Repeated blood instillation into the airway of the horse does not cause pulmonary fibrosis

Reasons for performing the study: Exercise‐induced pulmonary haemorrhage (EIPH) occurs in nearly all strenuously exercising horses. Recent studies have attempted to identify the role of free blood within the airspaces, in the lung fibrosis that develops within the lungs of EIPH horses. Hypothesis: Repeated exposure of the equine lung to autologous blood results in lung fibrosis similar to that observed in spontaneous EIPH. Methods: Forty ml of autologous blood from the jugular vein was instilled into preselected lung regions of 6 horses one, 2, 3, 4 or 5 times at 2 week intervals, with 40 ml of saline instilled into the contralateral lung serving as a control. The time interval between instillation of the first blood and euthanasia ranged from 2–10 weeks. The lung from each instillation site was harvested, and the histopathology was scored from each region based upon the presence and abundance of blood, haemosiderin and interstitial collagen. Consequently, at the time of euthanasia, the time since instillation of the first blood ranged from 2–10 weeks. Results: Beyond retention of blood, and the accumulation of haemosiderin, there was no visible increase in perivascular and interstitial collagen within the blood‐instilled lung sites. In a small number of regions, there were foci of bronchiolitis obliterans organising pneumonia with collagen accumulation within these foci, but no collagen accumulation with the characteristic perivascular and interstitial histological distribution seen in EIPH. Conclusions: Free blood within the airways of horses does not result in a qualitative increase in the amount of interstitial collagen within 8–10 weeks, and is therefore an unlikely aetiological factor in the lung collagen accumulation that occurs in EIPH. Potential relevance: This study emphasises the efficiency of the equine lung in clearing blood from the airspaces. Further, it suggests that the aetiopathogenesis of EIPH is not driven by events within the airspace lumen, but rather emanates from within the vasculature and lung interstitium.     

Pulmonary response to airway instillation of autologous blood in horses

REASONS FOR PERFORMING STUDY: Exercise-induced pulmonary haemorrhage (EIPH) occurs in the majority of horses performing strenuous exercise. Associated pulmonary lesions include alveolar and airway wall fibrosis, which may enhance the severity of EIPH. Further work is required to understand the pulmonary response to blood in the equine airways. OBJECTIVES: To confirm that a single instillation of autologous blood into horse airways is associated with alveolar wall fibrosis, and to determine if blood in the airways is also associated with peribronchiolar fibrosis. METHODS: Paired regions of each lung were inoculated with blood or saline at 14 and 7 days, and 48, 24 and 6 h before euthanasia. Resulting lesions were described histologically and alveolar and airway wall collagen was quantified. RESULTS: The main lesion observed on histology was hypertrophy and hyperplasia of type II pneumocytes at 7 days after blood instillation. This lesion was no longer present at 14 days. There were no significant effects of lung region, treatment (saline or autologous blood instillation), nor significant treatment-time interactions in the amount of collagen in the interstitium or in the peribronchial regions. CONCLUSION: A single instillation of autologous blood in lung regions is not associated with pulmonary fibrosis. POTENTIAL RELEVANCE: Pulmonary fibrosis and lung remodelling, characteristic of EIPH, are important because these lesions may enhance the severity of bleeding during exercise. A single instillation of autologous blood in the airspaces of the lung is not associated with pulmonary fibrosis. Therefore the pulmonary fibrosis described in EIPH must have other causes, such as repetitive bleeds, or the presence of blood in the pulmonary interstitium in addition to the airspaces. Prevention of pulmonary fibrosis through therapeutic intervention requires a better understanding of these mechanisms.     

Exercise-induced pulmonary haemorrhage in the horse: results of a detailed clinical, post mortem and imaging study. II. Gross lung pathology

Gross post mortem examinations were performed on the lungs of 26 Thoroughbred horses of known exercise-induced pulmonary haemorrhage (EIPH) status. The most consistent finding was a variable degree of bilaterally symmetrical, dark discolouration of the dorsocaudal regions of the caudal lung lobes. In more severely affected lungs, the stained areas extended cranially along the dorsal surfaces of the lungs, and in some cases affected approximately one third of the lung surface. Discoloured areas of lung were denser than normal, collapsed less readily, often contained trapped air and were slow to inflate. The subpleural bronchial arteries were more prominent in the discoloured regions. Pleural adhesions were noted in two horses but were not related to the discoloured lung regions. It was concluded that the discoloured lesions have a complex pathogenesis and were related directly to previous bouts of EIPH. Associated with them were signs indicating probable partial small airway obstruction, decreased tissue compliance and direct involvement of the bronchial arterial circulation.     

Evaluation of leukotriene biosynthetic capacity in lung tissues from horses with recurrent airway obstruction

OBJECTIVE: To evaluate leukotriene (LT) biosynthetic capacity in lung tissue from healthy horses and horses with recurrent airway obstruction (RAO). SAMPLE POPULATION: Lung parenchyma and airway specimens from 8 RAO-affected and 5 healthy horses. PROCEDURE: Horses were stabled for > or = 72 hours. Blood was drawn before euthanasia, after which lung specimens were collected. Tissue strips from small airways and parenchyma were incubated in organ baths with the precursor LTA4 or stimulated with calcium ionophore A23187 or the tripeptide N-formyl-Met-Leu-Phe (fMLP), with or without exogenous arachidonic acid, in the presence of isolated blood neutrophils. RESULTS: Stabling induced typical clinical signs of airway obstruction in RAO-affected horses but not control horses. When lung parenchyma or airway specimens from both groups of horses were incubated with calcium ionophore, with or without arachidonic acid, they did not form LT. In contrast, addition of LTA4 to both tissues resulted in conversion to LTB4, although concentrations of LTC4 were negligible in airways and parenchymal strips from healthy and RAO-affected horses. Incubation of airway and parenchymal strips with suspensions of autologous neutrophils did not influence formation of LT stimulated by calcium ionophore or fMLP, with or without exogenous arachidonic acid. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that lung parenchyma and airway tissues themselves are not of substantial importance for LT formation in the lungs, although these tissues possessed some LTA4 hydrolase activity, enabling LTB4 formation. It may be speculated that LTB4 originates primarily from neutrophils and may play a role in the inflammatory events of RAO.     

Exercise-induced pulmonary haemorrhage in the horse: results of a detailed clinical, post mortem and imaging study. IV. Changes in the bronchial circulation demonstrated by C.T. scanning and microradiography

The purpose of this study was to use radiographic contrast techniques and special imaging methods to identify and high-light bronchial arterial involvement in lung lesions associated with exercise-induced pulmonary haemorrhage (EIPH) in horses. The lungs from four horses with histories of EIPH were prepared for computerised tomographic scanning and microradiography by perfusing the broncho-oesophageal artery with a mixture of red latex and either barium or iodine contrast materials while the pulmonary supply received only blue latex. Computerised tomographic scan slices of the prepared inflated lungs were obtained from the caudal tip of the lung to the hilus. Microradiography of selected lung slices was also performed on a Faxitron. Diffuse areas of increased density, with preferential bronchial arterial supply noted on the computerised tomographic scans were confirmed by microradiography. Dense focal and diffuse plexuses of markedly hypertrophied and highly branched bronchial arterial networks were identified, centred around certain small airways. The vascular supply to these plexuses was recruited predominantly from neighbouring bronchial vessels, and in some cases, from the enlarged vasa vasorum of pulmonary arteries sending anastomoses to the affected areas. The authors conclude that bronchial vascular lesions in EIPH cases are the likely origin of haemorrhage; that small airway disease is the probable initiating stimulus for bronchial vascular proliferation in these lesions; and that the morphology and nature of the neovascular tissue in these lesions provides the conditions leading to haemorrhage in the lungs of horses with EIPH.     

Pulmonary distribution of aerosolized technetium Tc 99m pentetate after administration of a single dose of aerosolized albuterol sulfate in horses with recurrent airway obstruction.

OBJECTIVE: To determine whether pulmonary distribution of aerosolized technetium Tc 99m pentetate is improved after inhalation of a single dose of albuterol sulfate in horses susceptible to recurrent airway obstruction (heaves). ANIMALS: 6 horses with heaves and 4 horses with normal respiratory tract function. PROCEDURE: Images were obtained during ventilation of horses at baseline (maximal change in pleural pressure during tidal breathing [deltaPpImax] >15 cm H2O) and after aerosolized albuterol sulfate (360 microg) administration, with a 24-hour washout period between experiments. The deltaPpImax was determined prior to the baseline scan, prior to albuterol sulfate administration, and 5 minutes after albuterol sulfate administration. Images were assessed by visual inspection (semi-quantitative scoring system) and histogram analysis. RESULTS: Images obtained from horses with heaves had nonuniform pulmonary distribution of radionuclide characterized by poor penetration in peripheral lung fields and excess deposition in large airways. Histogram analysis of images of the caudal portions of the lungs revealed nonuniform radionuclide deposition in horses with heaves and uniform radionuclide deposition in control horses. CONCLUSION: Administration of a single dose of aerosolized albuterol sulfate improved pulmonary distribution of aerosolized radiolabeled pentetate suspension in horses with heaves but did not alter pulmonary distribution in clinically normal horses. CLINICAL RELEVANCE: Precedent bronchodilator administration may improve pulmonary distribution of aerosolized, surface-active anti-inflammatory preparations.     

Differential association of MUC5AC and CLCA1 expression in small cartilaginous airways of RAO‐affected and control horses

Reasons for performing study: Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. Objectives: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO‐affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. Methods: We performed quantitative RT‐PCR of eqMUC5AC, CLCA1 and EGFR; volume density measurements of intraepithelial mucosubstances; and cytological differentiation of intraluminal inflammatory cells in small cartilaginous airways from cranial left and right and caudal left and right lung lobes of 5 clinically healthy and 5 RAO‐affected horses that had been exposed to indoor stable environment for 5 days before euthanasia. Results: Neutrophils were increased in RAO‐affected horses compared to clinically healthy controls. EqMUC5AC mRNA levels were positively correlated with both CLCA1 and EGFR mRNA levels in RAO‐affected horses but only with CLCA1 in controls. The relationship between eqMUC5AC and CLCA1 differed in the 2 groups of horses with RAO‐affected animals overexpressing CLCA1 in relation to eqMUC5AC. Conclusions: These data implicate CLCA1 as a signalling molecule in the expression of eqMUC5AC in horses but also suggest differential regulation by CLCA1 and EGFR between horses with RAO and those with milder degrees of airway inflammation.     

Airway inflammation is associated with mucous cell metaplasia and increased intraepithelial stored mucosubstances in horses

This study was performed to determine if a peripheral sample of lung from the site where biopsy is conducted is representative of the rest of the lung and to investigate the relationship between airway inflammation and intraepithelial mucous production in the peripheral airways. Lung parenchyma samples were collected from five different regions of the lung in five control and five heaves-affected horses. Horse groups were defined by clinical response to stabling. Tissue sections were used for semi-quantitative scoring of lesions, to count the number of airways, to quantify the amount of stored mucosubstances (Vs) within the epithelium, and to count the number of epithelial cells in terminal airways. No significant differences were found between lung regions or between groups of horses. Lack of regional differences in airway structures means that a biopsy sample can be used for diagnosis and investigation of diffusely distributed diseases. Airway inflammation was correlated with mucous cell metaplasia and Vs. Therefore, in horses, mucus accumulation is partly caused by increased number of mucous cells and is associated with airway inflammation. Therapy targeted to reduce airway inflammation will help reduce the excessive mucous accumulation in horses.     

Pulmonary gas exchange correlated to clinical signs and lung pathology in horses with chronic bronchiolitis

Eight horses (mean weight 438 kg) with chronic obstructive pulmonary disease (COPD) were studied for clinical signs, ventilation/perfusion relationships (VA/Q) and lung morphology. Four horses were killed and necropsied after the study. In horses with COPD, minute ventilation was almost twice as high as normal, whereas PaO2 was significantly decreased. Cardiac output was normal, but pulmonary artery pressure and pulmonary vascular resistance were significantly increased. The VA/Q distribution was abnormal with an increased scatter of VA/Q ratios. However, shunt (VA/Q = 0) was increased in one horse only. Ventilation of dead space and high VA/Q regions (VA/Q greater than 10) were increased markedly and comprised 74 per cent of minute ventilation. On lung biopsy, all horses showed chronic bronchiolitis with epithelial hyperplasia and metaplasia. Diffuse acinar hyperinflation also was evident at necropsy. There was a significant inverse correlation between the respiratory rate and the fractional ventilation of normal VA/Q regions. Also, there was a significant agreement between the extent of bronchiolar epithelial hyperplasia in necropsy specimens of lungs and the degree of ventilation of high VA/Q regions and dead space. The findings suggest that hyperinflation of the lung due to obstructed airways may be the common denominator of increased ventilation of high VA/Q regions and dead space ventilation and of elevated pulmonary artery pressure in horses with COPD from chronic bronchiolitis.     

Assessment of leukotriene B4 production in leukocytes from horses with recurrent airway obstruction

OBJECTIVE: To determine the ex vivo leukotriene (LT) biosynthesis in peripheral blood neutrophils (PBNs) and inflammatory cells in bronchoalveolar lavage fluid (BALF) obtained from horses affected with recurrent airway obstruction (RAO). ANIMALS: 6 RAO-affected and 6 control horses. PROCEDURES: Before and 6, 24, and 48 hours after stabling, disease severity was determined subjectively by clinical and mucus scores and measurement of the maximal change in pleural pressure (deltaPpl(max)); PBNs were isolated and BALF samples were examined cytologically. The PBN and BALF cells were activated with a calcium ionophore in the presence of arachidonic acid, and production of LTC4 and LTB4 was measured per 10(6) cells. RESULTS: Clinical and mucus scores and deltaPpl(max) increased during stabling in RAO-affected horses, but not in control horses. In neutrophils and BALF cells from both groups, production of LTB4 exceeded that of LTC4. At all times, LTB4 production by PBNs was less in RAO-affected horses than it was in control horses. Before stabling, LTB4 production by cells in BALF was low in RAO-affected horses, but increased considerably after 6 hours of stabling. This increase coincided with the migration of neutrophils into the airways. In control horses, production of LTB4 did not change during stabling. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested increased production of LTB4 in airways of RAO-affected horses, compared with control horses, that may contribute to the infiltration of neutrophils into the lungs and the sustained inflammation associated with RAO.     

Observations on the mechanism of functional obstruction of the nasopharyngeal airway in the horse.

Fibreoptic endoscopy was used to study the movements of the larynx and pharynx during nasal occlusion in 10 horses, which showed signs consistent with functional pharyngeal obstruction (FPO) on exercise. Cine-endoscopic films were made on 3 such horses. Consideration of the anatomy of the region indicates that FPO may best be regarded as a subluxation of the nasopharyngeal and larygneal airways and it was found that a constant component of the movements which brought about this subluxation was a marked caudal retraction of the larynx. It is suggested that this caudal retraction of the larynx occurred as a result of the action of th sternothyrohyoid and omohyoid muscles and that removal of a portion of these muscles might prevent subsequent airway subluxation in horses prone to FPO on exercise.     

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.     

High-resolution computed tomography of the mammalian lung.

High-resolution computed tomography (HRCT) was performed in 21 isolated animal lungs, from 4 mammalian species (pigs, rabbits, dogs, sheep). Gross and subgross central and peripheral lung morphology was determined by HRCT. Three distinct types of lungs can be identified, principally based on the extent of interlobular septal development; the relationship of major vessels to airways; and the thickness of the visceral pleura. Type-I lung is found in pigs, sheep, and cattle; type-II lung is found in rabbits, dogs, cats, and monkeys; and type-III lung is found in human beings and horses. These mammalian lungs were compared with human lungs. The potential use of HRCT to investigate specific human lung diseases in the aforementioned species also was considered.     

Regional distribution of collagen and haemosiderin in the lungs of horses with exercise‐induced pulmonary haemorrhage

Reasons for performing study: Regional veno‐occlusive remodelling of pulmonary veins in EIPH‐affected horses, suggests that pulmonary veins may be central to pathogenesis. The current study quantified site‐specific changes in vein walls, collagen and haemosiderin accumulation, and pleural vascular profiles in the lungs of horses suffering EIPH. Hypothesis: In the caudodorsal lung regions of EIPH‐affected horses, there is veno‐occlusive remodelling with haemosiderosis, angiogenesis and fibrosis of the interstitium, interlobular septa and pleura. Methods: Morphometric methods were used to analyse the distribution and accumulation of pulmonary collagen and haemosiderin, and to count pleural vascular profiles in the lungs of 5 EIPH‐affected and 2 control horses. Results: Vein wall thickness was greatest in the dorsocaudal lung and significantly correlated with haemosiderin accumulation. Increased venous, interstitial, pleural and septal collagen; lung haemosiderin; and pleural vascular profiles occurred together and changes were most pronounced in the dorsocaudal lung. Further, haemosiderin accumulation colocalised with decreased pulmonary vein lumen size. Vein wall thickening, haemosiderin accumulation and histological score were highly correlated and these changes occurred only in the caudodorsal part of the lung. Conclusion: The colocalisation of these changes suggests that regional (caudodorsal) venous remodelling plays an important role in the pathogenesis of EIPH. Potential relevance: The results support the hypothesis that repeated bouts of venous hypertension during strenuous exercise cause regional vein wall remodelling and collagen accumulation, venous occlusion and pulmonary capillary hypertension. Subjected to these high pressures, there is capillary stress failure, bleeding, haemosiderin accumulation and, subsequently, lung fibrosis.     

Development of equine upper airway fluid mechanics model for Thoroughbred racehorses

REASON FOR PERFORMING STUDY: Computational fluid dynamics (CFD) models provide the means to evaluate airflow in the upper airways without requiring in vivo experiments. HYPOTHESIS: The physiological conditions of a Thoroughbred racehorse's upper airway during exercise could be simulated. Methods: Computed tomography scanned images of a 3-year-old intact male Thoroughbred racehorse cadaver were used to simulate in vivo geometry. Airway pressure traces from a live Thoroughbred horse, during exercise was used to set the boundary condition. Fluid-flow equations were solved for turbulent flow in the airway during inspiratory and expiratory phases. The wall pressure turbulent kinetic energy and velocity distributions were studied at different cross-sections along the airway. This provided insight into the general flow pattern and helped identify regions susceptible to dynamic collapse. RESULTS: The airflow velocity and static tracheal pressure were comparable to data of horses exercising on a high-speed treadmill reported in recent literature. The cross-sectional area of the fully dilated rima glottidis was 7% greater than the trachea. During inspiration, the area of highest turbulence (i.e. kinetic energy) was in the larynx, the rostral aspect of the nasopharynx was subjected to the most negative wall pressure and the highest airflow velocity is more caudal on the ventral aspect of the nasopharynx (i.e. the soft palate). During exhalation, the area of highest turbulence was in the rostral and mid-nasopharynx, the maximum positive pressure was observed at the caudal aspect of the soft palate and the highest airflow velocity at the front of the nasopharynx. CONCLUSIONS AND CLINICAL RELEVANCE: In the equine upper airway collapsible area, the floor of the rostral aspect of the nasopharynx is subjected to the most significant collapsing pressure with high average turbulent kinetic during inhalation, which may lead to palatal instability and explain the high prevalence of dorsal displacement of the soft palate (DDSP) in racehorses. Maximal abduction of the arytenoid cartilage may not be needed for optimal performance, since the trachea cross-sectional area is 7% smaller than the rima glottidis.     

Humoral and cellular immune responses of pigs inoculated with Mycoplasma hyopneumoniae

Cellular and humoral immune responses of pigs inoculated with Mycoplasma hyopneumoniae were investigated at postinoculation weeks (PIW) 2, 4, and 6. The response of blood lymphocytes (BL) and bronchial lymph node lymphocytes (LNL) to stimulation by M hyopneumoniae antigens was evaluated by a lymphocyte-stimulation test. Specific antibodies in serum and lung washing samples were assayed by ELISA. Immunoglobulin-positive cells in lungs and bronchial lymph nodes were identified by indirect fluorescent antibody test, using isotype-specific monoclonal antibodies. At PIW 0 to 6, BL from control and M hyopneumoniae-inoculated pigs were stimulated by M hyopneumoniae cells; however, BL from inoculated pigs generally had higher stimulation indices, especially at PIW 6. The response of LNL was influenced by previous exposure to M hyopneumoniae, as indicated by higher stimulation indices (P less than 0.01) of LNL from inoculated pigs killed at PIW 2 and 6. Specific ELISA antibodies to M hyopneumoniae in lung washings from inoculated pigs consisted mainly of IgG and IgA isotypes. Examination of lung sections by indirect immunofluorescence revealed that cells producing IgM and IgA were in controls as well as M hyopneumoniae-inoculated pigs, but IgG-positive cells were only in lungs of inoculated pigs. Resolution of pneumonia appeared to correlate with development of increased sensitization of BL, as well as development of marked increases in immunoglobulins, particularly IgG in lung washings at PIW 6.     

Immunofluorescent evaluation of the lower respiratory tract of healthy horses and of horses with chronic bronchiolitis

Pulmonary parenchymal tissues from 6 healthy horses and from 9 horses with chronic bronchiolitis were evaluated by use of an indirect immunofluorescent technique. In horses of both groups, the diffuse interstitial immunofluorescence was most intense for immunoglobulin (Ig)G, moderate for IgA, and minimal for IgM. Intensity of fluorescence was increased markedly in diseased lungs exposed to anti-IgA and anti-IgG. Around small bronchi and large bronchioles of healthy horses, IgA-containing cells generally were more numerous than were IgG-Fc fragment (Fc)-containing cells; in small bronchioles, however, IgA-containing cells and IgG (Fc)-containing cells were identified with approximately equal frequency. Immunoglobulin-containing cell types usually were located in the lamina propria and submucosa of airways, with occasional Ig-containing cells seen interepithelially and intraluminally; IgG (T)- and IgM-containing plasma cells were observed infrequently in the same regions. The number of Ig-containing cells became fewer as the diameter of the airway decreased; therefore, at the level of the terminal bronchiole, Ig-containing cells only were observed occasionally. This pattern of Ig-containing cell distribution was similar in horses with chronic bronchiolitis; however, horses with chronic bronchiolitis had more IgA-containing cells forming perivascular and peribronchiolar cuffs than did healthy horses. The number of IgG (Fc)-containing lymphocytes and non-Ig-stainable lymphocytes also were higher in horses with chronic bronchiolitis than in healthy horses, but the number did not approach the magnitude of that observed for IgA-containing cells. One horse had deposits of complement C3 and IgG (Fc) within alveolar septa.     

Response of calves to exposure with swine influenza virus

In calves inoculated with live swine influenza virus (SIV) A/sw/IL/75 (H1N1) intranasally, SIV was isolated for 7 days, and respiratory tract disease was observed. Antibody was detected in serum of inoculated calves from postinoculation day 9, and virus-neutralization antibody was demonstrated on postinoculation days 14 and 21. The primary response was low, but readily differentiated from the secondary response after calves were challenge exposed with homologous SIV. Pneumonic lesions were observed at necropsy, and histopathologic changes in airways and lungs were consistently found. Fluorescent staining revealed viral activity in epithelial cells of airways. The virus was transferred to healthy calves housed with inoculated calves.     

Effects of the bronchoalveolar lavage procedure on lung function in horses with clinical exacerbation of recurrent airway obstruction

OBJECTIVE: To evaluate whether bronchoalveolar lavage (BAL) alters respiratory mechanics of horses with recurrent airway obstruction (ie, heaves) over a 48-hour period. ANIMALS: 6 horses affected with heaves. PROCEDURES: Horses were subjected to a complete BAL procedure, which included sedation with xylazine and butorphanol, intratracheal administration of lidocaine, and instillation and aspiration of two 250-mL boluses of saline (0.9% NaCl) solution through an endoscope (study 1). To evaluate the effects of saline solution, horses were subjected to the same procedure without saline solution instillation and aspiration (study 2). Lastly, the endoscope was similarly introduced into the lower airways, without sedation or saline instillation and aspiration (study 3). Respiratory mechanics were performed at baseline (time 0) and at 3, 6, 12, 24, and 48 hours after each procedure. RESULTS: In study 1, BAL induced a significant decrease in pulmonary resistance lasting up to 6 hours. This may have resulted from clearance of mucus in large airways. We also observed a significant increase in lung elastance and transpulmonary pressure at 12 hours after BAL in all 3 studies, which may be attributed to a circadian effect. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that the temporal effects of BAL procedures on lung mechanics should be taken into account when designing research protocols involving horses with heaves. Future studies should address the immediate effects of BAL on lung function.     

Plasma and bronchoalveolar fluid concentrations of nitric oxide and localization of nitric oxide synthesis in the lungs of horses with summer pasture-associated obstructive pulmonary disease

OBJECTIVE: To determine concentrations of nitric oxide (NO) in plasma and bronchoalveolar lavage fluid (BALF) and localize nitric oxide synthesis in the lungs of horses with summer pasture-associated obstructive pulmonary disease (SPAOPD). ANIMALS: 7 adult horses with SPAOPD and 6 clinically normal adult horses. PROCEDURE: Severity of SPAOPD was determined by use of clinical scores, change in intrapleural pressure (APpl) during tidal breathing, cytologic analysis of BALF, and histologic evaluation of lung specimens obtained during necropsy. Nitric oxide concentrations in plasma, BALF and epithelial lining fluid (ELF) were determined by use of a chemiluminescent method. Inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) were localized in formalin-fixed lung specimens by use of immunohistochemical staining, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) activity was localized in cryopreserved specimens by use of histochemical staining. RESULTS: Plasma concentration of NO in affected horses was slightly but not significantly greater than concentration in nonaffected horses. Nitric oxide concentrations in BALF or ELF did not differ between groups. Immunoreactivity of iNOS in bronchial epithelial cells of 3 of 5 lung lobes was greater in horses with SPAOPD, compared with nonaffected horses. However, staining for NT and NADPHd activity did not differ between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Expression of iNOS was greater in bronchial epithelial cells of horses with SPAOPD, compared with nonaffected horses, suggesting that NO may play a role in amplifying the inflammatory process in the airways of horses with this disease.