The experiments complied with current Hungarian legislation and were authorised by the Food Chain Safety and Animal Health Directorate of the Somogy County Agricultural Office (Ref: 406-6/2002) based on the approval of the Ethics Committee of the Ministry of Agriculture.
Twenty-eight new-born piglets from a single closed herd were selected for each experiment. Clinical signs of AR had never been observed in the herd and toxigenic P. multocida had never been identified. No vaccination against AR had ever been undertaken on the farm. The P. multocida-free status of the selected animals was confirmed prior to initiating the study. Medicated early weaning was applied to obtain B. bronchiseptica-free piglets. The sows delivering the piglets had been treated with tulathromycin (Draxxin inj., Pfizer, New York, USA) administered in a dose of 1 ml/40 kg one week before farrowing. The piglets were moved to isolated units at 1–2 days of age where they were kept in heated compartments. Pigs were fed with Sprayfo milk replacer (Sloten, Deventer, Netherlands) until the age of 28 days using Mambo milk applicator device (Sloten, Deventer, Netherlands) according to the manufacturer’s recommendations. At the age of 28 days the piglets were weaned from milk replacer and then consumed only prestarter feed. From 6 to 10 weeks of age they were fed with starter diet, and then fattening diet until the termination of the study. Animals were fed ad libitum from self-feeders and had free access to drinking water. No treatments were permitted that could have interfered with the outcome of the trial, such as the use of immunosuppressive drugs or antibiotics affecting the respiratory tract.
Toxigenic strain KM22 of B. bronchiseptica was isolated from a herd with clinical AR. LFB3, a toxigenic isolate of P. multocida, was kindly supplied by Dr JM Rutter (Institute for Animal Health, Compton, UK). Both strains are known to produce the disease . B. bronchiseptica strain KM22, cultured on Bordet–Gengou (BG) agar (Oxoid, Basingstoke, UK) at 37C for 24 hours, was diluted in phosphate-buffered saline (PBS), pH 7.2, to give a suspension of about 106 Colony Forming Units/ml (CFU/ml). P. multocida strain LFB3, cultured on 5% sheep blood agar (BA) at 37C for 24 hours, was diluted in Brain Heart Infusion (BHI) broth (Difco, Detroit, USA) to give a suspension of about 108 CFU/ml. The toxin-producing ability of P. multocida strain LFB3 was confirmed by a membrane assay .
Two experiments were performed. Each experiment consisted of two groups of 14 randomly selected piglets. Group 1 piglets were infected with either B. bronchiseptica alone or B. bronchiseptica and P. multocida together. Group 2 piglets remained uninfected (negative) controls. The day of B. bronchiseptica infection was study day 0 (D0) of the study.
Piglets in Group 1 in Experiment 1 were infected with B. bronchiseptica at 4 days of age by instilling 0.5 ml of bacterial suspension into each nostril. Piglets in Group 1 in Experiment 2 were infected with B. bronchiseptica at 4 days of age by instilling 0.5 ml of bacterial suspension into each nostril, and with P. multocida at 8 days of age by the same method.
Experiment 1 was terminated at 132 days of age and Experiment 2 at 128 days of age. At termination, the pigs were slaughtered and examined in a blind manner for turbinate bone atrophy (TA), nasal septum deviation (NSD) and lung lesions.
Collection and bacteriologic examination of nasal secretions
Nasal swabs were taken from piglets at each CT examination. The samples were tested for the presence of B. bronchiseptica on MacConkey agar (BBL, Sparks, USA) as described elsewhere . The presence of P. multocida toxin in the primary cultures of nasal swabs was determined using a PMT ELISA (Oxoid, Hampshire, UK) according to the manufacturer’s recommendation.
To immobilise the pigs for the scanning procedure, 4 mg/kg azaperone (Stresnil, Janssen Pharmaceutica) was administered intramuscularly for sedation and 9 mg/kg ketamine hydrochloride (SBH-Ketamin inj., SelBruHa) was administered intravenously to anaesthetise the pigs.
The anaesthetised pigs were fixed in stretched position, lying flat in a purpose-designed container during the CT examination. CT images from each pig were acquired using a Somatom Plus 40 (Siemens, Erlangen, Germany) third generation scanner. The starting position of the scanning was set approximately 10 mm anterior from the level of the first upper premolar teeth, and then consecutive three mm scans were taken with a table feed of five mm. The imaging protocol was a so-called “high” algorithm, which is extremely sensitive in visualising tissues of high-density differences. Zoom factor was set to 3.5.
In Experiment 1, CT images of all pigs were acquired at study days 0, 9, 41, 70, 101, and 132 while in Experiment 2 they were obtained at study days 0, 4, 18, 25, 32, 60, 88, and 128. Non-infected pigs were always imaged prior to the infected pigs to avoid cross-contamination.
Interpretation of CT images
Visual scoring of the nasal turbinate bones was done on CT scans at the level of the first premolar teeth. Each of the four scrolls of the ventral turbinate bones was scored according to the following criteria (TA score): 0, no lesion; 1, a small part of the turbinate bone (nearly half a scroll) is absent; 2, slight atrophy – more than half a scroll is absent; 3, moderate atrophy – the turbinate bone is straightened; 4, severe atrophy – total disappearance of the turbinate bone. NSD was scored on a scale of 0–2: 0, normal; 1, slight deviation; 2, severe deviation. TA and NSD scores were summed for each individual to a maximum value of 18 (nasal lesion score, NLS).