Assessment of poultry red mite effects on infested hens

Weekly assessments of bird behaviour were performed from Week -6 through Week 6 during daytime by direct observation, and during night-time by video recording. In Weeks 0 and 1, these observations were completed three and four days following the first and second fluralaner administrations, respectively (Table 1).

*Points from which observations were made;

¹Removed 2 days after placement;

²First fluralaner administration, 2 or 3 days before trap placement (Weeks 0 and 1), 3 or 4 days before behavioural observations (Weeks 0 and 1), 1 day before blood sampling (Week 1)

Before beginning any daytime behavioural observation, the investigator would remain stationary in front of each cage for 10 minutes, after which bird behaviours would be recorded over a 30-minute period between 9 am and 2 pm. Six observational points were randomly selected throughout the building, focusing on the perch area of two adjacent cages. Behaviours were counted by focusing on a subset of no more than 12 hens, with observations recorded on a group, rather than on any individual bird [21]. On each day of assessment, the investigator would begin observations at 9 am, starting at the same point (Point 1) on each day, changing every 30 minutes to the next observational point and so on until reaching the final point (Point 6). The same cages were used for each observational period.

Two infrared equipped cameras were installed on two of the previous observation points, focusing on the perch area of two adjacent cages per observation point, the same cages for each observational period. Recordings started three hours after the onset of darkness (at midnight) when mites are most active.

Behavioural observations covered a 30-minute period, combining scan samplings and continuous behaviour recordings of selected variables (Table 2) [14, 22–24]. Specific active behaviours were recorded at group level through samplings for one minute every two minutes, meaning that 15 continuous behaviour recordings of 1-minute duration were recorded over a 30-minute period. For each 1-minute observation, the number of hen behaviours observed in the perch area was counted. Each behavioural category was considered as being mutually exclusive and was measured as the number of times the behaviour appeared in the group of hens observed. Any behaviour repeated by the same hen within 5 seconds of the previous observation of that behaviour was counted as one bout. The data were further expressed as incidence/bird/15 min-period. General activity was assessed through scan sampling every two minutes and expressed as the percentage of active hens in proportion to the total number of hens observed (active and resting). A total of 39h daytime and 13h night-time observations were made. All behavioural observations were carried out by the same investigator from the Universitat Autonoma de Barcelona.

Prior to beginning the study, 5 birds per cage from 10 cages were randomly selected and identified by leg rings. To avoid any interference of the sampling on bird behaviour, the cages sampled for physiological parameters were different from those recorded for behavioural observations. Blood samples were obtained from these birds one week prior to the first fluralaner administration (Week -6) and at 1 and 6 weeks following that administration. As blood sampling procedure can greatly influence stress hormone levels, particular care was taken when catching and manipulating the birds [25]. Three weeks prior to the start of the experiment the birds were habituated to the presence of the observers and manipulation. When the experiment started, the birds did not show any fear response when the observer opened the cages, touched and caught them gently. Blood was collected by venepuncture from the wing vein into a heparin tube and into a plain tube (1 ml per tube).

Red blood cells, haematocrit, haemoglobin concentration, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration were measured using an automated hematology analyzer (ADVIA 120 Hematology System, Siemens Healthineers, Spain). White blood cells and heterophil to lymphocyte ratio were counted manually by examination of blood smears using a modification of the Wright-Giemsa stain (Diff-Quik). For each smear, 60 white blood cells were manually counted using a light microscope at 100x magnification [26]. Heparin tubes were centrifuged for 10 minutes at 3000 g to obtain plasma. Plasma concentrations of ovotransferrin (Chicken Ovotransferrin ELISA Kit, 157694 Abcam^®, Cambridge, UK) and IgG (Chicken IgG ELISA Kit; Bethyl Laboratories, Montgomery, TX, USA) were determined using commercial enzyme immunoassay (ELISA) kits specific for chickens. Corticosterone was measured by a high sensitivity ELISA kit (Corticosterone HS (High Sensitivity) EIA, IDS^® Immunodiagnostic Systems, Boldon, UK) following manufacturer instructions. The intra-assay and inter-assay variability for this kit were less than 10% and the limit of detection was 0.17 ng/ml. The ELISA used for quantitative determination of adrenaline (epinephrine) (Adrenaline Research ELISA^™) had intra-assay and inter-assay variability less than 10% and a limit of detection of 0.25 ng/ml. Total oxidant status, paraoxonase 1 (PON1) and total antioxidant capacity (TAC; measured by trolox equivalent antioxidant capacity) were analysed following published methods [27–29]. All determinations of oxidative stress biomarkers were performed in an automated biochemistry analyser (Olympus AU600 Automatic Chemistry Analyzer, Olympus Europe GmbH, Germany).

Changes to a bird’s HPA axis were estimated from corticosterone levels measured in 5 to 8 fully regrown feathers pulled from the interscapular area of 25 of the 50 birds selected for bleeding for the pre- and post-treatment study periods. Feather samples were all collected on the same day at Week -1 and Week +6 and individually stored at room temperature until analysis. In order to obtain the same range of length (30 cm) and minimum mass of 10 mg, four to six feathers were analyzed per bird. Feather corticosterone was extracted following a modified methanol-based technique and measured as for blood corticosterone [30]. All samples were analysed at the Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU, University of Murcia, Spain).

At weekly intervals, clinical observations were assessed in an additional 50 birds from the house. Observations were scored using the following scales.

Feather score: 0 = no damage; 1 = slight; 2 = moderate; 3 = severe) for neck, breast, vent, back, wings and tail (modified [31])

Comb pecking wounds: 0 = none; 1 = ≤ 3; 2 = > 3 [32]

Comb colour: 0 = red; 2 = very pale comb

Red mite visibility on the hen’s body: 0 = no mites; 1 = a few; 2 = many

General observations of flock health were completed daily by the study investigator or by a qualified and trained staff member. Any bird dying during the study that had been identified for feather or blood sampling was not replaced. Carcasses and eggs were disposed of in accordance with legal requirements, including applicable withholding periods.

The impact of PRM infestations on flock performance and the effects of acaricidal treatment were assessed at weekly intervals using farm-recorded data from Week -6 to Week 6. Parameters that were assessed included weekly bird mortality rates, rate of egg laying (ratio of the number of eggs laid to the number of hens present) and egg weight.