Volume-controlled bronchopulmonary lavage of normal and pneumonic calves

Saline bronchopulmonary lavage of the right lung of 16 anesthetized calves was performed using a single-lumen cuffed endotracheal tube. The initial volume of saline introduced was based on the functional residual capacity (FRC) of the right lung lobes as determined from the proportional weights of the right (58% of total FRC) and left (42% of total FRC) lung lobes. Calves were divided into "pneumonic" and "normal" groups based on clinical signs. Five sequential washes were done on each calf. There was no difference in the percentage of total lavage fluid volume recoverable between normal (83.8 +/- 4.2%) and pneumonic (81.1 +/- 8.2%) calves. Cell yield in the initial wash was consistently greater than in subsequent washes for both normal (12.7 +/- 6.6 X 10(6) cells/kg body weight) and pneumonic (58.1 +/- 37.6 X 10(6) cells/kg body weight) calves, and constituted 62.0% (normal) and 75.4% (pneumonic) of the total recoverable cell yield. Total cell yields were higher (P less than 0.05) in pneumonic calves, primarily due to neutrophil leukocytes (PMN). Neutrophils constituted 53.7 +/- 25% of the total cell yield in the pneumonic calves, but only 12.3 +/- 9.5% in the normal calves. The pulmonary alveolar macrophage (PAM) was the major recoverable cell in normal calves (85.7 +/- 8.7% of total lavage cells). Macrophages constituted a smaller (42.9 +/- 23.5) percentage of the total lavage cells in the pneumonic group due to increased PMN numbers. Viability of recovered cells from the pneumonic calves (91.5 +/- 4.8%) was lower than for the normal calves (94.1 +/- 2.5%), but the difference was not significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnosis of bovine viral respiratory diseases

Enzootic bronchopneumonia (EBP) is an infectious, multifactorial respiratory disease of cattle. Different viruses may be involved in its pathogenesis. In this study an adapted method of endoscopic bronchoalveolar lavage (BAL) of caudal parts of the right cranial lung lobe was established and evaluated. The obtained bronchoalveolar lavage fluid (BALF) served as template for the detection of BRSV, BPIV-3 and BCoV specific nucleic acids by RT-PCR. BALF samples of 44 cattle affected with respiratory disease were compared to nasal swabs in their reliability to detect the causative agent(s). In 6/7 animals tested positive for BRSV, RNA of this virus was detected in the BALF, in 4 animals it could be found in the nasal swabs. In two of the three BPIV-3 positive animals, the BALF was the only material that tested positive. The most reliable samples for detection of 15 BCoV positive animals were the nasal swabs. BAL was easy to perform, it led to severe coughing in one case and moderate worsening of dyspnoe in three cases. In conclusion this study shows that BAL of the right cranial lung lobe is in many cases the only tool to detect BRSV and BPIV-3, major viral triggers of EBP.     

The pulmonary clearance of Pasteurella haemolytica in calves given Corynebacterium parvum and infected with parainfluenza-3 virus.

Four control calves were aerosolized with parainfluenza-3 and one week later with Pasteurella haemolytica. Three calves were given Corynebacterium parvum at a dose of 15 mg/m2 body surface area, infected with parainfluenza-3 virus one week later, and aerosolized with P. haemolytica two weeks after C. parvum injection. All calves were killed four hours after P. haemolytica exposure and the bacterial retention in the lung was determined. Parainfluenza-3 viral infection did not exert any suppressive effect on pulmonary clearance of P. haemolytica in six out of seven calves used. However, the bacterial colony counts in the lungs of control calves were higher (P less than 0.05) than those in calves given C. parvum. Hence, C. parvum appeared to enhance bacterial clearance. Despite the marked influx of neutrophils into the lungs after the bacterial inoculation, the neutrophil:macrophage ratio in lavage samples was less in calves given C. parvum than in the control calves. The alveolar macrophages in C. parvum treated calves were generally larger but did not differ significantly (P less than 0.05) from those in the controls. There was no significant (P less than 0.05) correlation between the percentages of alveolar macrophages and the bacterial clearance. In calves given C. parvum, bacterial clearance was enhanced in those calves which had larger macrophages.     

The effect of isoprinosine and levamisole on factors relevant to protection of calves against respiratory disease

Experiments to characterize the effects of two immunomodulators, namely, isoprinosine and levamisole, on factors relevant to the resistance of calves to respiratory infection were undertaken. Daily oral doses of isoprinosine decreased the influx of neutrophils into the respiratory tract, increased membrane immunoglobulin and complement receptor expression on cells from bronchoalveolar lavage samples and decreased the severity of respiratory disease. Additional intravenous doses produced similar effects on neutrophil migration to the respiratory tract and on membrane receptor expression, but the changes were no greater than those seen with oral isoprinosine alone. No significant changes in the anti-bacterial activity of cells in bronchoalveolar lavage samples followed isoprinosine treatment. In vitro incubation of pulmonary alveolar macrophages harvested from normal calves with isoprinosine increased their expression of immunoglobulin and complement receptors. Levamisole did not affect neutrophil migration to the lower respiratory tract or membrane receptor expression by pulmonary alveolar macrophages after in vivo or in vitro treatment. The immunomodulatory effects of isoprinosine beneficially increase the resistance of calves to respiratory disease, and are potentially useful in the control of infectious diseases of farm animals.     

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.     

Morphometric and ultrastructural study of postnatal lung growth and development in calves

The objectives of this study were to characterize basic patterns of postnatal lung growth in 1- to 150-day-old Holstein calves and to identify periods of accelerated lung growth and times of epithelial cell development that might correlate with periods of heightened susceptibility to pulmonary injury by infective agents and dietary toxins. Calves had fully developed alveoli at birth. There was an age-associated increase in total alveolar surface area and alveolar number, which was most marked in calves older than 30 days. Lung volume and body weight increases were also most marked when calves were older than 30 days. Mean bronchiolar cross-sectional area increased significantly (P less than 0.05) with age, and there was a significant (P less than 0.05) decrease in the percentage of terminal bronchioles with diameters less than 3 x 10(4) microns between 1 and 150 days. In newborn calves, nonciliated bronchiolar epithelial cells retained fetal characteristics of differentiation, including abundant cytoplasmic glycogen and sparse agranular endoplasmic reticulum. Glycogen deposits were depleted, and agranular endoplasmic reticulum was developed in nonciliated cells by the time calves were 30 days old. We concluded that bronchiolar epithelium in calves is well differentiated by the time they are 30 days old and that lung growth in calves raised in semi-isolation begins most rapidly when calves are approximately 30 days old.     

Eosinophilic pulmonary granulomatosis in a young dog with prolonged remission after treatment

A two‐year‐old Jack Russell terrier was presented for evaluation of chronic cough and exercise intolerance. Previous treatment with antibiotics and glucocorticoids had only partially ameliorated the clinical signs. During investigation, hypoxaemia, peripheral eosinophilia and an eosinophilic bronchoalveolar lavage fluid were noted. Thoracic radiographs revealed two ovoid clearly delineated soft‐tissue opacities, one in the caudal segment of the left cranial lung lobe (diameter 26 mm) and the other in the right cranial lung lobe (diameter 20 mm). These findings were verified by computed tomography, which identified an additional smaller lesion (diameter 16 mm) dorsally in the right caudal lobe. Ultrasound‐guided fine‐needle aspiration samples confirmed the diagnosis of eosinophilic pulmonary granulomatosis and treatment with prednisolone and azathioprine was initiated. Within 1 month, granulomas were no longer detectable radiographically. All medication was discontinued after 7 months and currently, after 2·5 years, the dog remains free of clinical signs. To the authors’ knowledge this is the first case report to describe prolonged remission from idiopathic canine eosinophilic pulmonary granulomatosis.     

Immunohistopathology of calf pneumonia induced by endobronchial inoculation with bovine adenovirus 3

Three 1-week-old and three 3-month-old Holstein calves that had received colostrum were inoculated endobronchially with bovine adenovirus 3 (BAV-3). The gross and histologic lesions in these six infected calves were localized mainly in the right caudal lobe of the lung and were closely associated with the site of the deposition of the inoculum. The pneumonic lesions were severe necrotizing bronchitis, bronchiolitis, and alveolitis, accompanied by infiltration of inflammatory cells and proliferation of type 2 pneumocytes. Intranuclear inclusion bodies, BAV-3 antigen, and virus particles were detected in the degenerated epithelial cells in the 1-week-old but not the 3-month-old calves. After infection, the total cell count in the bronchoalveolar lavage (BAL) fluid cells was increased. The results of BAV-3 isolation from BAL fluid were correlated with the detection of intranuclear inclusion bodies in the desquamated epithelial cells in the BAL fluid cells from the right caudal lobe but not in cells from the left caudal lobe. CD8+ T lymphocytes in the pneumonic lesion were found only in the 3-month-old infected calves. The difference in the immunopathologic reactions between the 1-week-old and the 3-month-old infected calves may be attributed to differences in immune system development.     

Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds

The aim of this study was to examine the occurrence of bacterial, mycoplasmal and viral pathogens in the lower respiratory tract of calves in all-in all-out calf-rearing units. According to clinical status, non-medicated calves with and without respiratory disease signs were selected of the 40 herds investigated to analyse the micro-organisms present in healthy and diseased calves. Tracheobronchial lavage (TBL) and paired serum samples were analysed for bacteria, mycoplasmas, respiratory syncytial virus (RSV), parainfluenza virus 3 (PIV3), bovine corona virus (BCV) and bovine adenovirus (BAV). Pasteurella multocida was the most common bacterial pathogen. It was isolated from 34% of the TBL samples in 28 herds and was associated with clinical respiratory disease (p < 0.05) when other pathogenic bacteria or mycoplasma were present in the sample. Mannheimia spp. and Histophilus somni were rarely found. Mycoplasma bovis was not detected at all. Ureaplasma diversum was associated with clinical respiratory disease (p < 0.05). TBL samples from healthy or suspect calves were more often negative in bacterial culture than samples from diseased calves (p < 0.05). No viral infections were detected in six herds, while 16-21 herds had RSV, BCV, BAV or PIV3. In the herds that had calves seroconverted to BCV, respiratory shedding of BCV was more frequently observed than faecal shedding. This study showed that the microbial combinations behind BRD were diverse between herds. M. bovis, an emerging pathogen in many countries, was not detected.     

Effect of sodium cromoglycate on light racehorses with elevated metachromatic cell numbers on bronchoalveolar lavage and reduced exercise tolerance

Some young horses with clinical signs of small airway disease demonstrate increased metachromatic cell numbers on bronchoalveolar lavage. The purpose of this study was to determine the effect of sodium cromoglycate treatment on clinical signs, bronchoalveolar lavage cytology and bronchoalveolar lavage histamine parameters in these horses. Twelve racehorses (age: 3.4 ± 1.6 years) with a history of respiratory embarrassment at exercise, clinical signs of obstructive airway disease and bronchoalveolar lavage metachromatic cell differential greater than 2% were selected. Horses were randomly assigned to receive either 200 mg sodium cromoglycate or saline placebo nebulized twice daily for 7 days. A clinical respiratory score was assigned and bronchoalveolar lavage was performed on each animal on days 0 and 7. Measurements were made of the following bronchoalveolar lavage fluid parameters: total nucleated cell concentration, differential cell percentage and concentration, supernatant and lysate histamine concentration, lysate: supernatant histamine ratio and metachromatic cell histamine content. Between the two evaluation periods, sodium cromoglycate treated horses demonstrated an improvement in respiratory score (P = 0.01) and a stabilizing of metachromatic cell histamine content (P = 0.04) when compared with placebo treated horses. We conclude that sodium cromoglycate is effective for the treatment of small airway disease in this population of young racehorses although the pharmacodynamics of this drug in the horse require further investigation.     

Pharmacokinetics and distribution in interstitial and pulmonary epithelial lining fluid of danofloxacin in ruminant and preruminant calves

The objective of this study was to compare active drug concentrations in the plasma vs. different effector compartments including interstitial fluid (ISF) and pulmonary epithelial lining fluid (PELF) of healthy preruminating (3‐week‐old) and ruminating (6‐month‐old) calves. Eight calves in each age group were given a single subcutaneous (s.c.) dose (8 mg/kg) of danofloxacin. Plasma, ISF, and bronchoalveolar lavage (BAL) fluid were collected over 96 h and analyzed by high‐pressure liquid chromatography. PELF concentrations were calculated by a urea dilution assay of the BAL fluids. Plasma protein binding was measured using a microcentrifugation system. For most preruminant and ruminant calves, the concentration–time profile of the central compartment was best described by a two‐compartment open body model. For some calves, a third compartment was also observed. The time to maximum concentration in the plasma was longer in preruminating calves (3.1 h) vs. ruminating calves (1.4 h). Clearance (CL/F) was 385.15 and 535.11 mL/h/kg in preruminant and ruminant calves, respectively. Ruminant calves maintained higher ISF/plasma concentration ratios throughout the study period compared to that observed in preruminant calves. Potential reasons for age‐related differences in plasma concentration–time profiles and partitioning of the drug to lungs and ISF as a function of age are explored.     

Effect of sex, age, number of bronchoalveolar lavages and quantitation methods on the bronchoalveolar cell counts in rats.

This study was conducted to investigate the effect of sex, age, number of bronchoalveolar lavages and method of quantitation on the number of bronchoalveolar cells in rats. Forty Long Evans rats were divided into four age-sex subgroups of ten animals each. Nine consecutive bronchoalveolar lavages were done in every rat and the number of bronchoalveolar cells/mL in lavages 1-3 (L1), 4-6 (L2) and 7-9 (L3) were determined by hemocytometer and electronic cell counts (Coulter Counter). The sex or age of the rats did not show a significant effect (p less than 0.05) in the number of bronchoalveolar cells recovered from the lungs; however, there was a significant difference (p less than 0.05) in the number of cells/mL among lavages 1-3, 4-6 and 7-9 (L1 approximately equal to L2 greater than L3). A discrepancy of approximately 8% in the counts of bronchoalveolar cells was found between the hemocytometer and the Coulter Counter; however, these two methods of cell quantitation showed a significant (p less than 0.01) positive correlation (r = 0.89). No significant (p greater than 0.05) differences were found in the percentage of fluid recovery (overall mean = 94.5%) among lavages L1, L2 and L3. It was concluded that the electronic cell counting of bronchoalveolar cells is as reliable as manual counting. Although sex or age did not significantly affect the number of cells recovered from the lung, caution should be used in the number of lavages done per rat since this variable may significantly affect the results.     

Relationship between cytologic changes in bronchoalveolar lavage fluid and weight gain in calves with gastrointestinal nematodes and lungworms

Two of three groups of 10 calves each were infected with either 100,000 infective larvae (L3) of Ostertagia spp. and 100,000 L3 of Cooperia spp. or with 4000 L3 of Dictyocaulus viviparus, respectively, at the age of 14 weeks. The third group was not infected. After treatment with an anthelminthic five calves from each group were challenged with either 100,000 L3 of Ostertagia spp. and 100,000 L3 of Cooperia spp. or 4000 L3 of Dictyocaulus at the age of 20 weeks. The calves were 25 weeks old when slaughtered. Total and differential cell counts were determined in bronchoalveolar lavage fluid and showed that neutrophils were the most frequent and eosinophils the least frequent cell present. There was a significant negative relationship between eosinophil levels and weight gain of the calves.     

Effect of steroidal and non-steroidal anti-inflammatory drugs on inflammatory markers in calves with experimentally-induced bronchopneumonia

The influence of treatment with steroidal (SAIDs) and non-steroidal (NSAIDs) anti-inflammatory drugs on inflammatory markers in thirty, 6-8 week old calves with induced bronchopneumonia was investigated. Animals received a single intravenous treatment with meloxicam (0.5 mg/kg body weight), flumethasone (0.05 mg/kg body weight) or sterile 0.9% NaCl (10 ml per animal). Body temperature, respiratory and heart rate, concentration of prostaglandins PGE2, PGF2alpha, thromboxane (TXB2), leukotriene (LTB4) and malonyldialdehyd (MDA) and proinflammatory cytokines i.e. tumor necrosis factor (TNFalpha) and interferon (INFalpha) were recorded in serum, bronchoalveolar lavage (BAL) and blood platelets (BP). A significant reduction of main inflammatory mediators PGE2, PGF2alpha,TXB2 and MDA after meloxicam treatment in calves with induced bronchopneumonia indicates a beneficial effect on the inflammatory processes. Contrary to effects observed by flumethasone, meloxicam induced an increase of LTB4 and INFalpha indicating that it is not immunosuppressive.     

Interaction of bovine respiratory syncytial virus with bovine alveolar macrophages in vivo: effects of virus infection upon selected cell functions.

The effect of bovine respiratory syncytial virus (BRSV) upon alveolar macrophage (AM) function was investigated using an in vivo calf inoculation model. Alveolar macrophages were collected sequentially from live calves at multiple time points during the 14 day period following viral inoculation. Alveolar macrophages from bronchoalveolar lavage fluids were purified by density gradient centrifugation (> 95% AM) prior to in vitro evaluation of cell functions. There were significant but variable and inconsistent differences in the functions of AM from the BRSV inoculated calves compared to the control calves. Fc-receptor mediated phagocytosis was either increased or unchanged by BRSV inoculation. Nonopsonized phagocytosis was decreased during the early postinoculation period and later increased. There was a variable effect on AM phagosome lysosome fusion with increased fusion activity on postinoculation days 2 through 5, 7 and 12 but reduced activity on days 6 and 10. The AM respiratory burst, as measured by nitroblue tetrazolium dye reduction, was essentially unaffected with a reduction in activity on day 10 only. In this model, BRSV inoculation of calves primarily resulted in an alteration of the membrane associated phagocytic functions of the alveolar macrophages (p < 0.05).     

Isolation of mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in Belgium

Between 1997 and 2000, a total of 150 healthy cattle and 238 animals with respiratory disease were examined for six Mycoplasma species. Attempts were made to detect Mycoplasma canis, Mycoplasma dispar and Ureaplasma diversum in calves with recurrent disease, and all three of these species were identified in calves with recurrent disease and in healthy lungs. In healthy calves, 84 per cent of bronchoalveolar lavage fluids were mycoplasma free; when cultures were positive, Mycoplasma bovirhinis was the only species isolated. Mycoplasmas were isolated from 78 per cent of animals suffering recurrent respiratory disease and from 65 per cent of acute respiratory cases. Mycoplasma bovis was isolated from bronchoalveolar lavages from 35 per cent of calves suffering recurrent respiratory disease, and from 50 per cent of acute cases, and from 20 per cent of pneumonic cases examined postmortem. M bovis was associated with other Mycoplasma species in 44 per cent of cases. M dispar was also isolated from 45.5 per cent of calves suffering recurrent respiratory disease, often in association with M bovis. M canis was identified for the first time in diseased Belgian cattle. Other mycoplasmas, including Mycoplasma arginini, Mycoplasma alkalescens and U diversum, were isolated less frequently. Associations between mycoplasmas and other pathogens were often observed. Among lungs infected with Pasteurella and/or Mannheimia species, more than 50 per cent were mixed infections with M bovis.     

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

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

The microbial flora of the respiratory tract in feedlot calves: associations between nasopharyngeal and bronchoalveolar lavage cultures.

The upper and lower respiratory tracts of 59 feedlot calves with clinical signs of naturally occurring respiratory disease (cases) and 60 comparison (control) animals were cultured before treatment, using nasopharyngeal swabs (NPS) and bronchoalveolar lavage (BAL). The most prevalent organisms were Pasteurella multocida and Mycoplasma bovis. Isolations of P. multocida from NPS and BAL fluid were found to be significantly associated with morbidity (p less than or equal to 0.05), but the frequency with which other organisms were isolated from the nasopharynx and lungs was similar in cases and controls. There was evidence of moderate agreement between NPS and BAL isolates at the individual calf level using the kappa statistic, (range of kappa values = 0.47-0.61) but the variability of the kappa statistics was large. Therefore, in an individual calf NPS cultures did not accurately predict BAL cultures. The NPS and BAL culture results were quite similar at the group level, however.     

Changes in leukocyte populations in pulmonary lavage fluids of calves after inhalation of Pasteurella haemolytica

The distribution of leukocytes in bovine bronchoalveolar lavage fluids was determined in 15 calves at various times after aerosol exposure to Pasteurella haemolytica. For comparison, 10 calves were exposed to aerosols of phosphate-buffered saline solution; 15 calves, to Staphylococcus epidermidis; and 10 calves, to Salmonella typhimurium endotoxin. At 10 minutes after inhalation exposure for each group, the predominant cell type was the macrophage. Macrophages remained the predominant cell type throughout each lavage interval for calves exposed to phosphate-buffered saline solution and Staph epidermidis. For calves exposed to P haemolytica, there was a decrease in the percentage of macrophages detectable by 30 minutes after exposure, with a corresponding increase in the percentage of neutrophils. Sixty minutes after the inhalation exposure to P haemolytica, the percentages of macrophages and neutrophils in the lavage fluid were equal. By 240 minutes after exposure to P haemolytica, greater than 90% of the cells in the lavage fluids was neutrophils. The increase in the percentage of neutrophils in lavage fluids from calves exposed to S typhimurium endotoxin was similar to that seen for the calves exposed to P haemolytica.     

Isotype-specific antibody responses to bovine coronavirus structural proteins in serum, feces, and mucosal secretions from experimentally challenge-exposed colostrum-deprived calves.

Five newborn isolation-reared colostrum-deprived calves were inoculated orally and intranasally when they were 20 to 30 hours old and challenge exposed when they were 21 days old with a suspension of virulent bovine coronavirus (BCV). Blood, feces, nasal swabs, tears, saliva, and bronchoalveolar lavage (BAL) fluids were collected from each calf prior to inoculation and then weekly for 5 post-inoculation weeks. An ELISA was used to quantitate the immunoglobulin isotype titers of BCV antibodies in all samples. An immunoblot assay was used to determine the antibody isotype responses to BCV structural proteins in all the samples, except saliva. At postinoculation days 2 to 3, all calves had severe watery diarrhea, shed BCV in their feces, and had evidence of BCV replication in their upper respiratory tract. After challenge exposure, no calves became ill and no evidence of BCV replication in the respiratory or intestinal tracts was detected. At postinoculation week 1, IgM responses to the N protein were seen in mucosal secretions (except nasal fluid) and feces. At postinoculation weeks 2 and 3, IgA was predominant in mucosal secretions and feces directed toward all the BCV proteins (except the E2 protein in BAL fluid). After challenge exposure, an increase (or failure to decrease) in most IgA and some IgG1 titers to BCV proteins was seen. The increases in IgA titers were to all viral proteins in all mucosal secretions and feces, except to the N and E1 viral proteins in feces. The IgG1 titer increases were to the E2 proteins in tears and BAL fluid and to the E3 protein in BAL fluid.(ABSTRACT TRUNCATED AT 250 WORDS)