Vol. 41, No. 2INFECTION AND IMMUNITY, Aug. 1983, P. 789-7940019-9567/83/080789-06$02.00/0Copyright X) 1983, American Society for Microbiology

Isolation of Chlamydia psittaci from Naturally InfectedAfrican Clawed Frogs (Xenopus laevis)

BURTON W. WILCKE, JR.,'* CHRISTIAN E. NEWCOMER,2t MIRIAM R. ANVER 2t JERRY L.SIMMONS,3§ AND GEORGE W. NACE4

Division of Virology, Michigan Department of Public Health, Lansing, Michigan 489091; Unit for LaboratoryAnimal Medicine, School of Medicine,2 and Department of Biology,4 University of Michigan, Ann Arbor,Michigan 48109; and Department of Pathology, Veterans Administration Hospital, Ann Arbor, Michigan

481043

Received 8 March 1983/Accepted 10 May 1983

An inclusion-forming agent was isolated from the livers of commercially raisedAfrican clawed frogs (Xenopus laevis) involved in an epizootic of high morbidityand mortality. Original isolation was made in McCoy cells. This agent wasidentified as Chlamydia psittaci based on the formation of typical intracytoplas-mic inclusions which developed within 48 h, were not stained by iodine, and wereresistant to sulfadiazine. The isolate from one particular frog (designated as strain178) was further studied and found to be lethal for 7-day-old embryonated chickeneggs after intra-yolk sac inoculation. This strain was demonstrated not to bepathogenic for mice when inoculated intraperitoneally. The cell culture isolate ofC. psittaci was transmitted to uninfected X. laevis, causing disease and death.

The genus Chlamydia consists of two species,Chlamydia trachomatis and Chlamydia psittaci(6). C. trachomatis is well known as a humanpathogen and has been associated with a varietyof ocular, urogenital, and respiratory infectionsin humans. Among these are trachoma, inclu-sion conjunctivitis, lymphogranuloma venere-um, urethritis, cervicitis, and pneumonia of in-fants (7, 8). Various strains of C. psittaci havebeen isolated from and implicated as pathogensin many different species of wild and domesticbirds and mammals. The disease spectrum of C.psittaci in animals is large and includes conjunc-tivitis, polyarthritis, enteritis, endometritis, pla-centitis, encephalitis, and pneumonitis (12). C.psittaci is capable of causing severe pneumoniaor systemic nonrespiratory infections in humans(8).The extent to which C. psittaci is associated

with disease in animals other than birds andmammals has not been determined. C. psittacihas been isolated from various naturally andexperimentally infected ectoparasites, but therole of the ectoparasites, if any, in the transmis-sion of disease has not been well established (7,12). Although C. psittaci has been successfully

t Present address: Division of Comparative Medicine, Mas-sachusetts Institute of Technology, Cambridge, MA 02139.

t Present address: Clement Associates, 1010 WisconsinAve., N.W., Washington, DC 20007.

§ Present address: Laboratory Services, Veterans Adminis-tration Medical Center, Sioux Falls, SD 57105.

grown in cell cultures from cold-blooded animalssuch as the tortoise (9, 10), there have been noreports of C. psittaci isolated as the causativeagent of a naturally occurring disease in cold-blooded animals.

(These results were presented at the 21stInterscience Conference on AntimicrobialAgents and Chemotherapy [Program Abstr. In-tersci. Conf. Antimicrob. Agents. Chemother.21st, Chicago, Ill., abstr. no. 864, 1981].)

MATERIALS AND METHODS

Frogs. The original six clinically ill African clawedfrogs (Xenopus laevis) were obtained from a commer-cial supplier whose colony of frogs was experiencingan epizootic. Healthy X. laevis, putatively Chlamydiafree, were obtained from the University of MichiganAmphibian Facility for subsequent Chlamydia trans-mission experiments. During experimental infections,the frogs were housed in polycarbonate cages (47.5 by26.3 by 20.0 cm) in ca. 10 cm of dechlorinated water.The water was changed three times weekly. Animalhousing conformed to the Centers for Disease Controland National Institutes of Health guidelines for han-dling animals infected with C. psittaci (1).

Necropsy and histopathology. The frogs were grosslyand microscopically examined after spontaneousdeath or after being killed with Metofane (Pitman-Moore, Washington Crossing, N.J.). Necropsy wasperformed shortly after death for those frogs that diedspontaneously or immediately after death for thosefrogs that were sacrificed. Livers, kidneys, hearts,lungs, and spleens were collected for histopathologicalexamination. Tissues were fixed in 10% buffered neu-tral Formalin, and 5-,um paraffin sections were made.

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TABLE 1. Necropsy of original six index cases of chlamydiosis in African clawed frogs (X. laei'is)Isolation of

Frog no. Age Sex Mode of death Gross appearance of liver C. psittacifrom liver

173 Adult ND` Spontanoeus Brown, mottled, friable Yes174 Adult F Spontaneous Gray-black, mottled, enlarged Yes175 Adult F Spontaneous Not remarkable Yes176 Adult F Sacrifice Not remarkable No177 Juvenile ND Sacrifice Dark brown, congested Yes178 Adult ND Sacrifice Gray-green, mottled Yesa ND, Not determined.

Staining was performed with hematoxylin and eosin,Brown and Brenn (tissue Gram stain), periodic acid-Schiff, or Giemsa stains.

Electron microscopy. Material was fixed in neutralbuffered Formalin, minced into 1-mm cubes, andplaced in phosphate-buffered 2% glutaraldehyde at 4°Cfor 1 h. The tissue was postfixed in 1% osmium,stained with uranyl acetate and lead citrate, and em-bedded in Epon for thin sectioning.

C. psiffaci isolation from index cases. Livers or kid-neys or both were collected for C. psittaci isolation.Tissues were collected aseptically and inoculatedwithin 2 to 4 h or stored frozen at -70°C untilinoculation. Tissues were transported and held in asucrose-phosphate buffer, 2 SP (2). The tissue speci-mens were ground with a sterile mortar and pestle toachieve an approximate 10% (wt/vol) tissue suspen-sion in 2 SP. The suspensions were centrifuged brieflyat 800 rpm (170 x g) to remove large tissue fragmentsand debris. The supernatant was further diluted 1:10 in2 SP. The inocula (0.2 ml) from the 1:10 dilutions wereoverlaid onto cover slip monolayers of McCoy cells.The McCoy cells had been pretreated for 72 h with 5-iodo-2-deoxyuridine by the method of Wentworth andAlexander (13). After centrifugation for 1 h at 3,200rpm (2,500 x g), the cover slips were rinsed brieflywith calcium- and magnesium-free phosphate-bufferedsaline (pH 7.5), and freshly made tissue culture medi-um (Earle minimal essential medium [pH 7.2 to 7.4]with 2% fetal bovine serum, gentamicin [10 ,ug/ml],and amphotericin B [2 ,ug/ml]) was added. The inocu-lated monolayers were incubated for 48 to 72 h at 35 to37°C. After incubation, the cover slips were stainedwith Jones iodine or Giemsa stain. Quantitation ofinclusion bodies was made by averaging the number ofinclusions in 20 representative fields and multiplyingby the number of high-power fields per cover slip, i.e.,620. Quantitation was based on triplicate or quadrupli-cate cover slips.

Characterization of isolates. The isolates from X.Iaevis were characterized by staining reactions (Jonesiodine versus Giemsa stain), the morphology of theinclusions, the susceptibility to 25 ,i.g of sulfadiazineper ml, (Lederle Laboratories, Pearl River, N.Y.), andthe effect of centrifugation on inclusion body forma-tion.

Experimental infection in frogs. Liver suspensionsfrom two index cases of chlamydiosis were inoculatedinto the dorsal lymph sac offour healthy X. Iaevis, andthe presence of inclusion bodies was determined byhistological examination. Liver homogenate from nor-mal healthy X. Iaevis was inoculated as a control.

Further studies were performed with one particular

isolate of C. psittaci from index case number 178. Thisisolate (strain 178) was first obtained in the McCoy cellisolation system and passed in the same system. Asuspension of strain 178 passage material was seriallydiluted in 10-fold dilutions to 106, and 0.2 ml of eachdilution was inoculated into the dorsal lymph sac ofhealthy frogs (six frogs per dilution). As a control, anundiluted suspension of McCoy cells was inoculatedinto six healthy frogs (0.2 ml per frog). All frogs weremonitored for 3 weeks. Necropsies were performed onfrogs that died spontaneously, and liver and kidneyspecimens were aseptically removed for reisolationattempts. At 3 weeks, all frogs still living were killed,and necropsies were performed as described above.Reisolation attempts for C. psittaci were made inMcCoy cells. Serum specimens from infected andcontrol frogs were collected for subsequent testing.Mouse inoculation studies. Mice used were of an

outbred stock of white mice designated Mdh (Car-worth Farms Webster) and were obtained from R.Myers of the Animal Care Section, Michigan Depart-ment of Public Health. The colony was predeterminedto be Chlamydia free by serological testing of sentinelmice by immune adherence hemagglutination (4). Serial10-fold dilutions of C. psittaci 178 were made (undilut-ed through 106, and 0.5 ml of each dilution wasinoculated intraperitoneally into six mice. UndilutedMcCoy cells and a known C. trachomatis strain (strainG29 [E/UW-17/Cx]; originally obtained from E. R.Alexander, University of Washington) were used ascontrols.After 12 days of observation, the mice were anesthe-

tized with Metofane, exsanguinated, and necropsied.The sera obtained from these mice were tested byimmune adherence hemagglutination against a Chla-mydia group antigen (obtained from the Centers forDisease Control). Sections of livers, spleens, andkidneys were collected and pooled for C. psittaciisolation attempts with the McCoy cell system asdescribed above.Embryonated chicken egg inoculation. The yolk sacs

of 7-day-old embryonated chicken eggs were inoculat-ed (0.2 ml per egg) with ca. 102 to 103 inclusion-forming units (IFU) of strain 178, and 0.2 ml of 2 SPwas inoculated into control eggs. The yolk was collect-ed after the embryos had died, and titration of C.psittaci was carried out in the McCoy cell system.

Direct fluorescent antibody detection of Chlamydia.The identity of strain 178 as Chlamydia was furtherconfirmed by using a fluorescein-labeled conjugateobtained from Bartels Immunodiagnostics, Bellevue,Wash. The conjugate used does not distinguish be-tween C. trachomatis and C. psittaci.

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CHLAMYDIA PSITTACI FROM AFRICAN CLAWED FROGS

RESULTS

Isolation from index cases. The necropsies on

the original six index cases are summarized inTable 1. The gross pathological observationswere unremarkable, with the exceptions notedpertaining to the appearance of the liver in fiveof the six cases. The histopathological descrip-tion of chlamydial lesions in these Africanclawed frogs has recently been published byNewcomer et al. (5). Case number 176 showedno remarkable gross pathological findings, andthe isolation attempts for C. psittaci were nega-tive in this particular frog. The other five frogsall had C. psittaci isolated from the liver tissuespecimens. Chlamydia-like inclusions were de-tected in the McCoy cell system. In each casethe inclusions were intracytoplasmic, non-iodinestaining, diffuse in character, and readily visibleat 48 h post-inoculation. All of these characteris-tics are typically those of C. psittaci inclusions.The average number of inclusions counted in theMcCoy cell monolayers indicated that the origi-nal liver specimens contained ca. 3.5 x 105 to7.0 x 105 IFU/g of liver tissue.

Characterization of strain 178. (i) Growth char-acteristics. C. psittaci 178 was isolated and pas-saced in McCnv cells- It was determined that

- F; t <

;4. 4

.~~~~~~

FIG. 2. Electron micrograph of strain 178 inKupffer cell of X. laevis demonstrating initial bodiesand an intermediate body. x35,000.

aCL,%, III IVA%--,V A-113o. IL WU

pretreatment with 5-iodo-2-not necessary for the growtladdition, centrifugation did

M.

t ;..A

t.

[.e.t~~A 'k

i.Z

FIG. 1. Inclusions of strain(Giemsa stained). x250.

deoxyuridine was necessary for infecting cell culture. Strain 178of this agent. In produces non-glycogen-containing inclusions, as

not appear to be demonstrated by the fact that the inclusionsstained only with Giesma stain and not iodine.Typical Giemsa-stained inclusions in McCoycells are shown in Fig. 1. Treatment of strain 178with 25 ,ug of sulfadiazine per ml had no effect onthe size or number of inclusions formed. Incontrast, a known strain of C. trachomatis wascompletely inhibited by the same concentrationof sulfadiazine.

(ii) Morphological characteristics. Electron mi-crographs demonstrated that the morphology ofisolate 178 in livers from experimentally infectedfrogs was characteristic of the Chlamydia genus.Two stages in the developmental cycle of the

9* Chlamydia genus, dividing initial bodies and*§ intermediate bodies, are shown in Fig. 2.

Isolate 178 was stained after passage in Mc-Coy cells by using a commercial fluorescein-labeled Chlamydia antibody prepared in goats(Bartels Immunodiagnostics). Both the frog iso-late (Fig. 3) and a control strain of C. trachoma-tis stained equally well with this conjugate. Theinclusions were easily detected within 48 h andshowed typical intracytoplasmic morphology.

(iii) Pathogenicity of strain 178. The cell cul-ture isolate from frogs was inoculated into nor-mal healthy frogs (X. laevis). The selection of a3-week cutoff period for this experiment wasbased on the previously observed morbidity and

178 in McCoy cells death of frogs inoculated with liver homogenatefrom C. psittaci-infected frogs. The results of

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792 WILCKE ET AL.

s D

.* bJ'&

< ......> . .'g; w .S.'.ffi:,' .s: v e;

t:. M e t

t ::r w

+.* . .f . -.S . '*Y

t Ah

FIG. 3. Inclusions of strain 178 in McCoy c

48 h (stained with fluorescein-labeled Chlamydibody). x400.

this experiment indicated that the 50%dose appears to be <10 IFU when inoculatthe dorsal lymph sac route (Table 2). Histcally, all groups of frogs inoculated with178 demonstrated the presence of inclusicliver, kidney, and/or spleen sections. Retion of C. psittaci was made from represenfrogs in the undiluted, 10-3, and 10-5 gr

One of the six frogs in the control group hpsittaci isolated from a pool of tissue collecnecropsy. This frog was not clinically ill,was assumed that, despite precautions tcross contamination from an infected froghave occurred late in the experimentalframe. Attempts to demonstrate a serol4conversion to the Chlamydia group antigthe experimentally infected frogs were ucessful because of the anticomplementary z

ty of the frog sera.Strain 178 was not pathogenic for mice v

intraperitoneal route when mice were inociwith as many as 106 IFU. None of theinoculated with strain 178 at this dosagedeveloped any overt sign of infection. Aftdays, the mice were sacrificed and exsangied, and necropsy specimens were takentempts to isolate C. psittaci from livers, spland kidneys were all negative. Controlinoculated with either C. trachomatis or Epension of McCoy cells were similarly negEvidence of infection was seen, howeveusing the immune adherence hemagglutirtest to compare preinoculation sera withtaken at death. The mice which received106 IFU of strain 178 demonstrated a rise itto the Chlamydia group antigen (Table 3)

mice inoculated with C. trachomatis did notshow such a rise.Approximately 102 to 103 IFU of strain 178

was inoculated into the yolk sacs of 7-day-oldembryonated chicken eggs (six eggs per group).One embryo from each group died within 3 days

:.'%%>. as a result of trauma. The remainder died at 7 or

8 days post-inoculation. All yolk sacs were

harvested at 8 days. All control eggs inoculatedwith 2 SP survived and yielded no C. psittaci.The infected eggs yielded .106 IFU of strain 178per ml of yolk when titrated in McCoy cells,indicating proliferation of the organisms in yolksacs.

DISCUSSION

We report here on the isolation of C. psittacifrom a naturally occurring infection of Africanclawed frogs (X. laevis). To our knowledge this

ells at is the first report of C. psittaci as a pathogen inSa anti- frogs. Although Chlamydia-like inclusions have

been histologically seen in mollusks (3) andother ectotherms (9, 10), we are unaware ofisolations being made from cold-blooded ani-

lethal mals.

ted by The C. psittaci isolate from X. laevis was

:ologi- typical in its inclusion morphology, stainingstrain characteristics, and resistance to sulfadiazine.)ns in Detection of inclusion bodies in cell cultures was,isola- best carried out with Giemsa stain or a fluores-tative cein-labeled anti-Chlamydia conjugate. It wasoups. possible, however, to see the inclusions oflad C. the isolate from frogs even with Jones iodineKtedat stain. The inclusions did not stain any darkerand it with iodine than did the cell nucleus, but thetaken, morphology of the inclusion was distinctive.

,must Although originally isolated in a 5-iodo-2-de-time oxyuridine-treated McCoy cell system by cen-

ogical trifugation, it was subsequently determined;en in that neither 5-iodo-2-deoxyuridine treatment norinsuc- centrifugation was necessary for isolation inactivi- McCoy cells. The electron microscopic exami-

nation of this organism further confirmed its-athe identity as a typical Chlamydia strain whichulatedmicelevel

ter 12uinat-i. At-leens,mice

a sus-

,ative.r, byiationl sera104 ton titerI. The

TABLE 2. Experimental infection of healthy frogs(X. Iaevis) with strain 178 of C. psittaci

Dilution Inoculum size No. of frogs dead/inoculated (IFU) total no. of frogs

inoculated

Undiluted 104 6/610-1 103 6/610-2 102 6/6o-3 10 6/610-4 1 2/6lo-5 <1 1/6

Control' 0/6

a McCoy cell suspension.

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CHLAMYDIA PSITTACI FROM AFRICAN CLAWED FROGS 793

goes through a normal developmental cycle in-volving initial body and intermediate body for-mation.Evidence to establish this isolate of C. psittaci

as the causative agent for this epizootic wasobtained by transmitting the disease to healthyuninfected X. laevis and reisolating the agentafter death. Unfortunately, a serological conver-sion in frogs could not be demonstrated becauseof the anticomplementary activity of frog sera.

It was possible, however, to demonstrate aserological conversion in mice. Although therewas no clinical evidence of infection or positivecultures, mice which had received 104 IFU orgreater responded with a rise in antibody to theChlamydia group antigen. This lack of virulencefor mice is unlike that of the C. psittaci strainsobserved from psittacine birds and humans,which normally can cause death in mice within10 days (7).

Similar to most strains of C. psittaci, thisisolate from frogs was highly virulent for embry-onated eggs. The titers obtained in eggs werequite high in comparison to cell culture. In thisrespect strain 178 is quite different from C.trachomatis, which is relatively less virulent ineggs than is C. psittaci.

Serotyping of C. psittaci strains has laggedbehind the serotyping of C. trachomatis, and atthis point there is no recognized system for theserotyping of C. psittaci (7). There have been,

TABLE 3. Serological response to C. psittacci 178 inmice after intraperitoneal injection"

Inoculum IAHA titerInoculum sizete CGA(IFU)toG

C. psittaci 106 1:8178 1:64

1:32105 1:32

1:321:16

104 1:32<1:8<1:8

103 <1:8<1:8<1:8

C. trachomatis 105 <1:8<1:8<1:8

McCoy cell <1:8suspension <1:8

<1:8

aThree mice per group. IAHA, immune adherencehemagglutination; CGA, Chlamydia group antigen.Results at 12 days post-inoculation are given.

however, attempts to biotype C. psittaci basedon inclusion morphology and response toDEAE-dextran and cycloheximide (11). Furtherstudies of strain 178 will involve such biotyping.The source of the infection in this epizootic

has not been determined. It was revealed thatthe colony ofX. laevis was being fed condemnedbeef liver at the time of the outbreak, and C.psittaci is known to be the cause of severaldifferent types of bovine infections. Anotherpossible source could have been wild animalcontact. There was a period of time before theepizootic during which the frogs were releasedinto a pond in which they would have hadcontact with many animals in nature. The frogshad been moved back indoors, however, over 2months before the onset of the epizootic.

Further studies are needed to characterize thisstrain of C. psittaci. Since we have demonstrat-ed that C. psittaci can be a pathogen for X.laevis, it remains to be determined whetherother genera of frogs can be infected. The wholearea of C. psittaci as a pathogen in cold-bloodedanimals must be studied if the ecology andepidemiology of this organism are to be under-stood.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Servicegrants RROO200 and RR07008 from the Division of ResearchResources, National Institutes of Health and by researchfunds from the Veterans Administration.

LITERATURE CITED

1. Centers for Disease Control-National Institutes of HealthInteragency Working Group, U.S. Public Health Service.1979. Proposed biosafety guidelines for microbiologicaland biomedical laboratories. Centers for Disease Control,Atlanta, Ga.

2. Gordon, F. B., I. A. Harper, A. L. Quan, J. D. Treharne,R. St. C. Dwyer, and J. A. Garland. 1969. Detection ofChlamydia (Bedsonia) in certain infections of man. I.Laboratory procedures: comparison of yolk sac and cellculture for detection and isolation. J. Infect. Dis. 120:451-462.

3. Harshbarger, J. C., and S. C. Chang. 1977. Chlamydiae(with phages), mycoplasmas, and rickettsiae in Chesa-peake Bay bivalves. Science 196:666-668.

4. Lennette, E. T., and D. A. Lennette. 1978. Immuneadherence hemagglutination: alternative to complement-fixation serology. J. Clin. Microbiol. 7:282-285.

5. Newcomer, C. E., M. R. Anver, J. L. Simmons, B. W.Wilcke, Jr., and G. W. Nace. 1982. Spontaneous andexperimental infections ofXenopus laevis with Chlamydiapsittaci. Lab. Anim. Sci. 32:680-686.

6. Page, L. A. 1974. Order II. Chlamydiales. p. 914-918. InR. E. Buchanan and N. E. Gibbons (ed.), Bergey'smanual of determinative bacteriology, 8th ed. The William& Wilkins Co., Baltimore.

7. Schachter, J., and H. D. Caldwell. 1980. Chlamydiae.Annu. Rev. Microbiol. 34:285-309.

8. Schachter, J., and C. R. Dawson. 1978. Human chlamydialinfections. PSG Publishing Co., Littleton, Mass.

9. Shindarov, L., N. Runevski, and V. Vassileva. 1969. Mor-phologic and cytochemic studies on the development ofChlamydia psittaci in a tissue culture of a cold-bloodedanimal. Pathol. Microbiol. 34:329-339.

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10. Shindarov, L., N. Runevski, and V. Vassileva. 1971. Mor-phologic and cytochemic studies on the development ofChlamydia psittaci in tissue culture of a cold-bloodedanimal at 20°C. Zentralbl. Bakteriol. Parasitenkd. Infek-tionskr. Hyg. Abt. 1 Orig. Reihe A 216:9-14.

11. Spears, P., and J. Storz. 1979. Biotyping of Chiamydiapsittaci based on inclusion morphology and response to

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diethylaminoethyl-dextran and cycloheximide. Infect. Im-mun. 24:224-232.

12. Storz, J. 1971. Chlamydia and Chlamydia-induced dis-eases. Charles C. Thomas, Springfield, Ill.

13. Wentworth, B. B., and E. R. Alexander. 1974. Isolation ofChlamydia trachomatis by use of 5-iodo-2-deoxyuridine-treated cells. Appl. Microbiol. 27:912-916.

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