The aim of the present work was to study the usefulness of cytology, bacteriological culture and PCR of the broncoalveolar fluid (BALF) in foals with abscesses of peripheral lung revealed by a thoracic ultrasound. Inclusion criteria: 14 trotter foals, 7 colts and 7 fillies, aged 1-4.5 months, with abscessation of peripheral lung diagnosed by thoracic ultrasound (5 MHz) were included in this study. Foals were born in the same stud farm and had the same management. Clinical signs: persistent cough, tachypnea, harshness of pulmonary sounds; some foals showed fever, nasal discharge, depression, anorexia. Broncoalveolar lavage (BAL) was performed in all the subjects, with the standing and not sedated foals. 180 ml of pre-wormed sterile saline was instilled and retrieved via a two stage sampling catheter passed through the biopsy channel of the endoscope. The external nostrils were cleaned to reduce contamination and cough during the procedure was avoided by instillation of local anesthetic. Samples were collected in EDTA tubes for cytology and in sterile tubes for microbiology and PCR; specimens were stored at 4°C and processed within 2h. EDTA samples were used for total cell count and cytocentrifugated. Smears were air-dried and stained with Diff Quick. Differential cell count was performed by counting 400 cells/smear. Diagnosis of pneumonia was carried out with cell total count/μL >300 and an increased neutrophils percentage with sign of toxicity.1 X±SD were calculated for total cell number/μl, macrophages, lymphocytes, neutrophils, eosinophils percentages. Samples with no anticoagulant were used for bacteriologic and biomolecular examinations. For bacteriology, Blood Agar with and without Streptococcus Selective Supplement, Mannitol Salt Agar and MacConkey Agar were used. The DNA for PCR was obtained from the enrichment broth inoculated with BALF.2 The DNA was used for PCR to identify Streptococcus equi subsp. equi, Streptococcus equi subsp. zooepidemicus, Streptococcus dysgalactiae subsp. equisimilis, Rhodococcus equi, Staphylococcus aureus e Staphylococcus intermedius.2-4 R. equi positive samples were subsequently tested by PCR for the detection of the vapA gene. The same PCRs were carried out to identify the bacterial colonies grown in culture and resembling β-hemolytic streptococci, R. equi or coagulase positive staphylococci. In foals with R. equi, X±SD for total cell/μl, macrophages, lymphocytes, neutrophils and eosinophils percentages were 1233.3±1270.2 cell/μL, 56.3±1.5%, 4±1%, 39.7±0.58%, respectively. In foals with bacterial pneumonia not caused by R. equi, X±SD for total cell/μl, macrophages, lymphocytes, neutrophils and eosinophils percentages were 477.3±173.7 cell/μL, 64.4±10.6%, 14.9±6.9%, 19.8±10.6%; 1.1±1.5%. Bacteriology and PCR detected S. zooepidemicus in 5/14, Coagulase Negative Staphylococci in 4/14 foals, and S. aureus, S. intermedius, α-haemolytic Streptococci, Corynebacterium spp., E. coli, Serratia marcescens, Proteus spp., Pseudomonas aeruginosa in 1/14 foal, respectively. R. equi was identified in 3/14 foals, and all the three strains resulted vapA positive by PCR. In 5/14 animals two ore more different bacteria were detected. In our study, foals with R. equi showed higher total cell number/μL (1233.3±1270.2 vs 477.3±173.7 cell/μL) and neutrophils percentage (39.7±0.58% vs 19.8±10.6%) than the subjects with pneumonia caused by other bacteria, while lymphocyte percentage resulted considerably lower (4±1% vs 14.9±6.9%). Bacteriology and PCR detected pathogenic R. equi in 3/14 foals considered “suspected” for rhodococcosis, while more frequently other bacteria potentially responsible for the clinical signs were identified. In these cases is thus suggested to carry out further laboratory investigations and to clarify the etiology in order to avoid the abuse of incorrect antibiotics and to increase antibiotic resistance. BALF culture is a reliable method to isolate bacteria responsible for pneumonia in foals, and it is required for a definitive diagnosis of R. equi. The PCR based on the vapA gene sequence is necessary to distinguish the so called “high patogenicity” strains from the environmental R. equi.5 The finding of S. zooepidemicus in BALF of 5/14 foals suggests that these bacteria could be pathogenic in horse.2 In conclusion, our results suggest that thoracic ultrasound may be considered only a good screening test to rapidly identify pulmonary abscessations. BALF total cell count and cytology seems to be able to grossly differentiate the inflammatory pattern of R. equi infection from other bacterial infections, but bacteriology and PCR are required to identify the etiological agents and to make the antibiotic sensitivity test.

BALF cytological, bacteriological and PCR evaluation in foals with peripheral lung abscesses revealed by a thoracic ultrasound

SGORBINI, MICAELA;CORAZZA, MICHELE;
2009

Abstract

The aim of the present work was to study the usefulness of cytology, bacteriological culture and PCR of the broncoalveolar fluid (BALF) in foals with abscesses of peripheral lung revealed by a thoracic ultrasound. Inclusion criteria: 14 trotter foals, 7 colts and 7 fillies, aged 1-4.5 months, with abscessation of peripheral lung diagnosed by thoracic ultrasound (5 MHz) were included in this study. Foals were born in the same stud farm and had the same management. Clinical signs: persistent cough, tachypnea, harshness of pulmonary sounds; some foals showed fever, nasal discharge, depression, anorexia. Broncoalveolar lavage (BAL) was performed in all the subjects, with the standing and not sedated foals. 180 ml of pre-wormed sterile saline was instilled and retrieved via a two stage sampling catheter passed through the biopsy channel of the endoscope. The external nostrils were cleaned to reduce contamination and cough during the procedure was avoided by instillation of local anesthetic. Samples were collected in EDTA tubes for cytology and in sterile tubes for microbiology and PCR; specimens were stored at 4°C and processed within 2h. EDTA samples were used for total cell count and cytocentrifugated. Smears were air-dried and stained with Diff Quick. Differential cell count was performed by counting 400 cells/smear. Diagnosis of pneumonia was carried out with cell total count/μL >300 and an increased neutrophils percentage with sign of toxicity.1 X±SD were calculated for total cell number/μl, macrophages, lymphocytes, neutrophils, eosinophils percentages. Samples with no anticoagulant were used for bacteriologic and biomolecular examinations. For bacteriology, Blood Agar with and without Streptococcus Selective Supplement, Mannitol Salt Agar and MacConkey Agar were used. The DNA for PCR was obtained from the enrichment broth inoculated with BALF.2 The DNA was used for PCR to identify Streptococcus equi subsp. equi, Streptococcus equi subsp. zooepidemicus, Streptococcus dysgalactiae subsp. equisimilis, Rhodococcus equi, Staphylococcus aureus e Staphylococcus intermedius.2-4 R. equi positive samples were subsequently tested by PCR for the detection of the vapA gene. The same PCRs were carried out to identify the bacterial colonies grown in culture and resembling β-hemolytic streptococci, R. equi or coagulase positive staphylococci. In foals with R. equi, X±SD for total cell/μl, macrophages, lymphocytes, neutrophils and eosinophils percentages were 1233.3±1270.2 cell/μL, 56.3±1.5%, 4±1%, 39.7±0.58%, respectively. In foals with bacterial pneumonia not caused by R. equi, X±SD for total cell/μl, macrophages, lymphocytes, neutrophils and eosinophils percentages were 477.3±173.7 cell/μL, 64.4±10.6%, 14.9±6.9%, 19.8±10.6%; 1.1±1.5%. Bacteriology and PCR detected S. zooepidemicus in 5/14, Coagulase Negative Staphylococci in 4/14 foals, and S. aureus, S. intermedius, α-haemolytic Streptococci, Corynebacterium spp., E. coli, Serratia marcescens, Proteus spp., Pseudomonas aeruginosa in 1/14 foal, respectively. R. equi was identified in 3/14 foals, and all the three strains resulted vapA positive by PCR. In 5/14 animals two ore more different bacteria were detected. In our study, foals with R. equi showed higher total cell number/μL (1233.3±1270.2 vs 477.3±173.7 cell/μL) and neutrophils percentage (39.7±0.58% vs 19.8±10.6%) than the subjects with pneumonia caused by other bacteria, while lymphocyte percentage resulted considerably lower (4±1% vs 14.9±6.9%). Bacteriology and PCR detected pathogenic R. equi in 3/14 foals considered “suspected” for rhodococcosis, while more frequently other bacteria potentially responsible for the clinical signs were identified. In these cases is thus suggested to carry out further laboratory investigations and to clarify the etiology in order to avoid the abuse of incorrect antibiotics and to increase antibiotic resistance. BALF culture is a reliable method to isolate bacteria responsible for pneumonia in foals, and it is required for a definitive diagnosis of R. equi. The PCR based on the vapA gene sequence is necessary to distinguish the so called “high patogenicity” strains from the environmental R. equi.5 The finding of S. zooepidemicus in BALF of 5/14 foals suggests that these bacteria could be pathogenic in horse.2 In conclusion, our results suggest that thoracic ultrasound may be considered only a good screening test to rapidly identify pulmonary abscessations. BALF total cell count and cytology seems to be able to grossly differentiate the inflammatory pattern of R. equi infection from other bacterial infections, but bacteriology and PCR are required to identify the etiological agents and to make the antibiotic sensitivity test.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/130397
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