescent-antibody assay (IFA) using MAbs showed 3 patterns of reactivity. 1D5 showed patchy fluorescence, 4F7 showed peripheral fluorescence and 6B6, 1E7 and 4G11 showed bright diffuse fluorescence. These patterns were observed for all 5 human Blastocystis isolates. The MAbs exhibited some cross-reactivity with 2 reptilian Blastocystis isolates but not with Giardia intestinalis, Trichomonas vaginalis or Entamoeba histolytica.

Detection of anti-Borrelia burgdorferi antibody responses with the borreliacidal antibody test, indirect fluorescent-antibody assay performed by flow cytometry, and western immunoblotting.

Borreliacidal antibodies participate in the resolution of Lyme disease by clearing Borrelia burgdorferi sensu lato from the host. Detection of borreliacidal antibodies is also valuable for determination of the specific serodiagnosis of Lyme disease. We show in this work that antibody detected by the borreliacidal antibody test did not correlate with antibody detected by the indirect fluorescent-antibody assay or Western immunoblotting. Detection of borreliacidal antibody decreased with elimination of the spirochete from the host in the presence or absence of therapy. By contrast, the antibody responses detected by the indirect fluorescent-antibody assay or Western immunoblotting remained elevated or continued to expand, respectively. This suggests that the borreliacidal antibody test is a prognostic indicator for clearance of the spirochete. Additional investigations with humans are needed to confirm the prognostic potential of the borreliacidal antibody test.

Serologic Responses to Bartonella and Afipia Antigens in Patients With Cat Scratch Disease

To assess the serologic response to Afipia and Bartonella, previously named Rochalimaea, in patients with cat scratch disease (CSD) and a healthy control group. Prospective, controlled trial. Referral clinic and hospitalized patients in a university medical center. Eighty patients with CSD and 57 healthy control subjects of similar age. The immune responses to Afipia felis and Bartonella henselae were evaluated by a newly developed enzyme-linked immunosorbent assay (ELISA) in patients with CSD and healthy control subjects. Responses to B henselae were also measured by indirect fluorescent antibody (IFA) tests. Antibody levels to Bartonella quintana were measured by ELISA and IFA in a limited number of patients and control subjects. Of the 80 patients with clinical CSD, 56 had positive results of CSD skin tests. ELISA antibody levels to A felis did not differ between patients and control subjects, but immunoglobulin M (IgM) and IgG ELISA antibodies to B henselae and B quintana were significantly higher in patients than in control subjects. IFA responses to B henselae and B quintana were also significantly higher in patients than in control subjects. Patients with CSD had significant serologic responses to B henselae and B quintana but not to A felis, suggesting that the causative agent of CSD is antigenically related to the Bartonella genus and not to Afipia. The Bartonella IgM ELISA and IFA assay were both sensitive and specific and may be used to establish the diagnosis of CSD.

Characterization of a 17-kilodalton antigen of Bartonella henselae reactive with sera from patients with cat scratch disease.

A library of Bartonella (Rochalimaea) henselae DNA was constructed in the cloning vector lambda ZAPII and screened for expression of antigenic proteins by using a pool of sera from patients who had been diagnosed with cat scratch disease (CSD) and had antibodies to Bartonella spp., as determined by indirect fluorescent-antibody (IFA) assay. Ten immunoreactive phages were subcloned as recombinant plasmids by in vivo excision. All 10 recombinants expressed a protein of approximately 17 kDa when they were examined by immunoblot with the pool of human sera. Restriction endonuclease digestion of each recombinant plasmid indicated seven profiles, suggesting that cloning bias was not the reason for repeated isolation of clones expressing the 17-kDa antigen. The gene coding for the 17-kDa antigen was sequenced and shown to code for an open reading frame of 148 amino acids with a predicted molecular mass of 16,893 Da. The amino terminus of the deduced amino acid sequence was hydrophobic in nature and similar in size and composition to signal peptides found in gram-negative bacteria. The remainder of the deduced amino acid sequence was more hydrophilic and may represent surface-exposed epitopes. Further subcloning of the 17-kDa antigen as a biotinylated fusion protein in the expression vector PinPoint Xa-2 resulted in a 30-kDa protein that was highly reactive on immunoblots with individual serum samples from patients with CSD. The agreement between reactivity with the 30-kDa fusion protein on immunoblot analysis and the results obtained by IFA assay was 92% for IFA-positive sera and 88% for IFA-negative sera. The recombinant-expressed 17-kDa protein should be of value as an antigen for serologic diagnosis of CSD and Bartonella infections and warrants further study in attempts to develop a subunit vaccine to prevent long-term Bartonella infection in cats and the potential for further spread of these organisms to humans.

Use of Bartonella Antigens for Serologic Diagnosis of Cat-scratch Disease at a National Referral Center

Bartonella henselae (formerly the genus Rochalimaea) has recently been isolated from patients with cat-scratch disease and their cats, and since September 1992 the Centers for Disease Control and Prevention has offered an indirect fluorescent antibody assay for Bartonella-specific antibody. Physicians submitted serum samples from patients suspected of having cat-scratch disease or other Bartonella-associated illness and completed a questionnaire that recorded clinical information. Indirect fluorescent antibody assay was performed with the use of antigen derived from three Bartonella species: B henselae, Bartonella quintana, and Bartonella elizabethae. During 16 months, 3088 serum samples were received. The largest numbers of specimens and the highest percentages positive (titer, > or = 64) were observed in the fall and winter. Clinical histories of the first 600 patients for whom serum samples and completed information forms were received were examined in detail; seropositivity was significantly associated with cat contact, cat age of less than 1 year, cat scratch, presence of an inoculation papule, and regional adenopathy. Of 91 patients whose illness met a strict clinical definition of cat-scratch disease, 86 (95%) had titers of 64 or greater to either B henselae or B quintana. A fourfold rise or fall in titer was observed in 87 of 132 patients with paired serum samples. The indirect fluorescent antibody assay for Bartonella-specific antibody is sensitive for the diagnosis of cat-scratch disease. Redefinition of cat-scratch disease on the basis of cause and use of this assay as a diagnostic criterion is recommended.

An assessment of the role of white-tailed deer in the epizootiology of anaplasmosis in the southeastern United States.

The role of white-tailed deer (Odocoileus virginianus) in the epizootiology of anaplasmosis in the southeastern United States was examined through retrospective and prospective serosurveys and by experimental infection studies. No serum antibody reactive to Anaplasma marginale was detected with an indirect fluorescent antibody (IFA) assay from any of 1,376 free-ranging deer sampled from 1968 through 1990 from 13 states and Puerto Rico. Thirty-one additional deer from three bovine anaplasmosis enzootic premises also were negative by IFA and Giemsa-stained blood films. Three captive deer given A. marginale intravenously developed antibodies 38 to 41 days post-inoculation (DPI) and remained seropositive for the duration of the study (161 to 287 DPI). At 42 DPI, rickettsemias of approximately 0.0001% infected erythrocytes were observed in all three deer using a DNA probe; low rickettsemias (maximum 0.01%) persisted through 56, 63, and 87 DPI, respectively. One deer had a recrudescent infection from 126 to 146 DPI (maximum rickettsemia 0.001%). We believe that white-tailed deer in the southeastern United States, even though susceptible to A. marginale infection, are not exposed naturally, even at enzootic sites. Furthermore, white-tailed deer did not develop rickettsemias sufficient to support mechanical transmission by biting flies, which is believed to be the primary means of anaplasmosis transmission in this region.

Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic Ehrlichia

Homology in the 16S rDNAs shows that the agent of human granulocytic ehrlichiosis (HGE) is closely related to the veterinary pathogens Erlichia equi and Erlichia phagocytophila. After HGE, patients develop antibodies reactive with E. equi and E. phagocytophila; thus, we hypothesized that these species are closely related and share significant antigenicity. Antisera from humans, horses, dogs, and cattle were tested by indirect fluorescent-antibody assay (IFA) for antibodies reactive with E. equi and other ehrlichiae and tested by immunoblot to identify the specific reactions with E. equi. All convalescent-phase sera from human patients with HGE and from animals infected or immunized with E. equi or E. phagocytophila had antibodies reactive with E. equi by IFA; no reactions with Ehrlichia chaffeensis occurred with these sera, and only one horse naturally infected with E. equi had a serologic reaction against Ehrlichia sennetsu. Human and animal sera obtained after infection or immunization with other Ehrlichia, Rickettsia, and Bartonella species did not react with E. equi by IFA. E. equi immunoblots revealed as many as 19 bands with equine anti-E. equi serum. All HGE agent, E. equi, and E. phagocytophila antisera tested reacted with a 44-kDa antigen of E. equi, while other anti-Ehrlichia spp. sera reacted with this antigen rarely or not at all. HGE agent, E. equi, and E. phagocytophila antisera but not other sera also reacted occasionally with 25-, 42-, and 100-kDa antigens. Most sera reacted with antigens between approximately 56 and 75 kDa, probably heat shock proteins. The HGE agent, E. equi, and E. phagocytophila share significant antigenicity by IFA and immunoblot. Coupled with the nearly identical nucleotide sequences of 16S rRNA genes, these data indicate that E. equi, E. phagocytophila, and the human granulocytic ehrlichia are closely related or identical species.

Analysis of the fine specificities of sheep major histocompatibility complex class II-specific monoclonal antibodies using mouse L-cell transfectants.

The fine specificities of two panels of monoclonal antibodies (mAbs) for sheep major histocompatibility complex (MHC) class II molecules were determined using five mouse L-cell transfectants, each expressing a defined sheep DQ or DR MHC class II A/B gene pair. Using the transfectants in an indirect fluorescence antibody assay, previous immunochemical characterization of the mAbs was confirmed for 16 of 23 mAbs tested. The MHC class II subtype specificity (DQ or DR) of each mAb was assigned without interference from the products of other expressed class II loci. This allowed the identification of both cross-locus specificities as well as defining fine specificities of mAbs previously only partially characterized by immunochemical techniques.

Serologic evidence of respiratory and rickettsial infections among Somali refugees.

Somali refugees living in a camp located in Djibouti were studied in October 1991 and May 1992. The refugees had been living at the camp for about two years. The median age of volunteers was 25 years, of whom 69% were female. Paired sera obtained seven months apart were evaluated by complement fixation, microimmunofluorescence, indirect fluorescent antibody, streptococcal antibody, and enzyme-linked immunosorbent assay techniques for evidence of pathogen infection. Fifty-two percent, 31.3%, 8.0%, 5.9%, and 25.4% of the volunteers had serologic evidence for pre-enrollment infection with Chlamydia pneumoniae, Mycoplasma pneumoniae, Rickettsia typhi, R. conorii, and Coxiella burnetti, respectively. Similarly, 43.5%, 5.2%, 6.1%, 10.7%, 15.8%, and 11.9% of the volunteers studied had serologic evidence for new infection with Streptococcus pyogenes, C. pneumoniae, M. pneumoniae, R. typhi, R. conorii, and Cox. burnetii, respectively. These data suggest that the studied pathogens may be endemic in displaced populations living in the Horn of Africa.

Response of the meadow vole (Microtus pennsylvanicus) to experimental inoculation with Borrelia burgdorferi.

The response of the meadow vole (Microtus pennsylvanicus) to infection by experimental inoculation with Borrelia burgdorferi was evaluated. Forty-two adult voles were inoculated subcutaneously with 0.5 x 10(6) spirochetes. Sera taken during the 196 day trial were tested by indirect fluorescent antibody (IFA) assay for antibodies to B. burgdorferi. Tissues from animals which died during the trial, and from animals killed at 28, 112 and 196 days post-inoculation (DPI), respectively, were cultured in BSK-II medium for < or = 6 weeks. They also were examined histologically for lesions and the presence of spirochetes. All inoculated animals developed antibodies by 14 DPI and maintained titers > or = 1:10 for the duration of the trial. Spirochetes were isolated from ears, bladder, and spleen. Spirochetes also were identified by Bosma-Steiner silver stain or tissue IFA assay in sections of ears, bladder, kidney and heart. Infection as confirmed by re-isolation persisted for < or = 111 days. No lesions were identified in association with the presence of spirochetes. No increase in mortality was observed in inoculated animals compared with controls. Sensitivity of the IFA test at a cut-off titer of 1:10 was 100% from > or = 14 DPI, but at 1:20 reached a maximum of 97%. Specificity at 1:10 was 84% and at 1:20 was 97%. Use of antiserum to Microtus immunoglobulin (Ig) in a double-layered test provided no significant advantages over use of a commercial fluorescein-conjugated anti-mouse Ig in a single-layered IFA test.

Comparison of four different immunoassays for detection of Toxoplasma-specific immunoglobulin G

Serum specimens from 246 women of childbearing age were tested for immunoglobulin G (IgG) antibodies to Toxoplasma gondii by four different commercial assays: Abbott IMx microparticle enzyme immunoassay (EIA), Mercia Toxo-G EIA, Bartels Prima EIA, and bioMerieux Vitek (VIDAS) enzyme-linked fluorescent assay. A total of 27 specimens were initially positive with all four assays, 202 specimens were negative, and 17 specimens were discrepant (disagreement among the assays). After repeating tests for the 17 discrepant samples, five resolved (one was positive and four were negative). The 12 remaining discrepant samples were tested by an indirect fluorescent antibody (IFA) assay for Toxoplasma IgG antibodies; five specimens were positive for Toxoplasma IgG by IFA. The resolved sensitivities of the various kits ranged from 88% (bioMerieux VIDAS) to 94% (Abbott IMx), and the specificities were all 98%-99%. These results show that the four serologic tests used for detection of Toxoplasma IgG give very similar results and can all be readily used by clinical laboratories for screening purposes.

Flow cytometric analyses of lymphocyte subsets in peripheral blood and lymphoid tissues of gnotobiotic calves during primary acute postnatal infections of bovine viral diarrhea virus.

Eight 1-day-old gnotobiotic calves were exposed to one of 2 noncytopathic isolates of bovine viral diarrhea virus (ncp-BVDV 7937 and ncp-BVDV 126) and 2 similar calves were not infected and served as controls. Phenotypic analyses of peripheral blood mononuclear leukocytes at 3, 7, and 10 days postinfection (PI), and cell preparations of thymus, Peyer's patch, mesenteric lymph node, spleen, and bone marrow collected at necropsy, 10 days PI, were conducted using flow cytometric techniques on cells stained by an indirect fluorescent antibody assay using monoclonal antibodies specific for mononuclear leukocyte subsets. Significant fluctuations in specific subsets of peripheral blood mononuclear leukocytes were observed in calves exposed to ncp-BVDV 7937 on day 3 PI (cells expressing MHC class II), day 7 PI (B-cells), and day 10 PI (B cells and cells expressing MHCII). Significant phenotypic differences between groups were also detected in cell preparations from Peyer's patch and thymus. Calves exposed to ncp-BVDV 7937 had a significantly higher percentage of B cells than calves exposed to ncp-BVDV 126 and calves not exposed to BVDV. Calves exposed to ncp-BVDV 126 had a significantly higher percentage of CD2 (BoCD2) positive T cells than calves not exposed to BVDV. Fragility of thymic cell preparations was attributed to infection with virus. These results highlight the importance of the tropism of BVDV for cells of the bovine immune system and its role as a significant immunosuppressive agent capable of predisposing affected animals to infection with other agents.

Detection of microsporidian spores in clinical samples by indirect fluorescent-antibody assay using whole-cell antisera to Encephalitozoon cuniculi and Encephalitozoon hellem.

Three polyclonal mouse antisera, to Encephalitozoon cuniculi, Nosema algerae, and Nosema corneum, and two polyclonal rabbit antisera, to E. cuniculi and Encephalitozoon hellem, were used in an indirect fluorescent-antibody assay (IFA) with Enterocytozoon bieneusi, E. cuniculi, and Encephalitgozoon. hellem spores (spores of the last two were taken from culture). Enterocytozoon bieneusi cannot be cultured. By IFA, antisera to E. cuniculi and E. hellem reacted strongly and equally with each other's spores. The mouse antisera reacted strongly with the homologous species, but for these there was segmental and particulate or "dot" staining of heterologous microsporidian spores, indicating cross-reactions with more selected antigens. In fecal samples, cross-reactions with both mouse and rabbit antisera were sometimes seen with different yeast species, with species of streptococci, and species of gram-negative rods. There were no cross-reactions to staphylococci. Enterocytozoon bieneusi was easily identified in duodenal and colonic biopsies, duodenal and colonic fluids, and feces of symptomatic AIDS patients by IFA. In a study of 12 AIDS patients with diarrhea, the new IFA identified microsporidia in all of 11 fecal samples, three colon fluids, six duodenal fluids, and three duodenal biopsy touch preparations. Although the fecal sample of 1 of the 12 was negative, the patient's duodenal fluid contained microsporidian spores by IFA.

Evaluation of agar gel immunodiffusion and indirect fluorescent antibody assays as supplemental tests for dourine in equids

Summary The agar gel immunodiffusion (agid) and indirect fluorescent antibody (ifa) assays were evaluated as supplemental tests to the complement-fixation (cf) test, the official US importation certification test for dourine in equids. The American stabilate (n = 10 animals) or the Canadian stabilate (n = 6 animals) of Trypanosoma equiperdum cultured in rat blood was administered by catheterization and infusion in the urogenital tract of 16 equids. To assess parasitemia and serologic responses by use of the cf, agid, and ifa tests, a total of 787 serum and blood samples were obtained from equids before exposure and 3 times a week after exposure to T equiperdum. Results of the ifa and agid tests were compared with the cf test results. The disease was diagnosed earlier by the ifa test than by the agid test, regardless of antigen preparation or exposure group. The mean number of days between exposure and positive result by the cf and ifa tests was the same when either homologous or heterologous antigen was used in the ifa test. In general, the ifa test was more sensitive than the agid test in diagnosing dourine, regardless of the antigen preparation used in the test or exposure group. Differences in test specificity were observed among both groups of exposed equids when either antigen was used (P &lt; 0.05). The agid test, using the American antigen, was more specific than the ifa test for sera from both groups of equids. When the Canadian antigen was used, the ifa test was a more specific test than the agid test (P &lt; 0.01). Although differences in the positive predictive value and negative predictive value of the ifa and agid tests were significant (P &lt; 0.01) for both groups of equids, the values were not entirely conclusive in determining which of the 2 assays was better as a supplemental test. It is recommended that the more sensitive ifa test be used for screening the few equids with anticomplementary cf test results for dourine. The agid test can be used to verify positive ifa results for dourine.

Isolation and transmission of the Lyme disease spirochete from the southeastern United States.

The isolation of the Lyme disease spirochete (Borrelia burgdorferi) from the southeastern United States is reported. Three isolates, two from cotton mice (Peromyscus gossypinus) and one from the black-legged tick (Ixodes scapularis), were recovered from Sapelo Island, Georgia, in July and September 1991. The spirochetes were characterized by indirect fluorescent antibody assay using a battery of five monoclonal antibodies, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of whole cell lysates, and by the polymerase chain reaction (PCR) assay using primers for three DNA target sequences found in B. burgdorferi reference strain B-31. Transmission experiments indicate that the three Georgia isolates can infect experimentally inoculated hamsters and mice. Tick transmission of one of the isolates has been attempted so far; I. scapularis transmitted isolate SI-1 from hamsters to mice, but the lone-star tick, Amblyomma americanum, did not.

Application of indirect immunofluorescence to detection of Dientamoeba fragilis trophozoites in fecal specimens.

An indirect fluorescent-antibody (IFA) assay was carried out to examine for the presence of Dientamoeba fragilis trophozoites in preserved fecal specimens. Antiserum to D. fragilis trophozoites was raised in a rabbit with a dixenic culture of D. fragilis (ATCC 30948) from the American Type Culture Collection. After absorption with Klebsiella pneumoniae and Bacteroides vulgatus, the immune rabbit serum was used for examination by the IFA assay. A total of 155 clinical samples were tested; 42 with no parasites, 9 with D. fragilis, and 104 with other parasites. The IFA assay identified seven D. fragilis organisms. Two specimens with doubtful IFA assay readings showed very scanty amounts of D. fragilis trophozoites on stained smears. There were no false-positive IFA assay readings. The IFA assay appeared to be a promising method because of its speed in screening. The specificity of the IFA assay indicates that other diagnostic tests such as an enzyme-linked immunosorbent assay could be developed to identify D. fragilis antigens in fecal specimens.