Tracheal Lavage Research Articles

Indigenous cost-effective method for tracheal lavage in canines

Indigenous cost-effective method for tracheal lavage in canines

A RARE CAUSE OF COMMUNITY-ACQUIRED BACTERIAL PNEUMONIA IN A PATIENT WITH POORLY CONTROLLED DIABETES

A RARE CAUSE OF COMMUNITY-ACQUIRED BACTERIAL PNEUMONIA IN A PATIENT WITH POORLY CONTROLLED DIABETES

Comparison of mucosal immune responses to African swine fever virus antigens intranasally delivered with two different viral vectors

Transmission of African swine fever virus (ASFV) in domestic swine occurs mainly via contact with mucosal surfaces. In this study, we constructed a pseudotyped surface-displaying BacMam-F1 vector expressing ASFV CD2v-p30-p54 fusion antigen, and compared its mucosal responses in pigs with that of rAd-F1 vector expressing the same antigen. From day 21 after intranasal immunization, the antigen-specific IgG and intranasal secretory IgA (S-IgA) antibody responses induced by BacMam-F1 were significantly stronger than that by rAd-F1. The significantly different S-IgA antibody responses were also detected in their tracheal washes and lung lavages. After stimulation with ASFV antigens, 4/6 S-IgA-promoting cytokine responses in porcine alveolar macrophages (PAMs) from BacMam-F1-immunized pigs were significantly stronger than that from rAd-F1-immunized pigs. The similar expression patterns of S-IgA-promoting cytokines were also detected in their lung lavages. After pretreating ASFV with different samples from immunized pigs, significant inhibitory effects were detected in tracheal washes, lung lavages and PAM cultures, but not serum samples with slight inter-group difference. These data suggest that the pseudotyped surface-displaying BacMam vector is more suitable for swine mucosal immunization.

Equine asthma in Brazil – a scoping review

Asthma is a chronic respiratory disease that can occur in horses of different breeds and ages. While its occurrence has been reported in many countries, its occurrence across Brazil is unclear. This review aimed to gather information about equine asthma from studies performed in Brazil by different groups and investigate a variety of horse populations. Articles published in peer-reviewed scientific journals, as well as information contained in dissertations and theses, were searched on PubMed, SciELO, Google Scholar, and the Brazilian government catalog of dissertations and theses. Fifteen studies were included in this review, and according to the published information, the authors identified equine asthma using clinical signs, endoscopic findings, and/or cytological examination of tracheal wash and/or bronchoalveolar lavage fluid. Studies have investigated horses of different breeds and activities (racehorses, police horses, riding horses, Crioulos horses, and others) and were performed in different regions with diverse climatic conditions. On the previous observed studies, the occurrence varied from 14.0% to 60.0%, with a mean occurrence of 24% for severe asthma. Other studies must be performed to further understand the occurrence of equine asthma in Brazil, according to different climatic regions.

Effects of Bedding Material on Equine Lower Airway Inflammation: A Comparison of Two Peat Beddings, Wood Pellet, and Straw Pellet.

The effects of bedding material on air quality are important amongst horses worldwide. Respiratory diseases, especially equine asthma, are highly prevalent with air hygiene playing a major role on the pathophysiology of these diseases. The objective of our study was to investigate the effects of four bedding materials on the respiratory signs, tracheal mucus score, and tracheal wash (TW) and bronchoalveolar lavage fluid (BALF) cytology in healthy adult horses. The study design was a prospective controlled cross-over study, and the subjects were healthy adult riding school horses (n = 32) from a single stable. Wood pellet, straw pellet, and loosely stored peat (Peat 3) were compared to peat packed in plastic-covered bales (Peat 2). Lower airway endoscopy and sampling (TW and BALF) for cytological examination were performed after each 35-day bedding period. The tracheal mucus scores (P = 0.014) and respiratory rate (P = 0.026) were higher during the straw pellet period compared to the Peat 2 period. The respiratory rate was lower during the wood pellet period compared to the Peat 2 period (P = 0.004). The TW neutrophil percentage during the straw pellet period was higher compared to the Peat 2 period (P = 0.0003). The BALF neutrophil percentage was higher during the straw pellet period (P = 0.005) and during the Peat 3 period compared to the Peat 2 period (P = 0.04). We conclude that baled peat (Peat 2) caused lower neutrophil percentages in the airway samples compared to straw pellet and loosely stored peat (Peat 3). No difference was observed between Peat 2 and wood pellet. The information gained from this study may assist veterinarians and horse owners in selecting appropriate bedding materials, especially for horses with equine asthma.

Rhodococcus in foal, from diagnosis to treatment: Case report

The present work reports a case of a foal affected by Rhodococcus equi, an optional intracellular gram-positive pleomorphic cocobacillus, which, when eliminated with the feces of infected animals, in the soil becomes a potential pathogen, capable of causing mainly piogranulomatous bronchopneumonia. The animal, a 23-day-old Brazilian equestrian foal, was sent to the veterinary hospital with hyperthermia, increased heart and respiratory rates, crackle in tracheal auscultation, loss of appetite and prostration. After hematological, ultrasound, radiographic examinations, and collection of tracheal lavages for culture and PCR, the definitive diagnosis of rhodococcosis was obtained, which is a bacterial disease that mainly affects foals, has a worldwide distribution and has a great economic impact on equine culture. Based on the results, treatment with azithromycin, rifampin, flunixin meglumine, sucralfate, ranitidine hydrochloride and amoxicillin with clavulanate was instituted, associated with nebulization with acetylcysteine diluted to 0.9% sodium hydrochloride solution. After a few weeks, there was no evolution in the results of the ultrasound and radiographic exams, however the hematological exams were within the reference levels, there was a significant clinical improvement, and the animal was discharged. It is noteworthy that early diagnosis was essential for the effectiveness of treatment and remission of clinical signs. Keywords: rhodococcosis; foals; rhodococcus equi.

Evaluation of the Respiratory Microbiome and the Use of Tracheal Lavage as a Diagnostic Tool in Kemp's Ridley Sea Turtles (Lepidochelys kempii).

Simple SummaryA tracheal lavage is commonly used to characterize the microbes that may be causing pneumonia in sea turtles, typically by culture-dependent methods. In this study, we characterized the tracheal lavage microbiome through culture-independent methods and compared the resulting sequence data to conventional cultures, the degree of radiographic lung abnormalities, and pathogens of sea turtles as previously reported in the literature. This study also evaluates the microbial communities at different sections of the respiratory tract from deceased sea turtles. We found that radiographic lung abnormalities do not correlate with the tracheal lavage microbiome, tracheal lavage cultures under-represent the microbial community as determined by culture-independent methods, many previously reported sea turtle pathogens are present in low abundance of the tracheal lavage microbiome, and tracheal lavages are not representative of other sections of the respiratory tract.Respiratory disease is a common cause of morbidity and mortality in sea turtles, including the Kemp’s ridley sea turtle (Lepidochelys kempii). Although culture-dependent methods are typically used to characterize microbes associated with pneumonia and to determine treatment, culture-independent methods can provide a deeper understanding of the respiratory microbial communities and lead to a more accurate diagnosis. In this study, we characterized the tracheal lavage microbiome from cold-stunned Kemp’s ridley sea turtles at three time points during rehabilitation (intake, rehabilitation, and convalescence) by analyzing the 16S rRNA gene collected from tracheal lavage samples. We retrospectively developed a radiographic scoring system to grade the severity of lung abnormalities in these turtles and found no differences in diversity or composition of microbial communities based on radiographic score. We also found that the culture isolates from tracheal lavage samples, as well as other previously reported sea turtle pathogens, were present in variable abundance across sequenced samples. In addition to the tracheal microbial community of live turtles, we characterized microbial communities from other segments of the respiratory tract (glottis, trachea, anterior lung, posterior lung) from deceased turtles. We found a high degree of variability within turtles and a high degree of dissimilarity between different segments of the respiratory tract and the tracheal lavage collected from the same turtle. In summary, we found that the pulmonary microbial community associated with pneumonia in sea turtles is complex and does not correlate well with the microbial community as identified by tracheal lavage. These results underscore the limitations of using tracheal lavage for identification of the causative agents of pneumonia in sea turtles.

Adjuvant effects of bacterium-like particles in the intranasal vaccination of chickens against Newcastle disease

Given that the respiratory mucosa is an important site for the initial replication of Newcastle disease virus (NDV), developing intranasal vaccines for chickens is an effective strategy to protect against this disease. The low immunogenicity of inactivated NDV administered by the mucosal route motivated us to identify a safe and potent adjuvant. Previous studies have shown that bacterium-like particles (BLPs), which serve as mucosal adjuvants, induce effective local and systemic immune responses through TLR2 signaling in both mammals and humans. Here, we report that BLPs could activate the innate immune system of chickens in a manner that was dependent on the combination of chicken TLR2 type 1 (chTLR2t1) and chicken TLR1 type 1 (chTLR1t1). The chicken macrophage-like HD11 cell line was stimulated with BLPs, resulting in the production of nitric oxide and the expression of the proinflammatory cytokines IFN-γ, IL-1β and IL-6. Chickens intranasally immunized with inactivated NDV vaccines mixed with BLP adjuvants exhibited significantly increased levels of local SIgA in their tracheal lavage fluid and as well as hemagglutination-inhibiting antibodies in serum samples. The strong systemic and local immune responses induced by BLP-adjuvanted vaccines provided 100 % protection against intranasal challenge with a lethal dose of virulent NDV without showing any signs of disease. These results indicate that BLPs should be considered for use as a potential mucosal adjuvant for inactivated NDV vaccines and other vaccines for poultry.

Asthmatic Disease as an Underlying Cause of Dorsal Displacement of the Soft Palate in Horses

It is important to treat the upper and lower respiratory tracts as a single unit, as lower respiratory tract diseases can often cause upper respiratory functional disorders, whereas upper respiratory obstructions could be a factor in lower respiratory problems. The present study aimed to investigate the hypothesis that asthmatic diseases may be an underlying cause of dorsal displacement of the soft palate in horses. Pleasure or sport horses (n = 57) with a history of asthmatic disease were incorporated in the study. All horses were examined in the exacerbation phase of the asthmatic disease. Bronchoalveolar cytology and tracheal lavage bacteriology were performed in all cases. The upper respiratory tract was evaluated at rest in all horses and during exercising endoscopy in 11/57 with severe equine asthma. Binomial tests with P ≤ .05 significance were used to establish estimated intervals of the measured frequencies of dorsal displacement of the soft palate (DDSP) occurring in the studied groups. It was observed that more than 60% of horses with mild or moderate equine asthma and more than 79% of horses with severe equine asthma are presented with DDSP during resting endoscopy examination. During the exercising endoscopy, DDSP was detected in all cases of severe equine asthma. These findings support the proposed hypothesis that DDSP was common in horses with equine asthma. Both increasing negative pressure in the airways due to bronchoconstriction and inflammatory processes could be factors in the development of DDSP. The consequent step would be to investigate the same population of horses in the remission phase of the equine asthma.

Detection of bovine herpes virus (BOHV-1) infection in respiratory tract of bovines

Infectious bovine rhinotracheitis (IBR) is considered commonest form of BoHV-1 respiratory tract infection. The current work was designed to study the incidence of IBR using molecular technique and antigen detection sandwich ELISA. In the present study, nasal swab samples (100) and tracheal lavages (6) were collected from bovines, which were showing respiratory symptoms from clinics of College of Veterinary Science, GADVASU, Ludhiana. Four lung tissues were also collected from dead animals. All nasal samples were tested for IBR virus antigen by using commercial sandwich ELISA kit. The antigenic incidence of BoHV-1 was reported to be 1% in tested animals. For molecular detection of BoHV-1 in respiratory tract, the DNA was extracted from all samples (nasal swabs, tracheal lavages and lung tissues) and subjected to gI gene specific PCR by using published primers. The overall incidence of BoHV-1 in nasal swabs of animals with the history of respiratory symptoms was found to be 3% and rest of the samples were found negative for IBR virus in PCR. Species wise evaluation of BoHV-1 infection revealed that out of 75 cattle, 3 (4.0%) cattle were found positive in PCR where as none of the buffaloes were found positive in present study. The animals older than 3 year of age showed the higher incidence (4.2%) than animal up to 3 years of age (2.8%). In this study, all positive cattle were crossbred females. According to sex wise status, it was observed that females are more prone to infection, which may be because of production stress in female animals.

Secretory IgA in Mucosa of Pharynx and Larynx Plays an Important Role against Influenza A Virus Infection in Kidney Yang Deficiency Syndrome Model.

Objective Influenza virus poses a major threat to human health and has serious morbidity and mortality which commonly occurs in high-risk populations. Pharynx and larynx of the upper respiratory tract mucosa is the first defense line against influenza virus infection. However, the ability of the pharynx and larynx organ to eliminate the influenza pathogen is still not clear under different host conditions. Methods In this study, a mouse model of kidney yang deficiency syndrome (KYDS) was used to mimic high-risk peoples. Two different methods of influenza A (H1N1) virus infection by nasal dropping or tracheal intubation were applied to these mice, which were divided into four groups: normal intubation (NI) group, normal nasal dropping (ND) group, model intubation (MI) group, and model nasal dropping (MD) group. The normal control (NC) group was used as a negative control. Body weight, rectal temperature, and survival rate were observed every day. Histopathologic changes, visceral index, gene expressions of H1N1, cytokine expressions, secretory IgA (SIgA) antibodies of tracheal lavage fluids in the upper respiratory tract, and bronchoalveolar lavage fluids were analyzed by ELISA. Results The MD group had an earlier serious morbidity and mortality than the others. MI and NI groups became severe only in the 6th to 7th day after infection. The index of the lung increased significantly in NI, MI, and MD groups. Conversely, indices of the thymus and spleen increased significantly in NC and ND groups. H&E staining showed severe tissue lesions in MD, MI, and NI groups. H1N1 gene expressions were higher in the MD group compared with the MI group on the 3rd day; however, the MD group decreased significantly on the 7th day. IL-6 levels increased remarkably, and SIgA expressions decreased significantly in the MD group compared with the NC group. Conclusions SIgA secretions are influenced directly by different conditions of the host in the pharynx and larynx in the upper respiratory tract mucosa. In the KYDS virus disease mode, SIgA expressions could be inhibited severely, which leads to serious morbidity and mortality after influenza A virus infection. The SIgA expressions of the pharynx and larynx would be an important target in high-risk populations against the influenza A virus for vaccine or antiviral drugs research.

2777. Live-Attenuated Vaccine Against RSV Generates Robust Cellular and Humoral Immune Responses

BackgroundIn people over 65, there are on average 177,000 hospitalizations and 14,000 deaths because of respiratory syncytial virus (RSV) each year. Elderly patients infected with RSV can suffer serious infections leading to pneumonia and congestive heart failure. RSV vaccines have failed in the elderly in part because they have been unable to mount a robust cellular immune response.MethodsRSV-MinL4.0 is a live-attenuated intranasal vaccine candidate that was generated by codon pair deoptimization of the L gene followed by the addition of four stabilizing mutations found via stress passaging. Four African Green Monkeys (AGMs) per group were vaccinated with RSV-MinL4.0 or wild-type (WT) RSV at 2 × 106 PFU, boosted on day 28 and challenged with wild-type (WT) RSV on day 104. Oropharyngeal swabs and tracheal lavage were collected daily and every other day, respectively, to evaluate virus shedding (qPCR) and blood was drawn on days 1, 14, 21, 28, and 49 for antibody titers (PRNT50), and PBMC activation (IFNγ ELISPOT with whole inactivated virus).ResultsMinL4.0 was 2 to 3 log10 attenuated when compared with WT RSV in AGMs. Despite the presence of antibodies on day 28, there was a “take” of the boost indicating the potential for this vaccine to be immunogenic in the elderly with pre-existing circulating antibodies (Figure 1A). MinL4.0 led to robust activation of PBMCs comparable to WT RSV (> 2,000 spots per 106 total cells, Figure 1B). Shedding of the vaccine and challenge viruses was minimal (data not shown).ConclusionMinL4.0 led to robust activation of cellular and humoral immune responses, which are critical for induction of protective immunity in the elderly. Animals were protected from WT challenge. Preliminary data in AGMs with pre-existing antibodies to RSV indicate that circulating antibodies do not prevent vaccine “take,” critical for a vaccine targeting sero-positive elderly individuals. Disclosures All authors: No reported disclosures.

Bacillus amyloliquefaciens spray improves the growth performance, immune status, and respiratory mucosal barrier in broiler chickens

Studies have shown that supplementing probiotics in a nasal spray can provide prophylaxis against upper respiratory tract infections. Previously, we found that spraying with Bacillus reduced the concentration of atmospheric ammonia and airborne microbiological aerosols in broiler houses. In the present study, we evaluated the effects of spraying with Bacillus amyloliquefaciens on the respiratory mucosal barrier in broilers. Two trails were conducted simultaneously in four environmentally controlled chambers. In each trail, 320 1-d-old male broilers were randomly assigned to control (CON) and B. amyloliquefaciens (BAQ) group. Eight cages containing 20 birds per cage were placed in two chambers. The BAQ group chambers were sprayed with B. amyloliquefaciens at 4 × 109 CFU/m2 each day for 42 d. Spraying with B. amyloliquefaciens increased average daily gain and average daily feed intake in broilers (P < 0.01), but had no effects on FCR (P > 0.05). The relative weights of the bursa (21 d) and spleen (42 d) were higher in the BAQ group (P < 0.05). The serum IgG, IgM (21 d), and IgA, Ig M (42 d) levels were improved in BAQ group (P < 0.05). The sIgA levels in tracheal lavage fluid were significantly higher in the BAQ group than the CON group (P < 0.05). Furthermore, treatment with BAQ downregulated IL-6 and IFN-γ (P < 0.05), but upregulated IL-2, occludin, and mucin-2 (P < 0.05). In addition, the number of acidic goblet cells was significantly higher in the tracheas of treated broilers than untreated broilers (P < 0.05). In conclusion, spraying with B. amyloliquefaciens may help to enhance the growth performance and improve the respiratory mucosal barrier in broiler chickens.

Laboratory findings of tracheal wash and bronchoalveolar lavage in normal adult dairy cattle

ABSTRACT Tracheal Wash (TW) and bronchoalveolar lavage (BAL) are not well documented in adult cattle and published studies were undertaken on diseased animals aiming at isolating the causative agent of respiratory diseases. This study was undertaken to establish the normal cytological and microbiological findings of TW and BAL in normal adult cattle and compare between the laboratory analysis findings of both techniques. Eighteen healthy adult cattle were divided into two groups. BAL was completed the first group (n = 10) and TW was completed the second group (n = 8). Normal cytological findings of TW in healthy adult cattle were found to be primarily neutrophils (42.7 ± 35.7)% intermixed with lower numbers of columnar epithelial cells (26.2 ± 34.8)%, alveolar macrophages (14.7 ± 15.4)%, squamous epithelial cells (10 ± 28.6)%, lymphocytes (6.8 ± 16.1)% and rarely seen eosinophils (0.1 ± 0.0)% with a total cell count of 457 ± 310.1 cells/µl. Cytological findings of BAL were found to be mainly alveolar macrophages (94.3 ± 3.9)%, intermixed with lower number of neutrophils (4.3 ± 3.9)% and lymphocytes (1.3 ± 1.2)% and rarely seen eosinophils (0 ± 0.1)% with a total cell count of 238 ± 169 cells/µl. There are remarkable differences in TW and BAL cytological findings and the degree of contamination was higher in TW samples than that seen in BAL lavage.

Inflammatory lung injury in rabbits: effects of high-frequency oscillatory ventilation in the prone position.

ABSTRACTObjective:To compare the effects that prone and supine positioning during high-frequency oscillatory ventilation (HFOV) have on oxygenation and lung inflammation, histological injury, and oxidative stress in a rabbit model of acute lung injury (ALI). Methods:Thirty male Norfolk white rabbits were induced to ALI by tracheal saline lavage (30 mL/kg, 38°C). The injury was induced during conventional mechanical ventilation, and ALI was considered confirmed when a PaO2/FiO2 ratio < 100 mmHg was reached. Rabbits were randomly divided into two groups: HFOV in the supine position (SP group, n = 15); and HFOV with prone positioning (PP group, n = 15). For HFOV, the mean airway pressure was initially set at 16 cmH2O. At 30, 60, and 90 min after the start of the HFOV protocol, the mean airway pressure was reduced to 14, 12, and 10 cmH2O, respectively. At 120 min, the animals were returned to or remained in the supine position for an extra 30 min. We evaluated oxygenation indices and histological lung injury scores, as well as TNF-α levels in BAL fluid and lung tissue. Results:After ALI induction, all of the animals showed significant hypoxemia, decreased respiratory system compliance, decreased oxygenation, and increased mean airway pressure in comparison with the baseline values. There were no statistically significant differences between the two groups, at any of the time points evaluated, in terms of the PaO2 or oxygenation index. However, TNF-α levels in BAL fluid were significantly lower in the PP group than in the SP group, as were histological lung injury scores. Conclusions:Prone positioning appears to attenuate inflammatory and histological lung injury during HFOV in rabbits with ALI.

Endotracheal intubation results in acute tracheal damage induced by mtDNA/TLR9/NF-κB activity.

Tracheitis secondary to placement of an endotracheal tube (ETT) is characterized by neutrophil accumulation in the tracheal lumen, which is generally associated with epithelial damage. Mitochondrial DNA (mtDNA), has been implicated in systemic inflammation and organ dysfunction following trauma; however, less is known about the effects of a foreign body on local trauma and tissue damage. We hypothesized that tracheal damage secondary to the ETT will result in local release of mtDNA at sufficient levels to induce TLR9 and NF‐κB activation. In a swine model we compared the differences between uncoated, and chloroquine (CQ) and N‐acetylcysteine (NAC) coated ETTs as measured by tracheal lavage fluids (TLF) over a period of 6 h. The swine model allowed us to recreate human conditions. ETT presence was characterized by neutrophil activation, necrosis, and release of proinflammatory cytokines mediated by TLR9/NF‐κB induction. Amelioration of the tracheal damage was observed in the CQ and NAC coated ETT group as shown in tracheal tissue specimens and TLF. The role of TLR9/NF‐κB dependent activity was confirmed by HEK‐Blue hTLR9 reporter cell line analysis after coincubation with TLF specimens with predetermined concentrations of NAC or CQ alone or TLR9 inhibitory oligodeoxynucleotide (iODN). These findings indicate that therapeutic interventions aimed at preventing mtDNA/TLR9/NF‐κB activity may have benefits in prevention of acute tracheal damage.

Tuberculous pneumonia-induced severe ARDS complicated with DIC in a female child: a case of successful treatment

BackgroundTuberculous (TB) pneumonia can induce acute respiratory distress syndrome (ARDS). Although TB pneumonia is one of the causes of disease and death among children worldwide, the literature on TB pneumonia-induced ARDS is limited. We report herein on the successful treatment of a two-year-old female child with TB pneumonia-induced severe ARDS complicated with disseminated intravascular coagulation (DIC).Case presentationA two-year-old Vietnamese female child with sustained fever and cough for 20 days was transferred to our hospital. She had severe dyspnea and a chest X-ray showed bilateral infiltration without findings of heart failure. After tracheal intubation, her oxygenation index (OI) and PaO2/FiO2 (PF) ratio were 29 and 60 mmHg, respectively. Mycobacterium tuberculosis was detected by real-time polymerase chain reaction (rPCR) assay of tracheal lavage fluid. She was diagnosed as having severe ARDS that developed from TB pneumonia. Anti-tuberculous therapy and cardiopulmonary support were started. However, her respiratory condition deteriorated despite treatment with high-frequency oscillating ventilation (HFO), vasopressor support, and 1 g/kg of immunoglobulin. On the third day after admission, her International Society on Thrombosis and Hemostasis DIC score had increased to 5. Recombinant human soluble thrombomodulin (rTM) was administered to treat the DIC. After the administration of rTM was completed, OI gradually decreased, after which the mechanical ventilation mode was changed from HFO to synchronized intermittent mandatory ventilation. The DIC score also gradually decreased. Plasma levels of soluble receptor for advanced glycan end products (sRAGE) and high mobility group box 1 (HMGB-1), which are reported to be associated with ARDS severity, also decreased. In addition, inflammatory biomarkers, including interferon-gamma (IFN-γ) and interleukin-6 (IL-6), decreased after the administration of rTM. Although severe ARDS (P/F ratio ≦ 100 mmHg) continued for 19 days, the patient’s OI and P/F ratio improved gradually, and she was extubated on the 27th day after admission. The severe ARDS with DIC was successfully treated, and she was discharged from hospital on day 33 post-admission.ConclusionsWe successfully treated a female child suffering from TB pneumonia-induced severe ARDS complicated with DIC using multimodal interventions. (338/350).

A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy

BackgroundCanine neutrophils release neutrophil extracellular traps (NETs) in response to lipopolysaccharide but NETs from clinical septic dogs had not been identified. The primary aim is to describe the methodology of identifying and quantifying neutrophil extracellular traps (NETs) in cytology samples of septic foci in dogs with sepsis using immunofluorescence microscopy. Cytology samples including endotracheal tracheal wash (ETW), bronchoalveolar lavage (BAL), abdominal and pleural effusion collected from 5 dogs (3 septic, 2 non-septic) were fixed, permeabilized and stained for myeloperoxidase (MPO), citrullinated histone H3 (citH3) and cell-free DNA (cfDNA). Fluorescence microscopy was used to identify and quantify NETs in 10 random views at 40× magnification. NETs were identified based on co-localization of MPO, citH3 and cfDNA. NETs were quantified as a ratio (number of NETs: number of neutrophils). Neutrophils were identified based on cytoplasmic MPO, cellular diameter and nuclear morphology.ResultsNETs were identified and quantified in all cytology samples collected from septic dogs. A small number of NETs was documented in one dog with sterile chronic bronchitis. No NETs were found in sterile abdominal effusion collected from one dog with congestive heart failure.ConclusionsImmunofluorescence microscopy could be a useful tool for the study of NETs in dogs with clinical sepsis.

Tracheal acid or surfactant instillation raises alveolar surface tension.

Whether alveolar liquid surface tension, T, is elevated in the acute respiratory distress syndrome (ARDS) has not been demonstrated in situ in the lungs. Neither is it known how exogenous surfactant, which has failed to treat ARDS, affects in situ T. We aim to determine T in an acid-aspiration ARDS model before and after exogenous surfactant administration. In isolated rat lungs, we combine servo-nulling pressure measurement and confocal microscopy to determine alveolar liquid T according to the Laplace relation. Administering 0.01 N (pH 1.9) HCl solution by alveolar injection or tracheal instillation, to model gastric liquid aspiration, raises T. Subsequent surfactant administration fails to normalize T. Furthermore, in normal lungs, tracheal instillation of control saline or exogenous surfactant raises T. Lavaging the trachea with saline and injecting the lavage solution into the alveolus raises T, suggesting that tracheal instillation may wash T-raising airway contents to the alveolus. Adding 0.01 N HCl or 5 mM CaCl2-either of which aggregates mucins-to tracheal lavage solution reduces or eliminates the effect of lavage solution on alveolar T. Following tracheal saline instillation, liquid suctioned directly out of alveoli through a micropipette contains mucins. Additionally, alveolar injection of gastric mucin solution raises T. We conclude that 1) tracheal liquid instillation likely washes T-raising mucins to the alveolus and 2) even exogenous surfactant that could be delivered mucin-free to the alveolus might not normalize T in acid-aspiration ARDS. NEW & NOTEWORTHY We demonstrate in situ in isolated lungs that surface tension is elevated in an acid-aspiration acute respiratory distress syndrome (ARDS) model. Following tracheal liquid instillation, also in isolated lungs, we directly sample alveolar liquid. We find that liquid instillation into normal lungs washes mucins to the alveolus, thereby raising alveolar surface tension. Furthermore, even if exogenous surfactant could be delivered mucin-free to the alveolus, exogenous surfactant might fail to normalize alveolar surface tension in acid-aspiration ARDS.

Pathogen screening and prognostic factors in children with severe ARDS of pulmonary origin.

BackgroundAcute respiratory distress syndrome (ARDS) is one of the most lethal diseases encountered in the pediatric intensive care unit (PICU). The etiological pathogens and prognostic factors of severe ARDS of pulmonary origin in children with respiratory virus infections were prospectively investigated.MethodsEnrolled children fulfilled the following criteria: (1) PICU admission; (2) age of 1 month to 16 years; (3) diagnosis of infectious pneumonia and respiratory virus infection; and (4) development of severe ARDS within 72 h after PICU admission. Pathogens were detected in the blood and tracheal lavage fluid using molecular techniques and a conventional culture system. The serum levels of inflammatory mediators on the day of PICU admission were examined.ResultsFifty‐seven patients (32 boys; median age, 9 months) were enrolled. Multiple virus infections, co‐infection with bacteria/fungus, and bacteremia/fungemia were observed in 60%, 49%, and 32% of children, respectively. Adenovirus‐B, measles virus, and cytomegalovirus were detected predominantly in tracheal lavage fluid. There were no statistically significant differences between non‐survivors and survivors regarding the types of pathogen, incidence of multiple virus infection, gender, age, clinical features, and treatment. The serum levels of interferon (IFN)‐γ and the IFN‐γ/interleukin (IL)‐10 ratio were higher in non‐survivors.ConclusionsIFN‐γ upregulation as detected on the day of PICU admission was found to be one of the possible prognostic factors affecting a fatal outcome. These results suggest that modulation of inflammatory responses is critical for the clinical management of children with ARDS.