Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 2. Evaluation of the diagnostic significance of neutrophil percentage

OBJECTIVE: To determine whether diagnosis of airway inflammation, using cut-off percentages for neutrophils, differs when based on samples from tracheal aspirate (TA) and bronchoalveolar lavage (BAL) collected concomitantly from the same racehorse. DESIGN: Retrospective case series of 48 young Thoroughbred and Standardbred racehorses in race training, but showing poor performance. PROCEDURE TA and BAL samples were collected from all horses 1 to 2 h after high-speed treadmill exercise. Aliquots of the retrieved fluid were cytocentrifuged and smears stained with Diff-Quik. The mean percentage of neutrophils was calculated. Diagnostic cut-off points were set at 20% for TA samples and 5% for BAL samples. Agreement in the interpretations between the two techniques was analysed. RESULTS: In 19 of 51 paired samples (37%) there were differences in diagnostic interpretation between TA and BAL samples. Of these, airway inflammation was indicated only by the TA sample in 13 and only by the BAL in 6. CONCLUSION: TA and BAL samples give important information about different regions of the airway, but neither should be used alone for the diagnosis of inflammation of the entire lung. The limitations of these procedures mean that both samples should be collected when it is desired to cytologically evaluate the entire lower airway.     

The Effect of Pasteurella haemolytica A2 Infection on Phagocytosis Efficiency of Caprine Broncho‐Alveolar Macrophages

An experiment was designed to study the in vivo effect of Pasteurella haemolytica A2 infection on the phagocytosis activity of caprine broncho‐alveolar macrophages and the extent of pneumonic lesions. Twelve healthy local Kacang goats, about 7 months of age, were divided into two groups of six. Goats in group 1 were inoculated intratracheally with 4 ml inoculum containing 2.8 × 10⁹ colony‐forming units (CFU)/ml of Staphylococcus aureus. Goats in group 2 were inoculated intratracheally with 4 ml of inoculum containing 9.5 × 10⁸ CFU/ml of Pasteurella haemolytica A2 isolated earlier from pneumonic lungs of goat. At intervals of 3 and 7 days post‐challenge five goats from each group were killed and the lungs were washed with sterile phosphate‐buffered saline. Smears were prepared from the lung washing fluid and the number of macrophages with phagocytic activity was determined. At day 3 post‐infection, goats of both groups showed a similar pattern of pneumonic lesion. The lung washing fluid of goats in group 2 was found to contain numerous neutrophils and macrophages. Goats in group 2 showed significantly (P < 0.05) higher extent of lung lesions than group 1. Similarly, the average extent of lung lesions was significantly (P < 0.05) more severe in group 2 at day 7 post‐infection. The lung washing fluid contained mostly macrophages. The phagocytic activity following S. aureus infection was more efficient and significantly (P < 0.01) higher compared with infection by P. haemolytica A2. There were weak correlations between the extent of pneumonic lesion and the phagocytic activity. Thus, goats with poor phagocytic activity were likely to develop more extensive lung lesions.     

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.     

Mononuclear phagocytes of transport-stressed horses with viral respiratory tract infection

Twelve horses comprised 3 treatment groups; all horses in 2 of the groups had recently been transported and had clinical and laboratory evidence of respiratory tract infection, with equine influenza type 2 virus being the principal pathogen. Mononuclear phagocytes and other leukocytes from blood, lung, and peritoneal cavity were studied in phagocytosis and erythrocyte-antibody (EA) rosette assays. Total numbers of pulmonary alveolar macrophages were increased over control values in bronchoalveolar lavage (BAL) fluid of group 3 horses after recovery from influenza (P less than 0.02), whereas the increase in neutrophils in the fluid of those horses approached significance. Lymphocytes in BAL fluid of group 3 horses (after recovery from influenza) were in larger proportion than those in fluid of group 1 horses during acute influenza (P less than 0.05). Pulmonary alveolar macrophages of group 1 horses formed a lower percentage of EA rosettes than did those of controls (P less than 0.01) or group 3 horses (P less than 0.02). The differential counts of peritoneal macrophages and neutrophils in horses of groups 1 and 3 were virtually identical at the first collection, but differed from controls at the second collection 4 weeks later; peritoneal macrophages were reduced (P less than 0.01), whereas peritoneal neutrophils were increased (P less than 0.01). Peritoneal macrophages and peritoneal neutrophils of horses with acute influenza were phagocytic in larger proportion than were those in controls at both collection times (P less than 0.01 and P less than 0.01 for peritoneal macrophages, and P less than 0.01 and P less than 0.05 for peritoneal neutrophils, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pasteurella haemolytica leukotoxin on neutrophils from white-tailed deer and several exotic ruminant species

Pasteurella haemolytica leukotoxin is a pore-forming cytolysin which acts as a virulence factor in pasteurellosis of domestic ruminants. Leukocytes from cattle, sheep and goats are susceptible to leukotoxin-induced lysis; however, leukocytes from non-ruminant species so far tested are resistant to leukotoxin-induced lysis. Neutrophils obtained from three white-tailed deer, four Saiga antelope, an Addra gazelle, a Grant's gazelle and a Sable antelope were tested for susceptibility to the lytic effects of P. haemolytica leukotoxin using lactate dehydrogenase release. Results were compared to those obtained using neutrophils from a steer and cultured bovine lymphoma cells. Neutrophils obtained from all these ruminants, except the Addra gazelle, were susceptible to P. haemolytica leukotoxin. Individual variation among the Saiga and the deer did not appear to be due to the percentages of neutrophils or the percentage of contaminating erythrocytes in the cell preparations.     

Diagnosis of pneumocystis pneumonia in a 2‐year‐old King Charles Cavalier Spaniel using the polymerase chain reaction

A 2‐year‐old, female spayed, King Charles Cavalier Spaniel was presented for evaluation of dyspnea, inappetence, and lethargy. Thoracic radiographs revealed a moderate diffuse interstitial lung pattern affecting the perihilar and caudodorsal lung fields, and an echocardiogram revealed severe pulmonary hypertension. A bronchoalveolar lavage (BAL) was performed, and cytology revealed mixed inflammation with cysts and trophozoites consistent with Pneumocystis. Pneumocystis infection was later confirmed with PCR. To the author's knowledge, this report represents the first case of canine pneumocystis pneumonia diagnosed antemortem with PCR from a BAL sample. Pneumocystis represents an important, but uncommon cause of afebrile pneumonia in immunosuppressed dogs.     

Pulmonary Eosinophilia Associated with Increased Airway Responsiveness in Young Racing Horses

Horses are known to acquire small airway disease (SAD), an allergen-induced naturally occurring syndrome of reversible obstructive lung disease accompanied by airway hyperresponsiveness and increased inflammatory cell numbers on bronchoalveolar lavage (BAL). This disorder has received scant attention in young racehorses. The purpose of the present report was to examine the effect of BAL eosinophilia in young racehorses on clinical examination, BAL, hematology, airway responsiveness, and on pulmonary function at rest and after a standardized exercise challenge. Five (3 males, 2 females; age 2.6 ± 0.9 years) with a history of respiratory compromise and BAL eosinophil differential count <5% and 6 controls (4 males, 2 females; age 3.5 ± 1.0 years) training and performing to expectation with normal BAL cell differential (eosinophils < 1%) were studied. Respiratory system clinical examination was performed and expressed as a clinical score. Arterial blood gas measurements, CBC, and pulmonary function testing were performed at rest. Pulmonary mechanics measurements were repeated 1 hour and 20 hours after a standardized treadmill exercise challenge. Incremental histamine inhalation challenge was performed and the concentration of histamine effecting a 35% decrease in dynamic compliance (PC35C_Dyn) was determined. Significant differences were noted between and controls with regard to clinical score ( P = .01), blood eosinophils ( P = .04), BAL cell count ( P = .04), BAL macrophage differential ( P = .04), PC35C_Dyn ( P = .008), and tidal volume and respiratory rate at 20 hours following exercise challenge ( P = .05). We conclude that pulmonary eosinophilia and airway hyperresponsiveness are manifest in some young horses without overt airway obstruction at rest. We speculate that these may be early events in the natural progression of heaves.     

Diversity of Mannheimia haemolytica and Pasteurella trehalosi Serotypes from Apparently Healthy Sheep and Abattoir Specimens in the Highlands of Wollo,North East Ethiopia

The prevalence and serotypic diversity of Mannheimia [Pasteurella] haemolytica and Pasteurella trehalosi from nasal swabs, sera and abattoir specimens from sheep in the highlands of Wollo, North East Ethiopia was investigated. Prevalence rates of 83% and 75% of these microorganisms were found in the serum samples and nasal swabs, respectively, from apparently healthy sheep. In a local abattoir, 205 lungs were investigated, 34% of which showed pneumonia, from which samples were collected from 51 lungs and the same number of corresponding tonsils. Mannheimia and Pasteurella species were isolated from 59% of these pneumonic lungs and 69% of the respective tonsils. M. haemolytica serotypes accounted for 41 (59%) and P. trehalosi for 11 (32%) of the isolates from the abattoir specimens. The majority (67%) of isolates from nasal swabs were P. trehalosi, M. haemolytica being isolated f rom 4 (13%) of the swabs. M. glucosida was isolated only from the tonsils. The predominant serotypes of the isolates from both the nasal swabs and the abattoir specimens were M. haemolytica A1 (17%) and P. trehalosi T4 (16%) and T3 (13%). P. trehalosi T15 was less commonly encountered, while M. haemolytica A9 and A13 were not isolated. Studies on sera from 100 sheep indicated that antibodies against M. haemolytica serotype A1 (14%) were most common, followed by A5 and A8 (each 10%) and A9 and P. trehalosi T3 (each 9%) and T4 (8%). Antibodies against M. glucosida or serotype A11 occurred in 2% of the sera. Multiple serotypes were common in all types of samples. The importance of including in vaccines the most prevalent serotypes involved in the pneumonia of sheep in the area is discussed.     

Bronchoalveolar lavage cytology in cynomolgus monkeys and identification of cytologic alterations following sequential saline lavage

Total and differential cell counts were determined on cytolytic specimens obtained by fiberoptic bronchoscopy and bronchoalveolar lavage (BAL) of five normal cynomolgus monkeys. Total nucleated cell counts ranged from 100 to 430 cells/microliters. Macrophages were approximately 91% of total nucleated cells, while lymphocytes were 3%, neutrophils 4%, and eosinophils 2% of the initial BAL from each monkey. Less than 1% of the cells were mast cells and ciliated or nonciliated epithelial cells. The effects of repeated saline BAL on pulmonary cell populations were evaluated. Saline lavage of individual lung lobes resulted in a marked rise in circulating blood neutrophils at 4 hr after BAL; there was a similar rise in neutrophils in lavage fluids 24 hr after the initial lavage. Differential and total cell counts of both blood and lavage fluid returned to normal if subsequent lavages were spaced at 48-hr intervals. Lymphocytes were not present in saline-lavaged lung lobes, and protein levels of lavage fluids did not rise significantly. BAL produced a transient, reversible, intra-alveolar influx of neutrophils which was preceded by mobilization of bone marrow-stored neutrophils. Neutrophilia in the lavage fluid and blood was not detectable if lavage and blood sampling procedures were done at 48-hr intervals (which did not alter Ia antigen expression among BAL cells). These observations indicate that BAL is a valid method for sampling and assessing pulmonary cellular and fluid constituents if the procedures are done at intervals of at least 48 hr.     

Altered endothelium-dependent α-adrenergic vascular reactivity following exposure toPasteurella haemolytica antigens

Rats were injected with sterile saline (controls), 10⁵ cfu ofPasteurella haemolytica (biotype A) obtained from a commercial vaccine, or a commercialPasteurella leukotoxoid vaccine. Three days after vaccination, the animals were killed and the thoracic aorta was removed. In some experiments the vascular endothelium was mechanically removed. Each isolated aorta was placed in a tissue bath and the biophysical responses to methoxamine (-1 agonist) were determined. In separate experiments the endothelial surface was examined by scanning electron microscopy. In endothelium-intact vessels both vaccines appeared to enhance the contractile response to methoxamine. On the other hand, in endothelial-denuded vessels, the methoxamine-mediated contractile response was enhanced in theP. haemolytica-treated group but not in animals vaccinated with leukotoxoid. Furthermore, scanning electron microscopy revealed deposition of a fibrin-like material on the endothelial surface of vaccinated animals. These results suggest that exposure to vaccine-derivedP. haemolytica antigens alters the morphology and adrenergic responsiveness of vascular smooth muscle.Abbreviations BPP bovine pneumonic pasteurellosis - cfu colony forming units - EC₅₀ concentration giving 50% of the maximum response - IP intraperitoneal - mN millinewtons - pD₂ -log(EC₅₀)     

Exercising Blood Gas Analysis, Dynamic Upper Respiratory Tract Obstruction, and Postexercising Bronchoalveolar Lavage Cytology—A Comparative Study in Poor Performing Horses

Respiratory abnormalities are common causes of decreased performance in horses presumably because of impaired pulmonary gas exchange. The objectives of the present study were to describe respiratory abnormalities in poorly performing horses and to investigate the relationships between dynamic upper respiratory tract (URT) videoendoscopy, postexercising bronchoalveolar lavage (BAL) cytology, and exercising arterial blood gas analysis. Medical records of 93 horses with exercise intolerance, which presented for treadmill evaluation, were reviewed. Relationships between horse demographics, treadmill endoscopic findings, exercising blood gas values, and BAL cytology results were examined. A total of 25 (27%) horses had a URT obstruction and 91 (98%) horses had abnormal BAL cytology; 73 (78%) had evidence of inflammatory airway disease (IAD) and 83 (89%) had exercise-induced pulmonary hemorrhage (EIPH). In all, 39 (42%) horses had abnormal blood gas values. Dynamic URT obstruction was significantly associated with exercising hypoxemia (P = .036). There were no significant relationships between gas exchange and IAD or between EIPH. Out of 24 (26%) horses with combined URT obstruction and abnormal BAL, horses with URT obstruction and EIPH were more likely to be hypoxic during exercise (P = .037). It was concluded that horses with dynamic URT abnormalities are likely to have exercising hypoxemia. Although IAD and EIPH were commonly indentified in poor performers, they were not significantly associated with abnormal exercising blood gas analysis.     

Comparison of Sampling Procedures for Isolating Pulmonary Mycoplasmas in Cattle

Three sampling procedures were compared to determine the optimal technique for isolating mycoplasmas in cattle with respiratory diseases. The prevalence of mycoplasmas isolated from these animals is also reported. In the first group, bronchoalveolar lavage (BAL) and nasal swab cultures were compared with the corresponding lung cultures from cattle necropsied for fatal respiratory diseases (n = 20). In a second group, nasal swabs were compared with corresponding BAL cultures in living animals with recurrent respiratory pathologies (n = 49). There was complete agreement between the paired BAL and lung cultures. In contrast, nasal cultures were not representative of the mycoplasmas present in the lower respiratory airways. The relative sensitivity and specificity of the nasal swab technique compared to BAL in living animals confirmed that the nasal swab cultures were not predictive of lower respiratory airway pathogens, such as Mycoplasma bovis. BAL is considered to be the best method for isolating M. bovis in cattle with respiratory diseases as it combines reliability and feasibility under field sampling conditions. In the present study, Mycoplasma dispar (43%) and M. bovis (29%) were mainly isolated in mixed infections. This confirms the need to search for mycoplasmas in routine examinations and to take them into account in therapeutic strategies for respiratory diseases in cattle.     

Cytotoxic effect of Pasteurella haemolytica on ovine bronchoalveolar macrophages in vitro

Live Pasteurella haemolytica A1 was shown to have a cytotoxic effect on suspensions of sheep bronchoalveolar macrophages. Cytotoxic activity was also demonstrable in bacteria-free supernatants from suspensions containing P. haemolytica. Heat-killed and ultraviolet killed organisms of P. haemolytica and live Staphylococcus aureus were not toxic to sheep BAM. These results suggest that a bacterial cell-free cytotoxin is produced by metabolically active P. haemolytica. Guinea-pig peritoneal macrophages, McCoy and pig kidney epithelial cell suspensions were unaffected by live P. haemolytica and supernatant from P. haemolytica cultures, indicating that the cytotoxin may only affect phagocytic cells of ovine or bovine origin.     

Effect of experimental infection of cattle with bovine herpesvirus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with Mannheimia (Pasteurella) haemolytica leukotoxin

Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the beta(2)-integrin CD11a/CD18 (LEA-1) on ruminant leukocytes. LKT binding induces activation, and subsequent cytolysis, of these cells. It is well known that active viral infection greatly increases the susceptibility of cattle to pasteurellosis. To better understand the mechanism by which this occurs, we investigated the effects of experimental in vivo infection of cattle with bovine herpes virus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with the M. haemolytica LKT. In this study, we demonstrated that active BHV-1 infection increased the expression of the beta(2)-integrin CD11a/CD18 (as defined by the mAb BAT75) on bovine peripheral blood neutrophils, enhanced the binding of LKT to bronchoalveolar lavage (BAL) leukocytes and peripheral blood neutrophils, and increased the killing of BAL leukocytes and peripheral blood leukocytes by LKT. In addition, BHV-1 greatly increased the number of BAL, resulting in many more LKT-responsive cells being present in the lungs. These findings might explain in part the increased susceptibility of BHV-1 infected cattle to pneumonic pasteurellosis.     

Pathophysiological and immune cell responses in calves prior to and following lung challenge with formalin-killed Pasteurella multocida biotype A:3 and protection studies involving subsequent homologous live challenge

Pneumonic pasteurellosis is a common respiratory infection in cattle that has major economic and welfare implications world-wide and the incidence in the UK due to Pasteurella multocida, currently the same as that associated with Mannheimia haemolytica, is increasing. Whereas much is known regarding the pathogenesis of M. haemolytica infections little information is available on the pathogenic process of pasteurellosis initiated by P. multocida. In the present work calf systemic and innate immune responses to intratracheal challenge with formalin-killed P. multocida biotype A:3 and to subsequent experimental lung infection with live P. multocida were investigated. Eight-week-old calves were challenged intratracheally on day 0 with either 10⁹ colony forming units (cfu) of formalin-killed P. multocida biotype A:3 in 300 ml saline (n=10) or 300 ml saline alone (n=10), followed, at day 21, by challenge with 10⁹ cfu live P. multocida. Pathophysiological and lung phagocyte responses were assessed by clinical monitoring, sequential lung lavage and blood sampling. Results for samples obtained before, during and after challenge showed clinical and acute phase protein responses to both bacterial culture and saline control treatments, although higher responses were associated with bacterial challenge. Phagocytosis of P. multocida during 1 h incubation periods with lavaged cells in vitro was unaffected by exposure in vivo to killed P. multocida and there was evidence that P. multocida was able to survive intracellularly during this assay. There was no indication that lung exposure to formalin-killed P. multocida conferred protection against subsequent homologous live challenge.     

The influence of age and Rhodococcus equi infection on CD1 expression by equine antigen presenting cells

There is a distinct age-associated susceptibility of horses to Rhodococcus equi infection. Initial infection is thought to occur in the neonatal and perinatal period, and only foals less than 6 months of age are typically affected. R. equi is closely related and structurally similar to Mycobacterium tuberculosis, and causes similar pathologic lesions. Protective immune responses to M. tuberculosis involve classical major histocompatibility complex (MHC)-restricted T cells that recognize peptide antigen, as well as MHC-independent T cells that recognize mycobacterial lipid antigen presented by CD1 molecules. Given the structural similarity between these two pathogens and our previous observations regarding R. equi-specific, MHC-unrestricted cytotoxic T lymphocytes (CTL), we developed 3 related hypotheses: (1) CD1 molecules are expressed on equine antigen presenting cells (APC), (2) CD1 expression on APC is less in foals compared to adults and (3) infection with live virulent R. equi induces up-regulation of CD1 on both adult and perinatal APC. CD1 expression was examined by flow cytometric analysis using a panel of monoclonal CD1 antibodies with different species and isoform specificities.

Results

Three CD1 antibodies specific for CD1b showed consistent cross reactivity with both foal and adult monocyte-derived macrophages (MDM). CD1b and MHC class II expression were significantly higher on adult MDM compared with foals. R. equi infected MDM showed significantly lower expression of CD1b, suggesting that infection with this bacterium induces down-regulation of CD1b on the cell surface. Histograms from dual antibody staining of peripheral blood mononuclear cells also revealed that 45–71% of the monocyte population stained positive for CD1b, and that the majority of these also co-expressed MHC II molecules, indicating that they were APC. The anti-CD1 antibodies showed no binding or minimal binding to bronchoalveolar lavage (BAL)-derived macrophages.

Conclusion

The CD1b isoform is evolutionarily conserved, and is present on equine MDM, as well as on circulating blood monocytes. The unique susceptibility of foals to R. equi infection may be due in part to lower expression of CD1 and MHC class II, as observed in this study. The data also suggests that infection with R. equi induces down-regulation of CD1b on equine MDM. This may represent a novel mechanism by R. equi to avoid detection and killing of infected cells by the immune system, similar to that observed when human APC are infected with M. tuberculosis.     

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.     

Co-migrating and shared antigens of selectedPasteurella haemolytica untypable strains

Bacterial cell envelope preparations from eight untypable strains ofPasteurella haemolytica were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting with rabbit antisera prepared against the eight untypable strains (one untypable strain per rabbit) and with cattle antisera prepared againstP. haemolytica serotypes 1, 2, 5, 6, 9 and against one heterologous untypable strain. Numerous comigrating and shared antigens were recognized by the eight rabbit antisera and theP. haemolytica serotype cattle antisera. Comigrating antigens at 43 and 30 kilodaltons (kDa) were recognized by all eight rabbit antisera. Shared antigens, detected by all eight rabbit antisera when reacting againstP. haemolytica serotype 1, were recognized at 43, 32, 30, 20 and 15 kDa.     

Physiologic effects of furosemide in combination with water restriction when administered at 4 and 24 hours prior to high-intensity treadmill training

Although controversial, due to its reported effectiveness in attenuating bleeding associated with exercise-induced pulmonary hemorrhage (EIPH), furosemide is currently a permitted race day medication in most North American racing jurisdictions. The objective of this study was to assess the efficacy of furosemide in reducing the presence and severity of EIPH when administered 24 hr prior to strenuous treadmill exercise. Eight exercised Thoroughbred horses received saline or 250 mg of furosemide either 4 or 24 hr prior to high-speed treadmill exercise in a balanced 3-way cross-over design. Blood samples were collected for determination of furosemide, lactate, hemoglobin, blood gas, and electrolyte concentrations. Heart rate and pulmonary arterial pressure were measured throughout the run and endoscopic examination and bronchoalveolar lavage (BAL) performed. Horses were assigned an EIPH score and the number of red blood cells in BAL fluid determined. Although not significantly different, endoscopic EIPH scores were lower in the 4-hr versus the 24-hr and saline groups. RBC counts were not significantly different between the treatment groups. Pulmonary arterial pressures were significantly increased at higher speeds; however, there were no significant differences between dose groups when controlling for speed. A small sample size and unknown bleeding history warrant a larger-scale study.     

Effects of an external nasal strip and frusemide on pulmonary haemorrhage in Thoroughbreds following high-intensity exercise.

The purpose of this study was to examine the effects of an external nasal strip (NS), frusemide (FR) and a combination of the 2 treatments (NS + FR) on exercise-induced pulmonary haemorrhage (EIPH) in Thoroughbred horses. It was hypothesised that both the NS and FR would attenuate EIPH as assessed by red blood cell count in bronchoalveolar lavage fluid. In random order, 8 horses completed each of 4 sprint exercise tests on a treadmill: 1) NS; 2) FR (0.5 mg/kg bwt i.v., 4 h pre-exercise); 3) NS + FR; and 4) control (C; no treatment). After a 5 min warm-up (4.5 m/s), horses completed 2 min running at 120% maximum oxygen consumption (VO2max) with the treadmill set at 3 degrees incline. Mean +/- s.d. running speed was 14.2+/-0.2 m/s. In the FR and NS + FR trials, horses carried weight equal to that lost as a result of frusemide administration. During exercise at 120% Vo2max, oxygen consumption (Vo2) and carbon dioxide production (Vco2) were measured at 15 s intervals. Plasma lactate concentration was measured in samples collected before exercise, at the end of the sprint and after 5 min cool-down at the trot. Thirty minutes after the run, bronchoalveolar lavage (BAL) was performed and the red cell count in the fluid quantified. Vo2 and Vco2 were significantly lower in NS and NS + FR trials than in the C and FR trials at the end of the sprint exercise protocol. However, plasma lactate concentrations did not differ among treatments. Compared with the C trial (61.1+/-30.5 x 10(6) red blood cells/ml BAL fluid), pulmonary haemorrhage was significantly (P<0.05) decreased in both the NS (15.9+/-4.0 x 106 RBC/ml) and FR (12.2+/-5.8 x 10(6) RBC/ml) trials. EIPH in the NS + FR trial (7.9+/-1.0 x 10(6) RBC/ml) was further diminished (P<0.05) compared to the NS trial, but not different from the FR trial. We conclude that both the external nasal strip and frusemide attenuate pulmonary haemorrhage in Thoroughbred horses during high-speed sprint exercise. The external nasal strip appears to lower the metabolic cost of supramaximal exertion in horses. Given the purported ergogenic effects of frusemide, the external nasal strip is a valuable alternative for the attenuation of EIPH.