INFECTION AND IMMUNITY, May 1977, p. 680-689Copyright © 1977 American Society for Microbiology

Vol. 16, No. 2Printed in U.S.A.

Mycoplasma-Induced Hydrocephalus in Rats and HamstersDENNIS F. KOHN,'* B. E. KIRK, AND S. M. CHOU

Departments of Microbiology* and Pathology, School of Medicine, West Virginia University, Morgantown,West Virginia 26506

Received for publication 13 September 1976

Mycoplasma pulmonis, a pathogen of the respiratory tract in rats, was

inoculated intracerebrally into neonate rats and hamsters to determine if itwould induce lesions in the ependyma. Hydrocephalus was induced in 116 of 120rats and in 23 of 28 hamsters. The severity of hydrocephalus was greater in therats than in the hamsters. Hydrocephalus induction occurred only subsequent toinoculation of viable M. pulmonis. At 2 weeks of age, rats became refractory toinduction of hydrocephalus. Light microscopy indicated that the hydrocephaluswas communicating without an inflammatory response in the ventricles andmeninges. Preliminary electron microscopy revealed that amorphous materialcovered portions of the ependymal surface and that cilia were sometimes mattedtogether. It was suggested that the hydrocephalus was due to ciliary dysfunctionor to an imbalance of cerebrospinal fluid secretion and absorption. This M.pulmonis-induced hydrocephalus may be a useful model for elucidating thepathogenesis of certain types of congenital hydrocephalus in humans.

Various animal models have been developedto study possible mechanisms involved in con-genital hydrocephalus of humans. Hydrocepha-lus has been induced in neonate rodents withmumps virus (11), influenza A virus (10), reovi-rus type 1 (18, 15), and suckling mouse cataractagent (6).Other models that are noninfectious have

been demonstrated. A communicating hydro-cephalus occurs spontaneously as an autosomalrecessive trait in oh inbred mice (3). Thepathogenesis of the hydrocephalus in thismouse strain is unknown. A deficiency of vi-tamin A (19) or vitamin B12 (16) in pregnantrats will induce hydrocephalus in their new-born. Elemental tellurium when added to thediet of pregnant rats causes hydrocephalus intheir young (5).The association of Mycoplasma with hydro-

cephalus induction has not been investigated.However, two reports have noted hydrocepha-lus production in Mycoplasma-inoculated ro-dents. Findlay et al. (7) reported that rats in-fected with lymphocytic choriomeningitis viruswill occasionally develop hydrocephalus afterintracerebral inoculation of Mycoplasma neu-rolyticum. Lemcke (14) observed hydrocepha-lus in three of seven mice inoculated intracere-brally with M. neurolyticum. In the presentstudy, it was observed that M. pulmonis often

I Present address: Department of Comparative Medi-cine, Medical School, The University of Texas Health Sci-ence Center at Houston, Houston, TX 77025.

localized in the brains of adult rats after intra-venous inoculation. Since this organism has atropism for the ciliated respiratory epithelia(12), it was hypothesized that M. pulmonis maybe capable of inducing pathological changes ofthe ciliated ependyma. Neonate rats and ham-sters, 3 to 7 days old, were inoculated intracere-brally with M. pulmonis to test this hypothesis.

MATERIALS AND METHODSM. pulmonis strain. The M. pulmonis inocula

used throughout this investigation were derivedfrom a strain obtained from Gail Cassell, Universityof Alabama, Birmingham. After receipt, the culturewas inoculated intranasally into Mycoplasma-freerats to restore any virulence that may have been lostdue to subculturing. The animals were killed 4weeks post-inoculation, and portions of trachea wereplaced in broth medium. It was subsequently clonedthree times on agar medium. This cloned isolate wasthen transferred to broth medium and, after 72 h ofincubation at 37°C, was transferred to 5-ml vials forstorage at -70°C. This cloned isolate was identifiedas M. pulmonis by the growth inhibition test de-scribed by Clyde (4). Its identity was confirmed byMicrobiological Associates, Inc., Bethesda, Md., asM. pulmonis by immunofluorescence. A mediumformulated by Olson et al. (17) was used to cultivateM. pulmonis.

Animals. Multiple rat strains and sources of new-born rats (see Table 1) were used to observe fordifferences in host response to M. pulmonis and asan indirect means to determine if latent viruseswere contributing to the pathogenicity of M. pul-monis. Eleven timed-pregnant Wistar rats were in-oculated intravenously with 107 to 109 colony-form-

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MYCOPLASMA-INDUCED HYDROCEPHALUS 681

ing units (CFU) of M. pulmonis to determine ifhydrocephalus could be induced via in utero infec-tion. Thirty-four newborn hamsters (Engle Labora-tory Animals, Inc., Farmersburg, Ind.) were used todetermine if M. pulmonis would induce neuropath-ological lesions that were comparable to those in-duced in rats.M. pulmonis inoculation. Rats and hamsters 3 to

7 days old were inoculated with 0.02 ml of a broth orsaline suspension of M. pulmonis. The inoculum inmost instances varied from 107 to 108 CFU/ml. M.pulmonis was inoculated, with a tuberculin syringewith a 25-gauge needle into the left cerebral hemi-sphere of the animals. Older rats, ranging in agefrom 2 to 7 weeks, were also inoculated intracere-brally to determine if hydrocephalus production wasrelated to the age of the host. Controls includedlittermates that received either 0.02 ml of sterilebroth or saline intracerebrally.

Necropsy schedule. Inoculated animals werekilled routinely 2 to 3 weeks post-inoculation; how-ever, some rat litters were killed as early as 4 daysand as late as 8 weeks post-inoculation. Brains withor without the surrounding cranium were preparedfor light microscopy and gross evaluation of hydro-cephalus. A portion of the brains was used for elec-tron microscopy or for M. pulmonis isolation.

Quantitation of hydrocephalus. Brains were re-moved from the cranium and cut transversely at thelevel of the optic chiasm to evaluate the extent ofthehydrocephalus. The relative diameter of the lateralventricles and thickness of the cerebral cortex man-tel overlying the ventricles were used to rate degreeof hydrocephalus from 0 to 3+. The degree of hydro-cephalus was rendered blindly by the same observerusing a template to delineate the severity of hydro-cephalus.

Inactivation of M. pulmonis inoculum. Newbornrats were inoculated intracerebrally with inacti-vated M. pulmonis suspensions to evaluate the hy-drocephalus-inducing potential of nonviable Myco-plasma.

(i) Heat inactivation of M. pulmonis. A 2-mlvial ofM. pulmonis was placed in a 56°C water bathfor 40 min. The resulting inactivated culture wasinoculated intracerebrally into 7-day-old rats. Theculture before inactivation contained 2 x 107 CFU/ml.

(ii) Ultraviolet inactivation ofM. pulmonis. Twomilliliters of a M. pulmonis broth culture was ex-posed to a General Electric germicidal ultravioletlight source at a distance of 5 inches (ca. 12.7 cm) for10 min. This sterile culture was inoculated intoeight, 4-day-old littermates by the intracerebralroute. The culture before ultraviolet light treatmentcontained 2 x 107 CFU/ml.

Preparation of a M. pulmonis membrane inocu-lum. A membrane suspension was prepared by themethod of Gabridge and Gamble (8). Twenty millili-ters of a 107-CFU/ml broth culture was centrifugedat 10,500 x g for 30 min. The pellet was suspended in10 ml of sterile, distilled water. This suspension wassubjected to 20 freeze-thaw cycles. It was then cen-trifuged five times at 34,000 x g for 45 min andsuspended alternately in 0.5 M NaCl or beta buffer.The pellet was suspended terminally in phosphate-

buffered saline (pH 7.4) and inoculated intracere-brally into 4-day-old rats.

Preparation of a membrane filtrate inoculum. Todetermine if a soluble mycoplasmal product wasimplicated, a litter of 10 7-day-old rats was inocu-lated with a sterile filtrate obtained by passing abroth culture of M. pulmonis through a membranefilter (Millipore Corp., Bedford, Mass.) of 0.05 poros-ity. The culture originally contained 4 x 107 CFU/ml.

Latent murine virus screen. Since laboratory ratsmay carry numerous latent viruses, sera of 15 rats,from the three sources that were used for intracere-bral studies, were tested by Microbiological Associ-ates, Inc. Each sample was surveyed by hemaggluti-nation inhibition or complement fixation tests forantibody to the following viruses: reovirus type 3,Pneumonia virus of mice, Theiler encephalomyelitisvirus, rat coronavirus, Lymphocytic choriomeningi-tis, Minute virus of mice, Sendai virus, mouse ade-novirus, mouse hepatitis virus, Toolan H-1 virus,and Kilham rat virus.

Histological preparation. Tissues were removedand immediately fixed in 10% buffered Formalin. Aportion of the hydrocephalic animals were perfusedwith 10% buffered Formalin. Fixed tissues wereembedded in paraffin; skulls and joints were decalci-fied with Decal (Scientific Products, Evanston, Ill.)prior to embedding. Sections were cut at 8 ,tm andstained with hematoxylin-eosin.

Preparation of tissues for electron microscopy.Anesthetized rats were perfused via a gravity-fedsolution containing 1% formaldehyde and 1.25% glu-taraldehyde for approximately 2 min followed byperfusion for a similar period with a 4% formalde-hyde-5% glutaraldehyde solution. Fixed tissueswere stored in 0.1 M cacodylate buffer at 4°C forvarying periods. Tissues were postfixed for 2 h in 2%OS04 at room temperature. Tissues were dehydratedin graded ethanol solutions. Tissues were thenplaced in a 1:1 mixture of propylene oxide and Eponfor 1 h, followed by Epon-Araldite-dodecenyl suc-cinic anhydride overnight. Final embedding wasdone in this mixture with the addition of dimethyl-aminoethyl phenol.

Thick sections (0.5 ,um) were cut on a Porter-Blum microtome MT-1 by using glass knives. Sec-tions were placed on glass slides and stained withParagon stain. Sections were reviewed at x 100 andx 400 on a Zeiss microscope, and a portion of eachblock was selected for electron microscopy. Thinsections were stained with uranyl acetate for 15min.

RESULTSIncidence and gross characteristics of the

hydrocephalic lesions. Except for Fischer 344rats, hydrocephalus occurred in a similar quan-titative manner in rats from all sources andstrains (Table 1). From 120 rats inoculated withM. pulmonis, 4 had no hydrocephalus, 18 weregraded as 1+, 39 were graded as 2+, and 59were graded as 3+. The hydrocephalus inducedby M. pulmonis was rapid in development since

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682 KOHN, KIRK, AND CHOU

TABLE 1. Severity of hydrocephalus in various ratstrains after intracerebral inoculation of M.

pulmonis

Hydro-Animals/ cephalusRat strain Sourc& ite malitter mean

scoreb

Wistar A 11 2.27Wistar A 9 2.66Wistar A 10 1.80Wistar A 13 2.38Wistar A 7 2.86Wistar A 9 2.66Wistar A 6 1.66Wistar A 7 2.43Wistar B 5 2.40Sprague-Dawley B 6 1.83Sprague-Dawley B, axenic 12 2.92Sprague-Dawley C 4 2.25Long-Evans B 8 1.75Wistar Lewis B 7 2.28Wistar Furth C 5 2.80Fischer 344 C 6 1.16

't A, Hilltop Lab Animals, Inc., Scottdale, Pa.; B,Charles River Breeding Laboratories, Inc., Wil-mington, Mass.; C, Sprague-Dawley Company,Madison, Wis.

b Hydrocephalus scored 0 to 3: 0, no hydrocepha-lus; 1, minimal; 2, moderate; 3, severe.

rats killed 4 to 7 days post-inoculation often hadsevere hydrocephalus. Severely hydrocephalicrats had dome-shaped heads due to expansionof the skull. Transverse sections of brains fromM. pulmonis-inoculated animals revealed dila-tation of the lateral ventricles but no dilatationof the aqueducts, third or fourth ventricles(Fig. 1). Although hydrocephalus was inducedin hamsters, the incidence and severity wereless than those observed in rats. Of 28 hamstersinoculated with M. pulmonis, 5 had no hydro-cephalus, 12 were graded as 1+, 9 were gradedas 2+, and 2 were graded as 3 +. As in rats, thedilatation of the ventricular system was re-stricted to the lateral ventricles. Sixty controlrats and eight control hamsters inoculated in-tracerebrally with either sterile broth or salinehad no hydrocephalus.

Histological characteristics of the hydro-cephalus. Whereas M. pulmonis characteristi-cally causes a massive neutrophilic responsewithin the respiratory tract (13) and joints (1) ofrodents, serial sections revealed a negligibledegree of inflammation within the meninges,brain parenchyma, and ventricles of the hydro-cephalic rats and hamsters. Unlike myxovirus-induced hydrocephalus (10), the rat and ham-ster brains in this study did not display epen-dymitis, subependymal gliosis, or obliterationof the aqueduct and foramina of Monro. The

INFECT. IMMUN.

most consistently observed lesion was smallnumbers of mononuclear cells overlying theependyma and choroid plexus (Fig. 2). Theependyma of the lateral ventricles was very flatrather than low cuboidal as in nonhydroce-phalic rats. Interestingly, these flattened cells,in most cases, retained their cilia. Residentmacrophages lined portions of the aqueduct lu-mens (Fig. 3), although the lumens appeared tobe patent. Whereas the choroid plexuses ap-peared normal in most instances, large, focal,round cell infiltrates were present in two ani-mals (Fig. 4).

Ultrastructural characteristics of the hy-drocephalic lesions. Preliminary electron mi-croscopy studies indicated changes in both thethird ventricle and aqueduct. In the formersite, amorphous material covered some areas ofthe ependymal surface. Mononuclear, residentphagocytes were present within the lumens(Fig. 5). Higher magnification of the third ven-tricle showed that the cilia and microvilli be-neath the amorphous material were com-pressed and that basal bodies were disoriented(Fig. 6). Multiloculated bodies were observedwithin the aqueduct lumens in several in-stances. These bodies compressed and groupedcilia into large bundles (Fig. 7). (Matted ciliaare shown in greater detail in Fig. 8.) Unequiv-ocal identification of M. pulmonis at the epen-dymal cell surface was not possible, althoughMycoplasma-like bodies were infrequentlyseen. The subependyma, choroid plexus, and

I 1-~~~~~~~~~~~~~~~~~~~~~~~~~FIG. 1. Left, hydrocephalic rat brain sectioned

into three transverse sections. Only lateral ventriclesare dilated. Arrows point to nondilated aqueduct(middle section) and fourth ventricle (lower section).Right, Control rat brain similarly sectioned.

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FIG. 2. Photomicrograph of lateral ventricle from hydrocephalic rat. Note mononuclear cells overlying thechoroid plexus and ependyma. Hematoxylin and eosin stain. x250.

vessels within the brain were surveyed byelectron microscopy for M. pulmonis localiza-tion, but the organism was not detected.

Isolation of M. pulmonis from hydroce-phalic animals. M. pulmonis was recoveredfrom the brains of 32 of 51 (63%) hydrocephalicrats surveyed at necropsy. A comparable isola-tion rate was obtained from the few animals inwhich cerebrospinal fluid (CSF) was cultured (6of 9). The degree of hydrocephalus or post-inoc-ulation time was not related to M. pulmonisisolation.

Relationship of age at inoculation to hydro-cephalus induction. Hydrocephalus inductionwas dependent upon the age at which rats wereinoculated intracerebrally with M. pulmonis.Whereas 97% of animals inoculated at 1 week ofage developed hydrocephalus, only 2 of 20 at 2weeks of age and 1 of 5 at 3 weeks old becamehydrocephalic. Rats 4 weeks old at inoculationappeared to be totally refractory to hydrocepha-lus induction.

Effect of inactivated cultures and M. pul-monis membrane preparations. Cultures inac-tivated by heat or ultraviolet light treatmentdid not induce hydrocephalus, nor did mem-brane preparations have any observable effect.No hydrocephalus was observed in the 10 rats

that had been inoculated with the M. pulmonisculture filtrate.Antibodies to latent viruses. Serological

tests revealed antibody to Kilham rat virus intwo litters, to Sendai virus in two litters, and topneumonia virus of mice in another two litters.Sera from eight litters showed no antibodies toany of the 11 viruses tested. No differences inthe onset or degree of hydrocephalus were ob-served between antibody-positive and -negativerats.

Effect of intravenous inoculation of preg-nant rats. Inoculation of 11 rats that were atvarious days of gestation did not result in theproduction of hydrocephalus in their newborn.However, five dams developed a yellow, M.pulmonis-induced vaginal discharge. Abortedfetuses, stillborn, and young dying within 4 hpostparturation were delivered by five rats. M.pulmonis was isolated from the brains, livers,and lungs from selected stillborn and fetuses.The organism was also isolated from the brainsand lungs of six apparently normal 3-week-oldrats delivered by an infected dam.

Polyarthritis subsequent to intracerebralinoculation. An unexpected sequela to intrace-rebral inoculation of newborn rats was polyar-thritis that occurred in 75% of the animals. The

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tibiotarsal joints were the most severely af-fected; however, all joints of the fore and hindlimbs were arthritic in some animals. The ar-thritis usually became noticeable at 10 to 14days after inoculation. The degree of inflamma-tion reached a maximum at 16 to 18 days. After1 month, only one-third of the animals had

mildly swollen joints. Histologically, the ar-thritis was characterized by a mixed infiltrateof macrophages, lymphocytes, and polymorpho-nuclear cells in the synovial and surroundingtissues. In addition to polyarthritis of thelimbs, inflammation occurred along the verte-bral articulations after 10 days post-inocula-

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tion. Arthritic lesions did not occur in hamstersinoculated intracerebrally with M. pulmonis.

DISCUSSION

To our knowledge, this is the first report ofMycoplasma-induced hydrocephalus in a path-ogen-defined host. The negligible degree of in-flammation produced within the brain is a sig-nificant characteristic of this hydrocephalusmodel since hydrocephalus in human neonatesis also often non-inflammatory in nature. Weclassify this hydrocephalus as of the communi-cating type since serial histological sectionsshowed the ventricular system to be patent.The histological characteristics of the M. pul-monis-induced hydrocephalus are similar tothose present in the suckling mouse cataractagent- (6) and tellurium-induced (5) hydroceph-alus models and to the oh mouse model (3).Interestingly, it has recently been shown thatsuckling mouse cataract agent is a spiroplasmaand is serologically related to Spiroplasma ci-tri, a species within the order Mycoplasmatales

(21). The hydrocephalus in these three modelswas initially of the communicating type. Incontrast, mumps (11), reovirus (15), and myxo-virus (10) models are characterized by gliosis orobliteration of aqueduct lumens.The pathogenesis of communicating hydro-

cephalus in both humans and animals is diffi-cult to interpret; however, preliminary electronmicroscopy results suggest that the M. pul-monis-induced hydrocephalus may be due topathological changes of the ciliated ependyma.The observed lesions of the third ventricle andaqueduct would interfere with the ciliary pro-pulsion of the CSF. This CSF stasis would thenproduce an increased CSF pressure within thelateral ventricles. An alternative explanation,or one that could be operative in concert withciliary dysfunction, is an imbalance in CSFsecretion and absorption produced by the amor-phous material overlying the ependymal sur-face.Rats inoculated at 14 days of age were refrac-

tory to severe hydrocephalus. This age-effectrelationship could be due to the loss of cranial

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FIG. 5. Electron micrograph of third ventricle from hydrocephalic rat. Note the mononuclear cells lyingwithin the lumen of the ventricle. Arrows point to amorphous material covering portions of the ependyma.x2,500.

elasticity of the neonate at 2 weeks of age. Thisassumption is based upon the premise that theCSF pressure is increased in infected animalsenough to cause ventricular dilatation only ifthe cranial resistance is minimal. Supportingthis hypothesis is the work of Hochwald et al.(9), who produced obstructive hydrocephalus incats by inoculating kaolin into the meninges.To evaluate the relationship of the skull withprogressive hydrocephalus, they hemicraniec-tomized or craniectomized cats. In the formeranimals, it was found that the lateral ventriclewith no overlying skull progressively dilated,whereas the other ventricle beneath the rigidskull dilated only moderately. This nonprogres-sive hydrocephalus was limited by a thresholdat which increased CSF pressure elicited trans-ventricular absorption ofCSF. However, in ani-mals that did not have intact skulls overlyingventricles, the increased CSF pressure never

reached this threshold due to the expansivecapacity of the skulls. The authors suggest thatin human infants with hydrocephalus the ex-pansile nature of the skull is partially responsi-ble for the progressive ventricular dilatationeven though the CSF pressure may be onlyslightly elevated.

Since inactivated cultures, M. pulmonismembranes, and a culture filtrate had no effectafter intracerebral inoculation, it appears thatviable M. pulmonis is required for induction ofhydrocephalus. Similarly Thomas and Kakla-manis (20), in a study of M. pulmonis toxicity,found that only viable organisms were capableof inducing toxic deaths.The incidence and severity of hydrocephalus

were greater in rats than in hamsters. Thesespecies differences may be attributable tolessened M. pulmonis replication in hamsters,since rats are natural hosts for this organism

FIG. 6. Electron micrograph of third ventricle that shows several degenerating ependymal cells beneathamorphous material. Arrows point to disoriented basal bodies. x6,000.

FIG. 7. (A) Aqueduct ofan hydrocephalic rat. Arrow points to one of the multiloculated bodies within thelumen of the aqueduct. Electron-dense areas between the bodies are composed of bundles of cilia. (B) Aque-duct from control rat. Note the cilia arranged singularly within the aqueduct lumen. x1,500.

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and hamsters are not. The lack of a polyarthri-tic response in hamsters further indicates thatM. pulmonis is unable to replicate well in thishost.The etiological association of Mycoplasma

with congenital hydrocephalus in humans isunknown. However, M. hominis has beenassociated in one instance with hydrocephalusin a human newborn (2). In the present study,M. hominis was also inoculated intracerebrallyinto newborn rats, but it did not induce hydro-cephalus.Further studies are needed to elucidate the

pathogenesis of this M. pulmonis-induced hy-

drocephalus model. Determination of the mech-anism operating in this model could be of signif-icance in defining the pathogenesis of commu-nicating hydrocephalus in humans.

ACKNOWLEDGMENTS

We thank Lois Brent, Annamae O'Neal and GeraldVoice for their technical assistance.

This work was supported by Public Health Service Re-search Resources Grant RR00853.

LITERATURE CITED

1. Barden, J. A., and J. G. Tully. 1969. Experimentalarthritis in mice with Mycoplasma pulmonis. J. Bac-teriol. 100:5-10.

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2. Boe, O., J. Diderschsen, and R. Matre. 1973. Isolationof Mycoplasma hominis from cerebrospinal fluid.Scand. J. Infect. Dis. 5:285-288.

3. Borit, A., and R. L. Sidman. 1972. New mutant mousewith communicating hydrocephalus and secondaryaqueduct stenosis. Acta Neuropathal. 21:316-331.

4. Clyde, W. A. 1964. Mycoplasma species identificationbased upon growth inhibition by specific antisera. J.Immunol. 92:958-965.

5. Duckett, S. 1971. The morphology of tellurium-inducedhydrocephalus. Exp. Neurol. 31:1-16.

6. Elizan, T. S., A. Fabiyi, and H. F. Clark. 1972. Suck-ling mouse cataract agent (SMCA)-induced hydro-cephalus and chronic brain infection in newborn rats.Proc. Soc. Exp. Biol. Med. 139:51-55.

7. Findlay, G. M., E. Klieneberger, F. 0. MacCollum,and R. D. MacKenzie. 1938. Rolling disease, newsyndrome in mice associated with a pleuropneumo-nia-like organism. Lancet 235:1511-1513.

8. Gabridge, M. G., and D. D. Gamble. 1974. Independ-ence of leukemoid potential and toxigenicity ofMyco-plasma fermentans. J. Infect. Dis. 130:664-668.

9. Hochwald, G. M., F. Epstein, C. Malhan, and J. Ran-sohoff. 1972. The role of the skull and dura in experi-mental feline hydrocephalus. Dev. Med. Child. Neu-rol. 14(Suppl. 27):65-69.

10. Johnson, R. T., and K. P. Johnson. 1969. Hydrocepha-lus as a sequela of experimental myxovirus infec-tions. Exp. Mol. Pathol. 10:68-80.

11. Johnson, R. T., and K. P. Johnson. 1968. Hydrocepha-lus following viral infection: the pathology of aque-ductal stenosis developing after experimental mumpsvirus infection. J. Neuropathol. Exp. Neurol. 27:591-606.

12. Kohn, D. F. 1971. Bronchiectasis in rats infected withMycoplasma pulmonis: An electron microscopy study.Lab Anim. Sci. 21:856-861.

13. Kohn, D. F., and B. E. Kirk. 1969. Pathogenicity ofMycoplasma pulmonis in laboratory rats. Lab. Anim.Care 18:321-330.

14. Lemcke, R. M. 1961. Association of PPLO infection andantibody response in rats and mice. J. Hyg. 59:401-412.

15. Nielsen, S. L., and J. R. Baringer. 1972. Reovirus-induced aqueductal stenosis in hamsters: phase con-trast and electron microscopic studies. Lab. Invest.27:531-537.

16. O'Dell, B. L., J. R. Whitley, and A. G. Hogan. 1951.Vitamin B-12, a factor in prevention ofhydrocephalusin infant rats. Proc. Soc. Exp. Biol. Med. 76:349-353.

17. Olson, N. O., K. M. Kerr, and A. Campbell. 1963. Con-trol of infectious synovitis-12, preparation of an ag-glutination test antigen. Avian Dis. 7:310-317.

18. Phillips, P. A., M. P. Alpers, and N. F. Stanley. 1970.Hydrocephalus in mice inoculated neonatally by theoronasal route with reovirus type I. Science 168:858-859.

19. Rokkones, T. 1955. Experimental hydrocephalus inyoung rats. Int. Z. Vitaminforsch. 26:1-10.

20. Thomas, L., and E. Kaklamanis. 1970. Toxins of myco-plasma, p. 493-505. In T. C. Montie, S. Kadis, andS. J. Ajl (ed.), Microbial toxins, vol. 3. AcademicPress Inc., New York.

21. Tully, J. G., R. F. Whitcomb, and D. L. Williamson.1976. Suckling mouse cataract agent is a helical wall-free prokaryote (spiroplasma) pathogenic for verte-brates. Nature (London) 259:117-120.

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