Consumer acceptance of irradiated meat and poultry in the United States.

Food manufacturers in the United States are currently allowed to irradiate raw meat and poultry to control microbial pathogens and began marketing irradiated beef products in mid-2000. Consumers can reduce their risk of foodborne illness by substituting irradiated meat and poultry for nonirradiated products, particularly if they are more susceptible to foodborne illness. The objective of this study was to identify the individual characteristics associated with willingness to buy irradiated meat and poultry, with a focus on five risk factors for foodborne illness: unsafe food handling and consumption behavior, young and old age, and compromised immune status. A logistic regression model of willingness to buy irradiated meat or poultry was estimated using data from the 1998-1999 FoodNet Population Survey, a single-stage random-digit dialing telephone survey conducted in seven sites covering 11% of the U.S. population. Nearly one-half (49.8%) of the 10,780 adult respondents were willing to buy irradiated meat or poultry. After adjusting for other factors, consumer acceptance of these products was associated with male gender, greater education, higher household income, food irradiation knowledge, household exposure to raw meat and poultry, consumption of animal flesh, and geographic location. However, there was no difference in consumer acceptance by any of the foodborne illness risk factors. It is unclear why persons at increased risk of foodborne illness were not more willing to buy irradiated products, which could reduce the hazards they faced from handling or undercooking raw meat or poultry contaminated by microbial pathogens.

Comparison of two recombinant major outer membrane proteins of the human granulocytic ehrlichiosis agent for use in an enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, two Babesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.

Cloning and expression of the 44-kilodalton major outer membrane protein gene of the human granulocytic ehrlichiosis agent and application of the recombinant protein to serodiagnosis.

A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in human infection. A gene encoding this protein was cloned and sequenced. Southern blot results revealed the existence of multigenes homologous to the P44 gene in the genome of the HGE agent. The recombinant 44-kDa protein (rP44) was expressed by using expression vector pET30a. The reactivity of the affinity-purified rP44 was evaluated by Western immunoblot analysis and dot blot immunoassay. Western immunoblot analysis showed that mouse anti-rP44 serum reacted with 44- to 42-kDa proteins in six different HGE agent strains tested except strain 2, in which three proteins of 42, 40, and 38 kDa were recognized. Eleven HGE patient serum samples, a horse anti-HGE serum, and a horse anti-Ehrlichia equi serum recognized the rP44 protein. This suggests that rP44 is an HGE-E. equi group-specific antigen. Neither human anti-Ehrlichia chaffeensis serum nor rabbit anti-Borrelia burgdorferi serum reacted with rP44. Sera from two patients coinfected with the HGE agent and B. burgdorferi reacted positively with rP44 and the HGE agent. Sera from 20 HGE patients with indirect fluorescent-antibody (IFA) titers ranging from 1:20 to 1:2,560 gave distinct positive reactions in a dot immunoblot assay. There was a positive correlation between the color densities of the dot reactions and the IFA titers when greater than 50 ng of recombinant antigen per dot was used. The use of the affinity-purified rP44 protein as antigen would provide a more specific, consistent, and simpler serodiagnosis for HGE than the use of whole infected cells or purified HGE agents.

Ultrastructural and antigenic characterization of a granulocytic ehrlichiosis agent directly isolated and stably cultivated from a patient in New York state.

A human granulocytic ehrlichiosis (HGE) agent with 16S rDNA sequence identical to the published sequence of HGE agents was isolated from a patient from New York State by inoculation of the blood leukocyte fraction directly into a human promyelocytic leukemia cell line HL-60. The HGE agent was also isolated from the leukocyte fraction of the blood and bone marrow of a mouse inoculated with the leukocyte fraction of the patient's blood. The isolate has been passaged in tissue culture 30 times over 8 months. Electron microscopy revealed pleomorphic coccobacilli with a thin and highly rippled outer membrane in the clear inclusion matrix. Comparison of IFA reactivity of antisera obtained from a variety of sources with the cell-cultured HGE agent revealed that 3 HGE agent strains (New York isolate, Wisconsin [BDS] isolate, and a tick-derived isolate) are highly cross-reactive and there are diverse antigenic cross-reactivities between HGE agent and Ehrlichia chaffeensis.

Structural, immunological and functional comparisons of factor H, rheumatoid arthritis protein (RHP), and its apparent normal counterpart (N-RHP).

The isolation and characterization of two human serum proteins, RHP and N-RHP, are described. N-RHP appears to be the normal counterpart of RHP which is found at elevated levels in sera of patients with rheumatoid arthritis [Rosano et al. (1988b) Inflammation 12, 351 - 360]. Although both proteins crossreact with anti-Factor H and have identical N-terminal amino acid sequences, they differ from Factor H in pI, solubility at low ionic strength, and in glycosylation. RHP differs from Factor H and N-RHP in antigenicity in the rabbit, in effect on the C1q-anti-C1q precipitin reaction, and in ability to disaggregate C1, the first component of the complement system. Removal of RHP, N-RHP and Factor H from binding to C1q is a prerequesite for separation of RHP and N-RHP from Factor H by anion exchange chromatography and isoelectric focusing. The finding of uniquely demonstrable RHP activity (enhancement of C1q-anti-C1q precipitin activity) in unfractionated sera from patients with rheumatoid arthritis, but not in normal sera, suggests that RHP is not an artefact of Factor H produced during isolation.

Implementation of a proficiency testing program for Lyme disease in New York State.

Nine proficiency test events for Lyme disease (Borrelia burgdorferi) antibody were carried out from October 1988 to January 1992 by the New York State Department of Health, Albany, Overall sensitivity for the 846 participants averaged 95.4%, with varying sensitivities of 98.7% for users (71 laboratories) of immunofluorescence assays, 97.4% for users (144 laboratories) of solid-phase fluorescence immunoassays, and 94.6% for users (631 laboratories) of enzyme immunoassays. Thirty percent of the enzyme immunoassay laboratories tested at greater than or equal to 98.4% sensitivity by the DiaMedix test kit (DiaMedix Corp, Miami, Fla) and MarDx test kit (MarDx Diagnostics Inc, Scotch Plains, NJ), while 7% tested at less than or equal to 83% by the Access test kit (Access Medical Systems Inc, Branford, Conn) and the Cambridge BioScience test kit (Cambridge BioScience, Worcester, Mass). Overall specificity was 98.8%, with specificities greater than 99% for both solid-phase fluorescence immunoassay and enzyme immunoassay users and 92.9% for immunofluorescence assay users. Cross-reactivity with Treponema pallidum antibody was high for the Hillcrest (Hillcrest Biologicals, Cypress, Calif) (30%) and Wampole (Wampole Laboratories, Cranbury, NJ) (25%) immunofluorescence assay test kit users and for the MarDx (30%) and 3M (3M Diagnostics Systems Inc, Santa Clara, Calif) (24%) enzyme immunoassay test kit users. Laboratories that tested by the Wampole immunofluorescence assay test kit had also high cross-reactivity (25%) against heterophile antibody.

Monoclonal antibody to native P39 protein from Borrelia burgdorferi.

We have produced, by using a sonicate of Borrelia burgdorferi, a monoclonal antibody (MAb), NYSP39H, that is specific for the P39 protein band. This MAb reacted with 13 isolates of B. burgdorferi but not with eight different spirochetes (four borrelias, two leptospiras, and two treponemas). Surface labeling of B. burgdorferi with biotin and subsequent treatment with Nonidet P-40 showed that P39 was not biotinylated but was extracted with Nonidet P-40, indicating that it is present within the outer membrane, but not on the surface of the spirochete. Immunoelectron microscopy revealed the immunogold probe primarily at the cytoplasmic membrane region of the spirochete. The MAb detected B. burgdorferi in the indirect fluorescent-antibody test only when the spirochetes from a culture or in a tick homogenate were fixed with polylysine and not with acetone. NYSP39H appears to be an appropriate probe for use in the specific detection of B. burgdorferi.

Three-dimensional reconstruction of Coxiella burnetii-infected L929 cells by high-voltage electron microscopy.

Previous examination of thin sections of L929 cells heavily infected with the Q fever Priscilla isolate by conventional transmission electron microscopy indicated that the rickettsiae resided within multiple vacuoles. The present study using high-voltage electron microscopy and three-dimensional reconstruction revealed that, in heavily infected cells, the rickettsiae, in fact, reside in one multilobed vacuole. As a result of asymmetric cell division, the multilobed vacuole containing the rickettsiae apparently segregates into one daughter cell, while the companion daughter cell emerges parasite free. This likely explains the appearance of naive uninfected cells in long-term-infected (i.e., ca. 2 years) cell populations that had not been supplemented with uninfected L929 host cells.

The distribution of canine exposure to Borrelia burgdorferi in a Lyme-Disease endemic area.

OBJECTIVES: A serosurvey of canine exposure to Borrelia burgdorferi, the causative agent of human Lyme disease, was conducted in Westchester County, New York, to determine the distribution of exposure in an area endemic for Lyme disease. METHODS: A total of 1446 blood samples was collected from resident dogs and tested by modified enzyme-linked immunosorbent assay. Equivocal samples were further tested by immunoblot. A mean number of 57.8 samples was collected from each of 25 towns and cities. RESULTS: Seroprevalence rates for municipalities ranged from 6.5% to 85.2%. County seroprevalence was 49.2%. There was a significant difference among the rates for the northern (67.3%), central (45.2%), and southern (17.3%) regions. Multiple range analysis indicated homogeneity between the southern and central regions and the central and northern regions. CONCLUSIONS: Canine exposure to B burgdorferi increases in a south to north gradient within the county. Intensity of exposure, measured by enzyme-linked immunosorbent assay titers, indicates a similar pattern. The close association between dogs and humans suggests that human risk of acquiring Lyme disease within Westchester County is equally disparate and is inversely related to the degree of urbanization.

Adherence and entry of Borrelia burgdorferi in Vero cells.

Adherence to and entry of the parasite into the host is one of the essential elements of microbial pathogenicity. We investigated the adherence to and entry into primate kidney epithelial (Vero) cells of Borrelia burgdorferi by radiolabelling techniques, immunofluorescence and electronmicroscopy. The attachment to and subsequent entry of both untreated and heat (50 degrees C)-treated B. burgdorferi into Vero cells occurred at cell-surface sites associated with aggregated coated pits. In contrast, there was minimal attachment of spirochaetes heated at 60 degrees C. Radiometric studies showed that, with untreated cells, there was incorporation of both 14C-glucose-1-phosphate and 14C-thymidine, whereas with the 50 degrees C-treated spirochaetes only glucose-1-phosphate was incorporated, and with the 60 degrees C-treated spirochates neither radionuclide was incorporated. Spirochaetes heated at 50 degrees C or 60 degrees C did not grow at 35 degrees C in culture medium. These results suggest that the presence of certain metabolic activities of the spirochaete but not viability (ability to grow) are necessary for the attachment process. After entry of untreated B. burgdorferi, most of the spirochaetes were either free in the cytoplasm or tightly bound to the host membrane. In contrast, 50 degrees C-treated spirochaetes remained bound to host membrane in large phagosome-like vesicles.

Immunoblot studies to analyze antibody to the Rickettsia typhi group antigen in sera from patients with acute febrile cerebrovasculitis.

In 1986, an unusual syndrome of acute febrile cerebrovasculitis in the Piedmont Region of Virginia was reported. All patients had encephalopathy and prior exposure to both a sylvan environment and flea-infested animals. The initial serological studies suggested a rickettsial origin, corroborating clinical, epidemiological, and histopathological findings. Sera from four of five patients were subsequently studied by immunoblotting. Unabsorbed and absorbed sera were tested with electrophoresed and electroblotted Rickettsia typhi, Legionella bozemanii, and Proteus vulgaris OX19 antigens. The unabsorbed sera reacted with all three antigens. The P. vulgaris- and L. bozemanii-absorbed sera reacted with R. typhi only and without significantly less intensity. In contrast, the reactivity of R. typhi-absorbed sera was significantly lower with all three antigens. These results indicate that these patients had specific antibodies to a typhus group antigen. Although our findings suggest that a rickettsia of the typhus group may have caused this syndrome, no definitive diagnosis could be achieved because a rickettsial organism was not isolated.

Analysis of Coxiella burnetii isolates in cell culture and the expression of parasite-specific antigens on the host membrane surface.

Coxiella burnetii isolates may be classified into several groups based on plasmid character. These groups may also be correlated with disease syndrome--chronic or short-term acute. L929 mouse fibroblast cells were exposed, independently, to two members of the three major C. burnetii groups, and their growth/morphological characteristics analysed by light and electron microscopy, including High Voltage Electron Microscopy. The fates of the isolates were followed. Two acute isolates [Nine Mile (RSA 493) and Henzerling (RSA 331), QpH1-type plasmids] and two chronic isolates (S Q217 and L Q216, plasmidless) readily infected L929 cells in static culture. Priscilla (Q177) and F (Q228) isolates (QpRS-type plasmid, and implicated in causing chronic Q fever) took longer to infect cells, and, unlike the members of the other two groups, gradually disappeared when shifted to suspension culture. Cells infected with Q177 and Q228 exhibited a higher degree of vacuolation than cells infected with the other isolates. Parasite-specific antigens on the surfaces of the host cells were analysed by immunofluorescence/flow cytometry. The acute and plasmidless isolates caused the display of significantly more C. burnetii-specific antigen on the host cell membrane than the two QpRS plasmid-containing isolates. This host cell model system clearly reveals biological differences among the C. burnetii groups.

Possible typhus-group infection in New York State: presentation of four suspect cases.

Epidemiologic investigations were recently conducted on four cases which were reported in New York State in 1986 and 1987, three of which were within one family. These included hospital chart reviews, case or family interviews, animal trappings, and ectoparasite surveys. Serologic tests and immunoblots were performed on blood samples obtained from these patients. All four patients had acute febrile illnesses; two required hospitalization and one died. Microimmunofluorescence test results using Rickettsia typhi and R. prowazekii antigens showed a greater than or equal to 4-fold increase in titer with paired sera from three patients. The remaining patient had a single serum titer of 4096 with both antigens. In addition, sera from all patients reacted with R. typhi in the immunoblot test and, from the three patients for whom sera were available, also with R. prowazekii. Results suggest that the four patients were exposed to the typhus-group rickettsiae or to an organism which shares a common epitope(s).

Cross-reaction with Borrelia burgdorferi antigen of sera from patients with human immunodeficiency virus infection, syphilis, and leptospirosis.

We have studied the cross-reaction with Borrelia burgdorferi of sera positive for leptospirosis, syphilis, or human immunodeficiency virus by using the microimmunofluorescence test (micro-IF). The percentage of sera reactive in the micro-IF before absorption varied from 7 to 37% and was reduced to 3 to 8% after absorption with a commercial Reiter treponemal antigen. The cross-reaction of sera positive for syphilis or human immunodeficiency virus was distinguished from the homologous reaction with sera from patients with Lyme disease in the immunoblot test results. However, the cross-reaction could not always be distinguished from the homologous reaction with sera from patients with leptospirosis whose sera scored positive in the micro-IF for B. burgdorferi.

Fluoroimmunoassay studies with solubilized antigens from Borrelia burgdorferi.

Sodium deoxycholate-solubilized Borrelia burgdorferi antigen was prepared for use in a solid-phase fluoroimmunoassay (FIA-L) to detect antibodies in Lyme disease. Serum specimens were tested by FIA-L and by a microimmunofluorescence test. The FIA-L results are comparable to those of the standard microimmunofluorescence test. The overall agreement was 0.98. Moreover, the FIA-L procedure is simple and rapid; fluorescence is objectively determined and is proportional to antibody titer.

Cross-reaction of immune sera from patients with rickettsial diseases.

Rickettsia rickettsii and Rickettsia conorii are the causative agents of two common and serious diseases, Rocky Mountain spotted fever and Mediterranean spotted fever, respectively. In patients naturally infected with either of these organisms, antibodies are produced which cross-react with antigens of the other so extensively that diagnostic tests usually cannot identify the causative agents. The results of this study indicate that serodiagnostic tests with antigen from one of these two organisms could be used to detect antibodies in patients with either of the two rickettsial diseases.

Production of antibody to and cellular localization of erythrocyte-sensitizing substance from Rickettsia rickettsii.

Antibodies to Rickettsia rickettsii erythrocyte-sensitizing substance (ESS) were raised in rabbits by using a derivatized ESS. The resulting antibodies reacted with R. rickettsii and cross-reacted with Rickettsia conorii, a member of the spotted fever group rickettsiae, but did not react with Rickettsia typhi, a member of the typhus group rickettsiae, Legionella bozemanii, or Proteus vulgaris OX19 or OX2. Immunoblot analysis indicated that ESS was present in more than one fraction and that the major haptenic fraction was proteinase resistant. Immunoelectron microscopy indicated that the antibodies to R. rickettsii were specific to components located on the cell surface and intracellularly to components between the cell wall and cytoplasmic membrane.

Evaluation of a latex agglutination test for Rickettsia conorii antibodies in seropositive patients.

A latex agglutination test for antibodies to Rickettsia conorii was compared with micro-immunofluorescence (the reference test for total antibodies); 179 sera were from 115 confirmed cases of Mediterranean Spotted Fever, and 101 were from pregnant women (control group) who had no detectable antibodies by the reference method. The micro-immunofluorescence test for specific IgM antibodies was used to clarify some discordant results. The agreement obtained between latex-R. conorii and micro-immunofluorescence was 95%. Sensitivity and specificity were 96% and 93% respectively. When micro-immunofluorescence results for specific IgM antibodies were included, these figures rose to 96 and 99%, and agreement was almost 97%. Latex agglutination is a simpler and more rapid technique than micro-immunofluorescence and is suitable for the screening as well as for the titration of R. conorii antibodies.