Cilia-associated Respiratory Research Articles

Respiratory diseases in rats

ABSTRACT:Respiratory disease due to infectious disease is one of the most common presentations in rats but the cause is in many cases multifactorial. Predisposing factors are generally related to poor husbandry and diet. Respiratory disease is caused primarily by infectious agents such as Mycoplasma pulmonis, Streptococcus pneumoniae, Corynebacterium kutscheri, cilia associated respiratory (CAR) bacillus, Sendai virus and coronavirus. Other causes are cardiovascular diseases, neoplastic diseases and trauma of the upper and lower respiratory tract. A presumptive diagnosis can be made by good history and physical examination. However, many diagnostic tests are best performed under general anaesthesia. Medical treatment generally includes oxygen therapy, fluid therapy, analgesia and administration of antibiotics, the use of mucolytic agents and bronchodilators and supportive care. Medications can be administrated orally, parentally or via nebulisation.

Evaluation of New Cage Lid with Partitioning Barrier Based on Transmission of CAR Bacillus in Mice

We developed a new cage lid made of stainless steel wire mesh and having a screen barrier for partitioning a small laboratory animal cage into compartments. To evaluate the effectiveness of the lid, we tested the transmissibility of Cilia-Associated Respiratory (CAR) bacillus from infected mice to uninfected sentinel mice, which were kept in separate compartments using this lid. Infection from the infected mice to the uninfected mice was confirmed by microbiological, serological, pathological, and molecular diagnostic examinations, as previously observed in an intra-cage contact route. The cage lid that we developed is very useful when uninfected mice are used in quarantine and contagion experiments to prevent fighting among the mice.

Cilia-associated Respiratory (CAR) Bacillus Infection in Adult Red Deer, Chamois, and Roe Deer

Cilia-associated respiratory (CAR) bacillus is an unclassified bacterium that colonizes the ciliated epithelium of airways in laboratory rats, laboratory mice, and laboratory and conventionally reared rabbits, cattle, goats, and pigs. Data on the prevalence of CAR bacillus infection in wild animals are lacking. The present study demonstrated the occurrence of the organism in wild red deer (Cervus elaphus hippelaphus), chamois (Rupicapra rupicapra), and roe deer (Capreolus capreolus) from the Val Fontana in northern Italy. Prevalence ranged from 26% for red deer to 56% for chamois, with a statistically significant negative correlation between CAR bacilli infection and the presence of lymphoid follicles.

Cilia-associated respiratory bacillus infection in rats in New Zealand

AIMS: To demonstrate cilia-associated respiratory (CAR) bacillus associated with chronic pneumonia in wild and pet rats in New Zealand. METHODS: A range of tissues from 4 rats were examined grossly and by light microscopy and affected lungs were examined by transmission electron microscopy. RESULTS: All 4 rats had moderate to severe cranioventral bronchopneumonia with bronchiectasis and large numbers of argentophilic bacteria resembling CAR bacillus, intimately associated with the bronchial epithelium. CONCLUSIONS: CAR bacillus infection should be considered as a differential diagnosis for pneumonia in rats in New Zealand.

Sequence of 16S rRNA Gene of Rat-Origin Cilia-Associated Respiratory(CAR) Bacillus SMR Strain.

The 16S rRNA gene of the SMR strain of cilia-associated respiratory (CAR) bacillus, which was isolated from a spontaneously infected rat at our institute, was sequenced. Its 1,521 nucleotides were determined. On the basis of the results of the sequence analysis, the SMR strain was found to be most closely related to members of the Flavobacter/Flexibacter group. This sequence was compared with the previously determined 16S rRNA gene sequences (rat-origin: three; mouse-origin: one; rabbit-origin: one) of CAR bacillus isolates. The SMR strain showed the highest sequence similarity (99.9%) to the rat-origin CARB-NIH strain (Schoeb et al., 1993), and it was concluded that the strains are identical.

Isolation of cilia-associated respiratory (CAR) bacillus from pigs and calves and experimental infection of gnotobiotic pigs and rodents.

Filamentous, gram-negative bacteria morphologically similar to cilia-associated respiratory (CAR) bacillus of rodents and rabbits were isolated from the tracheas of 5 pigs and 4 calves. All pigs but none of the calves had histologic lesions of chronic tracheitis. In silver-stained histologic sections, CAR bacilli were adhered to the tracheal epithelium of each pig but were not found in the calves. Like CAR bacillus of rats, the bacteria displayed gliding motility and grew only in cell culture or cell culture medium supplemented with fetal serum. Initially, all isolates were contaminated by Mycoplasma spp. This contamination was eliminated from 4 pig isolates by limiting dilutions, and mycoplasma-free isolates were used to intranasally inoculate gnotobiotic pigs and CAR bacillus-free mice and rats and to immunize guinea pigs. The gnotobiotic pigs remained healthy, and when they were necropsied 4 and 7 weeks after infection no macroscopic or microscopic lesions were found in the respiratory tract. However, CAR bacillus was isolated at both times from the nasal cavities and tracheas of inoculated pigs, and the ciliated tracheal epithelium of infected pigs necropsied 7 weeks after infection was colonized by low numbers of CAR bacillus-like bacteria. The rats and mice remained healthy through week 12 postinoculation, and evidence of short- or long-term colonization was not detected by histologic examination or culture. When used as primary antibody for immunohistochemical staining, sera from guinea pigs immunized with pig CAR bacillus specifically stained CAR bacilli colonizing the respiratory epithelium of naturally infected pigs, whereas sera collected prior to immunization failed to react with the bacteria. These results indicate that CAR bacilli are unlikely to be primary pathogens of pigs or cattle and that rodents do not act as reservoirs.

A viral epizootic in cultured populations of juvenile goldfish due to a putative herpesvirus etiology.

bits and analysis of the 16S rRNA gene sequence. Lab Anim Sci 45:22–26. 3. Fernandez A, Oros J, Rodriguez JL, et al.: 1996, Morphological evidence of a filamentous cilia-associated respiratory (CAR) bacillus in goats. Vet Pathol 33:445–447. 4. Ganaway JR, Spencer TH, Moore TD, Allen AM: 1985, Isolation, propagation, and characterization of a newly recognized pathogen, cilia-associated respiratory bacillus of rats, an etiological agent of chronic respiratory disease. Infect Immunol 47: 472–479. 5. Hastie AT, Evans LP, Allen AM: 1993, Two types of bacteria adherent to bovine respiratory tract ciliated epithelium. Vet Pathol 30:12–19. 6. MacKenzie WF, Magill LS, Hulse M: 1981, A filamentous bacterium associated with respiratory disease in wild rats. Vet Pathol 18:836–839. 7. Nietfeld JC, Franklin CL, Riley LK, Zeman DH, Groff BT: 1995, Colonization of the tracheal epithelium of pigs by filamentous bacteria resembling cilia-associated respiratory bacillus. J Vet Diagn Invest 7:338–342. 8. Schoeb TR, Davidson MK, Davis JK: 1997, Pathogenicity of cilia-associated respiratory (CAR) bacillus isolates for F344, LEW, and SD rats. Vet Pathol 34:263–270. 9. Schoeb TR, Dybvig K, Davidson MK, Davis JK: 1993, Cultivation of cilia-associated respiratory bacillus in artificial medium and determination of the 16S rRNA gene sequence. J Clin Microbiol 31:2751–2757. 10. Schoeb TR, Lindsey JR: 1996, Cilia-associated respiratory bacillus infection: rat, mouse, and rabbit. In: Pathology of laboratory animals: respiratory system, ed. Jones TC, Mohr U, Hunt RD, 2nd ed., pp. 325–331. Springer-Verlag, Berlin, Germany.

Cilia-associated respiratory (CAR) bacillus infection in conventionally reared rabbits.

This study was designed to investigate the prevalence of Cilia-associated respiratory (CAR) bacillus infection in rabbits reared for meat production in Italy and to correlate the presence of CAR bacillus with inflammatory lesions of the respiratory tract. Seventy health, 3-month-old, New Zealand White rabbits, raised in 10 different rabbitries in Northern Italy were randomly selected at slaughter. No gross lesions were found at necropsy in any rabbit. In each animal, the trachea and lungs were sampled, fixed in 10% formalin, embedded in paraffin and stained with the Warthin-Starry method to evaluate the presence of CAR bacillus, and with haematoxylin and eosin to evaluate the presence of inflammatory lesions. CAR bacillus was present in 50 out of 70 rabbits (71.4%) with a prevalence of the infection that varied from 30% to 100% in the seven rabbitries. CAR bacillus was present both in the trachea and bronchi in 23 cases (32.8%), only in the trachea in 24 cases (34.3%) and only in the bronchi in three cases (4.3%). Inflammatory lesions were found in the trachea (22 cases, 31.4%) and the bronchi (58 cases, 82.8). There was a strong, statically significant correlation between the presence of CAR bacillus in the bronchi and bronchial inflammatory lesions (P < 0.0001). This study indicates that CAR bacillus infection is widespread in conventionally reared rabbits in Italy and that a possible correlation exists between the presence of CAR bacillus and bronchial inflammatory lesions.

Association of cilia-associated respiratory (CAR) bacillus with natural chronic tracheitis in goats

A histological, histochemical and immunohistological study of the respiratory tract of 83 slaughtered goats (50 adults and 33 kids) is described. Cilia-associated respiratory (CAR) bacillus was detected by means of the Warthin Starry method in the tracheal epithelium of seven (21.2%) of the kids and 16 (32%) of the adult goats. A chronic diffuse tracheitis characterized by mixed lymphocyte and plasma-cell infiltration was found in all seven kids and in 17 adults, including the 16 infected with the CAR bacillus. Although not proved, it is possible that the CAR bacillus caused the chronic tracheitis. Immunohistochemical results suggested that the caprine CAR bacillus was closely related to the rabbit CAR bacillus.

Pathogenicity of cilia-associated respiratory (CAR) bacillus isolates for F344, LEW, and SD rats.

We conducted experiments to test whether rats of F344, LEW, and SD strains differ in susceptibility to mycoplasma-free isolates of cilia-associated respiratory (CAR) bacillus, whether Mycoplasma pulmonis can affect expression of CAR bacillus disease, and whether isolates of CAR bacillus differ in virulence for rats. In the first experiment, 24 rats of each strain were inoculated intranasally with 10(7) bacilli of CAR bacillus X1428D/AS, and 24 rats of each strain were inoculated with sterile medium (controls). Eight weeks later, eight inoculated rats and eight control rats of each strain were euthanatized, eight inoculated and eight control rats were given 10(6.5) colony-forming units of M. pulmonis X1428D, and eight inoculated rats and eight control rats were sham inoculated. Four rats of each group were euthanatized 4 or 8 weeks after the second inoculation. Severity of lesions in nasal passages, middle ear, trachea, and lungs was assessed by scoring. Rats of all three strains given CAR bacillus had typical lesions of similar severity; M. pulmonis X1428D was avirulent and did not exacerbate CAR bacillus disease. In the second experiment, groups of eight rats of F344 and SD strains were given 10(5) or 10(7) CAR bacillus X1328E, X1428D/AS, or X2450D and euthanatized 8 or 16 weeks later. Isolates X1428D/AS and X2450D caused similar lesions in rats of both strains and at both doses, but CAR bacillus X1328E was avirulent. Rats of the tested strains are similarly susceptible to CAR bacillus disease, but CAR bacillus isolates differ in virulence.

Detection of Mycoplasma pulmonis in cilia-associated respiratory bacillus isolates and in respiratory tracts of rats by nested PCR.

To improve the detection of Mycoplasma pulmonis contamination of isolates of cilia-associated respiratory (CAR) bacillus, we developed a nested PCR method using primers for 16S rRNA gene sequences. Of 140 samples of 16 different CAR bacillus isolates, 73 (52%) were inhibitory in the first PCR, as indicated by the absence of amplicons of the internal control, but only 11 of 140 (7.9%) were inhibitory in the second PCR. Of 27 samples known to contain M. pulmonis, only 12 (44%) were positive in the first PCR, but 25 of 27 (93%) were positive in the second PCR. Nested PCR also detected M. pulmonis in 21 of 61 (34%) CAR bacillus samples from which M. pulmonis could not be cultured and identified 2 additional M. pulmonis-contaminated CAR bacillus isolates. Of 359 respiratory and reproductive tract lavage samples from rats and mice, 35 (9.8%) were inhibitory in the first PCR, but only 15 (4.2%) were inhibitory in the second PCR. Of 72 lavage specimens from rats inoculated with an avirulent, poorly infective M. pulmonis strain, 14 (19%) were positive by nested PCR, but only 2 of 72 (2.8%) were positive by culture. Nested PCR also detected M. pulmonis in 14 of 20 (70%) paraffin sections of lung and trachea from rats and mice inoculated with CAR bacillus isolates known to contain M. pulmonis, whereas single PCR gave no positive results. We conclude that nested PCR is superior to single PCR or culture for detecting M. pulmonis, and that M. pulmonis is present in all but four CAR bacillus isolates in our collection that were from naturally infected rats; the four isolates that were exceptions were obtained from rats from a single colony.

Bacteria associated with enzootic pneumonia in goats.

A histological and microbiological study of lung samples from 83 slaughtered goats (33 kids and 50 adults) drawn from a flock with a history of pleuropneumonia caused by mycoplasmas of the M. mycoides group was carried out. A total of 82% (27/33) of kids and 36% (18/50) of adult goats presented pulmonary lesions characteristic of enzootic pneumonia: lesions took the form of bronchointerstitial pneumonia with peribronchial and peribronchiolar proliferation of lymphocytes. Microbiological analysis confirmed a range of mycoplasma species, including Mycoplasma mycoides ssp. mycoides Large Colony (MmmlC) (3.70%; 1/27), Mycoplasma mycoides ssp. capri (Mmc) (7.40%; 2/27), Mycoplasma putrefaciens (22.2%; 6/27), Mycoplasma arginini (3.70%; 1/27) and Mycoplasma sp. (7.40%; 2/ 27), as well as Pasteurella multocida (14.8%; 4/27), associated with enzootic pneumonia lesions in younger animals, whereas Mycoplasma sp. was associated with enzootic pneumonia in adult goats (22.0%; 4/18). Cilia-associated respiratory (CAR) bacillus found by histochemical examination was associated with enzootic pneumonia in kids (25.9%; 7/27) and goats (44.4%; 8/18), being the first description of this bacterium in adult goats.

Natural cilia-associated respiratory bacillus infection in rabbits used for elaboration of hyperimmune serum against Mycoplasma sp.

Cilia-associated respiratory (CAR) bacillus was identified in lung lesions of rabbits used for elaboration of hyperimmune serum against Mycoplasma mycoides ssp. capri (Mmc). Numerous Warthin Starry (WS) positive filamentous bacteria aligned perpendicularly to the surface of bronchial epithelial lining were observed. Immunoperoxidase staining of these bacteria was detected using a serum anti-rabbit CAR bacillus. Ultrastructural morphology corresponds to that of CAR bacilli previously reported in rabbits. The desirability of monitoring laboratory rabbits for CAR bacillus infection as part of the health programme is reinforced, especially in rabbits used for raising sera against respiratory pathogens of animal species in which CAR bacillus infection has been described. This is the first report of natural CAR bacillus infection in rabbits in Europe.

Morphological evidence of a filamentous cilia-associated respiratory (CAR) bacillus in goats.

A filamentous cilia-associated respiratory (CAR) bacillus was discovered in 12 3-4 month-old goats experimentally infected with two different strains of mycoplasmas belonging to the Mycoplasma mycoides type. The CAR bacilli were always arranged parallel to the cilia, and the morphology of these bacilli is very similar to that of other bacilli described previously in other species from various parts of the world.

Colonization of the tracheal epithelium of pigs by filamentous bacteria resembling cilia-associated respiratory bacillus.

Warthin Starry staining revealed filamentous bacteria colonizing the tracheal epithelium of 41 of 88 (46.6%) pigs submitted for necropsy at 2 midwestern veterinary diagnostic laboratories. The bacteria were interspersed between and oriented parallel to the cilia. In 4 of 4 colonized pig tracheas, filamentous bacteria were demonstrated by transmission electron microscopy. The bacteria were approximately the same length and diameter as cilia, and in areas of heavy colonization the bacteria outnumbered cilia. The filamentous bacteria were similar in location and morphologic characteristics to cilia-associated respiratory (CAR) bacilli of rats, mice, rabbits, and cattle. Results of immunoperoxidase staining and polymerase chain reaction analysis indicated that the pig CAR bacillus is a different bacterium than the rat CAR bacillus. Rat CAR bacillus causes chronic respiratory disease in rats and mice. The association, if any, between pig CAR bacillus and swine respiratory disease is unknown.

Detection of cilia-associated respiratory bacillus by PCR.

The cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative, motile bacterium that has been implicated as an etiologic agent of respiratory disease in laboratory rodents. In the present study, approximately 1,200 bases of the 16S rRNA gene from three CAR bacillus isolates were sequenced. CAR bacillus-specific primers were designed on the basis of the 16S rRNA gene sequence and used in a PCR assay. The PCR assay detected as little as 500 fg of purified CAR bacillus DNA. The expected 267-bp DNA fragment was amplified from respiratory tissue of frozen, formalin-fixed, and paraffin-embedded samples from experimentally and naturally infected rats and mice. In contrast, no product was amplified from respiratory tissues of sham-infected experimental animals or animals that were serologically or histopathologically negative for the CAR bacillus. Our findings indicate that this PCR assay is a rapid, specific, and sensitive detection method for the diagnosis of CAR bacillus infection in rats and mice.

Cultivation of cilia-associated respiratory bacillus in artificial medium and determination of the 16S rRNA gene sequence

Cilia-associated respiratory (CAR) bacillus, an unclassified gliding bacterium associated with respiratory disease in rats, mice, and rabbits, has previously been cultivated only in embryonated chicken eggs, cell culture, or cell culture medium supplemented with conditioned medium from cultured tracheas. A reference strain of CAR bacillus, originally isolated in eggs, grew in cell culture flasks as adherent individual bacilli and ropy, whorled fascicles in cell culture media supplemented only with fetal calf serum. Using Dulbecco's minimal essential medium, we isolated CAR bacillus from naturally infected rats and a naturally infected rabbit and from experimentally inoculated mice and rats. Isolates were maintained for up to 20 passages. Isolates from rats were similar in morphology to the reference strain, but most were more actively motile and formed pincushion-like aggregates. The rabbit bacilli were smaller and formed fewer aggregates. DNAs of rat isolates differed only slightly in restriction fragment patterns from that of the reference strain, whereas that of the rabbit isolate was distinctly different. Cultures of CAR bacilli of all strains from rats contained Mycoplasma fermentans, Mycoplasma pulmonis, or both, and cultures of the CAR bacillus from the rabbit contained an unidentified arginine-utilizing mycoplasma. The sequence of the 16S rRNA gene of the reference strain was determined by amplification by polymerase chain reaction, cloning of the product, and sequencing by the dideoxynucleotide chain termination method. Comparison of the sequence with sequences in the GenBank data base indicated that CAR bacillus is a unique organism most closely related to Flavobacterium ferrugineum and Flexibacter sancti.

Cilia-associated respiratory bacillus in wild rats in central Iowa.

Twenty-eight wild rats were live-trapped in central Iowa (USA) to estimate the prevalence of the cilia-associated respiratory (CAR) bacillus. Both light and electron microscopy were used to look for the Gram-negative, filamentous bacterium among cilia in tracheal and lung tissue sections. The organism was observed in the trachea of 20 rats with chronic respiratory disease and in the trachea of three of eight normal rats. Therefore, the organism appears to be common among wild rats in central Iowa.

Two types of bacteria adherent to bovine respiratory tract ciliated epithelium.

Two hundred sixty tracheas were obtained from a Philadelphia abattoir under permit from the Department of Agriculture; the tracheas were excised from predominantly Holstein calves of both sexes that weighed approximately 250 kg. Tracheas were transported in normal saline to the laboratory at Thomas Jefferson University, Philadelphia, Pennsylvania. Evidence of bacteria adherent to the tracheal epithelium was found in specimens from 20/24 of these tracheas. The epithelium from each of five tracheas was placed in glutaraldehyde fixative for transmission electron microscopic examination. Epithelium from each of 12 other tracheas was placed in formaldehyde fixative for light microscopic examination. Microscopically, 13 of these 17 bovine tracheal epithelia were observed to contain bacteria located longitudinally parallel to and between cilia and microvilli of ciliated cells. Preparations of ciliary axonemes isolated from the epithelium of seven additional bovine tracheas also contained these bacteria in sections viewed by a transmission electron microscope. These bacteria had two different ultrastructural morphologies: filamentous with a trilaminar-structured cell wall and short with a thick, homogeneously stained cell wall beneath a regularly arrayed surface layer. The short bacillus had surface carbohydrates, including mannose, galactose, and N-acetylgalactosamine, identified by lectin binding. The filamentous bacillus was apparently externally deficient in these carbohydrates. Immunogold staining revealed that the filamentous bacillus was antigenically related to cilia-associated respiratory (CAR) bacillus, which has been identified in rabbit and rodent species. Significantly decreased numbers of cilia were obtained from tracheal epithelium heavily colonized by the filamentous bacilli, suggesting a pathologic change in ciliated cells.

Pathology of rats intranasally inoculated with the cilia-associated respiratory bacillus

Five-week-old Wistar/Ms rats were inoculated intranasally with a lung homogenate containing a strain of cilia-associated respiratory (CAR) bacillus and were examined on days 4, 7, 14, 21, 28 and 56 postinoculation (PI). Some rats showed clinical signs with wheezing and considerable body weight loss from day 21 PI. Gross lesions, including enlargement of lungs with focal atelectasis, bronchiectasis and emphysema, were observed from day 21 PI. Histologically, round cell infiltration was first present in the lamina propria of the nasal respiratory mucosa on day 7 PI. From day 14 PI, colonization of the CAR bacillus (4-8 micron in length), associated with round cell infiltration in the lamina propria and the peripheral regions, was observed in the ciliated mucosa of the bronchioles, bronchi, trachea and nasal cavities. Generally, the lesions progressed and expanded from upper to lower airways with time. Sporadic mucopurulent bronchopneumonia was observed from day 21 PI in some rats. The CAR bacilli (0.2-0.25 micron in diameter) were also demonstrated electron-microscopically in the ciliated epithelium of the intrapulmonary airways. The CAR bacillus antigen was demonstrated on the ciliated mucosa of the affected airways by the indirect immunofluorescence assay technique. Microbiological examination revealed that the rats used in this study were free from other known respiratory pathogens throughout the experimental period. Thus, it is suggested that the CAR bacillus alone can produce a murine respiratory disease. Fourteen days were needed for pathological lesions to develop.