Karim E. Hechemy

Toxoplasma gondii: purification of trophozoites propagated in cell culture.

Abstract A new procedure is described for the purification of trophozoites from the virulent RH strain of Toxoplasma gondii propagated in baby hamster kidney (BHK-21) cell cultures. The culture medium containing host cell debris and trophozoites was filtered through glass-wool filtering fiber, which removed most host cell material. The filtrate containing trophozoites was centrifuged, and the trophozoite pellet was resuspended and washed in phosphate-buffered saline. An average of about 75% of the original number of trophozoites was recovered. No loss of trophozoite viability was observed as determined by the rate of host cell culture monolayer destruction. The amount of host cell material contamination in the final trophozoite fraction was negligible as determined by measuring radioactivity in the trophozoite fraction after cofiltration with noninfected host cell material which had been prelabeled with radioactive precursors.

A Century of Rickettsiology: Emerging, Reemerging Rickettsioses, Clinical, Epidemiologic, and Molecular Diagnostic Aspects and Emerging Veterinary Rickettsioses

Abstract:  This overview summarize the salient features of advances in the epidemiology, vectors, and clinical and laboratory diagnoses of rickettsiology. Presentations on veterinary rickettsiology highlight the importance of the rickettsiae in animal husbandry.

Comparison of Two Recombinant Major Outer Membrane Proteins of the Human Granulocytic Ehrlichiosis Agent for Use in an Enzyme-Linked Immunosorbent Assay

ABSTRACT 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, twoBabesia 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.

Rickettsiology: Present and Future Directions

It has been 12 years since the New York Academy of Sciences last published an Annals volume dedicated to the rickettsiae and rickettsial diseases. e Since that time, the rickettsial field, as well as the field of microbiology in general, has undergone a significant evolution, due to advances in molecular genetics and in technologies. However, as expected for scientific advances, more findings have led to more questions. The presentations at the International Conference on Rickettsiae and Rickettsial Diseases held in Ljubljana, Slovenia on September 4–7, 2002, have endeavored to answer, at least in part, these new questions on issues—past and present—that remain unresolved. The Conference also attempted to push the limits of our understanding of rickettsiology since the inception of the field with the discovery of the agent of Rocky Mountain spotted fever by Howard Ricketts (in whose honor the order Rickettsiales was named). In the past 12 years, the taxonomy of rickettsias has undergone a significant reorganization. Genetic analyses of 16S rRNA genes, groESL, and surface-protein genes have indicated that the earlier taxonomic classification, based on the heterogeneous mix of characters previously used to group bacteria into the order Rickettsiales ( e.g., the obligate intracellular nature of most of these organisms, their geographical origins, and their vectors) could not endure in today’s molecular technology–based environment. Each of these previously used characters had exceptions. The reorganization has helped to provide a more homogeneous characterization of the order Rickettsiales. The family Bartonellaceae was moved out of the order Rickettsiales and placed in the order Rhizobiales, leaving the order Rickettsiales with two families, the Rickettsiaceae and Anaplasmataceae, and no tribes. The genus Coxiella was moved

History and Prospects of Coxiella burnetii Research

Coxiella burnetii, the causative agent of Q fever, is the only known intracellular pathogenic bacterium that replicates within the acidic, degradative confines of a eukaryotic phagolysosome. It appears that the bacterium nullifies or thwarts the toxic elements of this vacuole and evades both innate and adaptive immune responses. C. burnetii is ubiquitous in its geographic distribution with the disease prevalence more widespread than previously considered. Recent molecular and cell biological advances, along with improved instrumentation, have provided unique insight into the host-parasite interrelationship and revealed previously unknown virulence strategies of C. burnetii. An example is completion of the genome sequences of several strains of C. burnetii that has improved our understanding of pathogenic mechanisms used by the organism to cause disease in mammalian hosts.

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).

Inhibition of C1q functions by RHP, a protein elevated in sera from patients with rheumatoid arthritis.

We have previously shown that serum levels of C1q, unbound to C1r X C1s, are elevated in rheumatoid arthritis. We have also shown that RHP, a newly described serum protein which affects the C1q-anti C1q precipitin reaction, is also present at elevated levels in rheumatoid arthritis. We now show that RHP inhibits the hemolytic activity of C1q, disaggregates C1, and inhibits the ability of C1q bound to latex beads or to aggregated IgG to enhance the oxidative metabolism of neutrophils.

Coating of polymeric surfaces for immunoassay by a forced adsorption technique.

A forced adsorption technique for coating polymeric surfaces is described. This technique is simple and allows us to use (i) relatively impure ligands to be coated on latex surface and (ii) the minimum amount of ligands for preparation of large-volume lots of latex reagent. The serologic results are highly reproducible from lot to lot of the reagent, in contrast to the variability found with passively adsorbed reagents. Use of this technique could be extended to coat flat surfaces for solid-phase immunoassays.

Rickettsioses: From Genome to Proteome, Pathobiology, and Rickettsiae as an International Threat

During the past decade, several major developments have occurred in rickettsiology. With the advent of the newly emerging infections caused by a number of rickettsias, the re-emerging of old pathogenic species of rickettsias that cause both old and new syndromes has helped redefine the level of rickettsial pathogenicity. The intracellular nature of most rickettsias remains a mystery although their genome size is close to that of the free-living neisserias.

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.