Effect of sex and age on the activities of lactate dehydrogenase and alkaline phosphatase in the lungs of rats.

Since toxicity studies among different laboratories generally involve rats of different sex and age, this study was conducted to investigate the effect of sex, age and animal to animal variation in the activities of lactate dehydrogenase and alkaline phosphatase from bronchoalveolar lavage fluid, bronchoalveolar cell lysate and lung homogenate. Correlation between numbers of bronchoalveolar cells recovered from lungs and enzyme activity in bronchoalveolar cell lysate or lung homogenate supernatant were also investigated. Male rats showed significantly (p less than 0.05) higher activities of alkaline phosphatase in the bronchoalveolar lavage fluid and lung homogenate. Animal to animal variation for lactate dehydrogenase and alkaline phosphatase was higher in lungs than in serum. The number of bronchoalveolar cells recovered from lungs revealed a significant (p less than 0.01) positive correlation with the activities of both enzymes in the supernatant of cell lysates but not in the bronchoalveolar fluid. These results indicated that in an inhalation study interindividual variation in the levels of pulmonary enzymes should be considered in order to minimize the numerous possible sources of experimental error.     

Using urea dilution to standardise cellular and non-cellular components of pleural and bronchoalveolar lavage (BAL) fluids in the cat

A technique to standardise the analysis of cellular and non-cellular components in epithelial lining fluid (ELF) collected during saline lavage of pulmonary and pleural cavities was developed using the urea dilution method. Bronchoalveolar lavage (BAL) and pleural lavage (PL) fluids were collected from 12 clinically healthy cats. Total and differential cell counts in BAL fluid were within normal ranges for the cat, while cell counts in PL fluid were assumed to be normal based on clinical health during examination, auscultation and lactate dehydrogenase (LDH) activities being comparable with other species. The major clinical implication of this study was that nucleated cell counts within feline ELF could not be predicted from analysis of lavage fluid which suggests that calculation of the proportion of ELF in lavage fluid by the urea dilution method may be necessary to avoid misdiagnosis of health or disease in pulmonary or pleural cavities.     

Bronchoalveolar lavage in the cat: cytological findings.

Cells recovered by bronchoalveolar lavage from the lungs of specific pathogen-free (SPF) cats from birth to maturity and from adult conventional cats were enumerated and identified. The predominant cell type recovered was the pulmonary alveolar macrophage from all ages of both SPF and conventional cats. Other cell types included eosinophils, neutrophils and lymphocytes. Lavage of conventional cats yielded significantly more eosinophils and neutrophils than were recovered from SPF cats.     

Alveolar lavage in dogs

Alveolar lavage was performed in 10 healthy dogs. After tracheal intubation was done, a sterile fiberoptic bronchoscope was wedged in a distant bronchus and the lungs were lavaged with sterile saline solution. An average of 140 ml of saline solution was flushed into the lungs of each dog, and an average of 79% of the solution was recovered. Examination of the recovered fluid revealed average total cell counts of 6.4 X 10(6) cells/dog. Average differential cell counts were as follows: macrophages, 50.5%; lymphocytes, 46.0%; and neutrophils, 3.5%. Results of bacterial culture of the recovered fluid were negative in 8 dogs and positive in 2; Bordetella bronchiseptica was isolated in 1 dog and Klebsiella pneumoniae was isolated in the other.     

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.     

Flexible bronchoscopy and bronchoalveolar lavage in 68 cats (2001-2006)

BACKGROUND: Bronchoscopy is an important tool for identifying an underlying etiology for respiratory disease in cats. However, the procedure is challenging, because feline airways are small and prone to bronchoconstriction. HYPOTHESIS: Bronchoscopy and bronchoalveolar lavage (BAL) are appropriate and safe diagnostic procedures in the cat. ANIMALS: Sixty-eight cats. METHODS: Flexible bronchoscopy was performed in all cats with the cats under propofol infusion with jet ventilation. The procedures were reviewed for BAL volumes instilled and recovered and for the number and type of complications with the use of 3 flexible endoscopes < 5.0-mm outer diameter. The BAL procedure was compared among scopes by using a one-way analysis of variance. Complication rates were compared by using chi-square analysis. Significance was set at P < .05. RESULTS: Clinical diagnoses included inflammatory airway disease in 46 of 68 cats, pneumonia in 10 of 68, neoplastic disease in 8 of 68, and other conditions in 4 of 68 cats. Mean lavage volumes instilled for the 3 scopes were 2.62-5.05 mL/kg (range, 0.77-9.38 mL/kg). Mean percent fluid recovered for the 3 scopes was 51-73%, (range, 0-140%). BAL cell counts were adequate for cytologic assessment (> 300 cells/microL) in 61 of 64 cats (97%), and in 107 of 120 samples (89%) collected. Complications occurred in 38% of procedures; however, these were mild in 24% of cats; 6% of cats died or were euthanized after the procedure. Complications were not associated with fluid volume instilled or recovered, and could not be related to the underlying disease process. CONCLUSIONS AND CLINICAL IMPORTANCE: Flexible bronchoscopy with BAL was well tolerated in most cats examined.     

Light microscopic and ultrastructural characterization of cells recovered by respiratory-tract lavage of 2- and 6-week-old chickens

This study characterized the cell population recovered by respiratory-tract lavage of 57 two-week-old and 59 six-week-old specific-pathogen-free chickens as a prerequisite to study the response of the avian respiratory tract to infectious agents. The respiratory tract of each bird was lavaged through the trachea with a series of three lavages of 10 ml of room-temperature, neutral phosphate-buffered saline per lavage. The three lavages per bird were pooled for analysis. Total recovery volumes were measured, lavage fluid cellularity was determined, and a 200-cell differential count of non-erythrocyte cells was performed. Lavage fluid recovery was greater from 2-week-old birds (91.3 percent) than from 6-week-old birds (86.3 percent). Total cells recovered were greater for 6-week-old chickens (6.79 x 10(5)) than for 2-week-old chickens (5.03 x 10(5)). Cells of epithelial origin included squamous cells, goblet cells, and both ciliated and non-ciliated columnar epithelial cells. Cells of non-epithelial origin consisted of heterophils, lymphocytes, macrophages, eosinophils, basophils, and erythrocytes. Cells of epithelial origin were the predominant cell type recovered from the 2-week-old chickens, followed by heterophils. In 6-week-old chickens, heterophils were the predominant cell type recovered, followed by cells of epithelial origin. In descending order of prevalence, the remainder of cell types recovered from chickens of both ages were lymphocytes, macrophages, eosinophils, and basophils.     

Inflammatory cerebrospinal fluid analysis in cats: clinical diagnosis and outcome

The medical records of 62 cats with clinical signs of central nervous system disease and accompanying inflammatory cerebrospinal fluid (CSF) analysis were examined retrospectively to determine if signalment, clinical signs, CSF analysis and ancillary testing could accurately predict the type of central nervous system disease that was present. An inflammatory CSF was defined as one in which a total nucleated cell count was greater than 5 cells/microl or one in which the total nucleated cell count was normal but the nucleated cell differential count was abnormal. Sex, degree of CSF inflammation, neuroanatomical location and systemic signs provided little contributory information to the final diagnosis. In 63% of the cases a presumptive diagnosis could be made based on a combination of clinical signs, clinicopathological data and ancillary diagnostic tests. CSF analysis alone was useful only in the diagnosis of cats with feline infectious peritonitis, Cryptococcus species infection, lymphoma and trauma. Overall, despite extensive diagnostic evaluation, a specific diagnosis could not be made in 37% of cats. The prognosis for cats with inflammatory CSF was poor with 77% of cats surviving less than 1 year.     

Effect of repetitive bronchoalveolar lavage on cytologic findings in healthy dogs

OBJECTIVE: To determine reference values for cytologic examination results of bronchoalveolar lavage fluid (BALF) and to investigate effects of repeated lavages on pulmonary health and on results of cytologic examination of BALF in dogs. ANIMALS: 16 healthy adult Beagles. PROCEDURE: All dogs underwent pulmonary lavage to obtain BALF. Eleven dogs were repeatedly lavaged 6 times at 5- to 7-week intervals. Analyses for total and differential cell counts and for viability of cells before and after cell processing were performed. Arterial blood gas analysis before and after bronchoalveolar lavage was used to study the safety of the lavage procedure. Histologic and radiologic examinations were used to study effects of repeated lavages on pulmonary health. RESULTS: Mean (+/- SD) cell count was 104 +/- 69 cells/microl, comprising 75 +/- 7% alveolar macrophages, 13 +/- 6% lymphocytes, 5 +/- 4% neutrophils, 4 +/- 5% eosinophils, 2 +/- 2% mast cells, 0.6 +/- 0.7% epithelial cells, and 0.3 +/- 0.4% plasma cells. Centrifugation of samples and washing of cells caused significant cell loss (59 +/- 13%). Repeated lavages did not cause significant variations in cell counts of BALF or results of arterial blood gas analysis, thoracic radiography, or histologic examination of pulmonary specimens. Only a moderate, although significant, decrease in arterial oxygen content was observed after bronchoalveolar lavage. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis indicated that several lavages performed at 5- to 7-week intervals can safely and reliably be used to study the kinetics of pathologic processes in pulmonary tissues or for evaluation of therapeutic efficacy.     

Rush immunotherapy in an experimental model of feline allergic asthma

Specific allergen immunotherapy represents the only curative treatment of allergy. No studies have evaluated its efficacy in feline allergic asthma. We hypothesized that an abbreviated course of immunotherapy (rush immunotherapy, RIT) would blunt eosinophilic airways inflammation in experimental feline asthma induced with Bermuda grass allergen (BGA). The 6-month study included asthmatic-RIT treated cats; asthmatic-no RIT treated cats; and non-asthmatic cats. RIT involved increasing parenteral doses (20-200 microg) of BGA over 2 days. Numbers of eosinophils in bronchoalveolar lavage fluid (BALF), serum and BALF immunoglobulins, lymphocyte blastogenesis assays, and cytokines in blood and BALF were evaluated. BALF eosinophils decreased (P=0.048) only in asthmatic-RIT treated cats (baseline 1.1 x 10(6); Month 6, 2.4 x 10(5)). Serum BGA-specific IgG was higher (P<0.001) at all time points after baseline within the asthmatic-RIT group, and was higher (P<0.001) than asthmatic-no RIT cats at Months 1 and 3. No differences (P=0.133) in BGA-specific IgE levels over time were noted among asthmatic-RIT cats, but this group had lower IgE levels (P<0.001) levels than asthmatic no-RIT cats at Months 3 and 6. Differences in BGA-specific IgA levels over time and between the two groups did not reach the traditional level of significance. The mean BGA stimulation index in the asthmatic-RIT cats was biologically insignificant at 6 months, reflecting BGA-specific lymphocyte hypoproliferation. Preliminary results of cytokine profiles were not significantly different; however, BAL cytokine profiles favoring a Th2 response prior to RIT shifted to increased IFN-g and IL-10 thereafter. RIT dampens eosinophilic airways inflammation in cats with experimental asthma. The mechanism of RIT may involve changes in allergen-specific immunoglobulins, induction of hyporesponsive lymphocytes, or alteration of cytokine profiles.     

Profiles of type-II pneumocytes in rats inoculated intratracheally with bacterial lipopolysaccharide

Ultrastructural and morphometric profiles of type-II pneumocytes (P-II) were investigated in rats killed 18 or 24 hours after a single intratracheal inoculation of bacterial (Escherichia coli) lipopolysaccharide (LPS). Inoculation with LPS induced pulmonary injury and inflammation, as measured by increased lactate dehydrogenase and alkaline phosphatase activities and increased numbers of polymorphonuclear neutrophils in fluid collected by bronchoalveolar lavage. Marked ultrastructural changes and desquamation of a few P-II developed at the time of high activity of lactate dehydrogenase and alkaline phosphatase in bronchoalveolar lavage fluid. Ultrastructural changes included swollen mitochondria and localized cisternal dilatation of the endoplasmic reticulum in which was contained membrane-bound homogenous material of medium electron density. Twenty-four hours after LPS inoculation, point-count stereologic analysis and digitizing morphometry revealed greater than 50% increase in P-II size. Changes in cell size corresponded with ultrastructural finding of swollen cells. Results obtained by point-count stereologic analysis and digitizing morphometry were highly correlated (r = 0.95). Lamellar bodies (LB) comprised 12 to 15% of P-II volume. Volume density and number of LB remained unaltered in LPS-injured P-II, and evidence of accelerated release of LB was not detected after LPS inoculation. Exudated polymorphonuclear neutrophils and pulmonary alveolar macrophages were involved actively in the phagocytosis of LB originating from necrotic and desquamated P-II. On the basis of measurement of enzyme activity (enzymes released into the bronchoalveolar space), considerable ultrastructural alterations developed in P-II when maximal LPS-induced pulmonary cell injury took place.     

Using urea dilution to standardise components of pleural and bronchoalveolar lavage fluids in the dog

AIM: To develop a technique to estimate the volume of epithelial lining fluid (ELF) obtained during bronchoalveolar lavage (BAL) and pleural lavage (PL) in the dog, using the urea dilution method. METHODS: BAL and PL fluids were obtained by saline lavage of pulmonary and pleural cavities of nine clinically healthy mixed-breed dogs immediately after euthanasia. Cell counts in the BAL and PL fluids were measured using standard techniques. The concentration of ELF in each lavage fluid was calculated from the relative concentration of urea in plasma and in each type of lavage fluid. Cell counts in ELF were then calculated. RESULTS: There were substantially higher cell counts in ELF compared to BAL or PF fluid. However, nucleated cell counts in ELF could not be predicted from cell counts in BAL or PL fluid. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggest that accurate assessment of cellular or non-cellular components in lavage fluids should include a calculation of the proportion of ELF recovered, using a method such as urea dilution.     

Reference intervals for feline cerebrospinal fluid: biochemical and serologic variables, IgG concentration, and electrophoretic fractionation

Reference intervals are reported for feline CSF biochemical and serologic variables, IgG concentration, and electrophoretic fractionation, derived from 58 clinically normal adult cats that did not have histologic lesions of the CNS. There was no apparent effect of age on any variable. The CSF total protein concentration was significantly (P = 0.012) greater in males than in females, but all other variables were unaffected by gender. The only variable that had a statistically significant correlation with its corresponding blood concentration was IgG. Blood contamination of the CSF affected the following CSF variables: total protein concentration, activities of lactate dehydrogenase and creatine kinase, IgG ratio, and gamma-globulin percentage. The reference intervals proposed for feline CSF were derived from 33 cats with CSF RBC count less than 31 cells/microliters. Reference limits for CSF with 31 to 1,700 RBC/microliters also are reported.     

Analysis of transtracheal aspirates and pleural fluid from clinically healthy llamas (Llama glama)

Seventeen clinically healthy adult llamas were used to study the characteristics of transtracheal aspirates (TTA) and pleural fluid samples. Results of complete blood counts, fibrinogen determination and thoracic radiographs were within normal limits prior to sampling. Cytologic evaluation of TTA revealed the majority of cells were vacuolated macrophages (60-100%), with 0-40% neutrophils, and fewer lymphocytes (0-1%), eosinophils (0-3%), and ciliated respiratory epithelial cells (0-10%). In TTA from 10 of 17 llamas, neither aerobic nor anaerobic bacteria were isolated. Bacteria isolated in pure culture from TTA were similar to isolates found in clinically healthy animals of other species, and included Acinetobacter sp., Staphylococcus sp. and Bacillus sp. Results (mean +/- SD) of pleural fluid analyses were: total nucleated cell count 576 +/- 361/microliter; specific gravity 1.0133 +/- 0.002; glucose concentration 135.1 +/- 9.02 mg/dL; and lactate concentration 2.95 +/- 1.34 mg/dL. Pleural fluid total protein concentrations determined by refractometry ranged from < 2.5 to 3.5 g/dL. The refractive index ranged from 1.3396 to 0.0013. In pleural fluid, small lymphocytes were the predominant cell type.     

Interstitial lung disease in feline immunodeficiency virus (FIV) infected cats

Postmortem bronchoalveolar lavage of feline immunodeficiency virus-infected cats indicated an alveolitis process, and histological examination of their lungs confirmed the occurrence of alveolitis, parenchymatous lymphoplasmocytic infiltration and myomatosis. Similar lymphoid interstitial pneumonitis has been described in human and animal lentiviral diseases: lymphocytic interstitial pneumonitis in hiv-1-infected human beings, and maedi in sheep infected by the maedi-visna virus. Such lymphoid interstitial pneumonitis may thus be a common feature of lentiviral infections.     

Interstitial lung disease in feline immunodeficiency virus ( ) infected cats

Postmortem bronchoalveolar lavage of feline immunodeficiency virus-infected cats indicated an alveolitis process, and histological examination of their lungs confirmed the occurrence of alveolitis, parenchymatous lymphoplasmocytic infiltration and myomatosis. Similar lymphoid interstitial pneumonitis has been described in human and animal lentiviral diseases: lymphocytic interstitial pneumonitis in -1-infected human beings, and maedi in sheep infected by the maedi-visna virus. Such lymphoid interstitial pneumonitis may thus be a common feature of lentiviral infections.     

Antemortem diagnosis and treatment of toxoplasmosis in two cats on cyclosporin therapy

Clinical toxoplasmosis was diagnosed antemortem in two cats being treated with therapeutic doses of cyclosporin. The diagnosis was made by detecting tachyzoites on cytological examination of bronchoalveolar lavage fluid from one case and pleural effusion from the other. Despite early diagnosis and aggressive treatment in both cases, only one cat survived. Reactivation of latent Toxoplasma gondii infection secondary to cyclosporin-induced immunosuppression was considered likely in both cases. The presence of respiratory signs in cats treated with cyclosporin should alert clinicians to the possibility of clinical toxoplasmosis. Consideration should be given to determining the serostatus of cats to T gondii prior to use of drugs which are potent inhibitors of cell mediated immunity, such as cyclosporin. Two cases of feline toxoplasmosis are presented.     

Using urea dilution to standardise components of pleural and bronchoalveolar lavage fluids in the dog

AIM: To develop a technique to estimate the volume of epithelial lining fluid (ELF) obtained during bronchoalveolar lavage (BAL) and pleural lavage (PL) in the dog, using the urea dilution method.

METHODS: BAL and PL fluids were obtained by saline lavage of pulmonary and pleural cavities of nine clinically healthy mixed-breed dogs immediately after euthanasia. Cell counts in the BAL and PL fluids were measured using standard techniques. The concentration of ELF in each lavage fluid was calculated from the relative concentration of urea in plasma and in each type of lavage fluid. Cell counts in ELF were then calculated.

RESULTS: There were substantially higher cell counts in ELF compared to BAL or PF fluid. However, nucleated cell counts in ELF could not be predicted from cell counts in BAL or PL fluid.

CONCLUSIONS AND CLINICAL RELEVANCE: These results suggest that accurate assessment of cellular or non-cellular components in lavage fluids should include a calculation of the proportion of ELF recovered, using a method such as urea dilution.     

Adjuvanted rush immunotherapy using CpG oligodeoxynucleotides in experimental feline allergic asthma

Allergic asthma is driven by relative overexpression of Th2 cell-derived cytokines in response to aeroallergens. In independent studies, both allergen-specific rush immunotherapy (RIT) and CpG oligodeoxynucleotides (ODN) showed promise in blunting eosinophilic inflammation in a model of feline allergic asthma. We hypothesized that RIT using allergen and CpG ODN would work synergistically to dampen the asthmatic phenotype in experimentally asthmatic cats. Twelve cats with asthma induced using Bermuda grass allergen (BGA) were studied. Of these, six were administered adjuvanted BGA RIT using CpG ODN #2142; six were administered placebo (saline) RIT and later crossed over to adjuvanted RIT. Over 2 days, subcutaneous CpG ODN (0.5ng/kg) with BGA (increasing doses every 2h from 20 to 200microg) was administered. Adverse events were recorded and compared with historical controls. Percentage of eosinophils in bronchoalveolar lavage fluid (BALF), % peripheral CD4+CD25+ T regulatory cells (Tregs), lymphocyte proliferation in response to ConA, and cytokine concentrations in BALF were measured over 2 months. Group mean BALF % eosinophils for the adjuvanted RIT cats were significantly lower at week 1 and month 1 (p=0.03 for both), and marginally significantly lower at month 2 (p=0.09) compared with placebo RIT cats. By the end of the study, 8/12 treated cats had BALF % eosinophils within the reference range for healthy cats. Adjuvanted RIT, but not placebo RIT, cats had significant decreases in the ConA stimulation index over time (p=0.05). BALF IL-4 concentrations were significantly higher at week 1 in adjuvanted RIT cats compared with baseline and month 2, and also with placebo RIT cats at week 1. No significant differences were detected between treatments or over time for IL-10 or IFN-gamma concentrations in BALF or for %Tregs cells in peripheral blood. Adjuvanted RIT using CpG ODN in experimental feline asthma dampens eosinophilic airway inflammation. Adverse effects associated with adjuvanted RIT were less severe compared with a historical, non-adjuvanted RIT protocol. The exact mechanism(s) by which adjuvanted RIT alters the aberrant allergic immune response were not elucidated in this study.     

The synergism of Actinobacillus pleuropneumoniae and influenza A virus in experimentally-infected mice

Models for infecting mice with Influenza A-Virus (A/PR 8/34, H0N1) and Actinobacillus pleuropneumoniae (serotype 9) were developed in Han: NMRI-mice. After infecting mice with sublethal doses of one of the infectious agents, or both together as a mixed infection, animals were subsequently exsanguinated and the lungs washed by bronchoalveolar lavage. Clinical symptoms were recorded daily, examination of lung lavage fluid and sera as well as histology of the lungs were done. An increase in mortality, weight reduction and total cell yield of lung lavage fluid was observed after mixed infection. Compared to mixed infections total protein content and elastase in sera and lung lavage fluid after singular ones were raised not as much. In lung lavage fluid the total cell yield was increased more marked. These alterations indicate a synergistic effect of viruses and bacteria, developed by mixed infection as well as a bacterial infection on top of a viral one. Histopathologically the lung alterations were found to depend on the infectious agent and the mode of infection.