Detection, molecular characterization and phylogenetic analysis of full-length equine infectious anemia (EIAV) gag genes isolated from Shackleford Banks wild horses

The genetically distinct wild horse herds inhabiting Shackleford Banks, North Carolina are probably the direct descendents of Spanish stock abandoned after failed attempts to settle mid-Atlantic coastal regions of North America in the Sixteenth Century. In a 1996 island survey, 41% of the gathered horses were discovered seropositive for Equine Infectious Anemia Virus (EIAV) with additional cases identified in 1997 and 1998. As a result of their unique genetic heritage, EIAV seropositive individuals identified in the two latter surveys were transferred to a quarantine facility on the mainland. In September 2008 two of the horses SB1 and SB2 after 10 and 11 years in quarantine respectively, developed clinical signs of EIA. In the case of SB2 these were so severe that the only humane option was euthanasia. Although SB1, survived it experienced a second clinical episode one month later. In May 2009, a third horse in quarantine, SB3, developed extremely severe clinical EIA and was euthanized. This demonstrates naturally infected long-term inapparent carriers can experience recrudescence of very severe disease many years after initial exposure to EIAV. Phylogenetic analysis of complete EIAV gag gene sequences obtained from each of three Shackleford horses demonstrated they were infected with very closely related viruses. Although these were distinguishable from all other strains examined, they belong to a monophyletic group comprising almost exclusively of New World isolates that is distinct from a number of recently characterized Central European EIAV strains.     

Fibrin/fibrinogen in lungs and respiratory secretions of horses with chronic pulmonary disease

The concentration of soluble fibrinogen derivatives (SFD) and protease and procoagulant activities were determined in cell-free supernatants of equine respiratory secretions obtained from horses with chronic pulmonary disease. The concentration of neutrophils was estimated from direct smears of the secretions. Lung specimens and smears of the secretions were evaluated for the presence of fibrin or fibrinogen by use of immunohistochemical methods. Thirty-five of 80 specimens tested contained SFD. Respiratory secretions from horses with moderate or severe chronic pulmonary disease contained SFD more frequently than did secretions from mildly affected horses (P less than 0.05). Respiratory secretions with vast numbers of neutrophils had significantly (P less than 0.05) higher SFD concentrations than respiratory secretions with fewer neutrophils. Protease and procoagulant activities in respiratory secretion specimens were positively correlated with neutrophil content, clinical diagnosis, and SFD concentration. Immunohistochemically, macrophages that stained for fibrin or fibrinogen were observed in direct smears of respiratory secretions from horses with moderate and severe chronic small airway disease, but not in smears from mildly affected horses. Fibrin or fibrinogen was detected in a few thickened alveolar septa from 10 horses with moderate or severe chronic small airway disease, but not in lungs from horses with mild or no evidence of chronic small airway disease. Fibrin or fibrinogen was detected in alveolar septa, granulomas, and on alveolar macrophages in lungs of all horses with chronic granulomatous and chronic bronchointerstitial pneumonia. The presence of SFD in equine respiratory secretions may be an indicator of pulmonary inflammation.     

Equine Infectious Anemia Virus (EIAV): what has HIV's country cousin got to tell us?

Equine Infectious Anemia Virus (EIAV) is a lentivirus, of the Retrovirus family, with an almost worldwide distribution, infecting equids. It causes a persistent infection characterized by recurring febrile episodes associating viremia, fever, thrombocytopenia, and wasting symptoms. The disease is experimentally reproducible by inoculation of Shetland ponies or horses with EIAV pathogenic strains. Among lentiviruses, EIAV is unique in that, despite a rapid virus replication and antigenic variation, most animals progress from a chronic stage characterized by recurring peaks of viremia and fever to an asymptomatic stage of infection. The inapparent carriers remain infective for life, as demonstrated by experimental transfer of blood to naive animals. The understanding of the correlates of this immune control is of great interest in defining vaccine strategies. Research on EIAV, this "country cousin" of HIV (Human Immunodeficiency Virus), over the last five decades has produced some interesting results on natural immunological control of lentivirus replication and disease and on the nature and role of virus variation in persistence and pathogenesis. These studies are of interest in the context of HIV and efforts to develop a vaccine. This review will focus on some of the most recent results.     

Respiratory syncytial virus pneumonia in young calves: clinical and pathologic findings

Nine young calves given respiratory syncytial virus by a combined intranasal and intratracheal route developed a severe respiratory tract disease in which coughing, tachypnea, and hyperpnea were prominent clinical features. Calves were euthanatized on postinoculation (initial) days (PID) 1 to 13. At necropsy, large areas of consolidation were present in the cranial, middle, accessory, and cranial parts of the caudal lung lobes of calves killed between PID 4 and 13. Histopathologic examination revealed widespread and severe lesions in small bronchi, bronchioli, and alveoli. Multinucleate epithelial syncytia on bronchiolar and alveolar walls, many containing eosinophilic intracytoplasmic inclusion bodies, were present in the lungs of calves killed on PID 4, 5, and 6. Necrosis and epithelial loss, hyperplasia, and metaplasia were also observed in the epithelium of small bronchi and bronchioli. The lumina of these airways were occluded to varying degrees with exudate. Exudate was present within alveoli, and interalveolar septa were markedly thickened. Collapse of the thickened septa produced large areas where alveolar air spaces were totally obliterated. Repair was evident in the lungs of calves killed at PID 10 and 13 with reepithelialization of damaged bronchiolar mucosa, organization of bronchiolar exudate leading to bronchiolitis obliterans, and peribronchial and peribronchiolar fibrosis. Inoculation of 3 calves by an intranasal route alone produced a less severe clinical disease with only minimal lesions present at necropsy.     

Chronic small airway disease in the horse: immunohistochemical evaluation of lungs with mild, moderate and severe lesions

The peroxidase anti-peroxidase technique was used to demonstrate free and intracellular immunoglobulin (Ig) within the lungs of 23 horses with chronic small airway disease. Histologically, all the horses had chronic bronchiolitis; however, the lesions varied in degree from mild in eight horses, to moderate in nine horses and severe in six horses. Lungs from three horses which had no gross or histopathological lesions were used as controls. In comparison with control horses, horses with mild chronic bronchiolitis had increased numbers of Ig A-containing and non-immunoglobulin staining cells around the vasculature and bronchioles. As the severity of the lesions increased so did the number of IgA-, IgG(Fc)- and in several cases non-immunoglobulin staining cells around the vasculature, bronchioles and in the alveolar septa. In severely affected horses, large amounts of free IgG(Fc) were observed interstitially and in alveoli. In areas of mucosal epithelial hyperplasia and metaplasia large amounts of free IgA and IgG(Fc) were sometimes observed interepithelially in a pattern which differed from that in control horses.     

Pulmonary lesions induced by 3-methylindole and bovine respiratory syncytial virus in calves

Our objectives were to describe the ultrastructural morphogenesis of pulmonary lesions induced by 3-methylindole in 30- to 45-day-old Holstein calves and to determine whether toxic exposure to 3-methylindole exacerbates pulmonary lesions induced by bovine respiratory syncytial virus. Administration of 3-methylindole (0.25 g/kg) to calves resulted in interstitial edema and ultrastructural swelling of type-I alveolar epithelial cells and nonciliated bronchiolar epithelial cells as early as 4 to 6 hours after intraruminal administration. More severe alveolar edema containing protein was associated with swelling of capillary endothelial cells at 2 days after administration. Proliferation of type-II alveolar epithelial cells was first observed at 2 days after 3-methylindole administration, and marked hyperplasia of type-II epithelial cells and nonciliated bronchiolar epithelial cells was evident by 4 days after administration. Pulmonary cytochrome P-450 monooxygenase concentrations decreased significantly (P less than 0.001) by 12 hours after administration and did not increase significantly again by 8 days after administration. Calves were inoculated with bovine respiratory syncytial virus 3 days after administration of 3-methylindole, and pulmonary lesions were assessed 5 days after viral inoculation. Viral replication was demonstrated by fluorescence microscopy for viral antigen or by transmission electron microscopy in ciliated and nonciliated airway epithelial cells. Viral antigen was identified infrequently in alveolar macrophages and in type-II alveolar epithelial cells. 3-Methylindole exposure in calves did not result in more widespread distribution of viral antigen in alveolar tissue of respiratory syncytial virus-inoculated calves or in significant enhancement of viral pneumonia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural findings in horses with chronic obstructive pulmonary disease (COPD). II: Pathomorphological changes of the terminal airways and the alveolar region

Extensive light and electron microscope studies (transmission and scanning electron microscopy) of the bronchioles and alveolar region, in 28 horses suffering chronic obstructive pulmonary disease (COPD) and eight control horses, revealed good correlation between clinical severity and morphological changes. In the bronchiolar epithelium the non-ciliated bronchiolar epithelial (Clara) cells, in particular, showed ultrastructural alterations and, even in the mild stages of disease, these presented degenerative changes and lack of differentiation. Together with loss of granulation in the Clara cells and metaplasia of the goblet cells, cells were seen with unusual intracytoplasmic lamellar inclusion, the number of which increased sharply with clinical severity. The focal changes in the alveolar region were necrosis of type I epithelial cells, alveolar fibrosis of varying degrees with type II epithelial transformation and emphysema or hyperinflation, with an increase in Kohn's pores. Some horse also showed morphological signs of interference with the surfactant system, in the form of marked cysts with lamellar structure. The alveolar changes were mostly in the peribronchiolar region and were, therefore, interpreted as reactive processes. No conclusions as to the aetiology of equine COPD can be derived from these morphological investigations.     

Light and electron-microscopic localization of CD9 and surfactant protein A and D in normal lungs of the horse

The lung is a complex organ, and its physiology and immunology are regulated by various immune molecules and cells. Lung surfactant, a mixture of phospholipids and proteins produced by the bronchiolar and type II alveolar epithelial cells, is one such important player in lung physiology. Compared to knowledge about the biology of the surfactant in rodents and humans, only limited data are available on the surfactant in the horse. Although there are data linking levels of surfactant proteins with respiratory disease in the horse, there are no data on the cellular localization of surfactant protein A (SP-A) and surfactant protein D (SP-D). A member of the tetraspanin family of proteins, CD9 is a cell-signaling and adhesion protein and its expression has been detected in both normal and cancer cells, including those in the lung. Because there are no immunolocalization data on SP-A, SP-D, and CD9 in the normal lungs of the horse, our objective was to conduct a light and electron microscopic immunocytochemical study on normal lungs of the horse. The data showed SP-A and SP-D in bronchiolar epithelial and type II alveolar epithelial cells. These proteins were also localized in type I alveolar epithelial cells, pulmonary intravascular macrophages, and neutrophils, which is likely an outcome of endocytosis of the proteins by these cells. CD9 was present in the airway and vascular smooth muscle cells, endothelium, and blood cells, but not in the airway epithelium. These new data provide a baseline to further examine the expression and functions of SP-A, SP-D, and CD9 proteins in inflammation associated with respiratory diseases in the horse.     

Pathogenesis of acute toxoplasmosis in specific-pathogen-free cats

Systemic toxoplasmosis was produced in specific-pathogen-free cats by intravenous inoculation with Toxoplasma gondii tachyzoites. Infectious organisms were recovered from all tissues studied, but the number of organisms recovered from liver, lungs and spleen was 10-fold to 10,000-fold higher than from heart and brain. The occurrence and severity of Toxoplasma-induced lesions correlated with the number of infectious organisms recovered from the various tissues. In nonlymphoid tissues, the Toxoplasma-associated lesions consisted of multifocal necrosis, usually with demonstrable organisms. Lesions in the spleen and mesenteric lymph nodes consisted of reticuloendothelial and lymphoid hyperplasia, with few demonstrable organisms. Pneumonitis was severe and sometimes fatal in the early stages of systemic toxoplasmosis. Light- and electron-microscopic studies showed that the earliest lung lesions were randomly distributed infiltrates of neutrophils, eosinophils, and mononuclear cells into alveolar walls. Later lesions were diffuse alveolar necrosis, pneumocytic hyperplasia, and extensive fibrinocellular exudates in alveoli. Tachyzoites were present in cytoplasmic vacuoles of fibroblasts, macrophages, type I and II pneumocytes, bronchiolar epithelial cells, bronchiolar smooth muscle cells, endothelial cells, neutrophils, and eosinophils, and circulating monocytes. Replication of organisms was found in all parasitized cell types except neutrophils and eosinophils.     

Alveolar type II cells expressing jaagsiekte sheep retrovirus capsid protein and surfactant proteins are the predominant neoplastic cell type in ovine pulmonary adenocarcinoma

Ovine pulmonary adenocarcinoma is caused by jaagsiekte sheep retrovirus. To gain insight into the histogenesis and viral pathogenesis of this neoplasm, the tumor cell phenotypes and differentiation state were correlated with the distribution of jaagsiekte sheep retrovirus capsid protein in neoplastic and normal cells of the lung in nine naturally occurring and 12 experimentally induced cases of ovine pulmonary adenocarcinoma. Overall, 82% of tumor cells had ultrastructural features consistent with alveolar type II cells, 7% of tumor cells had features of Clara cells, and 11% of tumor cells were insufficiently differentiated to classify. The proportion of the neoplastic cell phenotypes varied within tumors, and no tumor consisted of a morphologically uniform cell population. To further characterize the neoplastic cell population, sections of tumors were immunostained with antibodies to surfactant protein A, surfactant protein C, and Clara cell 10-kd protein. Overall, surfactant proteins A and C were expressed in 70% and 80% of tumor cells, respectively, whereas Clara cell 10-kd protein was expressed in 17% of tumor cells. Jaagsiekte sheep retrovirus capsid protein was detected in 71% of tumor cells and in macrophages (5/21 tumors examined) and in nonneoplastic alveolar and bronchiolar cells (6/14 tumors). Expression of this viral protein in neoplastic cells, classified morphologically and by immunophenotyping primarily as of the alveolar type II lineage, implies an important role for specific virus-cell interactions in the pathogenesis of ovine pulmonary adenocarcinoma.     

Expression of cyclooxygenase-2 in swine naturally infected with Actinobacillus pleuropneumoniae

Cyclooxygenase-2 (COX-2) was detected and localized in 15 pigs with naturally occurring pleuropneumonia using a 437-base pair digoxigenin-labeled cDNA probe in an in situ hybridization protocol. Histopathologic changes in the acute stage were characterized by coagulative necrosis of lung parenchyma, hemorrhage, vascular thrombosis, edema, fibrin deposition, and infiltration of lung parenchyma by neutrophils and alveolar macrophages in nine pigs. In chronic lesions, a thick layer of granulation tissue surrounded foci of pulmonary necrosis in six pigs. All 15 pigs infected with Actinobacillus pleuropneumoniae, confirmed by bacterial isolation, had distinct positive hybridization signals for COX-2 in bronchial, bronchiolar epithelial cells, alveolar macrophages, neutrophils, and type I pneumocytes. COX-2 expression was detected primarily in neutrophils from pigs with acute lesions and primarily in alveolar macrophages from pigs with chronic lesions. The results suggest that a prostanoid product of COX-2 is an important component of the inflammatory response to acute and chronic A. pleuropneumoniae infection.     

Sequential lesions of experimental bovine pneumonic pasteurellosis

A strain of Pasteurella haemolytica biotype A serotype 1, which had been isolated from a pathologically-confirmed outbreak of bovine pneumonic pasteurellosis, was used successfully to reproduce the disease in conventional calves. The development of the various pathological features was studied at regular intervals following infection. The acute inflammatory reaction which had developed by day 2 after initial infection was characterised by flooding of the alveoli by oedema and neutrophils together with a mild degree of bronchiolar epithelial necrosis. This progressed to an acute exudative fibrinous pneumonia with extensive involvement of the interlobular septa and often with pleurisy. Subsequently, these pulmonary lesions became walled off by fibrous tissue which became infiltrated by plasma cells and lymphocytes. At this stage organisms could be demonstrated only within these nodules in the lung tissue.     

Serum concentration of surfactant protein D in horses with lower airway inflammation

Reasons for performing study: Surfactant protein D (SP‐D), mainly synthesised by alveolar type II cells and nonciliated bronchiolar cells, is one important component of innate pulmonary immunity. In man, circulating concentrations of SP‐D are routinely used as biomarkers for pulmonary injury. To date, serum SP‐D levels have only been investigated in horses in an experimental model of bacterial airway infection. Objectives: To compare serum SP‐D concentrations at rest and after exercise in horses with and without inflammatory airway disease (IAD). Methods: Venous blood samples were collected from 42 Standardbred racehorses at rest and 60 min after performing a standardised treadmill exercise test. Tracheal wash and bronchoalveolar lavage fluid (BALF) samples were collected after exercise. Based on BALF cytology, 22 horses were defined as IAD‐affected and 20 classified as controls. Serum SP‐D concentrations were assessed using a commercially available ELISA kit and statistically compared between groups of horses and sampling times. Results: Serum concentrations of SP‐D in IAD‐affected horses were significantly higher than those of control horses, both at rest and after exercise. Within the IAD‐affected group, no significant correlation was found between serum SP‐D concentrations and BALF cytology. Within each group of horses (IAD and control), no significant influence of exercise was found on serum SP‐D levels. Conclusions: This is the first study determining serum SP‐D concentrations in a noninfectious, naturally occurring form of lower airway inflammation in horses. The results highlight that IAD is associated with a detectable, though moderate, increase of circulating SP‐D levels. Potential relevance: Serum concentration of surfactant protein D could represent a potentially valuable and readily accessible blood biomarker of equine lower airway inflammation.     

Lesions and retroviruses associated with naturally occurring ovine pulmonary carcinoma (sheep pulmonary adenomatosis)

Five sheep with ovine pulmonary carcinoma were markedly dyspneic and had sporadic coughing; two had copious watery nasal exudate. In four, lesions consisted of multifocal nodules of neoplastic cuboidal epithelial cells in acinar or papillary patterns. Electron microscopically, cells had microvilli, tight junctions, and cytoplasmic lamellar bodies typical of alveolar type II cells. One sheep had a single lung tumor of nonciliated bronchiolar epithelial cells. Vacuolated alveolar macrophages surrounded adenomatous foci. One sheep had a metastatic lesion in the caudal mediastinal lymph node. All sheep had histologic lesions of lymphoid interstitial pneumonia (LIP, ovine progressive pneumonia) consisting of peribronchiolar and interstitial lymphoid hyperplasia, and fibromuscular proliferation; all had serum precipitating antibodies to ovine lentivirus. Lung fluids or tumor homogenates contained a 26-kd peptide that crossreacted with a primate-derived type D retrovirus as detected by immunoblotting or interspecies competition radioimmunoassay. Ovine lentivirus was isolated from concentrated lung fluids or tumor tissues of four sheep tested and from tumor cell DNA of one animal transfected into ovine muscle cells. These studies document the presence of type D-related retrovirus antigen in ovine pulmonary carcinoma (OPC) in the United States and indicate that lentivirus-induced LIP is a lesion frequently associated with this disease.     

Ultrastructural morphogenesis of 4-ipomeanol-induced bronchiolitis and interstitial pneumonia in calves

The two objectives of this research were 1) to describe the ultrastructural morphogenesis of pulmonary damage and repair induced in calves after treatment with 4-ipomeanol and 2) to characterize infiltrating pulmonary inflammatory cells by bronchoalveolar lavage. Interstitial edema was observed as early as 4 hours after intravenous injection of 4-ipomeanol (5 mg/kg body weight) and progressed to severe alveolar edema by 72 hours. Damage to type I alveolar epithelial cells and terminal bronchiolar nonciliated cells included dilation of endoplasmic reticulum and perinuclear envelopes and was present at 4 hours after treatment. Necrosis and sloughing of these cells from basement membranes occurred at times from 12 to 96 hours after treatment. Alveolar capillary endothelial cells had mild dilation of endoplasmic reticulum at times from 12 to 72 hours after treatment. Necrosis of endothelial cells was not observed. Inflammatory cell infiltrates in bronchioles and alveoli were dominated by macrophages and neutrophils. Significant elevations (P less than 0.05) in numbers of neutrophils and macrophages were recovered by bronchoalveolar lavage at times from 24 to 96 hours after 4-ipomeanol-treatment. Hyperplasia of nonciliated bronchiolar epithelial cells and of type II alveolar epithelial cells were observed at 72 and 96 hours after treatment. The results indicate that type I alveolar epithelial cells and nonciliated bronchiolar epithelial cells are most susceptible to 4-ipomeanol-induced damage and necrosis in calves. 4-ipomeanol-induced pulmonary edema in calves occurs prior to ultrastructurally-demonstrable, mild, alveolar capillary endothelial cell damage.     

Pulmonary expression of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-8 in the acute phase of bovine pneumonic pasteurellosis

Inflammatory cytokines are suspected to contribute to the pathogenesis of bovine pneumonic pasteurellosis (BPP) through neutrophil recruitment, leukocyte activation, and the induction of a broad array of soluble inflammatory mediators. An in vivo experimental model of BPP was used to characterize the pulmonary expression kinetics of tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) genes and proteins during the acute phase of disease development. Cytokine expression in bronchoalveolar lavage (BAL) fluid, BAL cells, and pneumonic lung parenchyma was quantitated by northern blot analysis, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization at 2, 4, 8, 16, and 24 hours after endobronchial inoculation of Pasteurella (Mannheimia) haemolytica. Expression of TNFalpha, IL-1beta, and IL-8 was significantly increased in the airways and lung lesions of infected calves as compared with mock-infected controls. Although kinetic patterns varied, peak levels of cytokine mRNA occured within 8 hours postinfection (PI), and peak cytokine concentrations occurred within 16 hours PI. In all samples, IL-8 was expressed to the greatest extent and TNFalpha was least expressed. Expression of TNFalpha was restricted to alveolar macrophages. Alveolar and interstitial macrophages produced IL-1beta and IL-8 in the first 4 hours; bronchial and bronchiolar epithelial cells were also significant sources of IL-8 during this period. By 8 hours PI, neutrophils were the dominant source of both IL-1beta and IL-8. These findings demonstrate a spatial and temporal association between pulmonary expression of inflammatory cytokines and acute lung pathology, supporting the hypothesis that cytokines contribute to inflammatory lung injury in BPP.     

Equine multinodular pulmonary fibrosis: a newly recognized herpesvirus-associated fibrotic lung disease

Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a gamma-herpesvirus of horses that has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout the lungs. Histologically, there was marked interstitial fibrosis, often with preservation of an "alveolar-like" architecture, lined by cuboidal epithelial cells. The airways contained primarily neutrophils and macrophages. Rare macrophages contained large eosinophilic intranuclear viral inclusion bodies; similar inclusion bodies were also found cytologically. The inclusions were identified as herpesviral-like particles by transmission electron microscopy in a single horse. In situ hybridization was used to detect EHV-5 nucleic acids within occasional macrophage nuclei. With polymerase chain reaction (PCR), the herpesviral DNA polymerase gene was detected in 19/24 (79.2%) of affected horses and 2/23 (8.7%) of the control horses. Virus genera-specific PCR was used to detect EHV-5 in all of the affected horses and none of the control horses. EHV-2 was detected in 8/24 (33.3%) of affected horses and 1/9 (11.1%) of the control horses. This disease has not been reported before, and the authors propose that based upon the characteristic gross and histologic findings, the disease be known as equine multinodular pulmonary fibrosis. Further, we propose that this newly described disease develops in association with infection by the equine gamma-herpesvirus, EHV-5.     

Detection and localization of Mycoplasma hyopneumoniae DNA in lungs from naturally infected pigs by in situ hybridization using a digoxigenin-labeled probe.

Mycoplasma hyopneumoniae DNA was detected in 20 naturally infected pigs by in situ hybridization using a nonradioactive digoxigenin-labeled DNA probe. A 520-base-pair DNA probe targeting a reiterative sequence of the M. hyopneumoniae genome was generated by the polymerase chain reaction. All 20 pigs infected with M. hyopneumoniae had distinct and positive hybridization signals without background staining. A strong hybridization signal was detected mainly in the luminal surface of bronchial and bronchiolar lining epithelial cells, whereas no hybridization signal was seen in the cytoplasm of bronchial and bronchiolar lining epithelial cells. When hybridization signal was detected in the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole had peribronchiolar lymphoid hyperplastic tissues. Hybridization signals were not seen in the peribronchiolar lymphoid hyperplastic tissues. A less intense signal was detected in the interstitial and alveolar macrophages randomly scattered in the thickened alveolar septa and spaces. Hybridization signal was rarely detected in the type I pneumocytes. The in situ hybridization technique developed in this study was useful for detection of M. hyopneumoniae nucleic acids in tissues taken from naturally infected piglets and may be a valuable technique for studying the pathogenesis of M. hyopneumoniae infection.     

Morphometric and ultrastructural study of postnatal lung growth and development in calves

The objectives of this study were to characterize basic patterns of postnatal lung growth in 1- to 150-day-old Holstein calves and to identify periods of accelerated lung growth and times of epithelial cell development that might correlate with periods of heightened susceptibility to pulmonary injury by infective agents and dietary toxins. Calves had fully developed alveoli at birth. There was an age-associated increase in total alveolar surface area and alveolar number, which was most marked in calves older than 30 days. Lung volume and body weight increases were also most marked when calves were older than 30 days. Mean bronchiolar cross-sectional area increased significantly (P less than 0.05) with age, and there was a significant (P less than 0.05) decrease in the percentage of terminal bronchioles with diameters less than 3 x 10(4) microns between 1 and 150 days. In newborn calves, nonciliated bronchiolar epithelial cells retained fetal characteristics of differentiation, including abundant cytoplasmic glycogen and sparse agranular endoplasmic reticulum. Glycogen deposits were depleted, and agranular endoplasmic reticulum was developed in nonciliated cells by the time calves were 30 days old. We concluded that bronchiolar epithelium in calves is well differentiated by the time they are 30 days old and that lung growth in calves raised in semi-isolation begins most rapidly when calves are approximately 30 days old.