Stress alters the cellular and proteomic compartments of bovine bronchoalveolar lavage fluid

The stresses of transportation, weaning and commingling are associated with an increased incidence of bacterial and viral pneumonia in cattle. Proteins expressed in the epithelial lining fluid (ELF) of the lungs, in conjunction with resident leukocytes, represent the first line of defence against opportunistic pathogens, and stress-induced alterations in their expression may reveal markers of disease susceptibility. Bronchoalveolar lavage fluid was sampled in weaned and transported calves and ELF protein expression was compared to a control group of calves using two-dimensional electrophoresis (2DE). Serum and pulmonary haptoglobin were increased following stress concurrent with the number of blood neutrophils. Using 2DE, significant changes in expression were observed in spots identified by mass spectrometry as annexin A1 and A5, odorant-binding protein (OBP), isocitrate dehydrogenase, fibrinogen, heme-binding protein, alpha-2-HS-glycoprotein, alpha-1-antichymotrypsin and albumin. Quantification of OBP mRNA by real-time RT-PCR and OBP protein by western blot revealed gender-dependent differences in relative OBP expression in response to stress. These findings reveal stress-associated protein changes in pulmonary ELF and suggest a mechanism through which stress alters respiratory disease susceptibility.     

A method for bronchoalveolar lavage in live pigs

In order to isolate porcine alveolar macrophages and to quantitatively study the components of recovered lung fluid, a bronchoalveolar lavage technique in living pigs was developed. Lung lavage was performed after introducing a catheter through the mouth via the trachea in the diaphragmatical lobe. Thirty ml of phosphate-buffered saline (PBS) was introduced into the lung and the fluid was aspirated after one minute. Following this, another 15 ml of PBS was introduced into the lung and aspirated after one minute. The recovered volume of the second lavage averaged 15 ml (+/- 0.4 S.E.M.). Cells thus obtained from specific-pathogen-free (SPF) pigs were composed of 98% macrophages. Lavage fluids from conventionally bred pigs contained 67% macrophages, 17% neutrophilic granulocytes and about 16% lymphocytes, demonstrated by their morphology and acid phosphatase activity. The viability of the recovered cells was over 98% in both SPF and conventionally bred pigs. The dilution of the aspirated lung liquid was determined by using methylene blue in the introduced fluid. The calculated dilution factor of the recovered lavage fluid was 0.58 (S.E.M. 0.02). No influence was noticed on the number or composition of cells nor on the dilution factor when lung lavage was done in SPF pigs twice a week during a four week period. The protein concentration in lavage fluid from SPF pigs was 142 (SD +/- 26) mcg/ml. In conventionally bred pigs, however, a wide variation (276 +/- 229 mcg/ml) in protein content was noted. Lavage fluid supernatant of some animals had a bactericidal effect on Haemophilus pleuropneumoniae strain 13261, whereas no bactericidal effect was noted in other lavage samples.     

Respiratory disease markers in porcine bronchoalveolar lavage fluid

In bronchoalveolar lavage fluid (BALF) of pigs originating from different herds bacteria, cells and the antibacterial peptide PR-39 were examined to gain information about the lung health status. In a high health nucleus herd 56% and in low health herds 20-100% of the examined pigs were found positive for potentially pathogenic bacteria. Based on these findings, a novel definition for bacterial respiratory tract disease was established using an 8% cut-off for the relative number of neutrophils in bronchoscopic and a 40% cut-off in transtracheal BALF in combination with the occurrence of potentially pathogenic microorganisms. The antibacterial peptide PR-39 was highly correlated to this definition of respiratory disease. An assessment of the bacteriological respiratory health status appears to be possibly based on the determination of PR-39 concentrations in BALF using different cut-off values according to the lavage method (2.5 nM for bronchoscopic and 5 nM for transtracheal BALF).     

Collagenolytic activity in bronchoalveolar lavage fluid in canine pulmonary eosinophilia

We studied and characterized the collagenolytic matrix metalloproteinases (MMP-8 and MMP-13) in the pathogenesis of canine pulmonary eosinophilia (PE). Twenty dogs with PE and 16 healthy control dogs underwent similar clinical examination and collection of bronchoalveolar lavage fluid (BALF). Analyses of total cell and differential cell counts and collagen I degradation with and without aminophenyl mercuric acetate (APMA) treatment were performed. Correlations between cell counts and percentage of degraded collagen I in BALF were studied. Collagenase activity detected in BALF was characterized by Western immunoblotting for collagenase-2 (MMP-8) and collagenase-3 (MMP-13), and their cellular location was studied by immunocytochemical means. Collagenolytic activity was significantly increased in cell-free and native BALF of PE dogs compared to healthy controls. APMA treatment had no significant effect on BALF collagenase activity, indicating that collagenolytic activity occurred in diseased BALF in vivo in active form. Western immunoblotting identified the presence of MMP-8 and MMP-13 immunoreactivities, of which the latter was converted to active form. Major immunoreactivity for MMP-8 was observed in macrophages and epithelial cells, and major immunoreactivity for MMP-13 was observed in macrophages. A significant positive correlation was noted between the percentage of degraded collagen I and the counts of eosinophils, macrophages, lymphocytes, and mast cells. These findings suggest that the up-regulation of collagenolysis eventually contributes to pulmonary tissue destruction in canine PE.     

Neutrophil migration induced by equine respiratory secretions, bronchoalveolar lavage fluids and culture supernatants of pulmonary lavage cells

Supernatants of equine respiratory secretions enhanced the migration of equine neutrophils into the lower compartments of Boyden chambers. Checkerboard analysis revealed that the neutrophil migration promoting activity (NMPA) of secretion specimens was in great part caused by chemokinesis, irrespective of the neutrophil score of the specimen. The NMPA of respiratory secretions was correlated neither with the neutrophil score of the secretion specimen nor with the severity of the chronic pulmonary disease. Respiratory secretions collected while horses were kept under low dust or under dusty housing conditions induced migration of neutrophils in the same order of magnitude. The number of migrated neutrophils and the procoagulant activity (PCA) within respiratory secretion specimens was positively correlated; however, the meaning of this finding is not yet clear. None of the nine cell-free supernatants of bronchoalveolar lavage fluid, which were assayed undiluted, induced significant neutrophil migration, although some samples contained up to 4.0 x 10(5) neutrophils/ml. In vitro culture of lung lavage cells, which mainly comprised macrophages and lymphocytes, without stimulation or with the addition of low doses of phytohemagglutinin (PHA) resulted in the secretion of NMPA which was in great part chemotactic. However, culture supernatants of lung cell preparations which were stimulated by lipopolysaccharide (LPS) or by PHA-prestimulated lymphocytes reduced the migration of neutrophils compared with the supernatants of control cells. NMPA within culture supernatants had a highly significant negative correlation with the PCA of macrophages within the lung cell preparations. Our results imply that a complicated and sophisticated regulation underlies neutrophil accumulation within the airways of horses affected with chronic pulmonary disease. Future experiments are required to assess the biological significance of the factors modulating neutrophil migration which are present in the respiratory secretions and in the culture supernatants of equine lung lavage cells.     

Association between quantitative bacterial culture of bronchoalveolar lavage fluid and antibiotic requirement in dogs with lower respiratory tract signs

BackgroundHistorically, positive bacterial cultures from the lower respiratory tract (LRT) have been considered clinically relevant when quantitative bacterial cultures of bronchoalveolar lavage fluid (BALF) were >1700 colony forming units (cfu)/mL. However, this threshold might not accurately predict a requirement for antibiotics.ObjectivesTo study whether quantitative BALF bacterial culture results were predictive of antibiotic requirement in dogs with LRT signs.AnimalsThirty‐three client‐owned dogs.MethodsCross‐sectional study. Dogs with positive quantitative bacterial culture of BALF were included. Dogs were divided into 2 groups, depending on whether they had a LRT infection requiring antibiotics (LRTI‐RA) or LRT disease not requiring antibiotics (LRTD‐NRA), based on thoracic imaging features, presence of intracellular bacteria on BALF cytology, and response to treatment. Predictive effect of cfu/mL and BALF total nucleated cell count (TNCC) on antibiotic requirement, adjusting for ongoing or prior antibiotic therapy and age, were studied using logistic regression.ResultsTwenty‐two and 11 dogs were included in the LRTI‐RA and LRTD‐NRA groups, respectively. The cfu/mL was not significantly predictive of antibiotic requirement, independent of ongoing or prior antibiotic treatment and age (LRTI‐RA: median, 10 000 cfu/mL; range, 10‐3 × 10⁸; LRTD‐NRA: median, 10  000 cfu/mL; range, 250‐1.3 × 10⁹; P = .27). The TNCC was not significantly predictive of antibiotic requirement when only dogs with bronchial disease were considered (LRTI‐RA: median, 470 cells/μL; range, 240‐2260; LRTD‐NRA: median, 455 cells/μL; range, 80‐4990; P = .57).Conclusion and Clinical ImportanceThe cfu/mL is an inappropriate measure for determining whether antibiotics are of benefit in dogs with LRT signs.     

Differential expression of non-cytoplasmic Actinobacillus pleuropneumoniae proteins induced by addition of bronchoalveolar lavage fluid

Actinobacillus (A.) pleuropneumoniae is the causative agent of a porcine pleuropneumonia occurring worldwide. In order to identify novel non-cytoplasmic putative virulence-associated proteins, we prepared fractions enriched in surface-associated proteins for differential proteome analysis by two-dimensional (2D) gel electrophoresis and quadrupole time-of-flight mass spectrometry (Q-Tof MS). Bacteria grown under standard culture conditions were compared to an ex vivo model based on the addition of bronchoalveolar lavage fluid (BALF) to the culture media. Twelve proteins were found to be upregulated upon induction with BALF, among them a superoxide dismutase, a parvulin-like peptidy-prolyl isomerase, a polynucleotide phosphorylase and the highly immunogenic lipoprotein OmlA. Four of the proteins upregulated by BALF were additionally constitutively expressed by an isogenic A. pleuropneumoniae fur deletion mutant and could be identified by Q-Tof MS as the heat shock protein GroES, a putative dipeptide transporter, a putative metal ion transporter and a conserved protein of unknown function. In silico analysis of the putative promoter regions of the encoding genes revealed putative Fur boxes upstream of two genes, one of which encodes part of a putative metal ion transporter. An isogenic mutant with a deletion in this protein was constructed and designated as A. pleuropneumoniae Deltafui. Analysis of the mutant in an aerosol infection model revealed symptoms indistinguishable from those seen upon infection with wild type A. pleuropneumoniae. This result implies that not all proteins upregulated by BALF are directly involved in A. pleuropneumoniae virulence.     

Collection of bronchoalveolar lavage fluid in cats, using an endotracheal tube

Bronchoalveolar lavage fluid was collected from 12 anesthetized cats by use of an endotracheal tube and syringe adapter. The safety of the technique was evaluated by monitoring mucous membrane color, capillary refill time, pulse rate, respiratory rate, ECG, and arterial blood gas tensions and by necropsy findings. Group A consisted of 3 cats that were administered (by lavage) 4 aliquots of 20 ml of saline solution during anesthesia for placement of femoral artery catheters. Group B consisted of 4 cats that were administered a smaller total volume of saline solution (3 aliquots of 5 ml/kg of body weight) during a separate anesthetic period, other than the one for placement of catheters. Group C consisted of 5 cats administered 3 aliquots (5 ml/kg) of saline solution during a separate anesthetic period and administered supplemental oxygen for 5 to 10 minutes before and for 20 minutes after the lavage procedure. Group-A cats had a prolonged recovery period that was attributed to the lengthy anesthetic period required for placement of femoral catheters. The effect was eliminated in the cats of the other groups in which the lavage procedure itself accounted for only 5 to 10 minutes of anesthetic time. Evaluation of mucous membrane color, capillary refill time, ECG, pulse, and respiratory rate revealed no persistent abnormalities. Transient increase in pulse and respiratory rate was seen in some cats. Blood gas analysis revealed noticeable decrease in arterial oxygen pressures (Pao2) after the lavage procedure. In group-C cats, oxygen supplementation allowed the maintenance of normal or above normal Pao2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular variables in bronchoalveolar lavage fluids (BALF) in selected healthy pigs

Reference values are essential for the interpretation of cytological findings in bronchoalveolar-specimens from healthy and diseased pigs, and also for studies on local immunity of the porcine lung. Values were calculated from 164 piglets out of six closed herds based on cellular bronchoalveolar lavage (bal) data. The piglets were preselected by criteria of pulmonary health and total as well as differential cell count were performed. The values established were: total cell count 1–4.5 Giga litre⁻¹, alveolar macrophages 90–99 per cent, lymphocytes 0–9 per cent, polymorphonuclear neutrophils 0–8 per cent, blastocytes 0–2 per cent, polymorphonuclear eosinophils and basophils 0–1 per cent.     

Effect of transportation stress on bronchoalveolar lavage fluid analysis in female horses

Four bronchoalveolar lavages were performed sequentially on 9 control and 8 transport-stressed female horses. Alterations in results of fluid cytologic analyses, microbial content, and phagocyte function of recovered pulmonary macrophages in all horses were determined. Seemingly, absolute and relative increase in the number of inflammatory cells detected in the second bronchoalveolar lavage fluid of control horses was the result of irritation of the first lavage. This increased response was not observed in transport-stressed horses until 5 days after transport (third lavage; 10 days after initial lavage). Seemingly, delayed inflammatory response was the result of the transport stress. Microbial content and macrophage function were not significantly different between the 2 groups (P greater than 0.05).     

Clinical signs, evaluation of bronchoalveolar lavage fluid, and assessment of pulmonary function in horses with inflammatory respiratory disease

OBJECTIVE: To evaluate the association among clinical signs, results of cytologic evaluation of bronchoalveolar lavage (BAL) fluid, and measures of pulmonary function in horses with inflammatory respiratory disease. ANIMALS: 9 healthy horses, 5 horses with inflammatory airway disease (IAD), and 9 horses with chronic obstructive pulmonary disease (COPD). PROCEDURES: Clinical examination, lung function tests, and BAL were performed on each horse. RESULTS: Standard lung mechanics of horses with exacerbated COPD differed significantly from those of healthy horses; however, there were few differences among horses with IAD, horses with COPD during remission, and healthy horses. Most variables for forced expiration (FE) in horses with COPD or IAD differed significantly from those for healthy horses. Results of clinical examination had low to moderate sensitivity and predictive values for a diagnosis of COPD (range, 67 to 80%). Results of FE tests had high sensitivity, specificity, and predictive values for a diagnosis of COPD (79 to 100%), and results of standard lung mechanics tests had low sensitivity and predictive values (22 to 69%). Percentage of neutrophils in BAL fluid was highly sensitive (100%) but moderately specific (64%) for a diagnosis of COPD. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical examination is moderately accurate for establishing a diagnosis of COPD. Forced expiration tests can specifically detect early signs of airway obstruction in horses with COPD and IAD that may otherwise be inapparent. Cytologic evaluation of BAL fluid allows early detection of inflammatory respiratory disease, but it is not specific for COPD.     

Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction.

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis-cell migration chamber. The number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAO-affected horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P = 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAOaffected horses.     

Influence of truck-transportation on the function of bronchoalveolar lavage fluid cells in cattle

The study sought to evaluate whether truck-transportation had an impact on the respiratory immune system of cattle. Six castrated 6-10-month-old Holstein calves were shipped approximately 100 km by road for 4 h. Plasma and bronchoalveolar lavage (BAL) fluid samples, collected immediately before transportation, at 4 h (soon after transportation), and on days 3 and 7 after transportation, were examined. A marked elevation of plasma cortisol concentration was observed at 4 h, but this level was unchanged in controls. The chemiluminescence (CL) response of phagocytes in BAL fluid cells, composed mainly of alveolar macrophage, decreased significantly after transportation (P<0.05). Transportation increased the CD3+ T cell population significantly (P<0.05), and a significant increase (P<0.05) in the ratio of CD4+ to CD8+ cells in BAL fluid was evident. We conclude that short-term road transportation alters pulmonary cells and their function, which may engender bovine respiratory disorders.     

Cytological analysis of bronchoalveolar lavage fluid acquired by bronchoscopy in healthy ferrets: A pilot study

The objective of this study was to investigate the normal cytological evaluation of bronchoalveolar lavage (BAL) fluid in healthy adult ferrets (N = 12). These ferrets underwent bronchoscopy and BAL using sterile saline [1.5 mL/kg body weight (BW)]. Percentage of fluid recovered, total leukocyte count, differential leukocyte count, and cell count of the epithelial lining fluid (ELF) were determined. The mean percentage of lavage volume recovered from the right lung and left lung were 67.8 ± 14.9% and 69.7 ± 20.0%, respectively. Gender (P = 0.12) and weight (P = 0.17) did not significantly affect the mean percentage of recovered volume. The mean percentage of recovered volume (P = 0.47) and the mean leukocyte count (P = 0.17) from the right and left lung were not significantly different. Macrophages were the main leukocyte component of the lavages, followed by neutrophils, lymphocytes, and eosinophils. The mean proportion of ELF in BAL fluid was 9.3 ± 3.7% v/v. Bronchoscopy is clinically useful for collecting good quality BAL samples for cytological analysis in ferrets. The leucocyte differential was established, which may help veterinarians to make better clinical decisions when treating respiratory disease. Further studies are required with a larger group in order to establish the healthy reference intervals for BAL values in ferrets.     

New transtracheal bronchoalveolar lavage technique for the diagnosis of respiratory disease in sheep

A new transtracheal bronchoalveolar lavage technique for the diagnosis of respiratory disease in sheep under field conditions was tested in 76 sheep. The sheep were divided into three groups, normal sheep, sheep with clinical signs of respiratory disease and housed sheep, on the basis of their respiratory disease history and husbandry conditions. The detection of Mannheimia haemolytica and Mycoplasma ovipneumoniae or parainfluenza virus type 3 and bovine respiratory syncytial virus antigen in the lavage samples was closely correlated with clinical disease. The sheep with clinical respiratory disease had a higher mean percentage of neutrophils in the lavage fluid than the sheep in the other two groups.     

Increased adenosine concentration in bronchoalveolar lavage fluid of horses with lower airway inflammation

Several reports have suggested a role for adenosine in the pathogenesis of chronic airway conditions and this has led to new therapeutic strategies to limit airway inflammation. In this study, detectable levels of adenosine in bronchoalveolar lavage (BAL) samples from 11 horses with non-infectious lower-airway inflammation and 14 healthy controls are reported, with significantly higher values in horses with airway inflammation. Although these increased levels did not correlate with changes in neutrophil percentage in BAL, a positive association between adenosine levels and signs of lower airway inflammation (clinical score) was observed. These novel findings support the hypothesis that adenosine may contribute to bronchoconstriction and also act as a pro-inflammatory mediator in the bronchoalveolar milieu of horses with airway inflammation. Further investigation of this axis could lead to new approaches for the treatment of highly prevalent lower airway inflammatory conditions in the horse.