Isolation and Continuous Culture of Neorickettsia helminthoeca in a Macrophage Cell Linet

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1991, p Vol. 29, No /91/ $02.00/0 Copyright C) 1991, American Society for Microbiology Isolation and Continuous Culture of Neorickettsia helminthoeca in a Macrophage Cell Linet YASUKO RIKIHISA,l* HAROLD STILLS,2 AND GARY ZIMMERMAN3 Department of Veterinary Pathobiology' and Department of Veterinary Preventive Medicine,2 College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210, and College of Veterinary Medicine, Oregon State University, Corvallis, Oregon Received 28 January 1991/Accepted 11 June 1991 Experimental evidence is presented supporting the development of a system for the isolation and propagation of a Neorickettsia sp. in a continuous canine macrophage cell line (DH82). To isolate a Neorickettsia sp. pathogenic to the canine species, three naive dogs were fed metacercaria-encysted kidneys of salmon caught in a river where infection of metacercariae with Neorickettsia helminthoeca has been circumstantially known for decades. Clinically, the classic course of salmon poisoning disease developed in all of the dogs. Parasitemia began on day 8 to 11 postinfection, when the dogs developed a febrile peak, and continued until euthanasia. At necropsy, characteristic gross and microscopic lesions of the disease were present. A Neorickettsia sp. was also isolated from liver and spleen samples of these animals. The isolates have been continuously propagated and passed in DH82 cells for more than 6 months. Electron microscopic examination confirmed that the rickettsial organisms multiplied in the membrane-bound compartment of DH82 cells and that they morphologically closely resembled rickettsia belonging to the genus Ehrlichia. An indirect fluorescent antibody test using Neorickettsia organisms cultured in DH82 cells showed that all dogs seroconverted 13 to 15 days postinfection. Finally, inoculation of the cell-cultured Neorickettsia organisms into a naive dog reproduced clinically typical salmon poisoning disease which was of greater severity and had a more rapid time course than that in the dogs from which the original isolation was made. On the basis of the clinical and pathologic responses of the dogs in our study, we believe that virulent N. helminthoeca was isolated and cultured in a continuous cell line. Neorickettsia helminthoeca is a minute, intracytoplasmic, rickettsial organism found in macrophages of Canidae with salmon poisoning disease. The organism varies from coccoid to crescentic, is nonmotile, stains readily with Giemsa stain, and is gram negative (5, 8, 11). N. helminthoeca is classified in the tribe Ehrlichieae in the latest Bergey's Manual of Systematic Bacteriology (11). The disease was recognized in the early 19th century by white settlers in the Pacific Northwest and named salmon poisoning disease because dogs became sick after eating salmon (8). N. helminthoeca is a rickettsia parasitic to a fluke, Nanophyetus salmincola, which has a complicated life cycle involving both snails (Oxytrema silicula) and salmonid fish as intermediate hosts and carnivores as the final host. Infection of dogs with N. helminthoeca occurs following ingestion of salmonid fish which harbor metacercariae containing Neorickettsia organisms (5, 8). N. helminthoeca is the first and only obligatory helminth-borne pathogen that has been discovered in the bacterial field (8). Nanophyetus salmincola harbors Neorickettsia organisms throughout its life cycle stages from egg to adult (5, 8, 11). The evidence for residence of N. helminthoeca in Nanophyetus salmincola is, however, based entirely on transmission experiments, and the organism has not been directly confirmed by either culture or microscopic evaluation in any developmental stages of Nanophyetus salmincola. Salmon poisoning disease in dogs is characterized by fever, depression, anorexia, weight loss, dehydration, vomiting, and watery, often bloody diarrhea. Mortality exceeds * Corresponding author. t Oregon Agricultural Experiment Station technical paper no % in untreated cases (2, 5, 8). Intestinal hemorrhage and inflammation are the most characteristic lesions and are more severe than those caused by the fluke alone (2, 5, 8). Generalized lymph node enlargement due to marked infiltration of macrophages is accompanied by severe depletion of small lymphocytes and a loss of germinal centers (2, 4, 5, 8). Splenic follicular central hemorrhage and necrosis and obliteration of thymic architecture by macrophage infiltration are common (4, 5). Coccoid bodies are found in the macrophages in most of the lymph nodes by both light and electron microscopy (4, 5, 8). Although the disease can be transmitted by injection of the blood or lymph node homogenate from infected to susceptible dogs, the organism has never been seen in blood smears (8). N. helminthoeca has not been grown in ordinary bacteriologic media, yolk sac, fibroblasts, or small laboratory rodents. Only two studies are known that describe in vitro culture of the Neorickettsia sp. (1, 3). The organism was isolated from leukocyte-enriched plasma of experimentally infected dogs at the acute stage of infection. The organism 1928 was maintained for up to 2 months by weekly passage in primary canine blood monocyte cultures in 2 ml of L-15 medium containing 40% canine serum (3) or in Eagle's minimal essential medium containing 20% canine serum and 0.1 mm L-glutamine (1). To the authors' knowledge, however, Neorickettsia cultures do not currently exist in any laboratory and the organism has never been cultured in a continuous cell line. Difficulties in culturing a sufficient quantity of this organism by using primary canine blood monocyte cultures and media containing canine serum have therefore limited studies on salmon poisoning disease and Neorickettsia species to the clinical, gross, and histopathologic levels. The antigenic, biochemical, molecular aspects of Neorickettsia spp. and the disease pathogenesis are

2 VOL. 29, 1991 ISOLATION AND CONTINUOUS CULTURE OF N. HELMINTHOECA 1929 unknown. Furthermore, there are currently no antigens readily available to serodiagnose Neorickettsia infection. The purpose of this study was to isolate and culture Neorickettsia organisms in an established canine macrophage cell line. Since no method for detection of Neorickettsia infection in the fluke has been described and our purpose was to isolate virulent N. helminthoeca, we fed dogs with metacercaria-encysted salmon caught in a river where infection of metacercariae with Neorickettsia spp. had been circumstantially known for decades. This study reports the isolation and cultivation of Neorickettsia organisms in a continuous cell line from the blood, liver, and spleen of dogs exhibiting classical clinical signs of salmon poisoning disease and characteristic gross and microscopic lesions at necropsy. The time course and severity of parasitemia and antibody titers are for the first time documented. Lastly, the reproduction of the disease by using cell-cultured Neorickettsia organisms was documented. MATERIALS AND METHODS Dogs. Four conditioned adult female mongrel dogs were purchased from Butler Farms, Clyde, N.Y. Salmon poisoning disease has never been reported east of the Rockies or continental divide; therefore, dogs originating from New York could be considered free of previous exposure to the agent and thus appropriate experimental subjects. These dogs had been vaccinated for canine distemper, infectious canine hepatitis, leptospira, parainfluenza, parvovirus, canine adenovirus II, rabies, and bordetella. The dogs had tested negative for Brucella canis and Dirofilaria immitis and were treated with a series of antihelminthics consisting of ivermectin (Merck & Co., Inc., Rahway, N.J.), pyrantel pamoate (Strongid-T; Pfizer, Inc., New York, N.Y.), and praziquantel (Droncit; Haver; Mobay Corp., Shawnee, Kans.) 1 to 2 months before use. Source of infectious Neorickettsia organisms. Five 2- to 3-lb (ca to 1-kg) salmon from the Roaring River State Fish Hatchery, near Lacomb, Oreg., were obtained through the Oregon Department of Fisheries and Wildlife. In western Oregon, most salmonid fish of this size category are parasitized by Nanophyetus salmincola and carry the etiologic agent of the disease. Fluke metacercariae, presumed to be Nanophyetus salmincola, were visually identified (x 160 magnification) in kidney tissue prior to shipping the fish on ice, by express mail, to the primary author's laboratory. The kidneys were removed from the fish upon arrival, and the presence of viable metacercariae containing moving juvenile flukes within the cysts was again confirmed. Selection of kidney tissue was based on tendency of the metacercariae to be concentrated in kidney tissue of fish, the ease of recovering this tissue, and the willingness of dogs to ingest this organ. Infection of dogs. Three dogs were fed approximately one and a half fluke-infested salmon kidneys per dog. Rectal temperature, pulse rate, respiratory rate, appetite, attitude (depression), and consistency of feces (diarrhea with or without blood) were recorded daily. Approximately 10 ml of heparinized blood was obtained from the cephalic vein every other day to isolate neorickettsia from the blood. The dogs were euthanatized at the acute stage of infection, and the tissue samples from the mesenteric lymph node, liver, and spleen were aseptically resected for neorickettsia isolation. Tissue specimens from the major organs were fixed in 10% neutral buffered formalin and processed for light microscopic observation after hematoxylin and eosin staining. Lymph node samples were prepared for electron microscopy. Isolation and culture of Neorickettsia organisms. Heparinized blood samples (approximately 10 ml) from each dog were overlaid on 10 ml of Histopaque 1077 (Sigma Chemical Co., St. Louis, Mo.) and centrifuged at 800 x g for 15 min at room temperature. The interface containing leukocytes was collected and centrifuged at 1,000 x g for 5 min. The pellet was suspended in Hanks balanced salt solution and washed by centrifugation two further times. These dog peripheral blood leukocytes were overlaid on a semiconfluent monolayer of canine macrophage DH82 cells (12) in Dulbecco's modified minimum essential medium (GIBCO Laboratories, Grand Island, N.Y.) containing 10% fetal bovine serum (Flow Laboratories, Inc., McLean, Va.) and 2 mm L-glutamine (GIBCO) and cultured in 5% C02-95% air at 37 C. The original seed of DH82 cells had been kindly provided by M. Wellman at The Ohio State University, and a relatively more adherent and larger cell population was selected after several passages in the senior author's laboratory before being used for this study. The liver, spleen, and mesenteric lymph node samples were immediately soaked in Hanks balanced salt solution after they were removed. These tissues were smashed with the blunt end of a 10-ml syringe through a Cellector (Thomas Scientific, Swedesboro, N.J.) to release the cells. The cell suspension was overlaid on Histopaque 1077, and the interface was collected, washed, overlaid on a monolayer of DH82 cells and cultured at 37 C in 5% C02-95% air. DH82 cells were dissociated from the bottom of the flask by tapping the flask, and an aliquot (about 0.2 ml) was centrifuged onto a glass slide in a Cytospin 2 cytocentrifuge (Shandon, Inc., Pittsburgh, Pa.) every 2 days. The cells were stained with Diff-Quik stain (Baxter Scientific Products, Obetz, Ohio) and examined for the presence of coccobacillary bodies by light microscopy. Positive cultures were confirmed by indirect fluorescent antibody (IFA) labeling using known positive canine anti-n. helminthoeca serum. These cultures were weekly passed for up to 1 month and 6 months for microorganism-negative and microorganism-positive cultures, respectively. IFA tests of the dog sera. The cells infected with Neorickettsia organisms (90% infection) were suspended in Hanks balanced salt solution and were air dried and acetone fixed onto Teflon (E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.)-coated 12-well slides (Cell-Line Associates, Newfield, N.J.) at 104 cells per well. Serial twofold dilutions of test sera in phosphate-buffered saline, ph 7.4 (PBS), starting at a 1:20 dilution, were made in 96-well microtiter plates, and a 100-,ul volume and 10-,lI volume of each dilution were applied to a well of antigen-coated IFA slides. The slides were incubated in a humidified 37 C incubator for 30 min and rinsed three times in PBS containing 0.002% Tween 20. After the slides were blotted to near dryness, 10 pal of fluorescein-conjugated goat anti-dog immunoglobulin (United States Biochemical Corp., Cleveland, Ohio) diluted at 1:200 in PBS was added to each well. The slides were incubated, washed, and counterstained with Evans blue (Sigma Chemical Co.), and a small drop of mounting fluid (70% glycerol in PBS) was placed in each well. Coverslips were added, and the slides were examined with a fluorescence microscope. Electron microscopy. Infected DH82 cells and lymph node specimens of infected dogs were fixed in a mixture of 2.5% paraformaldehyde, 5% glutaraldehyde, and 0.03% trinitrophenol in 0.41 M cacodylate buffer (ph 7.4) and postfixed in

3 1930 RIKIHISA ET AL. J. CLIN. MICROBIOL. o 40.6 Dog 1 0 * Dog I 2 0~~~~~~~~~~~~~~ Dog 2 diarrhea Do _Dog 3 diarrhea djr Days Post Infection FIG. 1. Clinical signs of dogs infected with N. helminthoeca. Three dogs were infected with N. helminthoeca by feeding them portions of Nanophyetus salmincola metacercaria-infested salmon kidneys on day 0. Eu, euthanasia. 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 Philips 300 electron microscope. Inoculation of a dog with cell-cultured N. helminthoeca. One of four naive dogs described above was intravenously inoculated with 2 x 107 N. helminthoeca-infected DH82 cells. Clinical signs, parasitemia, and immunoglobulin antibody titer were monitored as described above. At necropsy, tissue specimens of the major organs were fixed for both light and electron microscopy as described above. RESULTS Clinical features of dogs. The clinical features of the experimentally infected dogs were consistent with those described previously for salmon poisoning disease (2, 5, 8). All infected dogs became febrile (39.8 to 40.9 C) beginning approximately 7 days after eating fluke-encysted kidney (postinfection) for a duration of approximately 5 days before their temperatures sharply dropped to below the normal level (Fig. 1). The dogs developed persistent depression, anorexia, and severe watery diarrhea beginning on days 8 (dog 3), 10 (dog 2), and 15 (dog 1) postinfection. Dehydration and weight loss occurred in all three dogs, and two of the dogs had a thick yellowish conjunctival discharge and serous nasal discharge. Necropsy lesions. The pathologic changes observed in experimentally infected dogs were compatible with those previously reported (2-4, 8). Briefly, gross necropsy lesions included enlarged lymph nodes, especially mesenteric lymph nodes, and splenomegaly. Microscopic lesions included hyperplasia of lymph node reticuloendothelial cells with smalllymphocyte depletion. Intestinal lesions were limited to lymphoid hyperplasia, macrophage infiltration of the lamina propria, and the presence of Nanophyetus salmincola in the small intestine of two of the dogs. Microscopically, blue- and lilac-stained small cocci were observed in the smears of the homogenate of the mesenteric lymph nodes of dogs 2 and 3 after Diff-Quik staining, as well by IFA labeling and by electron microscopy. In other tissues stained with Diff-Quik, microorganisms were not identifiable. Isolation of Neorickettsia organisms. Neorickettsia organisms were consistently isolated and continuously propagated in DH82 cells from the blood monocytes of all three dogs. Light microscopic examination of Diff-Quik-stained blood films consistently failed to detect Neorickettsia organisms in monocytes. If we define parasitemia as positive isolation and culture of the organism from the blood by using this culture system within 30 days of in vitro culture, two dogs became parasitemic on day 8 postinfection, with the remaining dog becoming parasitemic on day 11 postinfection (Fig. 2). Detectable parasitemia persisted until euthanasia. Incubation periods before the DH82 cell cultures were identified as positive decreased as the disease progressed. Cocultivation of monocyte-rich fractions of lymph nodes, spleen, and liver samples with DH82 cells resulted in Neorickettsia isolation. Once isolated, Neorickettsia organisms from the liver and spleen but not from the lymph node continuously multiplied in DH82 cells (Table 1). No viruses, other bacteria, or parasites were detected in any cell-cultured specimens or light and electron microscope specimens. Morphology of Neorickettsia organisms in culture. Neorickettsia organisms were distributed in DH82 cells as individual cocci as well as morulae of various sizes, and more than 100 organisms were often found per cell (Fig. 3). The presence of intact organisms was confirmed by electron microscopy. By electron microscopy, the organisms were very tightly enveloped by the host membrane (Fig. 4). Two layers (outer and inner) of membrane, ribosomes, and fine DNA strands were evident in the organism (Fig. 4). There was no thickening of the inner or outer leaflet of the outer membrane (Fig. 4). IFA titration of dog sera. All three dogs were seronegative at the time of infection (titer, <1:20) and showed a greater TABLE 1. Organ Isolation of Neorickettsia organisms from organs of dogs orally infected with metacercaria Culture result (day)" for dog no.: Lymph node - (0) + (0-1) + (0-8) - (after 3)b - (after 10)b Liver + (after 17) + (after 14) + (after 26) Spleen + (after 10) + (after 7) + (after 23) ' Days in culture after euthanasia; day 0, day of euthanasia. b Neorickettsia organisms died.

4 VOL. 29, 1991 ISOLATION AND CONTINUOUS CULTURE OF N. HELMINTHOECA 1931 C = 2 0._ 0 0 o as C Do 0zZ 30 o Neg Days Post Infection FIG. 2. Neorickettsia isolation from the peripheral blood of three dogs infected with N. helminthoeca. Blood was collected every other day from each dog, and monocyte fractions were overlaid on DH82 cell monolayers. The infectivity of cultured cells was evaluated every other day after staining cytospin-prepared cells with Diff-Quik. For each blood monocyte culture from each dog, the first day when the culture became clearly positive with Neorickettsia infection was recorded. The culture period (days) required for positive Neorickettsia identification for each blood collection is indicated on the vertical axis. than fourfold rise in immunoglobulin G IFA titer (1:160 to 1:1,280) at day 13 to 15 postinfection (Fig. 5). Reproduction of the disease with cell-cultured Neorickettsia organisms. A dog inoculated with cell-cultured N. helminthoeca developed severe clinical signs of salmon poisoning disease. The dog became febrile beginning 3 days after inoculation. Neorickettsia organisms were reisolated from the blood of this dog starting at day 2 postinoculation. Necropsy lesions were consistent with, but much more severe than, those seen previously, including generalized lymph adenopathy, hemorrhages in the small intestinal mucosa, and hyperemia in the brain. DISCUSSION This study demonstrated that N. helminthoeca can be isolated from dogs exhibiting the clinical signs of salmon poisoning disease and continuously propagated in a canine macrophage cell line. Clinical signs and gross and histopathologic lesions were consistent with previous reports (2, Downloaded from on November 7, 2018 by guest FIG. 3. Light micrograph of N. helminthoeca-infected DH82 cells. The organisms are seen in the cytoplasm as small morulae (arrows), as individual organisms (arrowheads), and outside the host cells. Diff-Quik stain. Magnification, x1,500. Bar = 10,um.

5 1932 RIKIHISA ET AL. J. CLIN. MICROBIOL. Downloaded from FIG. 4. Electron micrograph of N. helminthoeca-infected DH82 cells. Note N. helminthoeca tightly enveloped by the host membrane (arrowheads). Fine DNA strands and ribosomes (R) are evident in the organisms. Magnification, x44,000. Bar = 0.1.m. 4, 8). Inoculation of cell-cultured Neorickettsia organisms reproduced severe disease in a dog, and Neorickettsia organisms were reisolated from this dog after the development of the clinical signs. N. helminthoeca was successfully isolated from the blood, liver, and spleen. Parasitemia was detected a few days after fever began and continued after the body temperature had dropped to below normal. It is not known for how long parasitemia persists. In nature, however, the disease is not transmitted via blood (8). While lymph node tissues have been used to reproduce the disease in the laboratory (1, 3) and cocci were evident in the lymph nodes, as previously reported (5, 8), the organisms eventually disappeared from the culture, probably because of activation of DH82 cells by lymphokines produced by the cells in the hypertrophied lymph nodes. We have shown that gamma interferon can eliminate Ehrlichia cells in murine macrophages infected with Ehrlichia risticii (7). At the light microscopic level, Giemsa-stained Neorickettsia organisms resemble Ehrlichia organisms (1, 3, 8-10). The morulae of Neorickettsia organisms are a smaller size and less compact than those of Ehrlichia canis (11), while the on November 7, 2018 by guest 1280 L- '0 a Neg DAYS POST INFECTION FIG. 5. IFA titers of dogs infected with N. helminthoeca.

6 VOL. 29, 1991 ISOLATION AND CONTINUOUS CULTURE OF N. HELMINTHOECA 1933 individual forms are similar to those of E. risticii (9, 10). A lack of antigenic cross-reactivity, however, has been previously reported between a Neorickettsia sp. and Ehrlichia sennetsu (6) and E. canis (1) by direct fluorescent antibody testing. These results should be reexamined with tissuecultured Neorickettsia organisms and specific antisera with a higher titer, since one study used infected lymph node tissue as the antigen and antisera with a titer of less than 1:32 (6). In the other study, the antibody titer was not reported (used at 1:3 dilution) (1). By using tissue-cultured Neorickettsia organisms as the antigen, an IFA technique was developed and temporal development of immunoglobulin G antibody against a Neorickettsia sp. was determined. The dogs seroconverted 13 to 15 days after infection. Thus, Neorickettsia organisms cultured in DH82 cells may be useful for serodiagnosis and serological determination of the disease distribution. Cell-cultured Neorickettsia organisms induced disease with much greater severity than that caused by the naturally infected fluke. Very interestingly, intravenous inoculation of Neorickettsia organisms induced severe hemorrhages throughout the small intestine. Thus, the intestinal lesions are produced not because of the natural portal of entry of Neorickettsia organisms, i.e., the oral route, but probably because of the intestinal tissue trophism of Neorickettsia organisms. In addition to N. helminthoeca, Neorickettsia elokominica, or Elokomin fluke fever agent, has been described previously (3-5). Both organisms have been cultured in primary dog blood monocytes by continuous passages for up to 2 months (3), and no morphological differences have been noted (3) at either the light or electron microscopic level. The organisms were not cross protective in dogs (4), but by a direct fluorescent antibody test, slight cross-reaction was seen by using antisera at a 1:1 dilution (6). Without more data to judge by, some researchers believe that N. elokominica is an isolate or a strain of N. helminthoeca (5). Elokomin fluke fever was originally reported in dogs along the Elokomin River in the state of Washington (5). Clinical signs of N. elokominica in dogs are much milder than those of N. helminthoeca (4, 5). With N. helminthoeca infections, dogs develop a fever with more of a temperature spike, whereas Elokomin fluke fever produces a longer plateautype fever (5). The lymph node pathology is different between two Ehrlichia species. Follicles are active in N. elokominica infection, but in N. helminthoeca infection, follicles disappear accompanied by histiocytosis (4). On the basis of the severe clinical signs in our four dogs, sharp peak-type fever, lymph node histopathology, and the fact that the salmon had been caught in an area in the state of Oregon where Elokomin fluke fever disease has not been recognized, we believe that the organism we cultured is N. helminthoeca, not Elokomin fluke fever agent. Further isolations and studies at the antigenic and genetic levels may clarify the still-elusive agent, N. elokominica, in the future. The success in propagation in continuous cell line could lead to clarification of the phylogenetic relationship of Neorickettsia spp. to other members of the tribe Ehrlichieae and the family Rickettsiaceae in the future. In a practical aspect, in the future a vaccine may be developed for salmon poisoning disease by using a tissue-cultured organism. ACKNOWLEDGMENTS We thank James Laird and Holly Ferrell for technical support in culturing Neorickettsia organisms and Saverit Capuano and Thomas Philips for technical help in collecting blood from the dogs and keeping clinical records. We also appreciate the technical assistance of Kathy Dailey in sample collecting at necropsy. This study was supported in part by the State of Ohio Canine Research Grant. Oregon efforts were supported in part by funds from the Northwest Canine Research Foundation. REFERENCES 1. Brown, J. L., D. L. Huxsoll, M. Ristic, and P. K. Hildebrandt In vitro cultivation of Neorickettsia helminthoeca, the causative agent of salmon poisoning disease. Am. J. Vet. Res. 33: Cordy, D. R., and J. R. Gorham The pathology and etiology of salmon disease in the dog and fox. Am. J. Pathol. 26: Frank, D. W., T. C. McGuire, and J. R. Gorham Cultivation of two species of Neorickettsia in canine monocytes. J. Infect. Dis. 129: Frank, D. W., T. C. McGuire, J. R. Gorham, and R. K. 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