Abstract:
The present invention relates to a method of continually propagating Ehrlichia canis in a microphage-monocyte cell line referred to as DH82.

Description:
The present invention is related generally to the method of culturing microorganisms. More particularly, the present invention is related to a method of continually growing an obligate intracellular bacterium such as Ehrlichia canis (E. canis), E. risticii, E. sennetsu, E. phagocytophila and Neorickettsia helminthoeca in a canine macrophage cell line. 
     Human ehrlichiosis is a newly recognized rickettsial disease that has been reported from 19 states in the USA. The etiologic agent has not been isolated from a human, but seems to be serologically and morphologically related to Ehrlichia canis, a white blood cell associated rickettsia long recognized to cause both acute and chronic illness in dogs around the world. 
     Current research on human ehrlichiosis is dependent upon the in vitro growth of E. canis. Ehrlichia canis can be grown in primary canine monocytes, however, the life span of these cells is approximately 2-3 weeks, and a continual supply of blood from a dog is, therefore, necessary in order to maintain a very limited quantity of E. canis organisms. 
     A continuous mouse-dog hybrid (MDH) cell line has been developed for the purpose of in vitro cultivation of a Florida isolate of E. canis. No other cell line has heretofore been known or described for continual propagation of E. canis, either the Florida or the herein described Oklahoma isolate. 
     SUMMARY OF THE INVENTION 
     It is, therefore, an object of the present invention to provide a new method for continual propagation of E. canis in a cell line distinctly different from the MDH cell line. 
     Other objects and advantages will become evident from the following detailed description of the invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The above and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawing wherein FIG. 1 represents a transmission electron micrograph of E. canis, Oklahoma isolate, in a membrane-bound vacuole in the cytoplasm of a DH82 cell. Note very pleomorphic structure of E. canis embedded in abundant capsular matrix. Ribosomes and fine DNA strands were visible. Bar=1 μm. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention comprises a demonstration of continual propagation of E. canis in a canine monocyte macrophage cell line DH82 in an in vitro medium that supports the growth of DH82 cells. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. 
     MATERIALS AND METHODS 
     The cultures of DH82 cell line employed in developing this invention, including those employed in the examples below, were obtained from the laboratory of Dr. Yasuko Rikihisa, Department of Veterinary Pathobiology, Ohio State University, Columbus, Ohio. 
     Isolation of E. canis 
     Primary canine monocyte cultures were established from a normal donor dog 48 hours before the arrival of the infected blood. Whole blood from the donor dog was transported in 2×15 cc heparinized tubes. After rinsing the tubes with 70% ethanol, the blood was transferred equally into 2 sterile erlenmeyer flasks and subsequently drawn into 2×60 cc syringes loaded with 20 ml of 2.5% dextran in saline. The needle was replaced and the syringe positioned needle-end up to allow for erythrocyte sedimentation. After 45 minutes, the leukocyte rich plasma from each syringe was transferred to 50 ml centrifuge tubes and spun at 50×g for 10 minutes. Each pellet was resuspended in 5 ml lysing buffer, held at room temperature (about 22°-24° C.) for 2 minutes and then centrifuged at 50×g for 10 minutes. The pellets were resuspended in 8 ml of culture medium (Minimum essential medium containing Earle&#39;s salts and sodium bicarbonate supplemented with 1% L-glutamine (200 mM) and 15% heat-inactivated homologous serum). Finally, the cell suspension was added equally to 4 25 cm 2  tissue culture flasks. The cultures were incubated at 37° C. for 24 hours at which time 5 ml of fresh culture medium was added. This procedure was repeated every 2 weeks in order to maintain a constant supply of primary canine macrophages. 
     Forty ml of whole blood drawn from a tick-infected carrier dog was sent to the Centers for Disease Control from Oklahoma State University. The infected blood was treated in a manner similar to the uninfected blood except that after the erythrocytes had sedimented, the leukocyte rich plasma was immediately transferred onto the 4 previously established primary canine monocyte cultures, and incubated at 37° C. for 24 hours. The leukocyte poor plasma was then decanted and 5 ml of culture medium was added to each flask and replaced every 3-4 days. Since primary canine macrophages only survive in vitro for a couple of weeks, the original cultures were harvested after two weeks and transferred to new primary canine macrophage cultures established 2-3 days earlier. 
     Starting 10 days after the addition of the leukocytes from the carrier dog, the cells were checked for infectivity. Briefly, 1 ml of supernatant from each culture was loaded into a cytospin (Shandon Inc., Pittsburgh, Pa.). The cells were centrifuged onto a glass slide, fixed in acetone for 15 minutes and then stained by direct immunofluorescence. This procedure was repeated twice weekly until organisms were observed. 
     Culture of Ehrlichiae 
     One 2 cc vial of frozen DH82 cells, established from the neoplastic progenitor cells of canine malignant histiocytosis (Wellman et al, 1988, In Vitro Cellular and Developmental Biology 24:223-229) was rapidly thawed in a 37° C. water bath and then transferred to a centrifuge tube containing 10 ml of culture medium. After 10 minutes of centrifugation at 400×g, the supernatant was removed and the pellet resuspended in 4 ml of culture medium. The cell suspension was then transferred into a 25 cm 2  tissue culture flask. 
     After 5 days of incubation at 37° C., the monolayer was split 1:2. Since a very limited amount of the bacteria was available, the supernatant from a 60-70% infected primary canine monocyte culture was added to the DH82 cells along with 2 ml of fresh medium. This process was repeated 10 days later and then again in 8 days with regular media changes every 3 to 4 days. Once the ehrlichia were established in the DH82 cell line, the 15% canine serum in the growth medium was changed to 15% FBS. 
     Preparation of antigen slides for IFA 
     Cells from E. canis (Oklahoma isolate) infected DH82 cultures (80-90% infection) were suspended in culture supernatant. Drops (10 μl) of the antigen suspension were placed onto 12 well teflon coated slides. The slides were air dried for 1 hour, wrapped in lint-free tissue and stored at -90° C. As slides were needed, they were thawed and then fixed in acetone for 15 minutes. 
     Serologic comparison of commercially prepared E. canis (Florida isolate) and DH82 (Oklahoma isolate) antigen slides 
     A total of 15 human sera previously tested at CDC were used in a blind comparison study. Nine of the 15 patients were positive for antibodies to E. canis (Florida isolate). The 15 serum specimens were split into 2 sets, each coded with different numbers. The IFA (indirect fluorescent antibody) test was performed as described by (Fishbein et al, 1989, J. Infect. Dis.160(5):803-809) on each serum set using the Oklahoma isolate in the DH82 cells and the commercially prepared slides with the Florida isolate in the MDH cells (E. canis antigen slides, Protatek, St. Paul, Minn.). Briefly, the serum sample was screened at a dilution of 64 in 0.15M PBS solution. When specific fluorescence was observed at this titer, serial two fold dilutions were made. Serologic results were reported as the reciprocal of the highest dilution at which specific fluorescence of E. canis morulae or individual bodies were observed. 
     Electron microscopy 
     Electron microscopy was performed as described by Rikihisa and Perry (1985, Infect. Immun. 49(3): 513-517). Briefly, infected macrophage cultures were fixed in a mixture of 2.5% paraformaldehyde, 5% glutaraldehyde, and 0.03% trinitrophenol in 0.1M cacodylate buffer (pH 7.4) and postfixed in 1% osmium tetroxide in 1.5% potassium ferrocyanide. After block staining in 1% uranyl acetate in maleate buffer (pH 5.2), cells were dehydrated in a graded series of ethanol and propylene oxide and embedded in Poly/Bed 812 (Polysciences, Inc., Warrington, Pa.). Thin sections (60 to 90 nm) were cut and stained with uranyl acetate and lead citrate and examined with a JEM 100 CXII electron microscope. 
     RESULTS 
     Cultivation of E. canis 
     Ehrlichia canis (Oklahoma isolate) morulae were first observed in the primary canine macrophage cells 30 days, and 3 blind passages, after the addition of the leukocyte rich plasma from the carrier dog. Similarly, morulae were observed in the continuous cell line 24 days after the first addition of infected supernatant. By day 29, 60% of the DH82 cells were infected and by day 37, 100% of the cells contained morulae. The supernatant was then used to infect normal DH82 cultures established 4 days previously. Also, infected cultures were split 1:4 and layered onto normal DH82 cells, or cultures that were 30-40% infected were split 1:2. All of these methods were determined to be equally effective in perpetuating the growth of E. canis. 
     Serologic comparison of commercially prepared and DH82 E. canis antigen slides 
     The IFA results for 12 of the 15 sera were identical on both the commercial antigen slides prepared with the Florida isolate and the DH82 slides prepared with the Oklahoma isolate. Sera from one additional patient was twofold higher on the commercial slide. The remaining two sera were an acute and a convalescent sample from the same patient. The acute sample was seronegative on the DH82 slide and positive at a titer of 256 on the commercial slide. The results on the convalescent sample were positive on both slides. However, the convalescent result on the commercial slide was sixfold higher than on the DH82 slide (Table I). 
     Since all previous serologic tests for human ehrlichiosis were performed on the commercial slides with the Florida isolate, the sensitivity and specificity of the DH82 slides with the Oklahoma isolate were calculated based upon this standard. The specificity of the DH82 slides was determined to be 100%, while the sensitivity was about 87.5%. 
     In summary, the results presented herein clearly show that E. canis can be continually propagated in DH82 cell line in vitro in a suitable growth medium. 
     Other bacteria such as E. risticil, E. sennetsu, E. phagocytophila, and Neorickettsia helminthoeca are similarly propagatable in the DH82 cell line. 
     A deposit of E. canis infected and uninfected DH82 cell lines has been made at the ATCC. Rockville, Md. on Mar. 20, 1990 under the accession numbers CRL 10390 and CRL 10389, respectively. The deposit shall be viably maintained, replacing if it becomes non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and upon issuance of the patent made available to the public without restriction in accordance with the provisions of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit. 
     It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. 
     
                       TABLE I______________________________________Indirect fluorescent antibody titers of 15 human sera testedwith the Oklahoma and Florida E. canis isolates from DH82and commercially prepared cells, respectively.Serum #   DH82/Oklahoma                  Commercial/Florida______________________________________1          64           1282         &lt;64          &lt;643         &lt;64          &lt;644         &lt;64          &lt;645         &lt;64          &lt;646          512         40967         &lt;64          &lt;648         &lt;64           2569          512          51210         256          25611        &lt;64          &lt;6412        &lt;64          &lt;6413         256          25614        8192         819215        2048         2048______________________________________