From 23 samples (41%) two or more bacterial species were detected. the presence and interaction of bacteria and virus in the lower respiratory tract in calves with and without clinical symptoms of respiratory disease from six Danish herds. In addition, the serum concentrations of haptoglobin and serum amyloid A (SAA) were determined to describe the correlation between the presence of infectious agents and acute phase protein response. In order to avoid contamination from bacteria resident in the upper respiratory tract, the sampling was performed by trans-tracheal aspiration. This method has been recommended as optimal for evaluation of the microbiological status of the lower respiratory tract in order to elucidate the etiology of pneumonia in an animal (Espinasse et al., 1991, Rebbun, 1995, Virtala et al., 1996, Pommier, 1999, Pommier and Wessel, 2002). Finally, an aim was to evaluate whether PCR tests are suitable for obtaining a reliable and quick diagnosis of pneumonia related to bacterial pathogens. 2.?Materials and methods 2.1. Herds and samples The investigation included six dairy herds, which in previous years had experienced Aripiprazole (D8) problems with calf pneumonia during the winter period. In September 2002, 56 trans-tracheally aspirated samples were taken from clinically healthy animals in Aripiprazole (D8) these herds (herds 1, 3, 4, and 6: 10 samples; herd 2: 9 samples; herd 5: 7 samples). In November 34 samples from calves suffering from respiratory distress were taken from 4 of the herds (herd 1: 10 samples; herd 3: 13 samples; herd 5: 6 samples; herd 6: 5 samples). No animals were treated by antibiotics at the time of sampling. In two herds (herds 2 and 4), respiratory disease was not observed and, consequently, no samples were taken. In total, 90 trans-tracheal aspirations were obtained. The first samples were taken during a warm and dry September from clinically healthy calves (rectal temperatures below 39.5?C, no nasal discharge, no coughing, and an unprovoked respiration frequency lower than 40?min?1). All calves sampled in November showed clinical symptoms of pneumonic disease. Clinical disease was defined as a rectal temperature above 39.5?C in connection with nasal discharge, coughing, or an unprovoked respiration FAS frequency higher than 40?min?1. The age of the calves ranged from 14 days to 4 months. The average age of the clinically healthy calves sampled in September was 2.1 months (S.D.?=?1.1) and for the pneumonic calves sampled in November 2.8 months (S.D.?=?0.9). Only four of the Aripiprazole (D8) calves were sampled on both occasions (one calf in each of herd 3 and 5, and two calves in herd 6). Blood samples were taken from all clinically healthy calves in connection with the first visit in September. In addition, paired blood samples (3 weeks interval) were taken in NovemberCDecember after onset of clinical disease in the four herds where pneumonia was observed. An area of 3?cm??3?cm located 7C10?cm distal to the larynx was shaved and decontaminated with 70% alcohol and iodophors. The Aripiprazole (D8) calves were sedated by intramuscular injection of 0.1C0.2?mg/kg xylasine. After injection of local analgesic (0.5?ml 2% lidocain), a longitudinal incision of 1 1?cm was made in the midline directly above the trachea. Perforation of trachea was done with an Intraflon? 12G between two cartilage rings. A male dog urinary catheter was inserted into the Intraflon? and pushed down into the airway until a slight resistance was felt. Between 20 and 40?ml sterile 0.9% NaCl were injected through the catheter and followed by immediate aspiration. This resulted normally in 5C7?ml aspirated fluid. Aripiprazole (D8) One milliliter aspirated fluid was centrifuged for 3?min at 16,000?? was performed by PCR.