Animals and parasite strain
Male calves (n = 22) of Swedish dairy breeds (Swedish Red and White breed, or Swedish Holstein) were used. They were purchased from conventional dairy farms at the age of 2–3 months. The dairy farms were all declared free from bovine viral diarrhoea virus (BVDV) and enzootic bovine leucosis (EBL), according to the Swedish eradication programmes for these diseases. Before the start of the experiments the animals were allocated in smaller groups and were given an adaptation period of at least 4 weeks. They were kept on straw beddings and fed hay ad libitum and supplement according to weight. The infective D. viviparus third larvae (L3) used in the experiments were obtained from Intervet Nederland bv (Boxmeer, Netherlands). The L3s used for inoculation were fresh and obtained from donor calves. These larvae were incubated at 15°C and they were less than 3 weeks old when they were used for experimental infection.
Experimental design
The Swedish National Board for Laboratory Animals, Uppsala, Sweden approved the 3 experimental studies (I-III) performed. The average age of the animals at the start of the experiments was 21 weeks (range 19–23) in experiment I, 14 weeks (range 12–15) in experiment II and 27 weeks (range 23–31) in experiment III. In experiment I, 11 penned calves were inoculated orally once daily with 250 D. viviparus L3 on day 0 and 1 of the experiment. On day 35 post inoculation (p.i.) the animals were slaughtered and the lungs examined for gross lesions and presence of adult lungworms. These calves had 10 weeks earlier been inoculated with 500 larvae for 2 consecutive days, but the infection never reached patency as determined by faecal larval counts. This was likely due to that the L3s had been stored in water in tissue culture flask at 4°C for almost a year. In experiment II, 5 animals were inoculated once daily with 100 D. viviparus L3 on 5 consecutive days (day 0–4) at the start of the experiment. This experiment was finished on day 30. The calves in experiment II were not slaughtered, but were kept for other studies. In experiment III, 6 calves were inoculated with 2000 D. viviparus L3 on day 0. The experiment was finished on day 28 p.i., when the animals were slaughtered and the lungs examined for gross lesions and presence of adult lungworms.
Rectal temperatures were recorded daily throughout the adaptation periods and the experiments. Clinical signs such as coughing and depression were also recorded daily. Blood samples were taken day 0, i.e. before inoculation with D. viviparus larvae, and at 8 (experiment I) or 6 (experiment II and III) occasions after inoculation. Blood samples were obtained from the jugular vein in Venoject tubes with EDTA and without additive (Terumo Europe N.V., Leuven, Belgium). Samplings were always performed between 8 and 11 a.m. Faeces samples were collected from the rectum at the start of the experiments and then once weekly.
Analyses
The EDTA samples were analysed at the Department of Clinical Chemistry, Swedish University of Agricultural Sciences (SLU), for eosinophil numbers, using Cell-Dyn 3500 (Abbot Diagnostic Division, Abbot Park, IL, USA), and for fibrinogen concentrations by a kinetic method according to [5], using an automated analyser (Konelab 30, Konelab Corporation, Espoo, Finland). The values of eosinophils measured by Cell-Dyn were 2% higher compared to manual differential count, the correlation was 0.86. Samples without additive were centrifuged and the serum was collected and kept at -20°C until analysis of haptoglobin and SAA using Tridelta Phase™ Range Haptoglobin Assay and Phase™ Range Serum Amyloid A Assay (Tridelta Development Limited, Greystones, Co. Wicklow, Ireland). For haptoglobin and SAA, the intra- and inter-assay coefficients of variation were <4% and <10%, respectively. The specific antibody response reflecting patent D. viviparus infection was measured in serum using a diagnostic ELISA kit (Ceditest, IDO-DLO, Lelystadt, The Netherlands). In experiments I and III, infections were also confirmed by demonstration of lungworms at slaughter of the calves according to procedures described by [2] and [6] and/or by demonstration of larvae in faeces according to [12].
Statistical evaluation
A general linear model (GLM) for repeated measures was used in SAS for making statistical inferences of the dependent variables, namely: eosinophils, haptoglobin, SAA and fibrinogen. The values of APPs and eosinophils of the different days were also tested pairwise with the values day 0 using Dunnett adjustment to avoid mass significances.