Tracheal Wash Research Articles

Aspiration From Dysphagia Associated With the Presence of Pantoea agglomerans in a Horse

Pantoea agglomerans is a gram-negative bacterium that usually lives in symbiosis in soil and plants. However, it may also lead to serious diseases in plants, animals and humans. We describe a case of aspiration from dysphagia associated with the isolation of P. agglomerans in a horse admitted to an Italian hospital. The patient was presented to the hospital for the onset of chronic cough after an acute episode of pulmonary hemorrhage during a race recorded two months before and was treated with dexamethasone and penicillin. The cough was accentuated during meals, leading to a suspicion of aspiration from dysphagia. A bronchoalveolar lavage was collected, and cytological examination showed the presence of vegetable fibers, rod bacteria, and a high macrophage population (89%). From the tracheal wash collected for bacteriological culture, P. agglomerans was isolated in monoculture, and the antimicrobial susceptibility test revealed that the isolate was susceptible to all tested antimicrobials except sulfonamide. The horse recovered well after 10 days of enrofloxacin treatment. The present report highlights the role of P. agglomerans as an opportunistic respiratory pathogen in horses, especially in the presence of plants or their parts.

Cytological diagnosis of exercise-induced pulmonary haemorrhage: Comparison of tracheal wash and bronchoalveolar lavage in standardbred racehorses.

Cytology of airway samples is sensitive for diagnosis of exercise-induced pulmonary haemorrhage (EIPH), but the association between tracheal wash (TW) and bronchoalveolar lavage fluid (BALF) is unknown. The objective of this study was to determine whether diagnosis of EIPH, using haemosiderophages/macrophages (H/M) ratio, differs when based on TW or BALF. A prospective cross-sectional study was conducted on 102 standardbred horses in training. TW and BALF were collected concomitantly from all horses at rest (at least 24hours after their last training or race), and their H/M ratios were calculated. Spearman's correlation, Cohen's kappa and Gwet's coefficient tests were performed to evaluate the association between TW and BALF samples. With BALF, 21 horses met the cytological inclusion criteria for an EIPH diagnosis from individual and/or pooled samples. With TW, 20 horses had occasional (H/M < 10%) haemosiderophages, and nine, one and three horses had small (10%-25%), moderate (25%-50%) and large (>50%) proportions, respectively. Poor correlations and inconsistent concordances between TW and BALF were found for H/M ratio. Limitations include the use of a single staining method and the absence of a total haemosiderin score. No association between TW and BALF was found for the cytological diagnosis of EIPH. Based on H/M ratio, BALF remains the sample type of choice for cytological diagnosis of EIPH.

Antimicrobial Prescription Behavior in Equine Asthma Cases: An International Survey

Equine asthma is considered a non-infectious respiratory disease characterized by inflammation and hypersensitivity. Given the importance of antimicrobial stewardship, an international survey was designed to investigate the use of antimicrobials in asthmatic horses and the factors influencing prescription behavior. An online survey was distributed in six languages by international mailing lists and social media from December 2020 to January 2022. Of the 434 responses recorded, 249 veterinarians working in 25 countries finished the survey. These included 79 ECEIM/ACVIM diplomats. A total of 204 respondents confirmed national regulations concerning antimicrobial use in their country. Knowledge of the asthma definitions as presented in the revised ACVIM consensus statement was greater in veterinarians treating over 95% of equine patients compared to veterinarians treating more species, based on 10 questions (answers consistent with the consensus statement in 7 (IQR 5–9) and 4 (IQR 3–6) questions, respectively, (p &lt; 0.001)). A total of 131 respondents stated to use antimicrobials (at least ‘sometimes’) in at least one of the three presented cases consistent with equine asthma. Trimethoprim-Sulfa combinations, penicillin(s), and tetracyclines were prescribed most (by 105, 53 and 38 veterinarians, respectively). Aminoglycosides, cephalosporins and fluoroquinolones were also selected (22, 15, 9 veterinarians respectively). Tetracyclines and cephalosporins are prescribed more often by veterinarians working without national regulations (p 0.019 and p 0.035, respectively). The most selected factors influencing prescription behavior were: ‘tracheal wash culture’ (62% of 131 veterinarians using antimicrobials in these cases), ‘whether other horses in the barn have similar complaints’ (60%), and ‘response to other forms of therapy’ (53%). In conclusion, insight into prescription behavior is the first step towards minimizing and optimizing antimicrobial use.

Feeding spray-dried plasma to broilers early in life improved their intestinal development, immunity and performance irrespective of mycotoxins in feed.

Fungi that produce mycotoxins can grow on certain food products, such as grains and feed, and can cause a variety of health issues if consumed by animals, including chickens. The use of spray-dried plasma (SDP) is one strategy for combating the health problems caused by mycotoxins. In the present study, Ross 308 chickens (n = 960) were divided into four treatment groups. T1 group was given a control diet (corn-soybean meal), T2 group was given a control diet +2% SDP, T3 group was given a control diet +2% SDP + mixture mycotoxins and T4 group was givena control diet + mycotoxin mixture. The presence of SDP resulted in weight gain and decreased feed efficiency, whereas mycotoxins resulted in weight loss and increased feed efficiency. SDP increased the thymus' relative weight. The presence of mycotoxins increased the heterophile/lymphocyte ratio. The presence of mycotoxins reduced the production of IL-2 and macrophage inflammatory protein-3 Alpha (MIP-3a), whereas the presence of SDP increased the production of macrophage colony-stimulating Factor (M-CSF). SDP resulted in higher IgA concentrations in the intestinal and tracheal washes than mycotoxin. Finally, adding SDP to broiler diets boosts weight gain, feed efficiency, and immune system development. Our results provide information supporting that SDP is a promising tool for improving poultry immunity and performance.

Increased freedom of head movement mitigates stress and bacterial load in the airways of horses during transport.

Protection of horse welfare during transport is crucial. The aim of this study was to determine the effect of head and neck restraint on behavior and airway bacteria. In a randomized crossover study, six healthy Thoroughbreds were transported by road for 22 h in an individual bay with tight head restraint (50 cm short-rope) or loose head restraint (95 cm long-rope). Behavioral parameters relating to head position, eating, and stress were monitored during transportation. Tracheal wash samples were obtained 6 days before and immediately after transport for bacterial culture and metagenomic analysis. Compared to before transport, bacterial load (CFU/mL) after transport was significantly increased in the short-rope group (p = 0.04), whereas no changes were observed in the long-rope group. Transport significantly reduced Simpson index at phylum, class, order, and family levels in both groups (p < 0.001) of tracheal microbiota. In both groups, this reduction was associated with increases in the dominant members of relative abundance at phylum (Firmicutes: +24% in long-rope and +14% in short-rope), class (Bacilli: +20% in long-rope and +22% in short-rope) and family (Streptococcaceae: +22% in long-rope and +23% in short-rope) levels. Licking behavior during transportation with short-rope restraint was more frequent than in horses with long-rope restraint. These results suggest loose head restraint during transportation is likely to ameliorate stress and mitigate the associated increased bacterial load in the lower airways associated with transport. Further, head position during transportation is likely a more important determinant of airway hygiene and distress than duration of travel.

Lower airway ultrasonographic, endoscopic, and cytological changes in horses undergoing inhalation anesthesia with controlled ventilation for minor elective surgeries

Effects of general anesthesia with controlled ventilation on the respiratory system have had limited evaluation in horses. A prospective observational study was performed with eleven client-owned horses undergoing elective surgery. Physical examination, auscultation with a rebreathing bag, complete blood cell count, lung ultrasound imaging, tracheal endoscopy imaging and transendoscopic tracheal wash were conducted before and 24 hours after anesthesia. Lung ultrasound imaging was also repeated just after recovery. A significant increase in blood neutrophil count between pre- and post-anesthesia (P=0.004) was observed. There was an increase in ultrasonographic score of the lungs at recovery (left P=0.007, right P=0.017). The score of the dependent lung was higher than the independent lung at recovery time (P=0.026) but no difference was observed 24 hours after anesthesia. The tracheal mucus score was higher after anesthesia (P=0.001); severe local inflammation was present in several horses at the site of endotracheal tube cuff. Neutrophil count was significantly higher on tracheal wash fluid cytology after anesthesia (P=0.016), without any significant changes on bacterial load. Increased tracheal mucus score and neutrophil count in tracheal wash samples were observed after general anesthesia in healthy horses without clinical evidence of pneumonia (fever, cough). Tracheal inflammation secondary to endotracheal intubation and cuff inflation was, therefore, suspected. Elective surgery without complications can induce inflammation of the trachea and changes in ultrasound images of the lungs in healthy horses and should be considered when evaluating respiratory system after a general anesthesia.

Epidemiology and pathogenicity of M. equirhinis in equine respiratory disorders

Mycoplasmas are pathogens involved in respiratory disorders of various animal hosts. In horses, Mycoplasma (M.) equirhinis is the species most frequently detected in clinical respiratory specimens, with a prevalence of 12–16%, but its clinical implication in equine respiratory disorders remains unclear. Here we screened 1948 clinical specimens for the presence of M. equirhinis. The samples were both tracheal washes (TW) and bronchoalveolar lavages (BAL) collected by veterinarians in France in day-to-day work between 2020 and 2022. The samples were associated with a standardized form that served to collect key general and clinical information, such as horse age, breed, and living environment. M. equirhinis was detected using a combination of culture and post-enrichment PCR. Other diagnostic data included virology and bacteriology as well as neutrophil counts, when available. Prevalence of M. equirhinis was examined as a function of a clinical score based on four significant clinical signs (nasal discharge, cough, dyspnoea, and hyperthermia). Multivariate logistic regression analysis was run to identify risk factors for the presence of M. equirhinis, and comparative prevalence analysis was used to test for association with other bacteria and viruses. TW and BAL were analysed independently, as we found that TW samples were associated with a higher prevalence of M. equirhinis. As prevalence remained steady whatever the clinical score, M. equirhinis cannot be considered a primary pathogen. M. equirhinis was more frequently isolated in thoroughbreds and trotters and in horses living exclusively stabled compared to other horses or other living environments. M. equirhinis was never detected in BAL specimens with a ‘normal’ neutrophil count, i.e. 5%, suggesting it could be associated with an inflammatory response, similar to that observed in equine asthma. Prevalence of M. equirhinis was shown to increase in the presence of other bacteria such as Streptococcus equi subsp. zooepidemicus (S. zoo) or viruses, and S. zoo load was higher in M. equirhinis-positive samples, suggesting a potential increase of clinical signs in the event of co-infection.

A real-time PCR assay for the quantification of Mycoplasma equirhinis in tracheal wash samples from Thoroughbred horses.

Mycoplasma equirhinis is the predominant equine Mycoplasma sp. isolated from clinically normal horses and is suspected to be associated with inflammatory airway disease in which cough is the primary sign. Quantitative evaluation of bacterial counts is useful in assessing the association between the bacteria in samples and observed clinical signs, but this evaluation has been difficult with conventional culture methods of M. equirhinis given the need for pre-enrichment using liquid cultures. We established a quantitative real-time PCR (qPCR) assay for the quantification of M. equirhinis, targeting the hypothetical protein FJM08_00025. We confirmed its high species-specificity for M. equirhinis and a limit of detection of 2.9 copies/reaction. We quantified M. equirhinis in tracheal wash samples from 20 clinically normal horses and 22 coughing horses. The copy numbers detected by qPCR in 18 of the 22 samples from clinically affected horses were within the range detected in the 20 clinically normal horses (0-84 copies/reaction). The remaining 4 samples had considerably higher copy numbers (734-1,620,000 copies/reaction), suggesting the likely involvement of M. equirhinis infection. Quantitative evaluation of M. equirhinis over time using our qPCR assay may allow a more accurate assessment of M. equirhinis infection in coughing horses compared to culture methods.

GLOBAL RETROSPECTIVE REVIEW OF SEVERE ACUTE RESPIRATORY SYNDROME SARS COV-2 INFECTIONS IN NONDOMESTIC FELIDS: MARCH 2020-FEBRUARY 2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in nondomestic felids have been documented in North America, South America, Africa, Europe, and Asia. Between March 2020 and February 2021, at nine institutions across three continents, infection was confirmed in 16 tigers (Panthera tigris), 14 lions (Panthera leo), three snow leopards (Panthera uncia), one cougar (Puma concolor), and one Amur leopard cat (Prionailurus bengalensis euptilurus) ranging from 2 to 21 yr old (average, 10 yr). Infection was suspected in an additional 12 tigers, 4 lions, and 9 cougars. Clinical signs (in order of most to least common) included coughing, ocular and/or nasal discharge, wheezing, sneezing, decreased appetite, lethargy, diarrhea, and vomiting. Most felids recovered uneventfully, but one geriatric tiger with comorbidities developed severe dyspnea and neurologic signs necessitating euthanasia. Clinical signs lasted 1-19 d (average, 8 d); one tiger was asymptomatic. Infection was confirmed by various methods, including antigen tests and/or polymerase chain reaction (PCR) of nasal or oral swabs, tracheal wash, and feces, or virus isolation from feces or tracheal wash. Infection status and resolution were determined by testing nasal swabs from awake animals, fecal PCR, and observation of clinical signs. Shedding of fecal viral RNA was significantly longer than duration of clinical signs. Postinfection seropositivity was confirmed by four institutions including 11 felids (5 lions, 6 tigers). In most instances, asymptomatic or presymptomatic keepers were the presumed or confirmed source of infection, although in some instances the infection source remains uncertain. Almost all infections occurred despite using cloth facemasks and disposable gloves when in proximity to the felids and during food preparation. Although transmission may have occurred during momentary lapses in personal protective equipment compliance, it seems probable that cloth masks are insufficient at preventing transmission of SARS-CoV-2 from humans to nondomestic felids. Surgical or higher grade masks may be warranted when working with nondomestic felids.

Detection of fungi in the airways of horses according to the sample site: a methodological study.

Fungal detection in equine airways may be performed on either tracheal wash (TW) or bronchoalveolar lavage fluid (BALF) by either cytology or culture. However, method comparisons are sparse. Our objective was to determine the prevalence of fungi in airways of horses according to the sample site and laboratory methodology. Sixty-two adult horses, investigated in the field or referred for respiratory disease, were included. Tracheal wash, and BALF collected separately from both lungs, were collected using a videoendoscope. Fungi were detected in cytologic samples examined by light microscopy, and by fungal culture. Hay was sampled in the field. Prevalence of fungi was of 91.9% in TW and 37.1% in BALF. Fungi were cultured from 82.3% of TW and 20.9% of BALF. Fungal elements were observed cytologically in 69.4% of TW and 22.6% of BALF. In 50% of horses, the same fungi were detected in both TW and hay, but fungi detected in BALF and hay differed in all horses. Poor agreement was found for the detection of fungi between TW and BALF and between fungal culture and cytologic examination (Cohen's kappa coefficient (κ) < 0.20). Moderate agreement was found between cytologic examination of left and right lungs (κ = 0.47). The prevalence of fungi detected cytologically on pooled BALF was significantly different (p = 0.023) than on combined left and right BALF. Fungi were more prevalent in the TW than BALF, and results suggest that hay might not be the primary source of fungi of the lower respiratory tract of horses.

Atrial fibrillation as a risk factor for exercise-induced pulmonary haemorrhage following a standardised exercise test.

Atrial fibrillation (AF) has been proposed as a risk factor for exercise-induced pulmonary haemorrhage (EIPH) due to increased pressure in the left atrium. To evaluate if AF was associated with EIPH following a standardised exercise test (SET) to fatigue. Two-arm controlled experiment. Ten untrained Standardbred mares mean (standard deviation [SD]) age 6 (2) years performed a SET on the treadmill in sinus rhythm (SR) (SET1) and 25-44 days after induction of self-sustained AF (SET2). AF was induced by tachypacing using a pacing device. Endoscopy, including tracheal wash and bronchoalveolar lavage (BAL), was performed 48-72 h before and 24 h after the two SETs. In addition, endoscopic grading of tracheal blood was performed 2 h after each SET. After SET1, none of the horses showed blood in the trachea, and two horses showed erythrophagocytosis. Following SET2, two horses had grade 1 blood in the trachea and free erythrocytes and erythrophagocytosis in the BAL, while another two horses had erythrophagocytosis in the BAL. In SET2, the overall performance on the treadmill was decreased with a lower maximum velocity (SET1 10.3 ± 0.8 m/s vs. SET2 8.9 ± 0.9 m/s, p = 0.004), a higher heart rate (284 ± 21 vs. 221 ± 18 bpm, p = 0.003) and more abnormal QRS complexes (p < 0.001) compared with SET1. Two horses showed signs of EIPH, resulting in visible blood in the trachea, when exercising in AF compared with SR. However, a possible link between EIPH, pulmonary pressure and AF needs to be further elucidated.

Co-Occurrence of Severe Equine Asthma and Palatal Disorders in Privately Owned Pleasure Horses.

According to the "unified or united airway disease" theory, diseases in the upper and lower airways frequently co-occur because they represent a single morphological and functional unit. Palatal disorders (PDs) and severe equine asthma (SEA) are frequent diseases that, respectively, affect upper and lower equine airways; however, clinical studies focusing on the co-occurrence of PDs and SEA are limited. The present study investigated the prevalence of PDs in horses affected by SEA, and whether prevalence decreased after SEA treatment. Forty-six privately owned horses affected by SEA in exacerbation were included. For each horse, the severity of the asthma clinical signs was assessed using a previously described scoring system, and the co-occurrence of palatal disorders was investigated using overground endoscopy, before and after treatment for SEA. Before treatment (in exacerbation), 67.4% of SEA-affected horses showed evidence of PDs, including 39.1% showing evidence of palatal instability (PI) and 28.3% of dorsal displacement of the soft palate (DDSP). Airway inflammation (neutrophil percentage in the tracheal wash and bronchoalveolar lavage fluid) was worse in horses with co-occurring PDs. After treatment (in remission), no horses showed evidence of PI, while DDSP was diagnosed in 8.7% of horses. These findings suggest that palatal disorders respond to asthma treatment, supporting the hypothesis that both diseases could be manifestation of a common underlying disorder.

Detection of Mycoplasma spp. in horses with respiratory disorders.

Bacteria belonging to the genus Mycoplasma are small-sized, have no cell walls and small genomes. They commonly cause respiratory disorders in their animal hosts. Three species have been found in the respiratory tract of horses worldwide, that is., Mycoplasma (M.) equirhinis, M. pulmonis and M. felis, but their role in clinical cases remains unclear. The aim of this study was to i) develop and validate tools to detect, isolate and identify different Mycoplasma spp. strains in clinical equine respiratory-tract specimens and ii) subsequently define the prevalence of the three species in France depending on sample types and horse characteristics (age, breed, sex). Validation of a workflow for mycoplasma diagnosis and subsequent prevalence study. Mycoplasma-free tracheal wash samples spiked with numerated strains and DNA dilutions were used to validate the culture methods and real-time PCR (rt-PCR) assay. Isolated strains were identified by 16S rRNA gene sequencing. Prevalences were determined on a population of 616 horses with respiratory disorders, sampled in France in 2020. In total, 104 horses (16.9%) were found to be positive for Mycoplasma spp. by at least one method. M. equirhinis was the predominant circulating species, accounting for 85% of the rt-PCR-positive samples and 98% of the 40 cultured strains. The proposed pre-enrichment procedure improves the sensitivity of detection but hinders the quantification of the initial mycoplasma load in the clinical specimens. Prevalence of mycoplasma varied with age, breed, and type of sample.

Evaluation of serum concentration of acute-phase proteins (haptoglobin and serum amyloid A) in the affected Arabian foals with rhodococcosis.

Early detection of Rhodococcus equi pneumonia in foals is essential for horse health and for veterinarians. This study aimed to demonstrate the usefulness of assessing the serum concentration of acute-phase proteins (APPs) in the early diagnosis of pneumonia. The study evaluated APPs in 19 Arabian foals with R. equi pneumonia and compared them with 18 normal Arabian foals in equestrian clubs in Tabriz, Iran. Affected foals were identified through history, clinical findings and bacterial culture of tracheal washing. Biochemical methods and polymerase chain reaction tests were performed by examining the 16S rRNA and vapA genes to confirm the diagnosis of bacterial isolates. Blood samples were taken from all sick and healthy horses, and their serum was isolated. APPs in the serum were measured in all the samples. Rhodococcosis increased the serum concentration of haptoglobin (Hp) and serum amyloid A (SAA) (p < 0.001). The relationship between SAA and Hp was meaningful in the infected group (r = 0.933) but not in the healthy group. In cases where there are clinical findings of R. equi pneumonia, the concentration of SAA and Hp can help the effectiveness of treatment. Serum concentration analysis of APPs can be helpful in early diagnosis and successfully treating foals with R. equi pneumonia.

Piezoelectric Gas Sensors with Polycomposite Coatings in Biomedical Application

When developing methods for diagnosing pathologies and diseases in humans and animals using electronic noses, one of the important trends is the miniaturization of devices, while maintaining significant information for diagnostic purposes. A combination of several sorbents that have unique sorption features of volatile organic compounds (VOCs) on one transducer is a possible option for the miniaturization of sensors for gas analysis. This paper considers the principles of creating polycomposite coatings on the electrodes of piezoelectric quartz resonators, including the choice of sorbents for the formation of sensitive layers, determining the mass and geometry of the formation of sensitive layers in a polycomposite coating, as well as an algorithm for processing the output data of sensors to obtain maximum information about the qualitative and quantitative composition of the gas phase. A comparative analysis of the efficiency and kinetics of VOC vapor sorption by sensors with polycomposite coatings and a set of sensors with relevant single coatings has been carried out. Regression equations have been obtained to predict the molar-specific sensitivity of the microbalance of VOC vapors by a sensor with a polycomposite coating of three sorbents with an error of 5-15% based on the results of the microbalance of VOC vapors on single coatings. A method for creating "visual prints" of sensor signals with polycomposite coatings is shown, with results comparable to those from an array of sensors. The parameters Aij∑ are proposed for obtaining information on the qualitative composition of the gas phase when processing the output data of sensors with polycomposite coatings. A biochemical study of exhaled breath condensate (EBC) samples, a microbiological investigation of calf tracheal washes, and a clinical examination were conducted to assess the presence of bovine respiratory disease (BRD). An analysis of the gas phase over EBC samples with an array of sensors with polycomposite coatings was also carried out. The "visual prints" of the responses of sensors with polycomposite coatings and the results of the identification of VOCs in the gas phase over EBC samples were compared to the results of bacteriological studies of tracheal washes of the studied calves. A connection was found between the parameters Aij∑ of a group of sensors with polycomposite coatings and the biochemical parameters of biosamples. The adequacy of replacing an array of piezoelectric sensors with single coatings by the sensors with polycomposite coatings is shown.

Virulence plasmids in clinical isolates of Rhodococcus equi from sick foals in the Netherlands.

Clinical samples from 123 foals with suspected rhodococcosis submitted to the Veterinary Microbiological Diagnostic Centre of the Faculty of Veterinary Medicine between 1993 and 2006 were tested for the presence of the virulence gene vapA. Of the 123 samples, 120 were vapA-positive and 3 vapA-negative Rhodococcus equi were isolated. The 120 vapA-positive R. equi were isolated from 70 tracheal wash, 19 lung tissues, 7 lymph nodes, 6 synovial fluids, 13 abscesses or pus and single isolates from the uterus, gut, cerebrospinal fluid, abdomen fluid and faeces. Of the 120 isolates, 46 were from Dutch warmblood horses, 23 from Friesian horses, 14 from Trotters, 4 from Holsteiners, 3 from Arab breed, 2 from ponies, 1 from a Welsh pony and 27 from undefined breed horses. Using plasmid profile analysis of the 120 isolates, 117 isolates contained the 85-kb type I plasmid, 2 contained the 87-kb type I plasmid and 1 contained the novel 52-kb non-mobilizable virulence plasmid reported recently. These results showed that the virulent R. equi strains harbouring a virulence plasmid of 85-kb type I or 87-kb type I, which have been detected in clinical isolates from five European countries, are widespread in the Netherlands. This is the first report of plasmid types of clinical R. equi isolates in the Netherlands.

Diagnostic Value of Tracheal Wash Cytology for Monitoring Exercise-Induced Pulmonary Hemorrhage in Thoroughbred Racehorses

Lower airway cytology has been proposed as a complementary diagnostic method to confirm the presence and quantifying the severity of exercise-induced pulmonary hemorrhage (EIPH). Bronchoalveolar lavage is usually preferred over tracheal wash (TW), yet the need for sedation imposes as a limitation for active sport horses in addition to be a highly invasive technique. This study aims to evaluate the diagnostic value of TW with Total Hemosiderin Score (THS) for monitoring EIPH in active Thoroughbred racehorses. A sampling of 47 randomly selected Thoroughbreds were endoscopically examined for EIPH grading 30 to 60 minutes after competing in an official race and then classified as Group 1 (EIPH-negative), Group 2 (EIPH-positive) or Group 3 (furosemide users). Tracheal wash fluids (TWFs) were collected from 24 to 30 hours later and smears were stained for differential cell count and hemosiderophage grading and counting to calculate THS. Differential cell counts were compared by the median test. Comparisons between mean THS values by EIPH grade and by group were compared by the Mann-Whitney test and ANOVA, respectively. Test performance criteria were determined with a contingency table. It was not possible to establish a THS cut-off point but statistical results showed that, at THS stand view, all groups had similar results despite their EIPH grades. Most animals showed no signs of neutrophilic inflammation, but haemosiderophages were found even on a first-time runner from Group 1. Thus, TW can detect evidence of lung bleeding even on horses with no history of EIPH. The implementation of TW analysis to diagnose EIPH in racehorses is promising, as TW is both low-cost and a less invasive tool.

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.

High-multiplexing quantitative CodePlex proteomic profiling of platelets in triple-negative breast cancer (TNBC) patients and healthy subjects.

e15017 Background: Platelets play significant roles in inflammation, immune regulation, and intracellular communication processes involved in tumor pathogenesis. Aberrant platelet activation has been associated with tumor progression and metastasis, however, cancer patient platelet deep profiling remains underutilized. New tools are needed for rapid high throughput quantification of potential altered platelet secretome in cancer patients. Such technology may provide novel insights into impactful cancer-associated biomarkers. Methods: We have developed a high throughput multiplexed proteomic technology to profile platelet cytokine content and releasates and performed a pilot study in healthy patients versus triple negative breast cancer (TNBC). Macrochambers of the CodePlex chip were pre-patterned with a 23-plex antibody IsoPlexis Human Adaptive Immune Panel: GM-CSF, Granzyme B, IFN-g, IL-10, IL-13, IL-15, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1, MIP-1a, MIP-1b, perforin, sCD137, TGF-b1, TNF-a, TNF-b. 2% BSA/PBS was used as background control. The chips are loaded into an IsoLight automation system, releasates measured by fluorescence ELISA, and analysis performed by IsoSpeak software. In a pilot study to test feasibility, platelets were meticulously harvested from triple negative breast cancer patients (n=2) and healthy controls (n=2) and stimulated for 30 minutes (ADP or TRAP-6) to promote cytokine release. Both the supernatants and platelet lysates were then loaded on CodePlex chips and analyzed. Results: The methodology of bulk proteomic measurements has previously been validated in a variety of biofluids such as serum/plasma, cerebrospinal fluid, tracheal washes, and cell culture supernatants. Freshly isolated platelets activated by TRAP6 generally released more cytokines than those activated by ADP stimulation in both healthy controls and disease samples. Interestingly, platelet releasates in supernatants from TNBC patients demonstrated increased pro-tumor IL-8 and IL-9, and reduced antitumor IFN-g compared to healthy controls. Higher levels of IL-8 and IL-9 and minimal IFN-g were detected at similar activation concentrations, in platelet lysates from both healthy and TNBC platelets. This suggests that TNBC may alter platelet content and/or activation. Increased numbers are being performed to validate these initial results. Conclusions: From this small pilot study, multiplexing quantitative proteomic analysis is potentially useful in detecting differential cytokine profile in platelet releasates and lysate contents between TNBC patients and healthy controls. Quantifying the platelet proteomes in cancer patients using high throughput proteomics may provide valuable insights to dissect cancer-platelet crosstalk and potential pathogenesis mechanisms.