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Chlamydia trachomatis is a clinically important bacterium that infects epithelial cells of the genitourinary and respiratory tracts and the eye. These differentiated cells are in a quiescent growth state and have a surface organelle called a primary cilium, but the standard Chlamydia cell culture infection model uses cycling cells that lack primary cilia. To investigate if these differences are relevant, we performed infections with host cells that have a primary cilium. We found that C. trachomatis caused progressive loss of the primary cilium that was prevented by disrupting Aurora A (AurA), HDAC6 or calmodulin, which are components of the cellular cilia disassembly pathway. Stabilization of the primary cilium by targeting this pathway caused a large reduction in infectious progeny although there were no changes in chlamydial inclusion growth, chlamydial replication or the ultrastructural appearance of dividing and infectious forms (RBs and EBs, respectively). Thus, the presence of a primary cilium interfered with the production of infectious EBs at a late step in the developmental cycle. C. trachomatis infection also induced quiescent cells to re-enter the cell cycle, as detected by EdU incorporation in S-phase, and Chlamydia-induced cilia disassembly was necessary for cell cycle re-entry. This study therefore describes a novel host-pathogen interaction in which the primary cilium limits a productive Chlamydia infection, and the bacterium counteracts this host cell defense by activating the cellular cilia disassembly pathway., Competing Interests: The authors have declared that no competing interests exists., (Copyright: © 2024 Ekka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This study was conducted to investigate whether eucalyptol plays a role in influencing bacterial growth in cigarette smoke-exposed lungs. Rats were exposed to air (control) and cigarette smoke (smoking) in the presence and absence of eucalyptol (260 mg/day). Morphological analysis of lung structures and status of airway mucous production were observed under microscope. Pathological changes of ciliated columnar epithelium in airways were examined using transmission electron microscopy. MUC5AC protein and messenger RNA (mRNA) expression in bronchoalveolar lavage fluid (BALF) and lungs were determined. Application of eucalyptol reduced pulmonary bullae formation and airway mucus overproduction in the smoke-exposed lungs. Treatment with eucalyptol attenuated ciliated cell damage in cigarette smoke-exposed lungs. Bacterial colonies of lungs were obviously lower in the eucalyptol-treated rats than that in the smoking rats (p < 0.01). Treatment with eucalyptol reduced the counts of bacterial colonization residing in the challenged lungs (p < 0.01). Application of eucalyptol not only decreased MUC5AC protein expression in BALF and tobacco-exposed lungs but also suppressed its mRNA expression in the lungs (all p < 0.05). Intervention of eucalyptol benefits elimination of bacterial organisms from tobacco-exposed lungs through attenuating ciliated cell damage and suppressing MUC5AC expression in the lungs., (© 2018 Wiley Periodicals, Inc.)

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP) and responsible for major economic losses in global swine industry. After colonization of the respiratory epithelium, M. hyopneumoniae elicits a general mucociliary clearance loss, prolonged inflammatory response, host immunosuppression and secondary infections. Until now, the pathogenesis of M. hyopneumoniae is not completely elucidated. This present study explores the pathogenicity of mhp390 (P68, a membrane-associated lipoprotein) by elucidating its multiple functions. Microtitrer plate adherence assay demonstrated that mhp390 is a new cilia adhesin that plays an important role in binding to swine tracheal cilia. Notably, mhp390 could induce significant apoptosis of lymphocytes and monocytes from peripheral blood mononuclear cells (PBMCs), as well as primary alveolar macrophages (PAMs), which might weaken the host immune response. In addition, mhp390 contributes to the production of proinflammatory cytokines, at least partially, via the release of IL-1β and TNF-α. To the best of our knowledge, this is the first report of the multiple functions of M. hyopneumoniae mhp390, which may supplement known virulence genes and further develop our understanding of the pathogenicity of M. hyopneumoniae., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Several species of the Gram-negative genus Bordetella are the cause of respiratory infections in mammals and birds, including whooping cough (pertussis) in humans. Very recently, a novel atypical species, Bordetella pseudohinzii, was isolated from laboratory mice. These mice presented no obvious clinical symptoms but elevated numbers of neutrophils in bronchoalveolar lavage fluid and inflammatory signs in histopathology. We noted that this species can occur at high prevalence in a mouse facility despite regular pathogen testing according to the FELASA-recommendations. Affected C57BL/6 J mice had, in addition to the reported pulmonary alterations, tracheal inflammation with reduced numbers of ciliated cells, slower ciliary beat frequency, and largely (>50%) compromised cilia-driven particle transport speed on the mucosal surface, a primary innate defence mechanism. In an in vitro-model, Bordetella pseudohinzii attached to respiratory kinocilia, impaired ciliary function within 4 h and caused epithelial damage within 24 h. Regular testing for this ciliotropic Bordetella species and excluding it from colonies that provide mice for lung research shall be recommended. On the other hand, controlled colonization and infection with Bordetella pseudohinzii may serve as an experimental model to investigate mechanisms of mucociliary clearance and microbial strategies to escape from this primary innate defence response.

Objective: The aim of this cross-sectional in vitro study was to evaluate the mucosal surfaces of healthy maxillary sinuses, explore different forms of bacterial microorganism colonies present on the mucous membrane, and determine a mucosal surface area they occupy., Methods: Samples of the maxillary sinus mucosa were collected from 30 healthy patients (M = 11; F = 19). The material was obtained during the Le Fort I osteotomy performed during corrective jaw surgery. The morphological and morphometric analysis of sinus mucosa and bacterial film that was grown on it was performed using scanning electron microscopy (SEM) as well as imaging software., Results: Scanning electron microscopy analysis showed the presence of different bacterium and bacteria-like structures in all the analyzed samples. In most cases, the bacterial film was mostly composed of diplococci-like and streptococci-like structures on the mucosa of the paranasal sinus. In any case, the mucous layer did not cover the whole lining of the evaluated sample. Each colony consists of more than 20 single bacterial cells, which has grown in aggregates., Conclusions: Under the conditions of normal homeostasis of the body, the maxillary sinuses present diverse bacterial colonization. The bacteria are dispersed or concentrated in single microcolonies of the biofilm on the border of the mucous covering the ciliary epithelium. There is no uniform layer of the biofilm covering the mucosa of the maxillary sinuses. Because the biofilm is detected on healthy individuals sinus mucosa, the clinical question if it may become pathogenic is unclear and require an explanation.

Diverse animal taxa metamorphose between larval and juvenile phases in response to bacteria. Although bacteria-induced metamorphosis is widespread among metazoans, little is known about the molecular changes that occur in the animal upon stimulation by bacteria. Larvae of the tubeworm Hydroides elegans metamorphose in response to surface-bound Pseudoalteromonas luteoviolacea bacteria, producing ordered arrays of phage tail-like metamorphosis-associated contractile structures (MACs). Sequencing the Hydroides genome and transcripts during five developmental stages revealed that MACs induce the regulation of groups of genes important for tissue remodeling, innate immunity, and mitogen-activated protein kinase (MAPK) signaling. Using two MAC mutations that block P. luteoviolacea from inducing settlement or metamorphosis and three MAPK inhibitors, we established a sequence of bacteria-induced metamorphic events: MACs induce larval settlement; then, particular properties of MACs encoded by a specific locus in P. luteoviolacea initiate cilia loss and activate metamorphosis-associated transcription; finally, signaling through p38 and c-Jun N-terminal kinase (JNK) MAPK pathways alters gene expression and leads to morphological changes upon initiation of metamorphosis. Our results reveal that the intricate interaction between Hydroides and P. luteoviolacea can be dissected using genomic, genetic, and pharmacological tools. Hydroides' dependency on bacteria for metamorphosis highlights the importance of external stimuli to orchestrate animal development. The conservation of Hydroides genome content with distantly related deuterostomes (urchins, sea squirts, and humans) suggests that mechanisms of bacteria-induced metamorphosis in Hydroides may have conserved features in diverse animals. As a major biofouling agent, insight into the triggers of Hydroides metamorphosis might lead to practical strategies for fouling control., Competing Interests: The authors declare no conflict of interest.

Bordetella bronchiseptica frequently causes nonfatal tracheobronchitis, but its role in fatal pneumonia is less recognized. Our study evaluated histologic identification of cilia-associated bacteria as a method for diagnosis of B. bronchiseptica pneumonia. Cases of fatal bronchopneumonia were studied retrospectively, excluding neonates and cases of aspiration pneumonia, minor lung lesions, or autolysis. The study population comprised 36 canine and 31 feline cases of bronchopneumonia. B. bronchiseptica was identified in 8 of 36 canine and 14 of 31 feline cases based on immunohistochemistry (IHC) using serum from a rabbit hyperimmunized with pertactin, PCR testing (Fla2/Fla12), and/or bacterial culture data when available. Of these, IHC was positive in 4 canine and 7 feline cases, PCR was positive in 8 canine and 14 feline cases, and B. bronchiseptica was isolated in 2 of 5 canine and 3 of 9 feline cases tested. Examination of histologic sections stained with hematoxylin and eosin revealed bronchial cilia-associated bacteria in 4 of 36 canine and 5 of 31 feline cases; these were all positive by IHC and PCR. The presence of cilia-associated bacteria had been noted in the pathology report for only 2 of these 9 cases. Thus, the presence of cilia-associated bacteria seems frequently overlooked by pathologists, but is a diagnostically significant feature of B. bronchiseptica pneumonia. A specific diagnosis of B. bronchiseptica pneumonia is important because it suggests primary or opportunistic bacterial pneumonia rather than aspiration pneumonia, and because of the risk of animal-to-animal transmission of B. bronchiseptica, the availability of vaccines for disease prevention, and the potential zoonotic risk to immunocompromised pet owners., (© 2016 The Author(s).)

Background: The pathogenesis of Mycoplasma pneumoniae infection involves cytoadherence of M. pneumoniae to the ciliated respiratory epithelium (CRE), followed by CRE injury caused by the M. pneumoniae. However, whether CRE abnormalities are related to the severity of M. pneumoniae pneumonia (MP) remains to be determined., Methods: Thirty-eight patients with MP and 8 controls who underwent fiber-optic bronchoscopy with bronchial biopsy were included in this study. Patients with MP were divided into 2 groups: a mild disease group (12 patients) and a severe disease group (26 patients). The clinical features, laboratory findings, chest radiographic findings, and CRE abnormalities were characterized., Results: Patients with severe pneumonia had a higher epithelial integrity score than those with mild pneumonia (5.1 ± 0.76 vs 3.8 ± 0.75; p < 0.01). Patients with severe CRE abnormalities had a longer duration of fever (p < 0.01), higher C-reactive protein (p < 0.01), and lower proportion of blood lymphocytes (p < 0.05) compared to those with mild abnormalities. Patients with a positive bacteria culture had a higher epithelial integrity score compared to those with a negative culture (6.0 ± 0.44 vs 4.8 ± 0.71; p < 0.01)., Conclusions: CRE abnormalities are closely related to the severity of MP. These findings extend our current knowledge of MP.

Background: Bacterial biofilms are thought to contribute to recalcitrance in chronic rhinosinusitis (CRS) patients. Manuka honey (MH) and its active component methylglyoxal (MGO) have demonstrated antibiofilm activity in vitro. This study evaluated the safety and efficacy of these agents in an in vivo model., Methods: To assess safety, ovine frontal sinuses were flushed twice daily for 14 days. In each sheep, 1 sinus was flushed with a panel of MGO concentrations ranging from 0.5 to 7.2 mg/mL alone and flushed with a panel of with 16.5% wt/vol MH enriched with MGO at the same range of concentrations (0.5-7.2 mg/mL; designated MH/MGO). Contralateral sinuses were flushed with saline control. Tissue morphology was assessed histologically and with scanning electron microscopy. Efficacy was tested by developing Staphylococcus aureus biofilms in sheep sinuses. Twice-daily irrigation for 5 days was commenced with either saline, MGO (0.5-3.6 mg/mL) alone, or MH/MGO (with 0.5-3.6 mg/mL MGO). Biofilm biomass was compared between the groups (n = 4) using LIVE/DEAD BacLight staining and confocal scanning laser microscopy., Results: The results of the safety assessment, for normal sinuses treated with MGO alone or with MH/MGO (≤1.8 mg/mL) showed normal pseudostratified epithelium and cilia structure; however, higher concentrations caused cilia denudation and squamous metaplasia. As for efficacy, when compared to saline flush, treatment with MH/MGO at 0.9 mg/mL (0.608 ± 0.110 vs 0.316 ± 0.197 μm(3) /μm(2) , respectively; p = 0.015) and 1.8 mg/mL (0.676 ± 0.079 vs 0.114 ± 0.033 μm(3) /μm(2) , respectively; p = 0.001) significantly reduced biofilm biomass., Conclusion: Sinus irrigation with MH/MGO at MGO concentrations between 0.9 and 1.8 mg/mL is both safe to mucosa and efficacious against S. aureus biofilm. MH/MGO irrigation could represent a viable treatment option for recalcitrant CRS., (© 2014 ARS-AAOA, LLC.)

We studied the Euprymna scolopes-Vibrio fischeri symbiosis to characterize, in vivo and in real time, the transition between the bacterial partner's free-living and symbiotic life styles. Previous studies using high inocula demonstrated that environmental V. fischeri cells aggregate during a 3 h period in host-shed mucus along the light organ's superficial ciliated epithelia. Under lower inoculum conditions, similar to the levels of symbiont cells in the environment, this interaction induces haemocyte trafficking into these tissues. Here, in experiments simulating natural conditions, microscopy revealed that at 3 h following first exposure, only ∼ 5 V. fischeri cells aggregated on the organ surface. These cells associated with host cilia and induced haemocyte trafficking. Symbiont viability was essential and mutants defective in symbiosis initiation and/or production of certain surface features, including the Mam7 protein, which is implicated in host cell attachment of V. cholerae, associated normally with host cilia. Studies with exopolysaccharide mutants, which are defective in aggregation, suggest a two-step process of V. fischeri cell engagement: association with host cilia followed by aggregation, i.e. host cell-symbiont interaction with subsequent symbiont-symbiont cell interaction. Taken together, these data provide a new model of early partner engagement, a complex model of host-symbiont interaction with exquisite sensitivity., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Chronic recurrent sinusitis (CRS) is one of the most common chronic conditions in the United States. There is a significant subpopulation of CRS patients who remain resistant to cure despite rigorous treatment regimens including surgery, allergy therapy, and prolonged antibiotic therapy. Antimicrobial photodynamic therapy (aPDT) is a noninvasive nonantibiotic broad spectrum antimicrobial treatment. Our previous in vitro studies demonstrated that aPDT reduced CRS polymicrobial biofilm and planktonic bacteria and fungi by > 99.9% after a single treatment. Prior to human treatment however, aPDT treatment must be demonstrated to not result in histologic damage to the sinus ciliated respiratory epithelium. The objective of this study was to demonstrate the safety of aPDT treatment on a living human ciliated respiratory mucosal model (EpiAirway)., Methods: A study of aPDT treatment of EpiAirway was performed. Treatment groups included a nontreatment control, laser light alone, photosensitizer alone, and therapeutic photosensitizer and light combination (aPDT). At completion of treatment, the EpiAirway tissue was fixed in 10% formalin, paraffin-embedded, sectioned, H&E stained and mounted. All samples were blinded and microscopically examined by a human pathologist to assess any effect of aPDT on the tissue, cilia, or mucosal glands. The results were correlated with the treatment parameters., Results: The EpiAirway histologic study demonstrated no histologic alteration of the respiratory cilia or mucosal epithelium in any of the treatment groups., Conclusions: aPDT is a safe treatment for CRS resulting in no histologic alteration of human ciliated respiratory mucosa as is found in the human sinuses., (Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.)

Neisseria gonorrhoeae encodes five lytic transglycosylases (LTs) in the core genome, and most gonococcal strains also carry the gonococcal genetic island that encodes one or two additional LTs. These peptidoglycan (PG)-degrading enzymes are required for a number of processes that are either involved in the normal growth of the bacteria or affect the pathogenesis and gene transfer aspects of this species that make N. gonorrhoeae highly inflammatory and highly genetically variable. Systematic mutagenesis determined that two LTs are involved in producing the 1,6-anhydro PG monomers that cause the death of ciliated cells in Fallopian tubes. Here, we review the information available on these enzymes and discuss their roles in bacterial growth, cell separation, autolysis, type IV secretion, and pathogenesis.

Objective: To observe the micromorphological characteristic of bacterial biofilm on mucosa of chronic rhinosinusitis (CRS)., Method: Mucosa samples of middle turbinate were obtained from 4 patients of CRS during ESS. The size of each sample was about 4 mm x 4 mm. The samples were fixed in 4% paraformaldehyde for 24 hours, then fixed in 1% osmium tetroxide for 2 hours, graded dehydration with ethanol, dried with carbon dioxide and sputter coated with gold. The ultrastructure of these samples was observed by scanning electron microscope., Result: Bacterial biofilms were found on samples in all 4 patients. The biofilms were mainly formed on the surface of cilia. The bacterial flagella and cilia were intertwined. The biofilms could be found in a lot kinds of bacterial infections or mixed infections which were caused by multiple bacteria and fungi., Conclusion: Bacterial biofilm could be formed on ciliated epithelia.

Background: Chronic rhinosinusitis is a multifactorial disease resulting in impaired mucociliary clearance. Recent literature suggests that different bacterial species are associated with varied disease severity. We examined the immediate effect of microbial secreted factors on sinonasal ciliary function., Methods: Murine primary sinonasal cultures were established in an air-liquid interface (ALI). Bacterial supernatants were isolated from H. influenza, S. pneumoniae, S. aureus, and P. aeruginosa cultures, as well as co-cultures of H. influenza/S. pneumoniae and S. aureus/P. aeruginosa. Controlling for pH and osmolarity, supernatants were administered at 50% concentration to the apical surface of the ALI culture. Basal ciliary beat frequency (CBF) was recorded for 20 minutes, at 5-minute intervals. Control groups were treated with culture broth. At minimum, experiments were performed in triplicate. Stimulated CBF was recorded after mechanical stimulation via short bursts of pressurized air (55 mmHg)., Results: All supernatants reduced basal CBF. S. pneumoniae and P. aeruginosa caused significant reduction in CBF at all time points, with the largest decrease of -46.3 ± 1.6% (p < 0.001) for S. pneumoniae and -27.1 ± 2.8% (p < 0.001) for P. aeruginosa. S. aureus caused the basal CBF to decline by -33.0 ± 2.8% (p < 0.001) at 5 minutes, which reversed by 20 minutes. Overall, H. influenza yielded the least change in CBF (-20.0 ± 2.8%, p < 0.002). Co-cultures (H. influenza/S. pneumoniae and S. aureus/P. aeruginosa) resulted in delayed CBF reduction compared with monocultures. P. aeruginosa also blunted stimulated CBF (p < 0.02)., Conclusion: Results demonstrated acute decreases in murine sinonasal CBF after exposure to bacterial supernatants. Moreover, P. aeruginosa resulted in diminished ciliary stimulation capacity., (Copyright © 2012 American Rhinologic Society-American Academy of Otolaryngic Allergy, LLC.)

Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45(683), P48(683), and P50(683). A peptide sequence (TTKF↓QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48(683) and P50(683), determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45(683), P48(683), and P50(683) reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1(683)-F5(683), spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1(683)-F5(683) also bound porcine epithelial cilia, and antisera to F2(683) and F5(683) significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45(683), P48(683), and P50(683) each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.

Background: Sinonasal mucosal biofilms are recognized as contributors to the pathogenesis of chronic rhinosinusitis (CRS). Attachment of bacteria to the sinonasal surface is an initial step in biofilm formation. A critical defense against this occurrence is mucociliary clearance (MCC). To ascertain whether the ciliary component of MCC is uniform throughout the airway we compared ciliary beat frequency (CBF) in the murine nasal septum and trachea at baseline and after challenge with Pseudomonas aeruginosa, a common pathogen of CRS., Methods: Murine septal and tracheal air-liquid interface cultures were evaluated for basal and stimulated CBF after exposure to control or conditioned media from Pseudomonas. Additionally, the attachment of Pseudomonas to nasal and tracheal cultures was assessed after pretreatment with control or conditioned media., Results: Basal CBF is significantly slower in primary nasal airway cultures compared with tracheal airway cultures. Tracheal airway cultures show resistance to Pseudomonas secreted ciliotoxins not evident in nasal septal cultures. Furthermore, after challenge with viable Pseudomonas, significantly more bacteria attach to the nasal cultures compared with the tracheal cultures., Conclusion: Using primary murine nasal and tracheal airway cultures we show inherent differences in cilia function and increased susceptibility of the upper airway to attachment by Pseudomonas. Understanding the differences between upper and subglottic airway mucociliary clearance should lead to novel approaches in the management of upper airway infection.

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, adheres to ciliated respiratory epithelia resulting in ciliostasis and epithelial cell death. The cilium adhesin P97 (Mhp183) contains two repeat regions, designated R1 and R2, that play key roles in adherence. Eight pentapeptide repeats in R1 are sufficient to bind porcine cilia; however, both R1 and R2 are needed to bind heparin. Mhp271, a paralogue of P97, is the only other M. hyopneumoniae protein to contain both R1 and R2 repeats. These repeats are arranged as a set of three pentapeptide repeats (designated R1A₂₇₁), two decapeptide repeats (designated R2₂₇₁), and a second set of six pentapeptide repeats (designated R1B₂₇₁). To determine their function, recombinant proteins containing R1A₂₇₁) (F1₂₇₁) and R2₂₇₁-R1B₂₇₁ (F2₂₇₁) were constructed and used in in vitro binding assays. F2₂₇₁, but not F1₂₇₁, bound heparin (K(D)=8.1 ± 0.4 nM), fibronectin (K(D)=174 ± 13 nM) and porcine cilia. Pre-incubation of F2₂₇₁ with 100 µM heparin blocked cilium binding by ~69%. Cell surface shaving with trypsin combined with two-dimensional liquid chromatography coupled to tandem mass spectrometry analysis identified Mhp271 as surface-exposed. Our data suggest that both R1 and R2 in Mhp271 are involved in binding to host molecules., (© 2010 Blackwell Publishing Ltd.)

Background: Ciliated ependymal cells line the cerebral ventricles and aqueducts separating the infected CSF from the brain parenchyma in meningitis., Principal Findings: Investigation of the interaction of Listeria monocytogenes with cultured rat brain ependymal cells showed that certain strains reduced the beat frequency of the cilia but all the strains studied significantly reduced the ciliary beat amplitude (the linear distance travelled by the tip of each cilium per beat cycle)., Conclusion: The presence of the ependyma caused aggregation of some listeria strains and in some cases extracellular material also was seen in association with bacterial aggregates. These observations were dependent on the expression of genes required for invasion, intracellular survival and listerial cell to cell spread that are regulated by the transcriptional activator, positive regulatory factor A (PrfA).

Influenza virus infections increase susceptibility to secondary bacterial infections, such as pneumococcal pneumonia, resulting in increased morbidity and mortality. Influenza-induced tissue damage is hypothesized to increase susceptibility to Streptococcus pneumoniae infection by increasing adherence to the respiratory epithelium. Using a mouse model of influenza infection followed by S. pneumoniae infection, we found that an influenza infection does not increase the number of pneumococci initially present within the trachea, but does inhibit pneumococcal clearance by 2 hours after infection. To determine whether influenza damage increases pneumococcal adherence, we developed a novel murine tracheal explant system to determine influenza-induced tissue damage and subsequent pneumococcal adherence. Murine tracheas were kept viable ex vivo as shown by microscopic examination of ciliary beating and cellular morphology using continuous media flow for up to 8 days. Tracheas were infected with influenza virus for 0.5-5 days ex vivo, and influenza-induced tissue damage and the early stages of repair to the epithelium were assessed histologically. A prior influenza infection did not increase pneumococcal adherence, even when the basement membrane was maximally denuded or during the repopulation of the basement membrane with undifferentiated epithelial cells. We measured mucociliary clearance in vivo and found it was decreased in influenza-infected mice. Together, our results indicate that exposure of the tracheal basement membrane contributes minimally to pneumococcal adherence. Instead, an influenza infection results in decreased tracheal mucociliary velocity and initial clearance of pneumococci, leading to an increased pneumococcal burden as early as 2 hours after pneumococcal infection.

Aims: Adherence of Mycoplasma hyopneumoniae to the ciliated epithelial cells of the porcine respiratory tract is considered an important first step in the pathogenesis of enzootic pneumonia. It was the aim of this study to verify the usefulness of in vitro adhesion as a virulence marker., Methods and Results: Adherence capacity to immobilized cilia from porcine tracheal epithelial cells of three low, two moderately and two highly virulent M. hyopneumoniae field isolates was determined by a microtitre plate adherence assay., Conclusions: No significant differences between the isolates were demonstrated., Significance and Impact of the Study: The results suggest that mechanisms other than adherence might be responsible for the observed differences in virulence of these field isolates or that the in vitro assay does not adequately reproduce in vivo adherence conditions.

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.

Background: It has been reported that Chlamydophila (C.) pneumoniae is involved in the initiation and promotion of asthma and chronic obstructive pulmonary diseases (COPD). Surprisingly, the effect of C. pneumoniae on airway function has never been investigated., Methods: In this study, mice were inoculated intranasally with C. pneumoniae (strain AR39) on day 0 and experiments were performed on day 2, 7, 14 and 21., Results: We found that from day 7, C. pneumoniae infection causes both a sustained airway hyperresponsiveness and an inflammation. Interferon-gamma (IFN-gamma) and macrophage inflammatory chemokine-2 (MIP-2) levels in bronchoalveolar lavage (BAL)-fluid were increased on all experimental days with exception of day 7 where MIP-2 concentrations dropped to control levels. In contrast, tumor necrosis factor-alpha (TNF-alpha) levels were only increased on day 7. From day 7 to 21 epithelial damage and secretory cell hypertrophy was observed. It is suggested that, the inflammatory cells/mediators, the epithelial damage and secretory cell hypertrophy contribute to initiation of airway hyperresponsiveness., Conclusion: Our study demonstrates for the first time that C. pneumoniae infection can modify bronchial responsiveness. This has clinical implications, since additional changes in airway responsiveness and inflammation-status induced by this bacterium may worsen and/or provoke breathlessness in asthma and COPD.

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.

Chronic sinusitis is a prevalent, debilitating condition, and a subpopulation of patients fails to respond to either medical or surgical intervention. Bacterial biofilms are 3-dimensional aggregates of bacteria that have special properties due to their group structure, including increased resistance to antibiotics in some forms. They have been shown to play a major role in many chronic infections, including cystic fibrosis, endocarditis, and otitis media. Evidence now suggests that they may play an important role in chronic sinusitis. Our laboratory has identified the presence of biofilms in sinonasal mucosa isolated from human patients and on stents removed after frontal sinus surgery. In addition, biofilms have been found on the sinus epithelium of rabbits infected with Pseudomonas aeruginosa, but not in rabbits infected with non-biofilm-forming P. aeruginosa mutants. This animal model can provide opportunities to address the functional significance of biofilm production in the sinus cavities. A further understanding of the role of bacterial biofilms may lead to the development of more appropriate therapies for the treatment and prevention of chronic sinusitis.

The aim of this study was to investigate by light microscopy as well as by scanning and transmission electron microscopy the deciliation process which takes place on the respiratory epithelium of tracheal explants after experimental infection with Mycoplasma fermentans strain incognitus. Time-point photography allowed distinguishing five phases which occurred during the infection on the epithelial cell surface: (1) Attachment of M. fermentans to the cilia causing clumping of the cilia tips; (2) matting of cilia into bundles; (3) formation of abnormally shaped and shorter cilia; (4) collapse of cilia onto the epithelial cell surface; and (5) widespread loss of cilia. Based on the photographic images, a schematic model of the deciliation process was developed. Various potential factors contributing to the cilia destruction are discussed, including the release of mycoplasmal toxins, the physical presence of a high number of M. fermentans cells attached to the cilia, and the depletion of culture medium components by the mycoplasmas. This model of M. fermentans strain incognitus infection of respiratory epithelium is important for understanding mycoplasmal pathogenicity on a comparative level.

Cystic fibrosis (CF) lung disease is characterized by persistent lung infection. Thickened (concentrated) mucus in the CF lung impairs airway mucus clearance, which initiates bacterial infection. However, airways have other mechanisms to prevent bacterial infection, including neutrophil-mediated killing. Therefore, we examined whether neutrophil motility and bacterial capture and killing functions are impaired in thickened mucus. Mucus of three concentrations, representative of the range of normal (1.5 and 2.5% dry weight) and CF-like thickened (6.5%) mucus, was obtained from well-differentiated human bronchial epithelial cultures and prepared for three-dimensional studies of neutrophil migration. Neutrophil chemotaxis in the direction of gravity was optimal in 1.5% mucus, whereas 2.5% mucus best supported neutrophil chemotaxis against gravity. Lateral chemokinetic movement was fastest on airway epithelial surfaces covered with 1.5% mucus. In contrast, neutrophils exhibited little motility in any direction in thickened (6.5%) mucus. In in vivo models of airway mucus plugs, neutrophil migration was inhibited by thickened mucus (CF model) but not by normal concentrations of mucus ("normal" model). Paralleling the decreased neutrophil motility in thickened mucus, bacterial capture and killing capacity were decreased in CF-like thickened mucus. Similar results with each mucus concentration were obtained with mucus from CF cultures, indicating that inhibition of neutrophil functions was mucus concentration dependent not CF source dependent. We conclude that concentrated ("thick") mucus inhibits neutrophil migration and killing and is a key component in the failure of defense against chronic airways infection in CF.

In the virulent state (Bvg+), Bordetella bronchiseptica expresses adhesins and toxins that mediate adherence to the upper airway epithelium, an essential early step in pathogenesis. In this study, we used a rabbit tracheal epithelial cell binding assay to test how specific host or pathogen factors contribute to ciliary binding. The host antimicrobial agent surfactant protein A (SP-A) effectively reduced ciliary binding by Bvg+ B. bronchiseptica. To evaluate the relative contributions of bacterial adhesins and toxins to ciliary binding, we used mutant strains of B. bronchiseptica in the binding assay. When compared to Bvg+ or Bvg- phase-locked B. bronchiseptica strains, single-knockout strains lacking one of the known adhesins (filamentous hemagglutinin, pertactin, or fimbriae) displayed an intermediate ciliary binding capacity throughout the coincubation. A B. bronchiseptica strain deficient in adenylate cyclase-hemolysin toxin also displayed an intermediate level of adherence between Bvg+ and Bvg- strains and had the lowest ciliary affinity of any of the Bvg+ phase strains tested. A B. bronchiseptica strain that was missing dermonecrotic toxin also displayed intermediate binding; however, this strain displayed ciliary binding significantly higher than most of the adhesin knockouts tested. Taken together, these findings suggest that virulent-state B. bronchiseptica expresses multiple adhesins with overlapping contributions to ciliary adhesion and that host production of SP-A can provide innate immunity by blocking bacterial adherence to the ciliated epithelium.

We have studied gonococcal infection in human endometrium organ culture and in human primary endometrial epithelial cells using various microscopic techniques including scanning electron microscopy, transmission electron microscopy, bright field light microscopy and laser scanning confocal microscopy. Here we describe the interactions between Neisseria gonorrhoeae and human endometrial luminal epithelial cells at the ultrastructural levels. N. gonorrhoeae attached to cilia but were not observed associated with the plasma membrane of ciliated epithelial cells or internalized into ciliated epithelial cells. N. gonorrhoeae could be found in intracellular vacuoles in secretory epithelial cells. N. gonorrhoeae have diverse interactions with endometrial epithelium. These include intimate association and colocalization with asialoglycoprotein receptor (ASGP-R) and CEACAM, lamellipodia and ruffle formation and colocalization with CR3, and microvillus engagement. These studies indicate that N. gonorrhoeae utilize multiple mechanisms to associate with endometrial epithelial cells and can associate with both ciliated and secretory cells. This diversity is consistent with a role of the endometrium as a transition zone between frequently asymptomatic cervical gonorrhoea and symptomatic pelvic inflammatory disease.

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.

Objectives: Biofilms are bacterial pathogens that organize in several chronic and recalcitrant infectious processes. We hypothesize that biofilms play a role in chronic rhinosinusitis (CRS). Our goal is to demonstrate biofilms in mucosal specimens of patients undergoing surgery for CRS., Study Design: A prospective study of the presence of biofilms in patients undergoing endoscopic sinus surgery for CRS compared with control patients without CRS., Methods: There were a total of 30 subjects and 4 controls enrolled. The samples of 24 subjects and 4 controls were cultured and then prepared using standard methods for scanning electron microscopy (SEM). The remaining six subjects' samples were treated using advanced cryofixation methods as preparation to preserve structure for SEM and transmission electron microscopy (TEM)., Results: Using strict SEM morphologic criteria, 24 (80%) of the 30 patients were found to have micrographic evidence of biofilms. All controls had healthy appearing cilia and goblet cells without biofilms. The six cryofixation samples showed biofilm structures on SEM micrographs that were correlated with bacterial structures seen at the mucosal surface on the corresponding TEM cross sections. Bacterial cultures were positive on all patients., Conclusions: Biofilms were demonstrated to be present in patients undergoing surgery for CRS; none of the patients without CRS had any evidence of biofilms. Although SEM is capable of demonstrating the biofilms' three-dimensional structure, glycocalyx, and water channels, it cannot clearly demonstrate the presence of bacteria within the biofilm. We were able to demonstrate evidence of bacteria in the biofilms on the subjects tested using TEM.

The primary site of infection for Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis is the ciliated respiratory epithelium. Previous studies have implicated adherence of bacteria to cilia, induction of mucus production, induction of ciliostasis and damage to the ciliated epithelium in Bordetella pathogenesis. This paper describes the use of an air-interface organ culture system using canine tracheal tissue infected with B. bronchiseptica to assess the temporal relationship between these pathologies. Ciliostasis occurs very early during the host tissue-pathogen interaction, before mucus production and obvious signs of epithelial damage occur. A B. bronchiseptica bvg mutant does not colonize the organ culture model, induce ciliostasis or cause damage to the epithelial cell layer, but it does induce similar amounts of mucus release as does infection by wild-type bacteria. The authors propose that ciliostasis is a key early event during the B. bronchiseptica-host tissue interaction that abrogates the muco-ciliary defences of the host tissue, renders it susceptible to colonization by the bacteria and allows subsequent damage to the epithelium. The organ culture model described offers a physiologically relevant tool with which to characterize the molecular basis for interactions between Bordetella and its primary site of infection, the ciliated respiratory epithelium.

Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes the respiratory ciliated epithelial cells. Cilium adherence is mediated by P97, a surface protein containing a repeating element (R1) that is responsible for binding. Here, we show that the cilium adhesin is proteolytically processed on the surface. Proteomic analysis of strain J proteins identified cleavage products of 22, 28, 66, and 94 kDa. N-terminal sequencing showed that the 66- and 94-kDa proteins possessed identical N termini and that the 66-kDa variant was generated by cleavage of the 28-kDa product from the C terminus. The 22-kDa product represented the N-terminal 195 amino acids of the cilium adhesin preprotein, confirming that the hydrophobic leader signal sequence is not cleaved during translocation across the membrane. Comparative studies of M. hyopneumoniae strain 232 showed that the major cleavage products of the cilium adhesin are similar, although P22 and P28 appear to be processed further in strain 232. Immunoblotting studies using antisera raised against peptide sequences within P22 and P66/P94 indicate that processing is complex, with cleavage occurring at different frequencies within multiple sites, and is strain specific. Immunogold electron microscopy showed that fragments containing the cilium-binding site remained associated with the cell surface whereas cleavage products not containing the R1 element were located elsewhere. Not all secreted proteins undergo multiple cleavage, however, as evidenced by the analysis of the P102 gene product. The ability of M. hyopneumoniae to selectively cleave its secreted proteins provides this pathogen with a remarkable capacity to alter its surface architecture.

Mycoplasma gallisepticum (MG) was used to infect chicken embryos, and scanning electron microscopy was used to examine the morphologic changes in the tracheae. Tracheae harvested from embryos infected with MG for 5 days showed extensive deciliation, surface erosion, and inflammatory cell infiltration. Embryonic tracheal explants infected with MG for 6 hr showed the same deciliation and surface erosion. The damage to the tracheal surface caused by MG at the embryonic stage might play a role in the pathogenesis of MG infection.

According to epidemiological studies, it is possible that some secondary metabolites of indoor airborne fungi could be responsible for health troubles which occupants suffer from. In our previous experiments, a model with tracheal rings of 1-day-old chicks in vitro was shown to be a very suitable method to study the ciliostatic chloroform-extractable endo- and/or exometabolites of filamentous fungi. In this study we isolated the filamentous fungi from walls of "mouldy" dwellings and schools (cultivation on dichloran 18% glycerol agar at 25 and 37 degrees C for 10 d) in Slovakia. We studied the ciliostatic effect of the chloroform-extractable endo- and exometabolites of 96 representative isolates (stationary cultivation on the liquid medium with 2% of yeast extract and 10% of sucrose at 25 degrees C for 10 days) on the cilia movement in tracheal organ cultures of 1-day-old chickens in vitro after 24, 48 and 72 hrs (incubation in the minimal essential medium according to Eagle with Earl s salts and 20 microg of extract of metabolites dissolved in dimethylsulfoxide per 1 mL). Strains of Penicillium Link: Fr. sp., Aspergillus versicolor (Vuill.) Tiraboschi, A. flavus Link, Cladosporium sphaerospermum Penzig and C. cladosporioides (Fres.) de Vries were isolated most frequently. Two A. flavus isolates were able to produce aflatoxins B1, B2, G1, G2 in vitro after cultivation on the liquid medium with 20% sucrose and 2% yeast extract. This is the first isolation of aflatoxigenic A. flavus strains from dwellings in Slovakia. All frequently isolated strains produced secondary metabolites with the strongest ciliostatic activity -- their exo- and endometabolites stopped tracheal ciliary movement in chicks till 24 h. There are some toxic fungal metabolites in the indoor air not only with the ability to destroy ciliary movement in the upper airways in vitro but, probably, during long-lasting exposure to cause general intoxication of macroorganism via lung tissue.

Cilia-Associated Respiratory (CAR) bacillus is a filamentous bacterium that colonizes the ciliated epithelium of the respiratory tract of many animal species and that has been associated with chronic inflammatory lesions in naturally and experimentally infected rats, mice and rabbits. In the present study, the prevalence of CAR bacillus infection and histological lesions of the trachea in veal calf and adult cattle were investigated. Forty five healthy veal calves and 45 adult cattle, raised in 18 different herds were selected at slaughter. From each animal, a tracheal sample was processed for histology, stain-ed 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 identified in 17 veal calves (37.7%) and in 7 adult cattle (15.5%). Inflammatory lesions were found in 42 veal calves (93.3%) and in 41 adult cattle (91.1%). Statistical analysis pointed out a significant correlation between the presence and number of CAR bacilli and the presence and number of lymphoid follicles (P = 0.0071) and the presence and severity of neutrophilic infiltrates (P = 0.0428). These results indicate that CAR bacillus infection is common in cattle and is correlated with tracheal inflammatory lesions.

During the examination of Brachionus plicatilis (Rotifera, Monogononta), ectosymbiotic bacteria were found in the ciliated buccal region. These have about the same dimensions as the cilia and possess a specific apical region that apparently serves to attach the bacterium to its host cell. This apical region resembles a vesicle but is interpreted as a specialized region of the bacterial nucleocytoplasm devoid of ribosomes and chromatin strands but containing a crystalline rod. The bacterium has two membranes (as have other Gram-negative bacteria) also over the apical region. Thin strands join the two membranes and similar strands extend from the bacterium to the cell membrane of the host cell. No intracellular bacteria were found in this study.

Nontypable Haemophilus influenzae is a common cause of otitis media and initiates infection by colonizing the upper respiratory tract. In this article, I review our current understanding of the molecular determinants of H. influenzae colonization and discuss the relationship between colonization and otitis media.

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.

An in vitro culture system for swine tracheal epithelial cells was developed to study the adherence of swine mycoplasmas. Swine tracheal epithelial cells were isolated by enzymatic digestion and cultured on microporous membranes. Growth medium was placed under the membrane support to create air-liquid interface feeding resulting in the cells growing cilia and microvilli on the apical surface. Two strains of Mycoplasma hyopneumoniae (pathogenic strain 91-3 and non-pathogenic type strain J) and two strains of Mycoplasma flocculare (type strain Ms42 and field isolate 7160T) were used in this study. The morphology of the cultured tracheal cells was evaluated by transmission electron microscopy. Adherence of M. hyopneumoniae and M. flocculare and damage to the cilia were demonstrated using scanning electron microscopy. The pathogenic M. hyopneumoniae strain 91-3 adhered to cilia inducing obvious damage. The non-pathogenic M. hyopneumoniae strain J did not adhere to mature cilia. Both M. flocculare strains Ms42 and 7160T adhered to mature and budding cilia. No obvious ciliary damage was observed with strain Ms42. Minimal damage consisting of a slight tangling of the cilia occurred after adherence by strain 7160T. This model will enable us to further study the role of adherence of mycoplasmas on the pathogenesis of swine pneumonia.

The system of mucociliary clearance has the important task to remove from the airways inhaled substances and locally formed secretions. Inborn disorders of the mucociliary transport are due to ciliary dysfunction (Primary Ciliary Dyskinesia) (PCD) or of increased viscosity of the bronchial secretions (Cystic Fibrosis). To differentiate PCD from the ultrastructural abnormalities found during or after injuries such as respiratory infections, the name of Secondary--or acquired--Ciliary Dyskinesia (SCD) was created. In controls, less than 4% of the cilia may show ultrastructural abnormalities. The most frequent of these are the compound cilia and the peripheral microtubular abnormalities. Compound cilia often appear after infection and therefore are thought to arise secondarily. Secondary ultrastructural abnormalities of cilia include also blebs of the axoneme membrane, ciliary disorientation, and absence of axoneme membrane. No increase in ultrastructural ciliary abnormalities has been found in a variety of respiratory disorders: smoking, asthma and allergic rhinitis, chronic rhinitis and sinusitis, chronic bronchitis, cystic fibrosis, and lung carcinoma. But severe modifications of the respiratory epithelium can be seen. Important for the secondary ciliary disorders is their local and reversible character. To distinct from ultrastructural images between primary and secondary ciliary dyskinesia is often uneasy because some of the findings in secondary ciliary dyskinesia obviously mimic those dedicated to primary ciliary dyskinesia.

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.

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.

Mouse monoclonal antibodies (MAbs) developed to a rat isolate (R-3) of cilia-associated respiratory (CAR) bacillus were used to assess antigenic relationships among three rat and five rabbit CAR bacillus isolates. Evaluation of MAbs by enzyme-linked immunosorbent assays (ELISAs) indicated that 87 of 241 hybridomas secreted CAR bacillus-reactive antibodies that could be grouped into four major groups. Group-I MAbs reacted with epitopes expressed by all CAR bacillus isolates and at least two or more nonrelated species of bacteria. Group-II, -III, and -IV MAbs reacted with only one or more of the rat CAR bacillus isolates; no MAbs reacted only with rat and rabbit CAR bacillus isolates. Western blot analyses indicated that 41-, 50-, and 105-kDa peptides of rat CAR bacillus isolates expressed rat CAR bacillus group- and isolate-specific epitopes. Hyperimmune anti-CAR bacillus antiserum and serum specimens from a CAR bacillus histologically positive mouse and rat also reacted with the 41-, 50-, and 105-kDa peptides. Sera from CAR bacillus histologically negative rats did not react with these peptides. These results suggest that the 41-, 50-, and 105-kDa peptides may represent suitable antigens for development of a specific ELISA for detection of rodent CAR bacillus infections. Furthermore, these data indicate that use of crude CAR bacillus preparations for either rat or rabbit CAR bacillus ELISAs is inappropriate.

Ovine tracheal ring explants were infected with four different Mycoplasma ovipneumoniae and one M. arginini field isolate and their ability to induce cytopathic effects was tested by measuring ciliary activity and intracellular calmodulin release. Infected tracheal rings showed significantly decreased ciliary activity as compared to the non-infected control rings. There were, however, marked differences between isolates in the onset and severity of the effects which correlated with their ability to produce hydrogen peroxide. Infected tracheal rings released more calmodulin than the non-infected controls. The amount of calmodulin released also varied between isolates, and somewhat reflected the degree of loss of ciliary activity in the corresponding rings induced by the different isolates. Light and electron microscopic examinations of infected tracheal rings revealed disorganisation and sloughing of the epithelium, and association of mycoplasmas only with the cilia. Following repeated in vitro passages, the organisms had reduced ability to inhibit ciliary activity which correlated with decreased hydrogen peroxide production. Addition of catalase to the organ cultures delayed loss of ciliary activity. These results suggest that M. ovipneumoniae induced ciliostasis in ovine tracheal ring explants which correlated with hydrogen peroxide production. Furthermore, these M. ovipneumoniae-induced injuries to respiratory epithelial cells could contribute to the role that this organism may play in sheep respiratory disease.

The ciliostatic activity of the chloroform-extractable endo- and exometabolites of 5 strains of filamentous fungi--Alternaria sp., Aspergillus glaucus group, Aspergillus versicolor, Cladosporium sphoerospermum, Penicillium sp. and Ulocladium sp.--isolated from molded walls of a dwelling--on tracheal cilia from 1-d-old chicks in vitro was evaluated. Endometabolites of Alternaria sp. and A. versicolor and exometabolites of Ulocladium sp. were the most active, these extracts stopped the ciliary movement within 1 d. The results are discussed in relation to the health status of people living in "moldy" dwellings.

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.