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As concern about the PRM grows, methods for control have become increasingly complicated. Limitations of these approaches include: difficulties of achieving miticidal levels in all the hard-to-reach sites that harbour mites; additional stress to the birds from pesticide applications; risks of residues and pesticide exposure of workers; and the emergence of resistance to the available acaricides [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ].

Therefore there is a long-recognized need for novel methods of reducing or even eliminating the threats that arise from PRM infestations [ 14 ]. Fluralaner, an isoxazoline compound for use as a systemic treatment for D. This mode of action is different from that of all other acaricides. A dose-ranging study in D. These preliminary experimental data and establishment of maximum residue levels to the satisfaction of regulatory authorities provided a basis for assessing the performance of fluralaner when administered under field conditions [ 23 ].

Studies were performed at 12 separate commercial production farms across France, Germany and Spain. The protocol was prepared in alignment with European guidelines for testing of antiparasitic products [ 24 ].

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At each farm fluralaner efficacy was determined by the reductions in mean mite counts from traps placed in houses containing only fluralaner-treated birds in comparison with counts from traps placed in houses holding untreated control birds. Birds had access to free range on two farms. In each study all personnel, except the treatment dispenser and the farm manager who took no part in study assessments, were masked to treatment groups.

At 11 of the 12 farms the studies were initiated during the months of the year spring through summer when ambient temperature and humidity favoured mite proliferation. On one farm the study began in October.

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For a farm to be included, units for each treatment group were required to be similar, to contain the same type of hybrid bird and to be comparable regarding flock size exceptions were made at farms B and DC2 , age of birds, and management practices. On all farms each study unit was required to have a proven infestation of D.

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Depending on the the housing arrangement and bird stocking density at each farm, the number of mobile D. This criterion was the same for each unit on any one farm.

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Flocks could not have been treated with products effective against D. Acaricide treatments with potential efficacy against D. At each farm, personnel involved in any study activities were instructed to change all handling equipment and clothing for any movement between units, and to wear single-use overall and overshoes or similar whenever entering a study unit. All working equipment was to be available in double, one set for each unit. Feed and drinking water provision, air conditioning and stocking density followed the routine procedure of the farm. Study chickens were provided periodic veterinary care according to farm procedure, and general health observations were conducted daily in each study unit from Day -1 to Day 2, on Days 6 to 9, and then weekly until the end of the study.

On study days without veterinary health observations, the farm manager documented the general impression of the flock and was to advise the dispenser immediately upon observation of any health abnormalities in study chickens. With the exception of farm B, the unit with the highest mite infestation was allocated to the fluralaner group, the other unit to a control group that was to remain untreated for mites unless rescue treatment was required for animal welfare or economic reasons.

The targeted fluralaner dose rate was 0. Medicated water was freshly prepared and administered via a medication tank or using a dosing pump. At each farm, within the week preceding Day 0, the inner surfaces of the drinking water distribution system of the fluralaner-treated unit were cleaned. Drinking water consumption in each of the units allocated to the fluralaner group was determined on 1 day between Days -3 and -1 and also on Day 6 at replacement farms.

To estimate the total flock body weight to be treated, on Days -1 or -2, 24 chickens were randomly selected from each unit to be medicated and weighed. Whenever cocks were present, the proportion of hens and cocks selected for weighing was representative of the broader population in each unit.

The average chicken body weight and total flock body weight were then calculated based on records of the number of chickens in the flock on each day of fluralaner administration. Immediately prior to fluralaner administration, except at farm DC2 because of risk of leakage, drinker lines were emptied, and at some farms flushed. The product was mixed with water in a medication tank or in a stock-solution container when a dosing pump was available. Medicated water was then dispensed continuously until the medication source was empty. Visual inspection of the medicated water supply and drinking nipples verified that there were no medication obstructions or irregularities at any farm.

Once empty, the medication sources were rinsed with unmedicated tap water. The pipes were then connected to the regular water distribution system to completely displace the remaining medicated water in the system. The tap-water rinse was then provided to the fluralaner-group birds.

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Records were also available for the percentage of downgraded eggs at farms DC2 and A and egg hatchability at the two breeder farms A, A. All study chickens that died or were culled between Day 0 and the end of the animal phase at a farm were collected and, if possible, stored in a deep freezer. If the weekly mortality rate exceeded the usual weekly mortality rate of each unit, all cadavers or examiner-selected cadavers, were necropsied by a qualified veterinarian.

The mite infestation level in each unit was determined using eight to 24 traps Fig. Traps were evenly distributed throughout units, with a similar distribution of traps in each corresponding unit. Traps were placed close to potential areas of mite aggregates, at fixed positions throughout the study, out of reach of the chickens, and fastened horizontally as far as possible from air ventilation systems Fig.

Mite traps. The mites in each trap and its plastic bag were poured into a Petri dish. Remaining mites or eggs on the cardboard of the trap or in the plastic bag were added to the mites in the dish. Mite stages were identified, differentiated and counted [ 26 ]. An adverse event AE was any observation, whether or not considered product-related, that was unfavourable and unintended and occurred after the use of fluralaner.

Other conditions including mortality commonly associated with commercial poultry husbandry e. The statistical units for antiparasitic efficacy evaluation were mite traps, and for production parameters the experimental unit was the poultry unit. Homogeneity of study groups was evaluated descriptively for the distribution of Day -1 D. The determination of primary efficacy was based upon the D. Percent efficacy was calculated separately for each farm for each post-treatment assessment time point using the Henderson-Tilton formula:. The mean number of mites denotes the arithmetic mean of all mobile stages, i.

Chicken mortalities including culls in each unit during the study were compared to the pre-treatment mortalities of each group and summarized. The weekly laying rate was calculated as the number of eggs collected in relation to the number of chickens in the unit. Pre-treatment production parameters weekly means of each unit were compared to the post-treatment parameters, and the percent changes in treated chickens were compared to those in control chickens. There were eight layer farms, two breeding farms and two replacement farms enrolled into the studies. The breeds and ages of birds in each group treated or control were the same or generally similar within each farm, with the greatest difference in age of birds at a layer farm A Tables 1 and 2.

Despite these acaricide treatments, mite counts from control units remained at levels that were sufficiently high to allow extended comparisons with the fluralaner-treated group beyond the dates of these treatments. These extensions were then concluded at either the end of the production cycle DC3, A or when mite regrowth was apparent in traps from the treated unit A, A. The post-Day 56 decline in effectiveness at Farms A and A was attributed to inadequate separation of the fluralaner-treated group units from the respective control units, leading to a rapid re-infestation of the treated unit Fig.

Sites A and A had inadequate separation of the treatment groups, resulting in increased risk of mite cross-contamination between units, leading to termination of study assessments. At farms A, DC2, A, B and C assessments were concluded at the end of the production cycle or transfer of chickens to another farm.

Mite count reductions from fluralaner-treated units at layer farms. Mean mite trap counts increased in the treated unit at Farm A after Week 16, while high efficacy was maintained until the end of the study in Week 20 Day at farm A. Mite count reductions from fluralaner-treated units at the replacement pullet and breeder farms.

The decline in efficacy at Week 16 on site 5-A was attributed to a decline in mite counts in the control group, rather than being caused by a resurgence in mite population in the unit housing fluralaner-treated chickens. Hatched eggs of D. Across farms, overall reductions in larvae detected in traps had been reduced by Across all study farms, the weekly mortality rates were comparable between treatment groups before and after Day 0.

At one farm DC1 there was a 0. In layers allocated to the fluralaner group, pre-treatment mean weekly laying rate ranged from Post-treatment improvement in egg laying was greater in the fluralaner-treated group at all layer farms, with the difference from the control group ranging from 0.

On the two breeder farms, the difference for fluralaner compared to controls was An observation reported from layer farms was that control-group chickens would sometimes avoid laying eggs in infested nests. At farms DC2 and A, the percent of downgraded eggs was also recorded, with an overall post-treatment compared to pre-treatment improvement in the treated group of 3. At Farm A, the overall hatchability rate over the complete laying period was higher in the fluralaner-group unit At the other breeder farm A , the difference in hatchability rates between the treated and control groups was identical pre- and post-treatment.

Apart from a colibacillosis outbreak at farm DC1 7 weeks following fluralaner treatment initiation, there were no abnormal general health observations in treated birds. There were no treatment-related AEs. Parasitic stages of D. The trap methodology has been reported previously and has been validated as a robust method to quantify mite infestations of poultry, showing that the number of mites collected in traps can be statistically correlated to the mite population in a cage [ 27 , 28 ].

Once collected, the traps were deep frozen to ensure that the mites were killed, thereby avoiding the formation of mite agglomerates that would have made counting unreliable.

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The resulting mite counts demonstrate that a substantial mite challenge of the untreated control birds was maintained throughout all but one of the study farms, despite, in a number of cases, repeated acaricide spray treatments. On this basis, the mite population control i. The major factor contributing to the resurgence of mite counts in units with the chickens receiving fluralaner was the reliability, or lack thereof, of the separation of the treated and untreated groups.

The presence of a non-hermetic door, fence, or ceiling was associated with a faster mite regrowth in the treated unit than when the groups were more substantially separated, as in units in two separate buildings. Because of the protocol requirement for an untreated control unit in close proximity to a treated unit, movement of study personnel and equipment, including conveyors, between the 2 units regardless of best efforts at protocol adherence and the rigour with which hygiene was practiced would have greatly increased the risk of mite cross-contamination to the treated units.

The results reported here therefore represent a worst-case scenario, as under normal commercial conditions all houses or rooms which are adjoining or in close proximity would be treated simultaneously, thereby substantially reducing or eliminating this risk of cross-contamination. The rapid removal of adults from the population prevents additional egg production by female mites. As eggs that were present before treatment hatch within two to 3 days under favorable conditions, the emerging larvae are progressively killed after they mature to the nymphal stages and begin feeding [ 14 ].

The second fluralaner administration, 1 week after the first, kills any mites originating from eggs present at the time of the first treatment. Therefore the fluralaner treatment regimen provides the opportunity to provide a substantial and sustained reduction, or total elimination of mites from a production system.

The results in our studies are consistent with the linkage of PRM infestation with welfare and productivity losses, as the acaricidal efficacy of fluralaner and the resulting relief of mite-infested birds was reflected by an overall increase in laying rate in the treated groups, and a reduction in the percentage of downgraded eggs.

An interesting finding was that in some cases, under conditions of mite challenge layers avoided their nests. Additionally, increased hatchability rates were apparent in the treated group at one of the two breeder farms. These numerical improvements in laying rates appear sufficiently promising to generate further investigation into the production benefits of fluralaner medication of PRM-infested poultry farms.

This is the first study in which the potential value of an isoxazoline has been demonstrated in food-producing animals, and the first report of successful elimination of D. Administration of fluralaner to the birds in this study followed the establishment of maximum residue limits for fluralaner that were adopted by the European Commission [ 23 ]. Consistent with findings from the use of fluralaner in dogs and cats, the tolerability of the fluralaner treatment in the present set of studies was excellent.