Brief communications


Acknowledgement. We thank the Diagnostic Virology Laboratory at the National Veterinary Services Laboratory, Ames, IA, for assistance provided.

Sources and manufacturers a. Kodak Surecello Chlamydia Test Kit, Clinical Products Division, Eastman Kodak, Rochester, NY. b. S/P Culturette, American Scientific Products, Division of Travenol Laboratories, McGraw Park, IL. c. Falcon 3047 Tissue culture plates, Curtin Matheson Scientific, Aurora, CO. d. Bellco Glass, Vineland, NJ. e. GIBCO Laboratories, Life Technologies, Grand Island, NY. f. CRL 1696 McCoy cells: American Type Culture Collection, Rockville, MD. g. National Veterinary Services Laboratory, Ames, IA.

References 1. Bovarnick MR, Miller JC, Snyder JC: 1950, The influence of certain salts, amino acids, sugars, and proteins on the stability of rickettsiae. J Bacterial 59:509-522. 2. Brown S, White J: 1982, Diagnosis of avian psittacosis using cell culture technique. Proc Annu Meet Am Assoc Vet Lab Diagn 25:151-158. 3. Hammerschlag MR, Gelling M, Roblin PM, et al.: 1990, Comparison of Kodak Surecell Chlamydia test kit with culture for the diagnosis of Chlamydial conjunctivitis in infants. J Clin Microbiol 28:1441-1442. 4. Kennedy GA: 1985, Laboratory diagnosis of Chlamydial diseases. Proc Annu Meet Am Assoc Vet Lab Diagn 28:421-436.

5. Kingston RS: 1989, Rapid detection of Chlamydia in pet birds and cats using the Kodak Surecell Chlamydia test kit. Proc Annu Meet Am Assoc Vet Lab Diagn 32:12. 6. Mahony JB, Chemesky MA: 1985, Effect of swab type and storage temperature on the isolation of Chlamydia trachomatis from clinical specimens. J Clin Microbiol 22:865-867. 7. Moore FM, McMillan MC, Petrak ML: 1991, Comparison of culture, peroxidase-antiperoxidase reaction, and latex agglutination methods for the diagnosis of chlamydiosis in pet birds. J Am Vet Med Assoc 199:71-73. 8. Moulder JW: 1991, Interaction of Chlamydiae and host cells in vitro. Microbiol Rev 55: 143-190. 9. Riggs JL: 1979, Immunofluorescent staining. In: Diagnostic procedures for viral, ricketsial, and Chlamydial infections, ed. Lennette EH, Schmidt NJ, 5th ed., pp. 141-170. American Public Health Association, Washington, DC. 10. Schachter JS: 1989, Reports from the symposium on avian chlamydiosis. J Am Vet Med Assoc 195: 1501-1576. 11. Schacter J, Dawson CR: 1979, Psittacosis-lymphogranuloma venerum agents. In: Diagnostic procedures for viral, ricketsial, and Chlamydial infections, ed. Lennette EH, Schmidt NJ, 5th ed., pp. 1021-1059. American Public Health Association, Washington, DC. 12. Spears P, Storz J: 1979, Chlamydia psittaci: growth characteristics and enumeration of serotypes 1 and 2 in cultured cells. J Infect Dis 140:959-967. 13. Storz J: 1971, Chlamydia and Chlamydial-induced diseases, 1st ed., pp. 79-88. Charles C Thomas, Springfield, IL. 14. Woods WW, Dotson JF, Castro AE: 1989, A rapid monoclonal immunofluorescence assay for Chlamydia psittaci in fecal smears from psittacine birds. J Vet Diagn Invest 1:150-153.

J Vet Diagn Invest 4:460-463 (1992)

Isolation of Chlamydia psittaci from pleural effusion in a dog Francisco Arizmendi, James E. Grimes, Roberta L. Relford Chlamydia psittaci is an obligate intracellular parasite that is responsible for producing various signs in domestic and wild mammals and birds. The organism can infect the respiratory, urogenital, and intestinal tracts and the central nervous system and is also responsible for feline conjuncti3,7,10,15,17,19 vitis and ovine and bovine polyarthritis. Diagnosis of C. psittaci infection is based on isolation of the organism, antigen detection and serologic testing, or preferably a combination of both. Isolation is accomplished by inoculation of chicken embryos, mice, guinea pigs, and cell cultures. 1,9,14,17 Serologic tests, such as complement fixation (CF) and microimmunoflourescence (IFA), and antigen detection tests, such as immunoperoxidase techniques and enzyme-linked immunosorbent assay (ELISA) are used to diagnose chlamydial infection in different species.2,5,7,8,11-13,16,18 From the Texas Veterinary Medical Diagnostic Laboratory, PO Drawer 3040, College Station, TX 77841-3040 (Arizmendi, Grimes), and the Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843 (Relford). Received for publication January 31, 1992.

In a natural Chlamydial infection in England,4 C. psittaci was isolated from feces of an adult dog that had eaten the carcasses of dead budgerigars from an aviary in which birds were diagnosed as positive for C. psittaci. That dog developed a CF titer of 128. In Germany, a Chlamydial agent was determined to be the cause of conjuctivitis in 1 dog,10 and antibodies against C. psittaci were detected by an ELISA in 20% of 1,127 serum samples from dogs. 18 In a seroepidemiological survey of C. psittaci in dogs and cats-in Japan, 34 of 359 dogs had CF titers ranging from 16 to 64.5 Chlamydiosis, as a naturally occurring infectious disease, has not been reported in the canine population of the United States. However, experimental evidence suggests that dogs are susceptible to C. psittaci. Clinical signs were induced in dogs inoculated parenterally with a Chlamydial agent of ovine polyarthritis. 11 The agent was reisolated from brain, liver, spleen, kidney, and lung tissue, the intestinal tract, and synovial fluid. The CF titer rose from < 4 to 256 at 9 days postinoculation. The antibody titer decreased to 16 by 1 year after the initial inoculation. Clinical responses, lesions, and frequency of Chlamydia reisolation from dogs inoculated with an ovine Chlamydia strain have also been studied.20 Focal

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Brief communications


Figure 1. Macrophage from pleural effusion of a dog contains intracytoplasmic inclusion (arrow) that resembles a Chlamydial inclusion. Modified Wright stain.

hepatitis, hyperplasia of spleen, leptomeningitis, and fibrinopurulent polyarthritis were found. Reisolation of the chlamydial agent was consistent from the liver and spleen but not consistent from other organs. Thoracic fluid collected by needle thoracentesis was submitted September 8, 1989, from a 5-month-old male Bull Mastiff dog that was presented to the Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University. The dog was referred to this clinic with a history of 5 days of pyrexia and shifting leg lameness. The material was submitted for Chlamydial culture because microscopic examination of the fluid by modified Wright stain revealed intracytoplasmic inclusions in mac-

rophage cells (Fig. 1). The fluid material, which was opaque and reddish in color, was mixed with Chlamydia transport medium in a ratio of 2:3 and kept at 4 C overnight. Cultures of mouse fibroblast (clone L-929) cell monolayers on coverslip, in 24-well plates were used for the isolation attempt.1,9 One milliliter of diluted thoracic fluid was used to inoculate each of 4 wells, and the plate was centrifuged at 1,000 x g for 30 minutes. After centrifugation, the plate was incubated stationary at 37 C for 3 hours, at which time excess inoculum was removed and replaced with cell growth medium. The plate was then incubated at 37 C for 72 hours. In addition, 0.25 ml of undiluted thoracic fluid was inoculated into the yolk sac of each of 5 7-day-old chicken embryos as previously

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Brief communications

Figure 2. Chlamydial microcolony (arrow) in L-929 fibroblasts at 72 hours postinoculation of cell culture with pleural effusion from a dog. Gimenez stain, 10,000 x .

described.9 Microscopic examination for Chlamydial inclusions in cell cultures and embryo yolk sacs was done following staining of cell culture monolayer and yolk sac impressions by the Gimenez method.6 Stained material was examined with a light microscope using a 100 x oil objective. A second sample of thoracic fluid was received on November 6. It was pinkish rather than red and dark, as was the first sample. The material was inoculated into cell culture but not into chicken embryos. Serum samples for Chlamydia CF test were received on September 8 and 19 and November 6, 1989, and April 10, 1990. The sera received on November 6 and April 10 were also tested by IFA, as previously described.13 At the time serum samples were taken, the health condition of the dog and how it responded to antibiotic therapy was different. The initial treatment consisted of ampicillin and trimethroprin sulfadiazine; a thoracostomy tube was placed and pleural fluid was removed. The dog responded dramatically within 12 hours after prednisolone sodium succinate and butorphanol were administered. Two days after the thoracic drain was removed, the dog became febrile and swelling of the right metacarpal joint was noticed. On day 12 of hospitalization, the dog relapsed with pleural effusion. At this time, all attempts to culture fungi and bacteria in pleural fluid were negative. After removing thoracic fluid, response to therapy was minimal, but after a single injection of dexamethasone was administered, marked improvement occurred within 24

hours and ampicillin and oral prednisolone were continued. Seven days after corticosteroid therapy was initiated, the dog was dismissed with continued prednisolone and ampicillin. The dog has remained clinically asymptomatic for 19 months after complete cessation of prednisolone. Chlamydiae were isolated from the first thoracic fluid sample in cell culture after 72 hours of incubation (Fig. 2). The chicken embryos did not die after 6 days of incubation; however, impressions made from the yolk sacs contained chlamydial inclusions. A blind passage was made into a second set of chicken embryos, and on the fifth day of incubation all embryos were dead and the yolk sacs were positive for elementary bodies. Acetone-fixed cell culture monolayers infected with the dog isolate were stained with a specific antiC. pneumoniae fluorescent antibody conjugate and were negative. However, a genus-specific fluorescent antibody stain reacted positively. The dog isolate also was identified by monoclonal antibodies as a virulent “turkey” C. psittaci. The second thoracic fluid sample was negative for Chlamydiae. The Chlamydial CF antibody titer (Table 1) rose from 8 to 128 and then decreased 8-fold to 16, indicating that a Chlamydial infection and apparent recovery from it had occurred. The IFA titers on sera collected on November 6, 1989, and April 10, 1990, were 20 and 10, respectively. Note that the highest CF and IFA titer occurred in the same serum sample. Natural Chlamydial infection in a dog that was exposed to

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Brief communications Table 1. Results of Chlamydia isolation attempts from thoracic fluids and Chlamydial direct complement fixation (DCF) and indirect fluorescent antibody (IFA) titers in sera of a naturally infected dog.

a flock of budgerigars with psittacosis4 and results from dogs experimentally inoculated with an ovine Chlamydial agent 11,20 have demonstrated that dogs are susceptible to Chlamydia. CF titers found in naturally and experimentally infected dogs and those in seroepidemiologic studies4,5,7,11,18 are evidence of Chlamydial infection occurring naturally in dogs. The isolation of C. psittaci, the rise and decline of CF titer, and the positive IFA results are incontrovertible evidence of the chlamydial infection in the dog in this report. The source of the Chlamydiae that infected this dog can only be inferred. The isolate was identified by monoclonal antibody as a virulent “turkey” strain, indicating only that it most likely came from an avian host. Its growth characteristics, rate of multiplication, cytopathogenicity, and inclusion size and compactness did not resemble that of the common laboratory turkey strain. The owner has denied any contact of the dog with shorebirds or seabirds or contact with or consumption of uncooked poultry. The main contact with birds was in the wooded backyard in which various small free-flying birds had been seen. Inca doves, which nested in the yard, have been shown experimentally to be capable of shedding Chlamydiae that could be transmitted to turkeys? However, there are many other potential avian vectors. A presumptive diagnosis of a Chlamydial infection may be made by serologic testing or recognition of intracytoplasmic inclusions in stained impressions of tissues or smears of exudates. A definitive diagnosis is made by isolation of the agent in a laboratory host system. Such a diagnosis should perhaps be considered more often in dogs with unusual respiratory syndromes. Moreover, the interest in testing dogs for C. psittaci will help to determine the incidence and pathogenicity of such infection in the canine population, which is now unknown. Acknowledgements. We thank Dr. Arthur A. Andersen, National Animal Diseases Center, Ames, IA, and Dr. C.-C. Kuo, School of Public Health and Community Medicine, University of Washington, Seattle, WA, for fluorescent antibody studies.


References 1. Allan I, Pearce JH: 1979, Modulation by centrifugation of cell susceptibility to Chlamydial infection. J Gen Microbiol 1:8792. 2. Evert KDE, Andersen AA, Plaunt M, Hatch T: 1991, Cloning and sequence analysis of the major outer membrane protein gene of Chlamydia psittaci 6BC. Infect Immun 59:2853-2855. 3. Fowler ME, Schulz T, Ardon A, et al.: 1990, Chlamydiosis in captive raptors. Avian Dis 341657-662. 4. Fraser G, Norval J, Withers AR, Gregor WW: 1969, A case history of psittacosis in the dog. Vet Rec 85:54-58. 5. Fukushi H, Ogawa N, Minamoto A, et al.: 1985, Seroepidemiological surveillance of Chlamydia psittaci in cats and dogs in Japan. Vet Rec 117:503-504. 6. Gimenez DF: 1964, Staining rickettsiae in yolk-sac cultures. Stain Technol 39: 135. 7. Green CE: 1990, Chlamydial infections. In: Infectious diseases of the dog and cat, pp. 443-445. WB Saunders Co., Philadelphia, PA. 8. Grimes JE, Owens KI, Singer JR: 1979, Experimental transmission of Chlamydia psittaci to turkeys from wild birds. Avian Dis 23:9 15-926. 9. Grimes JE, Daft BM, Grumles LC, et al.: 1986, A manual of methods for laboratory diagnosis of avian chlamydiosis, pp. 1114. American Association of Avian Pathologists, University of Pennsylvania, Kennett Square, PA. 10. Krauss H, Schmeer N, Wittenbrink MM: 1988, Significance of Chlamydia psittaci infections in animal in the F.R.G. Proc Eur Soc Chlamydia Res, Bologna, Italy, May 30-June 1, p. 65. 11. Maierhofer CA, Storz J: 1969, Clinical and serologic responses in dogs inoculated with the Chlamydial (psittacosis) agent of ovine polyarthritis. Am J Vet Res 30: 1961-1966. 12. Perez-Martinez JA, Storz J: 1985, Antigenic diversity of Chlamydia psittaci of mammalian origin determined by microimmunofluorescence. Infect Immun 50:905-910. 13. Richmond SJ, Caul EO: 1982, Antibodies to Chlamydia: a single antigen test. In: Immunofluorescent techniques in diagnostic microbiology. Public Health Laboratory Service (PHLS) Monograph Series; v 18. London, Her Majesty’s Stationary Office (HMSO), p. 77. 14. Robinson GW, Anderson IE: 1979, Isolation of Chlamydia psittaci ovis in sheep thyroid cell culture. Vet Rec 104:55-56. 15. Rude TA: 1986, Feline chlamydiosis. Feline Pract 16:31-35. 16. Ruppanner RD, et al.: 1984, Enzyme immunoassay of chlamydia in birds. Avian Dis 28:608-615. 17. Storz, J: 1971, Chlamydia and Chlamydia-induced diseases, pp. 71-88. Charles C Thomas, Springfield, IL. 18. Werth D, Schmeer N, Muller H, et al.: 1987, Detection of antibodies to Chlamydia psittaci and Coxiellla burnetii in dogs and cats: comparison between enzyme-linked immunosorbent assay, immunoperoxidase-technique, complement fixation text and immunodiffusion test. Zentralbl Veterinaermed B 34: 165176. 19. Wyrich PB, Richmond J: 1989, Biology of Chlamydiae. J Am Vet Med Assoc 195:1507-1512. 20. Young S, Storz J, Maierhofer CA: 1972, Pathologic features of experimentally induced Chlamydial infection in dogs. Am J Vet Res 33:377-383.

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Isolation of Chlamydia psittaci from pleural effusion in a dog.

Brief communications 460 Acknowledgement. We thank the Diagnostic Virology Laboratory at the National Veterinary Services Laboratory, Ames, IA, for...
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