Petersen et al. Parasites Vectors (2021) 14:181 https://doi.org/10.1186/s13071-021-04685-7 Parasites & Vectors RESEARCH Open Access Assessment of fluralaner as a treatment in controlling Dermanyssus gallinae infestation on commercial layer farms and the potential for resulting benefits of improved bird welfare and productivity Ivo Petersen1*, Katharina Johannhörster2, Eric Pagot3, Damian Escribano4,5, Eva Zschiesche1, Déborah Temple6 and Emmanuel Thomas1 Abstract Background: Poultry red mite (PRM) (Dermanyssus gallinae) infestations are a cause of anaemia, impaired productiv- ity and stress-related behaviours linked to reduced hen welfare. A study investigated the potential health, welfare and productivity benefits following fluralaner treatment to eliminate PRM from infested hens. hens). Fluralaner ­(Exzolt®; 0.5 mg/kg body weight) was administered twice, 7 days apart (Weeks 0 and 1), via drink- Methods: A PRM-infested layer house was selected on a free-range farm (5400 hens) and an aviary farm (42,400 ing water. Mite populations were monitored by traps. Cameras recorded nighttime hen behaviours weekly, pre- and post-treatment. On the free-range farm, daytime behaviours were also recorded weekly. For pre- and post-treatment corticosterone assessments, eggs were randomly collected on both farms, and blood samples were collected from 50 randomly selected aviary farm hens. Production parameters were assessed using farm records. Results: Throughout the post-treatment period, fluralaner efficacy against PRM was > 99% on both farms. On the aviary and free-range farms, treatment was followed by significant nighttime increases in the proportion of resting hens (P < 0.0001; P = 0.0175, respectively). Significant post-treatment versus pre-treatment nighttime reductions were observed in head shaking (aviary, P < 0.0001; free-range P = 0.0233) and preening (P = 0.0032; P = 0.0018) and on the aviary farm in bouts of body shaking (P = 0.0108), vertical wing shaking (P = 0.0002), head scratching (P = 0.0335), and gentle feather pecking (P < 0.0001). On the free-range farm there were significant daytime reductions in head scratch- ing (P < 0.0001), head shaking (P = 0.0492) and preening (P = 0.0012). Relative to standard production parameters, no differences were detected on the aviary farm, but on the free-range farm the laying rate decline with increasing age was less than expected and the increase in egg weight greater than expected. Post-treatment increases in egg and plasma corticosterone were suggestive of stress factors in addition to mite infestation. Red blood cell counts and haematocrit increased following treatment. *Correspondence: ivo.petersen@msd.de 1 MSD Animal Health Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​ mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​mmons.​org/​publi​cdoma​in/​ zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Petersen et al. Parasites Vectors (2021) 14:181 Page 2 of 10 Conclusion: Fluralaner treatment eliminated mite challenge, leading to improved hen welfare and health, based on reductions in stress-related behaviours and restoration of the anaemia-inducing effects of mite blood feeding. Keywords: Dermanyssus gallinae, Fluralaner, Hen welfare, Hen health, Isoxazoline, Poultry red mite, Layer hens Background respectively [12]. In vitro studies have demonstrated Infestations with Dermanyssus gallinae, the poultry red that field isolates of the northern fowl mite (Ornithonys- mite (PRM), an obligatory blood feeder, have a negative sus sylviarum) and the PRM that are highly sensitive to effect on all types of poultry production, ranging from fluralaner are relatively insensitive to other compounds, backyard and organic farms to intensive, enriched cage including spinosad, phoxim, propoxur and deltamethrin or barn systems [1]. Anaemia can be a consequence of [13, 14]. The mite-killing activity of fluralaner is evident heavy infestations, and the PRM has been shown to be a within 4 h of the first administration and is maintained vector of important viral and bacterial pathogens, includ- for at least the next 14 days [15]. Adult mites are killed ing Salmonella enteritidis and avian influenza virus [2– quickly, while any egg or non-feeding larvae present at the 4]. Laboratory infestations of poultry have been found time of treatment are subsequently killed as they mature to be a cause of stress and impaired welfare, manifested to nymphs and take a blood meal, with mite populations by behavioural changes such as an increase in grooming reported to decline by as much as 99% within 3 days of and head scratching [5], increased levels of plasma corti- the first fluralaner administration and up to 100% within costerone, adrenaline and β-globulins, and reductions in 2 days of the second administration [16]. Elimination of γ-globulins in infested hens compared to uninfested con- the mites in this way may reduce or remove the welfare trols [6, 7]. Thus, the findings of moderate somatic and and productivity effects of infestations [4, 16, 17]. A study high psychogenic stress factors (corticosterone secretion was conducted in Europe to generate further evidence of and adrenaline secretion, respectively) in infested birds the potential benefits of elimination of PRM infestations. indicate that PRM infestations are a cause of impaired welfare, which in turn can lead to impaired productivity. Methods Annual PRM control and production losses in laying The study was conducted on two commercial egg produc- hens was estimated in a 2005 paper to be €130 million tion layer sites, one free-range farm in Germany housing [8]. In 2021 terms this is likely to be an underestimate LSL hens, the other an aviary farm in France with Lohm- because: Infestation rates in European flocks are now ann Brown hens. The objective was to assess the effects believed to be much higher [4]; until the 2017 introduc- of mite elimination by an acaricidal treatment with flu- tion of fluralaner there was a declining efficacy in treat- ralaner on behavioural welfare parameters in chickens ments [3, 4]; for safety reasons many treatments that naturally infested with D. gallinae. The potential ben- were used have been withdrawn [3, 4]; perhaps associ- efits of mite elimination on bird health and productivity ated with those changes, a larger proportion of flocks are were monitored by comparing pre- and post-treatment now believed to be infested (in 2013, in some European parameters and by assessing production parameters for countries, flock infestation rates were reported at 100%) each flock against the published standards for the age and [8]. Additionally, there has been a substantial increase breed of hen on each farm. All procedures were in align- in the number of laying hens in Europe [4]. These losses ment with the principles of Good Clinical Practice VICH are attributed to the negative impact of PRM infestations GL9 (GCP) [18]. An informed consent was completed by on the feed conversion ratio, egg production, proportion the farm owners prior to any enrolment and initiation of of downgraded eggs, susceptibility to intercurrent dis- treatment. ease and mortality rate [1, 3, 4, 9]. The problem of flock To qualify for the study, a farm had to have a known infestations with the PRM has been exacerbated by the history and current presence of PRM infestation, along difficulties of implementing measures that are effective in with a facility in which a PRM challenge could be moni- the face of increasing resistance to registered chemicals, tored, to have suitable equipment for accurate delivery while being safe for birds, workers and the environment of fluralaner via drinking water (dosing pump or medi- and avoiding egg and meat residues that could have a det- cation tank) and to accept the installation of cameras to rimental effect on human health [1, 4, 10, 11]. monitor bird behaviours. On each farm, a single house Fluralaner is an isoxazoline compound that is approved was used for the study. To avoid cross-contamination to for the treatment of poultry red mite infestations by two the study house, the farmer was required to accept flu- drinking-water administrations with a 7-day interval and ralaner administrations of all other farm houses concur- having egg and meat withholding times of 0 and 14 days, rently with study bird administrations, and to avoid use Petersen et al. Parasites Vectors (2021) 14:181 Page 3 of 10 of any acaricidal product, other than the scheduled flu- 6. On the aviary farm, 24 traps were placed under perches ralaner treatment, during the 2 weeks prior to starting at weekly intervals from Week -5 to Week 6. After 1 day and throughout the study period. (free-range farm) or 2 days (aviary farm), traps were col- The study was initiated with pre-treatment observa- lected and individually sealed in small plastic bags, each tions during July and August of 2018 in the free-range of which was then placed in a large plastic bag and stored and aviary farms, respectively, and concluded in Sep- at − 18 to − 20 °C or colder before being sent to a labora- tember and October of the same year. The study house tory with experience in identifying mites by species and in on the free-range farm was naturally ventilated, with counting and differentiating mite stages. The mites in each both natural and artificial light from 04:00 until 20:00 and trap and its plastic bag were poured into a Petri dish, and contained 5400 chickens, with a mean body weight of 1.8 mites or eggs remaining on the cardboard of the trap or in (± 0.1) kg. Birds were allowed to roam outside during the plastic bag were thoroughly collected and added to the daytime. On the aviary farm, the study house contained mites in the dish. Mite eggs, larvae and nymphs and adults 42,400 birds, 66 weeks old with a mean body weight of were differentiated and counted separately. In traps with 1.8 ± 0.2 kg at Week − 5. For both farms, feed was pro- up to 250 mg of mites (total weight of eggs and mobile vided according to standard procedure and water was stages), all D. gallinae were differentiated and counted. If provided ad libitum. Formal health assessments of study the weight of collected mites exceeded 250 mg, a subsam- birds were completed by a veterinarian at weekly inter- ple of approximately 100 mg was used. vals, and general observations were made by farm staff throughout the study, with any abnormal observations to Behavioural observations be notified to the veterinary investigator. On each farm, two cameras were used to monitor hen behaviours once per week. On the free-range farm, one Treatment camera recorded daytime behaviours, the other (infra- At the time of initial treatment, in August on the free- red) nighttime behaviours. Two infra-red cameras were range farm and September on the aviary farm, birds used on the aviary farm, where only nighttime behav- were 62 and 71 weeks of age, respectively. Before treat- iours were monitored. Selection of the observation field ment it was ensured that the drinking water system for each camera was based on the expected number of worked and was free of leaks and that there was no other birds present during the observation times; approxi- ­(Exzolt®, MSD Animal Health, Germany) was adminis- drinking water source available. The fluralaner solution mately 50 birds were observed at the aviary farm and approximately 30 birds at the free-range farm. Each tered according to label directions to achieve a dose rate recording began 3 h after the onset of darkness, when of 0.5 mg/kg body weight, twice with a 7-day interval mites are most active, at the same time on the same day (Weeks 0 and 1) (Table 1) [12]. The required volume of in each week, beginning at Week − 6 (free-range farm) or product was calculated using the body weights of 20 rep- − 5 five (aviary farm) and continuing to Weeks 5 and 6, resentative birds to estimate the total body weight of the respectively. All camera recordings were evaluated by a group of chickens to be treated. That volume was added single trained observer. Hens were scored, higher scores to 1 day’s water consumption, based on farm records of indicating greater stress, for the behaviour categories the previous day’s consumption. On the aviary farm a of body shaking, vertical wing shaking, gentle feather review of records indicated that an overestimation of pecking, severe feather pecking, aggression, head shak- the number of hens in the study house resulted in the ing, head scratching and preening [17]. Behavioural cat- labelled dose rate being exceeded by 11%. The duration egories were mutually exclusive. Observations included of treatment administration ranged from 7 h 45 to 10 h. continuous recordings of each targeted behaviour every 2 min for 1 min over a 60-min period and scan samplings Assessments of challenge with Dermanyssus gallinae of hen resting time. The percentage of resting hens at To determine the level of mite challenge, PRM traps each observation point was expressed in proportion to (Avivet) were used. These traps comprised a black Tylene the total number of observed hens. The number of events tube, 50 mm in length, with inner and outer diameters of for a behaviour was expressed as the number of bouts 12 and 16 mm, respectively. The tube contains rolled cor- (number of times a behavioural element was observed) rugated cardboard. These commercially available traps per hen within 15 min in proportion to the total number have been validated for quantitative assessment of PRM of hens observed. The number of resting and active hens infestations and were placed in the study houses accord- was assessed by scan sampling every 2 min. Each cat- ing to the manufacturer’s guidance [19]. On the free-range egory was measured as the number of events or animals farm, 20 traps were placed near the lowest pole of the performing the behaviour. perches, above the manure pits, in Weeks − 6, − 1, 0 and Petersen et al. Parasites Vectors (2021) 14:181 Page 4 of 10 Table 1 Schedule of study activities Data collected Units Study week −6 −5 −4 −3 −2 −1 0 1 2 3 4 5 6 Mite traps ≥ 20 traps Xa Xb Xb Xb Xb X X Xb X Xb Xb Xb X d Treatment House X X Observations Behaviour (day) Video recording Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa c a b Behaviour (night) Video ­recording X X X X X X X X X X X X Xb a Health 100 birds X X X X X X X X X X X X X Samplings Blood n = 50 (randomly selected in flock) Xb Xb Xb a b b a Eggs n = 40 (randomly selected) X X X X Xb Xa a Production House X X X X X X X X X X X X X a Free-range farm only b Aviary farm only c Two cameras used on aviary farm d First fluralaner administration, 2 or 3 days before trap placement, 3 or 4 days before behavioural observations (Weeks 0 and 1), 1 day before blood sampling (Week 1) Haematology and stress indicators Production parameters Blood samples for complete blood counts and blood Farm production records were accessed to determine the chemistry measurements were collected during Weeks hen weekly mortality rate (%), mean (%) laying rate and − 2, 0 and 6 from 40 randomly selected hens on the avi- mean egg weights for each study week. ary farm. On the aviary farm, 40 eggs were randomly col- lected during Weeks − 3, − 1 and 6 and on the free-range Statistical analysis farm during Weeks − 6 and 0, and 31 eggs were collected Statistical units were: the trap for the assessment of PRM on this farm during Week 6. Corticosterone concentra- infestation, the house for general health observations tions in blood samples were estimated from hens on the and performance evaluation, the observational point for aviary farm and in egg samples from both study farms. behavioural evaluation, the individual animal for assess- The red blood cells, haematocrit, haemoglobin concen- ment of body weight and blood sampling and health tration, mean corpuscular volume, mean corpuscular status evaluation, and the egg for egg collection and cor- haemoglobin and mean corpuscular haemoglobin con- ticosterone determination. centrations were measured using an automated hematol- The antiparasitic efficacy was calculated for each post- ogy analyzer (ADVIA 120 hematology system, Siemens treatment time point using the formula: Healthineers, Spain). Stained blood smears (Diff-Quik) Xpre − Xpost were examined under 100× light microscopy [20] to %efficacy = .100 manually count 60 white blood cells and determine the Xpre heterophil to lymphocyte (H:L) ratio. Heparin tubes where Xpre is the arithmetic mean pre-treatment mite were centrifuged for 10 min at 3000 g to obtain plasma. (mobile stages: larvae, nymphs, adults) count per trap, Corticosterone levels of egg extracted albumin and yolk and Xpost is the mean post-treatment mite count per trap. and also of the plasma samples were measured by a high- Product efficacy was claimed if the percentage efficacy EIA, ­IDS® Immunodiagnostic Systems, Boldon, UK) fol- sensitivity EIA kit (Corticosterone HS [High Sensitivity] exceeded 90% and if the post-treatment mite counts were significantly less than pre-treatment counts (two-sample lowing the manufacturer’s instructions. The extraction t-test). procedure for egg albumin and yolk corticosterone used Changes in production data (weekly mortality rate, a procedure previously described by Cook et al. [21], and mean laying rate and mean egg weight) were analysed for results were expressed as nanograms per gram of the significant changes over time using a linear regression freeze-dried sample taken for extraction. All tests were model. The slope of the regression line was compared to performed at the Interdisciplinary Laboratory of Clinical zero, a positive slope indicating an increase with time, a Analysis (Interlab-UMU, University of Murcia, Spain). negative slope a decrease with time. Laying rates and egg weights were assessed in relation to published standard Petersen et al. Parasites Vectors (2021) 14:181 Page 5 of 10 production data for the breed of bird used on each farm P = 0.0018) (Table 2; Figs. 3, 4). Nighttime body shak- [22, 23]. Pre- and post-treatment behavioural observa- ing (P = 0.0108), vertical wing shaking (P = 0.0002), tions were compared using a mixed linear model. The head scratching (P = 0.0335) and gentle feather pecking number of bouts (or % activity of hens) was the depend- (P < 0.0001) were also significantly reduced on the avi- ent variable; study phase (pre- or post-treatment) and, ary farm, where there was no significant interaction of for the free-range farm, day and night observation point study phase and observation point (P = 0.6859). Daytime interactions were the main effects to be investigated; observations from the free-range farm found significant study week was the repeated factor and observation point post-treatment versus pre-treatment reductions in head the random factor. The physiological parameters were scratching (P < 0.0001), head shaking (P = 0.0492) and analysed by comparing each post-treatment value with preening (P = 0.0012). the pretreatment value using a two-sided two-sample t-test. The statistical tests were conducted using a thresh- old of α = 0.05. Physiological and haematological analyses Corticosterone levels in egg yolk samples from the free- range farm were 9.3 (± 6.5) ng/ml prior to treatment Results (Week − 6), 17.7 (± 6.7) during the week of treatment There were no adverse events reported from either farm (Week 0) and 17.6 (± 6.7) in samples collected in Week following treatment. 6. The difference in corticosterone levels between Week 0 and Week 6 was not significant (P = 0.9321). On the Counts of Dermanyssus gallinae aviary farm corticosterone in egg yolk and albumen Prior to treatment, the mite counts on the aviary farm and plasma increased at each assessment from pre- comprised approximately 70% adults and 30% nymphs treatment through the week of treatment (Week − 1) to and on the free-range farm approximately equal propor- reach their highest levels at the final assessment (Week tions of adults and nymphs, with only a few larvae (≤ 1% 5) (Table 3). of motile mite life stages) identified on each farm. On Haematology results (only monitored on the aviary both farms, there was a sharp and statistically significant farm) revealed significant increases from Day 0 to Day decrease in mite counts following the first administra- 42 in erythrocytes, haematocrit, leukocytes, heterophils tion of fluralaner (Fig. 1). Efficacy was 100% by Week and lymphocytes (Table 4). The heterophil:lymphocyte 1 on the aviary farm (P < 0.0001) and remained at that ratio at Week 6 was significantly higher than at Week 0. level until Week 6 when a single mite was identified in one trap, while on the free-range farm the reductions in Production assessments mite counts following treatment were > 99% at Week 0 The weekly average mortality rate did not significantly (P = 0.0014) and at Week 6 (P = 0.0014), when the mean change with time on either the free-range farm (0.04% mite count was 1.4 (range 0–19). The antiparasitic effi- to 0.24%) (P = 0.3583) or the aviary farm (0.21% to cacy of fluralaner against blood-feeding stages of D. gal- 0.44%) (P = 0.6509). Consistent with the industry stand- linae was therefore achieved, and the post-treatment ard, the laying rate of the LSL hens on the free-range challenge to the birds is considered to have been elimi- farm showed a significant decline as the birds aged nated or reduced to clinically irrelevant levels. (P = 0.0054), but this decline occurred at a lower rate (0.27% per week) than the published standard for LSL Behavioural observations hens (0.42% per week) of the same age and time in pro- On both farms, fluralaner treatment and mite elimina- duction. Egg weights on this farm on Weeks − 6, 0 and tion were followed by significant reductions in night- 6 were 63.3, 65.9 and 66.6 g, an increase that was sig- time activity (i.e., increase in the percentage of resting nificant with time (P = 0.0008), and a mean increase of hens) (Fig. 2: aviary farm, P < 0.0001; free range farm, 0.28 g per week, while, according to the breed standard, P = 0.0175). Following fluralaner treatment of birds on an increase of 0.10 g per week would be expected. The the aviary farm, the percentage of resting hens increased rate of decline of the laying rate of the Lohmann Brown quickly, from 86.7% and 79.5% (Cameras 1 and 2, respec- hens on the aviary farm (0.56% per week; P = 0.0288) was tively) at Week − 2 to > 95% from Week 1 through the similar to the mean decrease of the standard laying rate remainder of the study in both cameras. Significant post- expected for birds of the same age and breed. Although treatment versus pre-treatment nighttime reductions the egg weights of study hens on that farm (range 65.1 were observed in both head shaking (aviary, P < 0.0001; to 65.6 g) did not change significantly as the birds aged free-range P = 0.0233) and preening (P = 0.0032; (P = 0.4065), throughout the study egg weights were Petersen et al. Parasites Vectors (2021) 14:181 Page 6 of 10 Fig. 1 Arithmetic mean and standard deviation of Dermanyssus gallinae counts from traps placed weekly on the aviary farm and during Weeks − 6, − 1, 0 and 6 on the free-range farm (aviary farm, Weeks 0 through 6 versus Week − 1, *P < 0.0001); free-range farm (points with no line, Weeks 0 and 6 versus Week − 1, ‡P = 0.0014). Arrows indicate fluralaner administrations (Weeks 0 and 1) Fig. 2 Percentage of resting hens observed in nighttime video recordings for each observation point. Arrows indicate fluralaner administrations (Weeks 0 and 1) approximately 3 g below the breed standard weight for mite was detected after Week 1 on the aviary farm, on hens of the same age. which a calculation error resulted in administration of a dose rate 11% higher than recommended. As fluralaner efficacy on the free-range farm was equivalent to that on Discussion this farm, and as efficacy at the recommended dose rate This study provides further confirmation of laboratory has been consistently shown to approximate 100% on and field studies showing that the rapid onset and high other layer farms, it can be concluded that this increase efficacy of fluralaner allows rapid control of the PRM in in dose rate did not affect the results, and there were no infested poultry flocks. On the free-range farm, the mean treatment-related adverse events [15–17]. Nonetheless, mite count at the end of the study was 1.4. Just a single Petersen et al. Parasites Vectors (2021) 14:181 Page 7 of 10 Table 2 Stress-associated behaviours of hens in which there was stress-related behaviours have been linked to markers of a significant improvement following fluralaner treatment somatic stress, including increases in plasma corticos- Free-range farm Aviary farm terone with the potential to lower humoral immunity, and increases in the heterophil:lymphocyte ratio [17, Night Day Night 24]. However, in the current study blood and egg lev- Body shaking * els of corticosterone increased from pre-treatment to Vertical wing shaking *** the week of treatment and increased further at the final Head scratching *** * assessments, as did the H:L ratio on the aviary farm. In Head shaking * * *** the study of layer hens in Spain, post-treatment reduc- Preening ** ** ** tions in stress-related behaviours were accompanied by Gentle feather pecking *** significant reductions in blood corticosterone levels and Resting * *** in the H:L ratio [17]. In that study, birds had been trained Arrows indicate fluralaner administrations (Weeks 0 and 1) to accommodate handling, and the same birds were sam- Significant difference from pre-treatment value *P < 0.05; **P < 0.01; ***P < 0.001 pled on each occasion. In contrast, in the current study, birds received no such training and samples were col- lected from different birds on each occasion. The stress of the error emphasizes the need for careful calculation of restraint and blood collection from untrained birds may the appropriate product volume to ensure accurate deliv- account for the increases in physiological stress markers ery of the correct dose rate. seen in the current study. Perhaps linked to that finding is In the current study, the finding on both farms that the observation that nighttime head shaking and preen- mite elimination or near-elimination was followed by an ing on the free-range farm were generally greater than improvement in nighttime hen resting behaviour, reduc- on the aviary farm, and the percentage of resting hens tions in preening and head shaking and, on the aviary was generally lower. The increase in stress-related behav- farm, head scratching aligns with similar findings from a iours on the free-range farm could be a result of unrec- recent study on an enriched cage farm in Spain [17]. Sim- ognized stress factors affecting study birds. For instance, ilarly, the current study and the Spanish study both align staff and management systems were different on the two with earlier work showing that PRM infestation results in farms, hens on the free-range farm may have been aware increases in those stress-related behaviours and provide of predator threats, or stress may have arisen due to further confirmation of the effects of PRM infestation in competition for access to feed or to differences between reducing bird welfare [5, 8]. As with the daytime findings hen breeds on the two farms. Those potential non-PRM- in our study, the report from Spain also described day- related stressors may have reduced the observed treat- time reductions in head scratching, head shaking and ment response, which was, nonetheless, still clearly preening following mite elimination. Physiologically, Fig. 3 Nighttime hen preening activity (preening bouts per hen in a 15-min period) at each weekly assessment. Arrows indicate fluralaner administrations (Weeks 0 and 1) Petersen et al. Parasites Vectors (2021) 14:181 Page 8 of 10 Fig. 4 Nighttime hen head-shaking activity (bouts per hen in a 15-min period) at each weekly assessment. Arrows indicate fluralaner administrations (Weeks 0 and 1) Table 3 Mean (± standard deviation) corticosterone concentrations in eggs collected from the free-range (Weeks − 6, 0 and 6) and aviary farms (Weeks − 6, − 1 and 6) and blood collected from hens on the aviary farm (weeks − 2, 0 and 6) Week Free-range farm Aviary farm Yolk (ng/g) Yolk (ng/g) Albumen (ng/g) Plasma (pg/ml) Pre-treatment 9.3 ± 6.5 5.9 ± 4.4 0.43 ± 0.06 16.2 ± 7.7 Treatment week 17.7 ± 6.7 7.2 ± 4.5 0.46 ± 0.04 23.2 ± 8.3 Post-treatment 17.6 ± 6.7 10.2 ± 5.5*a 0.5 ± 0.08**a 27.9 ± 10.3*a Significant difference from pre-treatment value *P < 0.05; **P < 0.01 a Comparison vs Week − 1 value Table 4 Mean (± standard deviation) haematology results of present in the free-range birds, albeit not as marked as in blood samples collected from 40 randomly selected hens on the the aviary birds. Thus, removing the irritant factor of an aviary farm infestation may have reduced external stress behaviours, Means Comparison but other factors may have driven the stress-physiology Day 0 vs Day 42 findings. Day − 14 Day 0 Day 42 Whether clinical or subclinical in appearance, anaemia Erythrocytes 2.3 (± 0.2) 2.2 (± 0.5) 2.4 (± 0.4) P = 0.0256 is a well-established consequence of PRM infestation. A ­(106/μL) laying hen may lose 3% of its blood volume every night Haemoglobin 7.9 (± 0.2) 7.8 (± 2.0) 8.1 (± 1.6) P = 0.4474 due to exposure to heavy infestations, while sub-acute (g/dl) anaemia up to death through severe anaemia has been Haematocrit (%) 17.6 (± 0.2) 15.3 (± 3.7) 17.5 (± 3.4) P = 0.0111 reported [11, 25]. The significant increase in red blood MCV (fl) 77.2 (± 5.8) 71.0 (± 7.5) 72.5 (± 7.2) P = 0.3978 cell counts and haematocrit from samples on the aviary MCH (pg) 35.2 (± 3.3) 34.7 (± 6.6) 33.3 (± 3.3) P = 0.2646 farm indicate that removal of the PRM challenge by the MCHC (g/dl) 38.9 (± 1.8) 38.8 (± 2.4) 39.4 (± 2.5) P = 0.2891 fluralaner administrations resulted in a restoration of Leukocytes ­(103/ 8.1 (± 1.1) 7.8 (± 1.1) 9.1 (± 1.5) P < 0.0001 μl) those blood values, providing further confirmation of the Heterophils (N) 3.7 (± 0.8) 3.7 (± 0.7) 5.7 (± 1.1) P < 0.0001 blood-draining impact of infestation. Lymphocytes 4.4 (± 0.7) 4.0 (± 0.7) 3.3 (± 3.9) P < 0.0001 The effects of PRM infestations on interfering with (N) layer productivity have been documented in a number Ratio H/L 0.9 (± 0.2) 1.0 (± 0.2) 1.8 (± 0.5) P < 0.0001 of studies. Affected parameters have included increases in hen mortality percentage and in the proportion Petersen et al. Parasites Vectors (2021) 14:181 Page 9 of 10 of downgraded eggs and reductions in egg weight Authors’ contributions ICP, EZ and ET wrote the study protocol, KJ and EP performed the on farm and laying rate [4, 9, 11, 16, 17]. There was no notice- activities, EP performed the observations, DE performed the laboratory analy- able change in mortality rates between pre- and post- sis, DT drafted the study protocol and supported the assessment of results, treatment periods on either farm. On the free-range and EZ conducted the statistical analysis. All authors read and approved the final manuscript. farm, based on the industry standard for LSL hens, the expected decline in laying rate over time was lower Funding than expected and the rate of increase in egg weights The study was funded by MSD Animal Health. was greater than expected. This was less the case on Availability of data and materials the aviary farm where, throughout the observation The datasets generated and analysed during the current study are available period, the laying rate paralleled the industry standard on reasonable request to MSD Animal Health Innovation, Schwabenheim Germany. for Lohmann Brown hens, while the rate of increase in egg weights remained below the breed standard, both before and after mite elimination. Overall, the results Declarations suggest that any production-impairing effects of the Ethics approval and consent to participate PRM infestation were removed by treatment. That con- The study was announced to the respective local governmental bodies and was in alignment with local, national and global guidelines. The farmers were clusion is consistent with a report in which egg laying fully advised of all study procedures prior to signing informed consent and rates on seven of eight layer farms improved by up to study start. 12.6% following PRM elimination by treatment with Consent for publication fluralaner compared with the laying rates observed in Not applicable. infested control birds that were either untreated or that received rescue treatments [16]. Competing interests IP, EZ and ET are employees of MSD Animal Health. The primary objective of demonstrating the benefits of PRM elimination in reducing the frequency or duration Author details of stress-related bird behaviours was achieved. Although 1 MSD Animal Health Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany. 2 Praxis Dr. Pöppel, Drubbelstrasse 2, 33119 Delbrück, Germany. production improvements were a secondary objective in 3 Centre Technique Des Productions Animales, Zoopole Dévelopment, 2 this study, positive benefits were suggested by the results rue Jean Rostand, 22440 Ploufragan, France. 4 Interdisciplinary Laboratory from the free-range farm, if not so clearly seen in results of Clinical Analysis (Interlab‑UMU), Veterinary School, Campus of Excellence Mare Nostrum, University of Murcia, Campus de Espinardo s/n, 30100 Mur- from the aviary farm. Importantly, the results of the cur- cia, Espinardo, Spain. 5 Animal Production Department, Regional “Campus rent study provide further confirmation of the beneficial of Excellence Mare Nostrum”, University of Murcia, 30100 Murcia, Espinardo, effects of PRM elimination on bird welfare. Spain. 6 School of Veterinary Medicine, Universitat Autonoma de Barcelona, Bellaterra, Spain. Received: 8 December 2020 Accepted: 16 March 2021 Conclusion On two commercial poultry farms affected with heavy PRM infestations, two drinking water administrations of fluralaner, 7 days apart, largely eliminated mite challenge References 1. Sparagano OAE, George DR, Harrington DWJ, Giangasparo A. Significance and reduced the incidence of stress-related layer hen and control of the poultry red mite Dermanyssus gallinae. Annu Rev behaviours. For the assessment of corticosterone a differ- Entomol. 2014;59:447–66. ent surrogate than blood is recommended in untrained 2. Sommer D, Heffels-Redmann U, Köhler K, Lierz M, Kaleta EF. Role of poul- try red mite (Dermanyssus gallinae) in the transmission of avian influenza birds. Further work is needed to determine the produc- A virus. Tierarztl Prax. 2016;1:47–54. tivity benefits suggested by the production data of treated 3. Tomley FM, Sparagano O. 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