Ultrastructural pathology of Bordetella avium infection in turkeys

One-day-old turkeys were infected intranasally with Bordetella avium, and tracheas were examined by scanning and transmission electron microscopy at 1 to 5 weeks post-inoculation (PI). The predominant ultrastructural lesions were progressive loss of ciliated epithelium with replacement by nonciliated cells, bacterial colonization of ciliated cells, membrane-bound crystalline inclusions in cytoplasma of epithelial cells, depletion of mucous granules, and distortion of tracheal rings and the mucosal surface. Tracheal surface exudates consisted of mucus, necrotic cells, heterophils, and fibrin. Ciliated cells were replaced by immature cuboidal cells characterized by abundant rough endoplasmic reticulum with small numbers of electron-dense mucous granules in the apical cytoplasm. Bacterial surfaces were rough and contained numerous pleomorphic, knob-like structures, 20-50 nm in diameter. Other changes included enlarged mucosal gland openings, cell extrusion marks, pleomorphic microvilli, and cells with small numbers of short cilia.     

Tracheal mucus transport rate in normal turkeys and in turkeys infected with Bordetella avium (Alcaligenes faecalis)

Using the radiopharmaceutical 99mtechnetium-sulfur colloid, the tracheal mucus transport rate (TMTR) was measured in healthy unanesthetized turkeys and in turkeys infected with Bordetella avium. The TMTR of uninfected turkeys was 35.6 +/- 14.4 cm/min. The TMTR of B. avium-infected turkeys was normal on days 0 through 14 postexposure (PE), despite heavy bacterial colonization of the tracheal epithelium. On day 21 PE, the TMTR of B. avium-infected turkeys was significantly depressed (P less than or equal to 0.01) compared with that of control turkeys. Depressed transport was associated with extensive loss of ciliated epithelium from the tracheal mucosa and replacement of the normal mucosa by immature nonciliated epithelium or metaplastic squamous epithelium.     

Experimental bovine respiratory syncytial virus infection in conventional calves: ultrastructural respiratory lesions

The morphogenesis and repair of airway and alveolar injury induced by bovine respiratory syncytial virus (BRSV) was studied ultrastructurally in conventional calves to characterize pulmonary cell types susceptible to viral infection and cytopathologic changes associated with infection. Viral nucleocapsids and budding virions were present in tracheal and bronchial ciliated and nonciliated epithelial cells and mucous cells 3, 5, and 7 days after inoculation and in bronchiolar ciliated and nonciliated epithelial cells 5 days after inoculation. Mild interstitial pneumonia was observed 5 days after inoculation and was characterized by swelling of type 1 and type 2 alveolar epithelial cells, interstitial edema, and infiltration by lymphocytes and macrophages. Viral assembly and release in tracheal and bronchial epithelial cells was associated with loss of cilia from ciliated cells, formation of syncytial epithelial cells, swelling of mitochondria and endoplasmic reticulum, and cell necrosis. Neutrophils, lymphocytes, and macrophages were present in close association with the viral-infected and damaged epithelial cells. There was intercurrent hyperplasia of basal epithelial cells that, in association with other epithelial lesions, resulted in the loss of normal ciliated epithelium in these airways 5 and 7 days after inoculation. Regeneration of airway epithelium was largely completed by 10 days after inoculation, except in 1 of 4 calves that had failure of epithelial repair and that developed secondary bacterial pneumonia. Pulmonary ultrastructure in BRSV-inoculated calves 30 days after inoculation was indistinguishable from that in controls. The results demonstrated that BRSV can induce reversible alterations in airway epithelium, which may cause depression of mucociliary clearance and thereby enhance susceptibility to bacterial infection.     

Epithelial cell kinetics in the inflammatory process of chicken trachea infected with infectious bronchitis virus

All stages of degeneration and regeneration in chicken tracheal epithelium were studied morphologically following an intratracheal inoculation of infectious bronchitis virus (IBV). Viral antigen was detected in the cytoplasm of tracheal epithelium from 1 to 7 days post-inoculation (d.p.i.) with a peak on 3 d.p.i. At 1 d.p.i., almost all epithelial cells were involved in the degeneration. At this time, labelling index of bromodeoxyuridine (BrdU) in the basal cells showed significantly high value compared with control. At 2 and 3 d.p.i., a great number of basal cells were recognized, but the BrdU labelling index tended to decrease. At 4 and 5 d.p.i., the BrdU labelling index of basal cells significantly decreased than 1 d.p.i., and a few number of regenerated immature ciliated epithelia appeared. At 6 to 11 d.p.i., the ciliated columnar epithelia increased rapidly in number, and returned to the normal appearance except for non-ciliated patch by 13 d.p.i. These results suggested that the tracheal epithelial cells infected with IBV degenerated within 24 hours and proliferating activity of basal cells functioned immediately, and 3 to 4 days later, these basal cells were differentiated to the ciliated epithelia.     

Antibiotic aerosolization: the effect on experimentally induced Alcaligenes rhinotracheitis in turkeys

Oxytetracycline hydrochloride was delivered by aerosol twice daily for 3 days to uninfected turkeys and to turkeys experimentally inoculated with Alcaligenes faecalis. The clinical, microbiological, and histological changes in the upper respiratory tracts were studied. No lesions were observed in the tracheas of uninoculated poults exposed to the aerosol. In experimentally infected poults, clinical signs included ocular and nasal discharges and open-mouthed breathing. Histologic lesions included progressive bacterial colonization of ciliated epithelium, loss of cilia, depletion of mucin from goblet cells, and accumulation of inflammatory cells within the tracheal lumen. Aerosolization of oxytetracycline effected a temporary decrease in bacterial colonization and a delay in clinical signs and histologic lesions in infected treated poults compared with untreated infected poults. Bacterial colonization and histologic lesions in the tracheas of both treated and untreated infected poults were similar by 4 days after the treatment was discontinued. This study indicates additional research with bactericidal antibiotics is needed to further evaluate antibiotic aerosolization as a treatment for alcaligenes rhinotracheitis.     

Ultrastructural changes of the tracheal epithelium after vaccination of day-old chickens with the La Sota strain of Newcastle disease virus

The progression of tracheal lesions induced by vaccination of day-old specific pathogen-free chicks with the La Sota strain of Newcastle disease virus (NDV) was examined by relating surface changes as observed by scanning electron microscopy with subcellular changes seen by transmission electron microscopy. NDV infection resulted in hypertrophy of goblet cells, their rupture, and the formation of excess mucus. Activation of goblet cells peaked within 4 days postvaccination. Afterward, the activation levels gradually decreased. At the level of the ciliated cells, a marked increase in the proportion of nonciliated to ciliated cells and later an almost complete deciliation of the tracheal surface were observed because a simple squamous to cuboidal epithelium replaced the original pseudostratified epithelium. Fifteen days postvaccination, all epithelial damage was restored. Because the observed vaccination-induced lesions are detrimental to epithelial integrity and function as a barrier against invading microorganisms, they might explain at the ultrastructural level the secondary complications of vaccination with the La Sota strain against NDV.     

An Alcaligenes faecalis isolate from turkeys: pathogenicity in selected avian and mammalian species

An Alcaligenes faecalis isolate of known pathogenicity for turkeys was examined for adherence and cytotoxicity in tracheal organ cultures of turkeys, chickens, Japanese quail, guinea pigs, hamsters, and mice, and for colonization and pathogenicity in these 6 species. Adherence and colonization were detected by fluorescent antibody staining. Infected and noninfected tracheal rings were examined by phase-contrast microscopy for cytotoxicity (ciliostasis, blebing of the cell membrane, and sloughing of the ciliated epithelium). Alcaligenes faecalis adhered to the tracheal rings of all species examined. Cytotoxicity was apparent in the tracheal rings of turkeys, quail, and chickens. Cytotoxicity was not detected in tracheal rings from the mammalian species. Alcaligenes faecalis colonization of turbinates and tracheas of intact turkeys and quail was detected. Clinical signs of alcaligenes rhinotracheitis were observed and histopathologic characteristics of the disease were detected. Chickens, guinea pigs, hamsters, and mice were refractory to infection with this isolate of A faecalis.     

Histological Characteristics of the Tracheobronchial Tree of the Least Shrew (Cryptotis Parva)

The least shrew (Cryptotis parva) is a small vomit‐competent insectivorous species which has recently been introduced as an emesis animal model in the laboratory. In this study, the respiratory system of the least shrew was examined and compared with the well‐established larger species routinely used in the laboratory. Five least shrews (4–5 g body weight, 45–60 days old) were used. Standard histological procedures were followed for light microscopic examination. The lining epithelium of the trachea was found to be pseudostratified ciliated columnar (PSCC). Three types of cells were easily identified, basal and ciliated as well as few goblet cells interspersed among the ciliated cells and they were not clearly recognizable. A few tracheal seromucous glands were located at the free end of the C‐shaped cartilaginous rings. The cartilaginous rings are replaced by smooth muscle cells before the bronchi enter into the lung. The lining epithelium of tracheobronchial tree gradually changes into simple cuboidal epithelium that lacks goblet cells. However, the division of the tracheobronchial tree is similar to other mammalian species. On the other hand, the principal bronchus lacks cartilaginous plaques as it becomes intrapulmonary bronchus. The wall of the bronchi is supported by thick layers of spirally arranged smooth muscles. Two types of cells were readily recognizable: basal and ciliated cells, with rarely observed goblet cells. In addition, the PSCC epithelium changes into simple cuboidal much earlier in the bronchial division relative to other species.     

Lectin histochemistry of trachea and lung of healthy turkeys (Meleagris gallopavo) and turkeys with pneumonia

Thirteen lectins were used to characterize lectin-binding specificity of glycoconjugates on sections of formalin-fixed lung and trachea from seven normal turkeys, two turkeys with acute pneumonia, and two turkeys with chronic pneumonia. Neuraminidase was used to digest sialic acid residues. One N-acetylgalactosamine-binding lectin and two N-acetylgalactosamine/galactose-binding lectins stained the apical membrane and cytoplasm of multifocal cells that lined air atria and hyperplastic granular cells. Other lectins in these groups stained ciliated cells of the trachea and bronchi and air capillary epithelial cells. Sialic acid residues were on apical surfaces of ciliated and nonciliated tracheal and bronchial lining cells, air capillary epithelial cells, and vascular endothelial cells. Mannose/glucose-binding lectins stained reticular and elastic fibers in the lamina propria of trachea, primary and secondary bronchi, and the tunica adventitia of arteries and veins. By transmission electron microscopy, colloidal gold-Arachis hypogaea (peanut agglutinin) labeled microvilli on the apical surface of mature granular cells. The L-fucose-binding lectin, in addition to several other lectins, stained nonspecifically in both trachea and lung. These studies show that granular cells that line air atria can be identified with lectins of N-acetylglucosamine and N-acetylgalactosamine/galactose groups, and that apical surfaces of epithelial cells and endothelial cells in the trachea and lung express terminal sialic acid residues.     

Pathological changes of tracheal mucosa in chickens infected with lentogenic Newcastle disease virus

Forty-day-old specific-pathogen-free chickens were exposed by aerosol to lentogenic Newcastle disease virus (NDV) and observed for 24 days for pathological changes in the tracheal mucociliary system. Specific fluorescence of NDV antigen was observed through day 5 postexposure (PE) in the cytoplasm of the tracheal epithelium and desquamated epithelium in the lumen. On day 1 PE, scanning electron microscopy revealed hypertrophy of goblet cells and small patches of the deciliated epithelium scattered mainly around the openings of mucous glands. The deciliated area of tracheal surface increased through day 4 PE. Light microscopy showed small vacuoles containing lymphocytes and heterophils in the epithelial layer. Immature epithelium proliferated in some areas. On days 5 and 6 PE, ciliated areas of the trachea tended to increase as a result of regeneration of the epithelium, still leaving many nonciliated patches of various sizes. On and after day 8 PE, there remained plaques with nonciliated flat epithelium, but most areas were covered with well-ciliated epithelium. Non-ciliated plaques were observed until day 24 PE, but they gradually decreased in size. These plaques were covered by a single layer of flat epithelium and were formed upon lymph follicles in subepithelial tissue.     

Light and electron microscopic studies of the trachea in the one-humped camel (Camelus dromedarius)

Histology of trachea of camel (Camelus dromedarius) was studied using light, scanning (SEM) and transmission electron microscopy (TEM). Tissue samples taken from the trachea (proximal, middle and distal part) were routinely prepared for histology (LM, EM) and stained with haematoxylin and eosin, Van Giesson (VG), Alcian blue, Periodic acid schiff (PAS), Masson's trichrome (MT), Verhof, PAS-VG and PAS-MT. The trachea of camel consists of 66-75 incomplete cartilaginous rings of hyaline. The lamina epithelium is composed of pseudostratified-ciliated columnar epithelium with many goblet cells. Submucosal layers were loose connective tissue with many elastic fibres. The mucosal and submucosal layers were 517.2 +/- 61.6 microm (n = 20) thick. Submucosal glands were tubuloalveolar with mucous (acidic and neutral) secretions. Trachealis muscle was attached to the inside sheet of tracheal cartilage. Ultrastructural studies showed that surface epithelium is pseudostratified with mucus-producing goblet cells, ciliated and basal cells, similar to other mammals. The ciliated cells contained many mitochondria, oval nucleus and many big granules. In scanning electron microscopy (SEM) studies, viscoelastic layers were observed on the epithelial surface of trachea, and there were highly condensed cilia under this layer.     

Tracheal mucus transport rate and bacterial clearance in turkeys exposed by aerosol to La Sota strain of Newcastle disease virus

Tracheal mucus transport rate (TMTR) and quantitative clearance of aerosolized Escherichia coli from the trachea, lung, and air sac were measured in healthy unanesthetized turkeys and in turkeys exposed by aerosol to a La Sota vaccine strain of Newcastle disease virus (NDV). The TMTR of uninfected turkeys was 42.4 +/- 14.7 cm/min. The TMTR of NDV-infected turkeys was depressed on days 3 through 7 postexposure (PE); depression was significant (P less than or equal to 0.05) on day 7 PE. Tracheal E. coli clearance in NDV-infected turkeys was reduced on days 4 through 9 PE, significantly so on day 5 PE (P less than or equal to 0.01). Depression of TMTR and tracheal E. coli clearance were associated histologically with replacement of normal pseudostratified columnar epithelium by 3 to 8 layers of immature nonciliated cells. E. coli clearance by the lung and air sac of NDV-infected turkeys was depressed on days 5 through 9 PE.     

Pneumonia in lambs inoculated with Bordetella parapertussis: bronchoalveolar lavage and ultrastructural studies

Eight colostrum-deprived lambs were inoculated intratracheally with ovine isolates of Bordetella parapertussis. Fluids obtained by bronchoalveolar lavage had a large increase in total cell counts 24 hours after inoculation; up to 93% of cells were neutrophils. From 3 days after inoculation, the number of alveolar macrophages in lavage samples was markedly increased. From 5 days onwards, many alveolar macrophages had moderate to severe cytoplasmic vacuolation. Topographically, tracheal and bronchial epithelium was covered by a large amount of inflammatory exudate 24 hours after inoculation. Later, the tracheobronchial epithelium showed focal extrusions from ciliated cells, which were occasionally associated with B. parapertussis organisms. Ultrastructurally, cytopathological changes associated with B. parapertussis infection were mild focal degeneration of airway epithelium with slight loss of cilia, moderate to severe degeneration of type I and type II alveolar epithelial cells, and focal inflammation in the lungs. These results suggest that the primary targets of B. parapertussis infection are alveolar macrophages and the epithelial cells of bronchioles and alveoli.     

Uptake of ferritin and Bordetella avium in bronchus-associated lymphoid tissue of turkeys

The uptake of macromolecular and particulate materials in bronchus-associated lymphoid tissue (BALT) in turkeys was examined using transmission electron microscopy. Tracer materials used were live and ultraviolet-killed (UV-killed) Bordetella avium and ferritin. Suspensions of bacteria and ferritin were instilled via intratracheal catheterization and allowed to remain in contact with the respiratory surfaces for 0, 10, 30, 60, 90, and 120 min. Ferritin and B. avium were taken up by both ciliated and non-ciliated cells of the epithelium overlying BALT (BALT epithelium). Ferritin was found in organelles associated with endocytosis (i.e. apical vesicles, endosomes, cytoplasmic vacuoles) and was apparently transported across epithelial cells, since it was also found in intercellular spaces. Bacteria were found in vacuoles within BALT epithelial cells, but not free in intercellular spaces. Some macrophages in BALT epithelium also contained bacteria. No differences were observed between uptake of live and UV-killed bacteria. We conclude that both ciliated and non-ciliated cells of BALT epithelium in turkeys are able to take up macromolecular and particulate materials. Bacteria are also accessible to intraepithelial macrophages, although whether they are taken up directly from the bronchial surface or whether they pass through epithelial cells first could not be determined. This evidence suggests that antigens, including respiratory pathogens, could gain access to cells of the avian immune system by transepithelial passage in BALT.     

Evaluation of cytopathologic changes induced in chicken tracheal epithelium by Mycoplasma gallisepticum in vivo and in vitro

Changes in tracheal epithelial surfaces induced by Mycoplasma infection in vivo and in vitro included release of mucous granules followed by exfoliation of ciliated and nonciliated epithelial cells. Light microscopy, scanning electron microscopy, and transmission electron microscopy confirmed that the loss of cilia from individual cells was infrequent. Epithelial cells typically lost their intercellular connections, rounded up, exfoliated, and then lysed--giving rise to a population of cellular organelles, such as mitochondria and cilia intermixed with mucus to form the exudate found within the tracheal lumen. Repair of the epithelial surface was effected by basilar epithelial cells differentiating and filling in the spaces formed by exfoliated cells. These cells were hypertrophied, nonciliated at 14 days after infection in vivo, and covered with microvilli. In sectioned material obtained during the infection, there was increasing epithelial thickness due to cellular infiltration and edema. Tracheal rings in vitro showed similar changes to those seen in vivo, except that exfoliation was more severe and occurred earlier. In addition, there were no cellular infiltration due to the lack of a vascular supply and only a small amount of mucus due to the smaller number of mucous cells available to release into the tracheal lumen.     

Growth of differentiated ovine tracheal epithelial cells in vitro

Culture of ovine tracheal epithelial cells is a useful tool for conducting various in vitro studies. We describe herein an in vitro technique and the conditions for culturing primary epithelial cells derived from tracheas of adult sheep. Ovine tracheas were surgically removed from 2- to 3-month-old healthy sheep and tracheal epithelial cells were isolated by 0.15% pronase digestion. After epithelial cells isolation, a Millicell insert with porous membrane was coated with 0.05% human placental collagen and the epithelial cells were added to the membrane. To create an air-liquid interface environment for the cells, the apical compartment of the membrane containing the tracheal epithelial cells was left exposed to 5% CO(2) at 37 degrees C for 2 days then increased to 9% CO(2) while cells in the basolateral compartment underneath the membrane contained the growth medium necessary for cells nourishment. Pepsin digestion was more effective in reducing the number of fibroblasts than other procedures. Cells were allowed to grow for 6-7 days to form a confluent monolayer and nearly 21 days for cilia formation on the apical surface as determined by light microscopy of haematoxylin and eosin-stained sections of membranes. In order to further confirm the epithelial origin of cells, cells were stained for cytokeratin antigen by immunohistochemistry. Most ciliated epithelial cells were immunoreactive for cytokeratin. This is the first report of differentiated ovine tracheal epithelial cells growth and isolation. This technique can be used in numerous in vitro investigative studies in ovine species as an animal model for human disease.     

Growth of Mycoplasma bovis in organ cultures of bovine foetal trachea and comparison with Mycoplasma dispar

Inoculation of tracheal organ cultures from bovine foetuses with Mycoplasma bovis resulted in a loss of cellular structure of the lamina propria, followed 20-22 days later by lifting and detachment of overlying epithelium. The effect was associated with large numbers of M. bovis, identified by immunoperoxidase labelling and electromicroscopy, infiltrating between the epithelial cells and amassing in the lamina propria, especially in the region of the basement membrane of the epithelium. Ciliary activity was undiminished for up to 18 days following inoculation and little or no cytopathic effect on the ciliated epithelium was seen in spite of the close proximity of large numbers of organisms. In contrast, M. dispar was restricted to the margin of the ciliated epithelium where, as previously reported, it caused pyknosis, sloughing and flattening of the epithelium with consequent loss of ciliary activity. The cytopathology observed for each mycoplasma bore a close similarity to the behaviour of the two mycoplasmas in vivo and it is suggested that the organ culture system may be a useful and relevant system to elucidate the pathogenic mechanisms for each mycoplasma.     

Respiratory bronchoscopy of subadult American alligators (Alligator mississippiensis) and tracheal wash evaluation.

Twelve healthy approximately 3-yr-old captive-born 4.5-9 kg American alligators (Alligator mississipiensis) each had bronchoscopy and tracheal washes performed four times during a 10-mo period to evaluate seasonal respiratory microbiology and cytology. Cytologic evaluation of most samples showed a small amount of mucus and low numbers of ciliated columnar epithelium, cubodial epithelium, and keratinized squamous cells. No bacteria or parasites were observed, and there was no seasonal variation in the cytology. No significant bacterial or fungal growth was identified in any season. Hematology performed in the spring and fall evaluations showed seasonal variation in the red blood cell count, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin concentration, eosinophil count, and thrombocyte count. The lower respiratory tract (at the tracheal level) of healthy subadult alligators appears to be sterile, and cytology is similar to that described in domestic mammals.     

A rapid method for preparing avian tracheal rings for organ culture

A simple, rapid method for preparing rings from chicken tracheas is described. Using a polyacrylamide disc gel slicer, up to 100 uniform tracheal rings were obtained from two tracheas with a single cut and without injury to the ciliated tracheal epithelium. The responses of rings cut with a disc gel slicer and those cut conventionally with a scalpel blade were compared by virus titration, virus neutralization, and viability of uninfected 18-day-old-chick embryo rings over a nine-day incubation period. Rings cut with a disc gel slicer did not differ significantly from those cut conventionally with respect to virus titer, neutralization indices, or viability. A disc gel slicer expedites the preparation of functional rings and offers an alternative to the present method of cutting tracheal rings with a scalpel blade.