Ureaplasma urealyticum Pneumonia: Experimental Production andDemonstration of Age-Related Susceptibility
PETER T. RUDD,"2 GAIL H. CASSELL,'* KEN B. WAITES,13 JERRY K. DAVIS,4 AND LYNN B. DUFFY'
Departments of Microbiology,' Pathology,3 and Comparative Medicine,4 University ofAlabama at Birmingham,Birmingham, Alabama 35294, and Department of Pediatrics, University of Cambridge, Cambridge, England2
Received 22 August 1988/Accepted 17 November 1988
Two different strains of Ureaplasma urealyticum isolated in pure culture from the lungs of newborn humaninfants were shown to produce an acute, self-limiting, interstitial pneumonia in newborn C3H/HeN andC57BL/6 mice that were free of other respiratory pathogens. Lesion severity peaked 3 to 6 days followingintranasal inoculation of ureaplasmas and was resolved by 12 days. Rhinitis and otitis also occurred but did soless frequently than pneumonia. Organisms were localized within the alveoli in areas of inflammation. Incomparison with newborn mice, 14-day-old mice were less susceptible to either colonization or disease.
The role of Ureaplasma urealyticum in respiratory diseaseof newborn human infants is unclear (3, 5, 30). Individualcase reports indicate that this organism causes congenitalpneumonia (6, 26, 36). Some serological studies (25, 27), butnot others (30), suggest that ureaplasmas are also associatedwith respiratory disease in newborns. Previous prospectivecultural studies do not indicate that there is a significantassociation (29, 35). Like many disease associations with U.urealyticum, there is reason to believe that only a subpop-ulation of infants may be at risk (3, 5). Recent prospectivestudies show that premature infants (weight, <1,000 g) fromwhom U. urealyticum is isolated from the endotrachealaspirate within 24 h after birth are more likely to developchronic lung disease or die than are infants of similarbirthweight but who are uninfected or infected infants whoweigh >1,000 g (8). These studies also indicate that manyureaplasmal respiratory infections are acquired in utero.The establishment and characterization of an animal
model could help delineate host and microbial factors thatpredispose newborn infants to U. urealyticum-induced res-piratory disease. The present study was designed, first, todetermine whether pure cultures of U. urealyticum isolatedfrom the respiratory tracts of diseased infants produce asimilar disease in experimental animals and, second, todetermine whether there is an age-related susceptibility toinfection and disease.Because respiratory disease research involving any infec-
tious agent is easily compromised by environmental factorsand indigenous infections (1, 17), the primary requirement ofan animal model should be the use of environmentally andmicrobiologically defined animals. We selected mice be-cause they are one of the few laboratory animals in which themicrobial flora (both commensal and pathogenic) of therespiratory tract is well defined (18, 20). The morphology ofthe lungs of normal mice is well characterized, as are thehistopathologic changes associated with murine respiratorypathogens (15, 19). Most importantly, pathogen-free miceare available and can be maintained to ensure their patho-gen-free status.We chose to infect mice with liquid inocula by the intra-
* Corresponding author.
nasal route as opposed to aerosol inocula to more closelymimic the natural exposure of the respiratory tract viainfected amniotic fluid (3, 5, 6, 8).
Results presented here prove that two different strains ofU. urealyticum isolated in pure culture from pneumoniclungs of human infants can produce a similar disease in thelungs of newborn mice proven to be free of other pathogensand that the age of the host is a critical determinant of thedevelopment and severity of disease. Preliminary resultsindicate that the genetic background of the host may also bean important determinant.
MATERIALS AND METHODS
Experimental design. Mice were inoculated within 12 to 24h or at 14 days after birth. One-day-old mice were inoculatedintranasally with 40 RI containing 2 x 107 CFU of U.urealyticum serotype 1, 3.7 x 105 CFU of U. urealyticumserotype 10, or sterile 10B broth which was incubated inparallel with inoculated broth. The pH was adjusted to thatof the inoculated broth at the time of organism harvest. The40 Rl of the respective inocula was divided into equal dosesand given over a 2-h period.
Fourteen-day-old mice were given numbers of organismsor volumes of sterile broth comparable to those given tonewborn mice based on lung weight. This usually was 2.5times the dose given to newborn mice. Uninoculated miceserved as additional controls. Between 1 and 12 days post-inoculation, animals were randomly selected from each litterand killed by sodium pentobarbital injection (22).At necropsy serum was collected for antibody determina-
tions. Ureaplasmal and bacterial cultures were performed ontracheobronchial and nasal lavages. Lungs, trachea, orophar-ynx, nasal passages, and middle ears were collected andprocessed for histopathology for determination of the pres-ence and severity of lesions. Lung tissue was evaluated byimmunofluorescent and electron microscopy to determineorganism localization.
Experimental animals. Two-month-old C3H/HeN orC57BL/6N pregnant dams with known gestation times andthat were previously shown to be free of murine mycoplas-mas, viruses, and pathogenic bacteria were obtained from
a NPL, Nasopharyngeal lavage; TBL, trachaeobronchial lavage.b Significantly different from 3 to 5 days, P < 0.05.
breeding colonies maintained in plastic film isolators at theUniversity of Alabama at Birmingham (9) or from barrier-maintained colonies at the Frederick Cancer Research Cen-ter (Frederick, Md.). The colonies were monitored monthlyfor the presence of mycoplasmas by enzyme-linked immuno-sorbent assay for immunoglobulin G (IgG) and IgM antibod-ies to Mycoplasma pulmonis and Mycoplasma arthritidis (7).The colonies were monitored quarterly for other murinepathogens by fecal cultures, necropsy, serologic tests forrodent viruses, and histopathologic examination of retiredbreeders. Serologic tests for rodent viruses for the mice usedin these experiments were conducted by MicrobiologicalAssociates (Bethesda, Md.) for pneumonia virus of mice,reovirus-3, Theiler's GDVII, polyomavirus, Sendai virus,minute virus of mice, ectromelia, mouse adenovirus, mousehepatitis virus, and lymphocytic choriomeningitis virus.Mice obtained from the Frederick Cancer Research Centerwere shipped to the University of Alabama at Birminghambehind bacteriological filter media.Dams were allowed to whelp, and each individual dam and
her pups were caged together in sterile shoebox cagesequipped with filter tops. All cages were provided withsterile hardwood chip bedding (P. J. Murphy Forest Prod-ucts, Rochelle Park, N.J.). Sterile food (Agway, Inc., Syra-cuse, N.Y.) and sterile water were provided ad libitum.
U. urealyticum. The serotype 1 strain used in this studywas previously isolated in pure culture from the lungs of anewborn human infant with documented congenital pneumo-nia (6). U. urealyticum serotype 10 was isolated in pureculture from an endotracheal aspirate collected within 24 hof birth from a preterm human infant with respiratorydisease (8). Serotype 1 was passaged three times on artificialmedia, four times in 6-week-old mice, and twice again onartificial media. Serotype 10 was cloned and passaged twiceon artificial media, once in newborn mice, and twice again onartificial media. Each passage in animals consisted of recov-ery by tracheobronchial lavage 3 days after intranasal inoc-ulation.Ureaplasmas were isolated and cultivated in lOB broth
and on A8 agar by the methods of Shepard (32). Serotypingof ureaplasma isolates was performed by immunofluores-
cence with rabbit antisera prepared as described previously(14).
Collection and culture of tracheobronchial and nasopharyn-geal lavages. Tracheobronchial lavages were collected with0.4 ml of lOB broth. Then the head was removed and theskin, lower mandible, and soft tissue of the nose removed. A27-gauge needle attached to a tuberculin syringe containing0.2 ml of lOB broth was then inserted into the posteriorchonae and the nasal passages were flushed anteriorly into asterile collection tube.The number of CFU of ureaplasmas was determined by
plating 10-fold dilutions of the lavages onto A8 agar. Forcultivation of other bacteria, lavages were plated onto bloodagar and inoculated into brain heart infusion broth contain-ing 0.1% agar.
Histopathology and lesion scoring. Lungs were fixed byintratracheal instillation of 95% cold ethanol and processedby the method of Sainte-Marie (31), which permitted thelocalization of ureaplasmas in tissues by indirect immuno-fluorescence and the simultaneous study of parallel sectionstreated with standard histologic stains.The trachea was removed and sectioned longitudinally.
Each lung lobe was separated and cut so that a single sectionwas obtained with the main stem bronchi cut in longitudinalsection. Three sections were taken from the nasal passages:(i) from a transverse plane immediately behind the develop-ing incisors, (ii) at the level of the third palatine ridge, and(iii) from a transverse plane bisecting the orbit. Sectionswere stained with hematoxylin and eosin and examined bylight microscopy.
Microscopic nasal passage lesions were scored subjec-tively by previously established criteria (9). Lung lesionswere also scored subjectively from 0 to 4, where 1 indicatesincreased numbers of mononuclear cells in the alveoli, 2indicates focal increase in mononuclear and polymorphonu-clear cells in the alveoli, 3 indicates extensive alveolarinflammation, and 4 indicates inflammatory cells in both thealveoli and larger airways.
Immunofluorescent and electron microscopy. Representa-tive lung sections were examined by immunofluorescentmicroscopy (31). Slides were reacted with rabbit anti-U.
TABLE 2. Histopathological results in C3H/HeN newbom mice inoculated intranasally with U. urealyticum serotype 1
Day after % Animals with microscopic lesions ina: % Animals with the following grade of lung inflammation':infection L T NP OP ME 0 1 2 3 4
TABLE 3. Cultural results of newborn and 14-day old C3H/HeN mice inoculated intranasally with U. urealyticum serotype 10
Mean ± SD CFU recovered byb:Day afterinfectiona NPL TBL
Newborns 14-day-olds Newborns 14-day-olds
3 5.6 x 104 8.9 x 104 3.8 x 102 1x 103c 1.5 x 106 1.6x 106 1.9 x 102 5 x 102c8 7.5 x 103 0.8 x 103 <lioc 3.0 x 104 4.5 x 104d 1.1 x 102 4 x 102c12 <lold 9 x 101 ± 3.8 x 102 <101d <101
a The number of newborn mice examined on days 3, 8, and 12 were 13, 12, and 16, respectively, and of 14-day-old mice 16, 15, and 17, respectively.b NPL, Nasopharyngeal lavage; TBL, tracheobronchial lavage.c Significantly different from newborn mice; P < 0.001.d Significantly different from newborn mice at 3 days; P < 0.05.
urealyticum antiserum that was prepared as described pre-viously (14). Adjacent sections reacted with normal rabbitantiserum served as controls. Fluorescein-labeled, affinity-purified goat anti-rabbit IgG (Cappel Laboratories, WestChester, Pa.) was used as the conjugate. Slides were exam-ined with a microscope (Orothoplan FA; Leitz/Opto-MetricDiv. of E. Leitz Inc., Rockleigh, N.J.).
Representative lung tissue was processed for electronmicroscopic examination after fixation in modified Kar-novsky buffer (2% paraformaldehyde and 2.5% glutaralde-hyde in 0.0667 M sodium cacodylate buffer; pH 7.4). Afterthe tissues were trimmed, they were postfixed in 1%5OS4 in0.0667 M sodium cacodylate buffer (pH 7.4) for 1 h at 4°C,dehydrated in acetone, embedded in Durcupan ACM(Fluka), and allowed to polymerize at 60°C for 48 h. Thicksections were cut and stained with toluidine blue 0 for lightmicroscopic examination. Thin sections cut on a ultramicro-tome (MT2-B; Dupont Sorvall, Inc.) were stained withalcoholic uranyl acetate and Reynold lead citrate and exam-ined in an electron microscope (Zeiss 9S-2 or JEOL 100CX).Antibody measurement. The serum from each animal was
analyzed for anti-ureaplasma antibodies by an enzyme-linked immunosorbent assay by using affinity-purified goatanti-mouse heavy-chain antisera conjugated with alkalinephosphatase (7, 14). Enzyme-linked immunosorbent assayvalues (expressed as A400 per 100 min) from experimentalanimals were compared with those from uninoculated orbroth-inoculated control animals.
Statistical analysis. Incidence values were analyzed bychi-square analysis. The Fisher exact test and the rank sumtest were used to compare nonparametric data.
100 - -Lung Newbom-U-* NP Newbom
80 -0-- Lung 14d-*@ NP 14d
60-% CulturePositive
40
20
2 4 6 8 10 12 14
Days Post-infectionFIG. 1. Incidence of infection in newborn and 14-day-old C3H/
HeN mice inoculated intranasally with U. urealyticum serotype 10(NP, nasopharynx). Infection in 14-day-old mice was significantlydifferent from that in newborns at 3 and 8 days (P < 0.001).
RESULTS
Ability of U. urealyticum to produce respiratory disease.Two serotypes representing each of the two genotypicallydistinct, but related, clusters (28) of U. urealyticum wereused for animal inoculations. Strains of both serotypes wereisolated from the respiratory tracts of human infants withpneumonia and were passaged at least once in microbiolog-ically defined mice. Both serotypes 1 and 10 colonized therespiratory tracts of newborn C3H/HeN mice and producedpneumonia which peaked in severity 3 to 6 days followinginoculation and which was resolved by 12 days (Tables 1 to3 and Fig. 1 and 2). There were no observable clinicalsymptoms or gross lesions and no deaths attributable to U.urealyticum infection.Development and resolution of lung lesions correlated
with the isolation of the organism from the lower respiratorytract (Tables 1 to 4 and Fig. 1 and 2). While strains of bothserotypes frequently colonized the nasopharynx, the occur-rence of rhinitis and otitis were rare, as were pharyngitis andtracheitis (Table 2 and Fig. 2). No direct comparisons can bemade between the infectivity and pathogenicity of the strainsof the two serotypes because the experiments were per-formed on different days.
Nasal passage and tracheobronchial lavages of infectedanimals were consistently negative for bacterial pathogens,as were sections of pneumonic lungs stained with Brown andBren. Nasopharyngeal and tracheobronchial lavages of allcontrol animals were consistently culture negative for U.
120
100
80
% Mice wilhMicroscopicLesions
60
0- Lung Newborn
U NP Newbom-0--O Lung 14d
* NP 14d
40 -
2012 4 6 8 10 12 14
Days Post-infectionFIG. 2. Incidence of microscopic lesions in newborn and 14-
day-old C3H/HeN mice inoculated intranasally with U. urealyticumserotype 10 (NP, nasopharynx). Infection in 14-day-old mice wassignificantly different from that in newborns at 3 and 8 days (P <0.001).
in bronchi; 1, increased numbers of mononuclear cells in the alveoli; 2, focalincrease in mononuclear and polymorphonuclear cells in the alveoli; 3,extensive alveolar inflammation; 4, inflammation in both the alveoli and largerairways.
b Different from newborns at 3 days; P < 0.01.c Different from newborns; P < 0.001.dDifferent from newborns; P < 0.005.
urealyticum and other pathogens, including murine myco-plasmas.
Influence of age. Fourteen-day-old mice were inoculatedon the same day as newborns, were given comparablenumbers of organisms per gram of lung weight, and were ofthe same microbiological and environmental background asnewborn mice. Yet the incidence and persistence of infec-tion, as well as the incidence and severity of lesions, was lessin 14-day-old mice as compared with that in newborn mice(Tables 3 and 4 and Fig. 1 and 2).
Production of pneumonia in mice with different geneticbackgrounds. C57BL/6 newborn mice matched with theC3H/HeN mice for microbiological and environmental fac-tors (9) and given comparable numbers of organisms devel-oped disease similar to that in C3H/HeN mice. Although nodirect comparisons can be made because the experimentswere performed on different days, C57BL/6 mice appearedto be no less susceptible to colonization or the developmentof pneumonia, but lung lesions appeared to be less severeand resolved more quickly (Tables 5 and 6).
Lesion character. Lung lesions were qualitatively similarin newborn mice of both strains inoculated with strains ofboth serotypes. Lung lesions were generally most severe at3 days and were characterized by a marked increase in thenumber of mononuclear cells and infiltration of neutrophilsinto alveoli (Fig. 3). Thickening of the alveolar walls was themost common lesion seen at all time points (Fig. 4), whereaspulmonary edema and hemorrhage were not observed at anytime point. In general, lesions began to resolve by 8 days,and fewer inflammatory cells were seen in the alveoli. Unlikeother mycoplasmal respiratory diseases (4), U. urealyticum
pneumonia in newborn mice was not accompanied byperivascular and peribronchiolar lymphocytic infiltration.However, this was a feature seen in 14-day-old animals.Another difference in the character of lesions seen in 14-day-old versus newborn mice was that in older animals,lesions were primarily limited to larger airways.
Control mice of all strains and all ages (total of 60) wereconsistently free of respiratory tract lesions (Fig. 4B).
Localization of organisms. Immunofluorescent micro-scopic examination of lungs of newborn mice at 3 and 8 daysconsistently revealed numerous U. urealyticum scatteredthrough the alveoli of affected areas (Fig. 5), but feworganisms were seen on the respiratory epithelium. In somemice, particularly those examined 8 days after inoculation,clumps of antigen were associated with macrophages. Ex-amination of lung tissue by electron microscopy confirmedthe results obtained by immunofluorescence and providedsuggestive evidence that organisms were located withinmacrophages. Small numbers of organisms were associatedwith ciliated cells in the bronchi.Immune responses. No anti-ureaplasma antibody was de-
tected in newborn mice at any time postinoculation; and onlya few 14-day-old mice were positive at 3, 8, and 12 dayspostinoculation. All control mice were serologically nega-tive.
DISCUSSIONFrom the reported cases of U. urealyticum pneumonia in
human infants (6, 8, 26, 36), the acute disease appears to beprimarily interstitial and is characterized predominantly by amononuclear cell infiltrate both in the alveolar walls and intothe alveolar and bronchiolar spaces. We have shown herethat ureaplasmas isolated from such infants produce a simi-lar disease in newborn mice. Localization of the organismsin the alveoli is compatible with the location of lesions in thatarea. In fact, one of the most impressive features of thedisease in mice is the extreme thickening of the alveolarseptae.That these organisms are indeed capable of colonizing and
producing disease in the respiratory tract is strengthened bythe fact that two different serotypes of U. urealyticum wereused to produce disease in two different strains of mice.Although no direct comparisons can be made between theinfectivity and the pathogenicity of the two serotypes, be-cause the experiments were performed on different days, it isinteresting that 2 log units less of serotype 10 appeared toproduce more severe lung lesions. However, the 50% infec-tious dose (ID50) and 50% pneumonia dose (PD50) for the twoureaplasma serotypes must be established before definiteconclusions can be drawn. Other investigators have reportedthat different clinical isolates of U. urealyticum differ inpersistence following inoculation into the kidneys of mice(12).That larger numbers of organisms were consistently iso-
TABLE 5. Cultural results of C357 BL/6 newborn mice inoculated intranasally with U. urealyticum serotype 1
Days after % Culture positive bya: Mean + SD CFU recovered by:infection NPL TBL NPL TBL
3-5 (n = 6) 17 50 1 x 102 + 0 1.4 x 104 + 2.5 x 1056-8(n =7) 0 14 1.3 x 103+2.5 x103 8 x 101 +9x1Olb9-10 (n = 4) 0 0 0 0
a NPL, Nasopharyngeal lavage; TBL, tracheobronchial lavage.b Significantly different from 3 to 5 days; P < 0.05.
9-10 (n = 4) 0 0 0 0 0 4 0 0 0 0a Abbreviations: L, Lung; T, trachea; NP, nasopharynx; OP, oropharynx; ME, middle ear.b Inflammation grades: 0, No microscopic changes; 1, increased numbers of nononuclear cells in alveoli; 2, focal increase in nononuclear and polymorpho-
nuclear cells in alveoli; 3, extensive alveolar inflammation; 4, inflammation in both alveoli and large airways.c Different from 3 to 5 days; P < 0.05.d Different from 3 to 5 days; P < 0.025.
lated from the tracheal aspirates as compared with thenasopharynx of mice suggest that prospective cultural stud-ies in humans based on sites other than the lower respiratorytract (8, 29, 35) might not correlate with the presence of lunglesions.
It is not yet clear whether the mouse model has significantrelevance to newborn human infants. However, if one wereto apply the findings of the mouse model, the lesions in thehuman lung would be expected to resolve spontaneously. Itmay be that prospective studies which have assessed thecolonization of newborn babies over a period of severalweeks (29) have failed to document evidence of the diseaseproduced by this organism because the lesions were tran-sient.The increased susceptibility of newborn mice compared
with that of 14-day-old mice is compatible with the apparentgestational age-related susceptibility seen in recent studies inhumans (8). The reasons for this are not known, but theyprobably have to do with the development of host defenses.The difference in the susceptibilities of newborn and 14-day-old mice is similar to the age-related susceptibility seenin group B streptococci that is related to macrophage num-
bers and function (16, 21, 34). Because ureaplasmas werefound within macrophages by 8 to 12 days of age and14-day-old mice did not develop alveolar lesions, it is sug-gested that differences in macrophage function may beimportant in causing susceptibility to U. urealyticum pneu-monia. However, 14-day-old mice were also more resistantto colonization of the upper respiratory tract, suggesting thatother factors may also be involved in the age-related suscep-tibility.The preliminary results presented here suggest that the
genetic background of the host may also be a criticaldeterminant of disease severity. We have previously shownthat C3H/HeN mice are more susceptible than C57BL/6mice to other mycoplasma-induced respiratory diseases (9)and that early nonspecific host responses involved in pulmo-nary clearance are in part responsible for these differences(24).
It might be argued that because we did not see deaths innewborn mice the disease in mice is unlike the disease inhumans, since human infants weighing <1,000 g are not onlymore likely to develop chronic lung disease but are also morelikely to die (8). Other variables that may influence disease
FIG. 3. Photomicrograph of lung tissue taken from a C3H/HeN mouse inoculated with U. urealyticum within 12 h after birth and killedat 3 days of age. Intraalveolar mononuclear and polymorphonuclear cells are present. Magnification, x25.
- u- Z-PU I x __ pFIG. 4. (A) Photomicrograph of lung tissue taken from a C3H/HeN mouse inoculated within 12 h after birth and killed at 5 days of age.
Intraalveolar accumulations of inflammatory cells are absent, but the alveolar walls are markedly thickened by interstitial cellularproliferation. (B) Photomicrograph of lung tissue from a C3H/HeN mouse inoculated with sterile 10B broth within 12 h after birth and killedat 5 days of age. Note the uniformly thin, delicate alveolar walls and the absence of intraalveolar cellular accumulations. Magnification, x 100.
severity in newborn versus older human infants may beassociated with the increased requirement for supplementaloxygen by premature infants weighing '1,000 g versus thoseweighing >1,000 g (23). Oxidants are thought to enhance
susceptibility to infectious lung disease (13) and in fact havebeen shown to enhance susceptibility to other mycoplasmalrespiratory diseases (2; R. F. Parker, J. K. Davis, G. H.Cassell, H. White, D. Dziedzic, D. K. Blaylock, and R. B.
FIG. 5. Photomicrograph obtained by immunofluorescent microscopy of lung tissue from a C57BL/6 mouse inoculated with U.urealyticum within 12 h of birth and killed after 8 days. Clumps of ureaplasmal antigen are present, primarily in association with alveolarmacrophages. Magnification, x400.
Thorp, Am. Rev. Respir. Dis., in press). Using the newbornmouse model described here, we have shown that newbornmice that are exposed to 80% oxygen are more susceptible toureaplasmal infection and death than are unexposed, in-fected mice (D. T. Crouse, G. H. Cassell, K. B. Waites, andG. Cassady, Zentralbl. Bakteriol. Parasitenkd. Infektionskr.Hyg. Abt. 1 Orig. Reihe A). Furthermore, exposure tooxygen increases the persistence of ureaplasmas in therespiratory tract.The mechanism(s) by which U. urealyticum produces
respiratory disease is unknown. These organisms are knownto induce ciliostasis in human fetal tracheal organ cultures(11) which could interfere with the clearance of these organ-isms. Also, the requirement for sterols by ureaplasmas (33)and their production of phospholipases (10) suggest thatinfection may alter lung surfactant levels. Other respiratorydisease-producing mycoplasmas are known to do so (4).Further characterization of the mouse model of U. urealyti-cum should help delineate both host and organism factorsthat are involved in disease susceptibility and disease patho-genicity.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grants HLBI19741 and HD 20928 from the National Institutes of Health (toG.H.C.). P.T.R. was supported by a fellowship from the WellcomeTrust.
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