Download Experimentally Induced Infection of Gerbils with Cilia

Laboratory Animal Science
Copyright 1999
by the American Association for Laboratory Animal Science
Vol 49, No 4
August 1999
Experimentally Induced Infection of Gerbils with
Cilia-Associated Respiratory Bacillus
Mark B. St. Clair, Cynthia L. Besch-Williford, Lela K. Riley, Reuel R. Hook, Jr., and Craig L. Franklin
Cilia-associated respiratory (CAR) bacilli are gram-negative, motile, nonspore-forming bacteria that colonize the respiratory tract of several mammalian species. CAR bacillus
was first described in 1980 in a group of aging rats by van
Zwieten et al. (1) and later by Ganaway et al. (2). In early
reports, CAR bacillus was often associated with Mycoplasma
pulmonis (1, 3); however, more recent studies have documented that CAR bacillus alone is capable of causing respiratory tract disease (4–11). Infections with CAR bacillus are
usually asymptomatic; in severe cases, clinical signs of infection may include weight loss, rough coat, and respiratory
signs of wheezing and rales (2, 4).
Bacteria morphologically consistent with CAR bacillus
have also been identified colonizing the ciliated respiratory
tract epithelium of wild rats, rabbits, cattle, goats, and swine
(3, 12–15). Experimentally induced infections with mouseorigin CAR bacillus indicated that the respiratory tract of
guinea pigs and Syrian hamsters can be colonized by this
bacterium, but naturally acquired infections have not been
reported in these species (16). To date, there have not been
reports of CAR bacillus infections in gerbils (Meriones
unguiculatus). This study was undertaken to investigate
whether gerbils could be infected with CAR bacillus and, if
so, whether this bacterium would induce respiratory tract
disease in this species.
CAR bacillus isolate designated R3 isolated from a rat
was selected because it has been well characterized previously (17). Isolate R3 was grown on 3T3 mouse fibroblasts in
Dulbecco’s modified Eagle’s medium (DMEM) (Hazelton,
Lenexa, Kans.) supplemented with fetal bovine serum
(Hazelton) and 2% L-glutamine (Sigma Chemical Co., St.
Louis, Mo.) as described (18). Cultures were incubated at
37⬚C until concentrations reached 105 to 106 bacteria/ml.
Flasks were scraped to dislodge adhered bacteria, and mammalian cells were removed by centrifugation at 1,000 X g for
20 min at 4⬚C. CAR bacilli were collected by passage of the
supernatant through 0.22-␮m cellulose acetate filters (Corning Incorporated, Corning, N.Y.). Bacteria retained by the filter were washed with phosphate-buffered saline (PBS) (pH
7.4), counted in a hemocytometer, and resuspended in PBS
at a concentration of approximately 106 bacilli/ml. Cultures
were periodically screened by use of a polymerase chain reaction analysis that detects all known Mycoplasma species
to ensure that CAR bacillus cultures were free of M.
pulmonis contamination (19).
Department of Veterinary Pathobiology, College of Veterinary Medicine,
University of Missouri, Columbia, Missouri
Gerbils were obtained from an in-house breeding colony.
Gerbils from this colony were monitored annually for mouse
hepatitis virus, pneumonia virus of mice, Sendai virus, lymphocytic choriomeningitis virus, and Clostridium piliforme
by performance of enzyme-linked immunosorbent assays
(ELISA) and were consistently found to be seronegative for
these agents. Gerbils were housed in Micro-Isolator™ cages
(Lab Products, Inc., Seaford, Del.) containing Sani-Chips
(Harlan Teklad, Madison, Wis.) with CareFresh bedding (Absorption Corp., Bellingham, Wash.) as nesting material and
were allowed ad libitum access to Laboratory Rodent Diet
5001 (Purina Mills, Inc., Richland, Ind.) and tap water. The
animal room was maintained at 24⬚C, 45% humidity, and
14.5 air changes/h, and a 12/12-h light/dark cycle was used.
All experimental manipulations of gerbils were approved by
the Institutional Animal Health and Use Committee.
Five 2-week-old gerbils were inoculated intranasally with
2 x 105 CAR bacilli in 20 ␮l of PBS. Two 2-week-old control
gerbils were inoculated with 20 ␮l of PBS obtained by washing uninfected 3T3 cells in a manner similar to that used to
harvest CAR bacilli. Twelve weeks after inoculation, gerbils
were euthanized by an inhaled overdose of carbon dioxide,
and portions of the nasal cavity, trachea, and lungs were collected for histologic examination. Trachea and lung biopsy
specimens were fixed in neutral-buffered 10% formalin. The
nasal cavity was subsequently decalcified in 5% formic acid.
Tissues were paraffin-embedded, sectioned, treated with hematoxylin and eosin (H&E) and Steiner silver stains, and
examined for lesions and CAR bacillus colonization.
Clinical signs of respiratory tract disease were not observed in any of the experimental or control gerbils. Gross
lesions were not apparent at necropsy of the experimentally
infected gerbils. Twelve weeks after inoculation, all experimentally infected gerbils had histologic evidence of tracheitis and bronchitis; relevant lesions were not seen in sections
of nasal cavity. Tracheal lesions were characterized by moderate hyperplasia of the ciliated trachea mucosa with
marked submucosal lymphocytic infiltration (Figure 1A).
Lung lesions consisted of moderate hyperplasia of the ciliated airway mucosa with marked peribronchial and
peribronchiolar lymphoid hyperplasia (Figure 2A). Examination of Steiner silver-stained sections revealed argyrophilic bacteria colonizing the ciliated epithelium of the nasal
cavity (5 of 5 gerbils), trachea (5 of 5), and lungs (2 of 5). Bacteria were parallel to the cilia in a pattern identical to that
described for CAR bacillus colonization of other rodents (2,
4–5). Colonization was heavy in the trachea, patchy and re-
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Vol 49, No 4
Laboratory Animal Science
August 1999
B
Figure 1. Photomicrograph of a section of trachea from a gerbil
experimentally infected with CAR bacillus isolate R3. (A) Notice
moderate hyperplasia of the ciliated trachea mucosa and marked
submucosal lymphocytic inflammation. Bar = 100 ␮m. (B) On silver-stained sections, abundant argyrophilic CAR bacilli colonize
the tracheal epithelium in a Steiner-stained section. Bar = 10 ␮m.
stricted to the ventral floor in the nasal cavity, and patchy in
the bronchioles (Figures 2A and B). Aside from small focal
peribronchiolar lymphoid aggregates, histologic lesions were
not detected on H&E-stained sections in the control gerbils,
and CAR bacilli were not detected on silver-stained sections
of tissues from these animals.
These findings indicate that gerbils are susceptible to experimentally induced infection with this strain of CAR bacillus. The bacilli colonized the entire respiratory tract,
including the nasal cavity, trachea, and lungs. Histologic lesions and respiratory tract colonization with CAR bacillus in
gerbils was similar to the tracheobronchitis and bronchopneumonia seen in laboratory mice, rats, and hamsters (2,
4–6, 16). This is in contrast to CAR bacillus infections in
guinea pigs where infection is associated with mild histologic lesions (16). The finding that gerbils develop histologic
lesions in the respiratory tract when inoculated with CAR
bacillus raises the concern that naturally acquired CAR bacillus infections present, but as yet undetected in gerbil colonies, may compromise experiments using infected animals.
Similar to rats and mice, infected gerbils would serve as
poor research subjects, especially in studies involving the
respiratory tract. Although clinical signs of infection were
not observed in gerbils of this study, CAR bacillus infection
in gerbils could potentially be of clinical relevance. The
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Figure 2. Photomicrograph of a section of a bronchiole from a
gerbil experimentally infected with CAR bacillus isolate R3. (A)
Notice moderate hyperplasia of the ciliated bronchiolar mucosa
and marked hyperplasia of the peribronchiolar lymphoid tissue.
Bar = 200 ␮m. (B) On silver-stained sections, few argyrophilic
CAR bacilli colonize the bronchiolar epithelium. Bar = 10 ␮m.
asymptomatic state of this infection is likely due to a lack of
severe pulmonary disease in the gerbils. Similarly, mice and
rats experimentally infected with CAR bacillus for 8 weeks
developed histologic lesions of comparable severity but remained asymptomatic (8, 16). It is possible that clinical disease would develop in gerbils if they were infected for longer
periods, allowing development of more severe disease. It is
also possible that the disease induced in this study was artificial, caused in part by inoculating gerbils with a high dosage of bacteria. To examine this possibility, studies are
ongoing in our laboratory to determine whether CAR bacillus can be naturally transmitted to gerbils by direct contact
with experimentally infected gerbils.
In conclusion, results of this study indicate that the respiratory tract of gerbils can be colonized by a rat isolate of
CAR bacillus, and this colonization results in tracheobronchitis. Determination of the relevance of this finding
as it relates to care and use of gerbils in research awaits
further studies.
Acknowledgement
We thank Howard Wilson for photographic assistance.
Note
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