Is the suckling period and application pattern relevant for uazuron ecacy against ticks in cows and their suckling calves?

Background: Fluazuron is a chitin synthesis inhibitor administered as a pour-on formulation in cattle for tick control. This study analyzes under endemic tick infestation, the incidence of the pour-on application pattern on the plasma levels of uazuron in cows and calves in the lactation period of the beef cow. Three hundred and thirteen beef cows around parturition were treated with commercial pour-on formulation of uazuron at a rate of 2.5 mg/kg of body weight. A total of 4 treatments were carried out with a dosing period set in a range of 32 to 45 days. At each administration time, the cows were grouped according to the pour-on administration pattern: long (~60 cm pour-on application surface) and short (~30 cm pour-on application surface). Fluazuron levels in cattle and calves plasma were determined before the third and fourth application for each subgroup (n=10) by HPLC-MS/MS. During the entire study cow–calf pairs were maintained under eld conditions and qualitatively examined for tick infestation on the day of each treatment. Both treatment (long and short) schemes were designed to prevent the annual persistence of ticks. Results: None of the groups presented ticks during the rst 117 days of the study. There were no differences after 40 days post-treatment of the second application (30±5 ppb vs 28.5±12 ppb, P>0.05) and 45 days after the third application (147±55 ppb vs 140±46 ppb, P >0.05) between groups of cows treated with the long or short pour-on application, respectively. Plasma concentration of uazuron at second and third application was increased (3.3 and 2.9 times, respectively) in calves under free suckling compared to cows. Nevertheless, both groups of cows (4.9 times) and calves (2.8 times) showed a signicant increase in plasma concentration of uazuron between times (P<0.05). In both groups, tick prevalence was 0% throughout the trial, except for day 77, which reached 1%.


Background
Tick infestations constitute one of the major problems in livestock production in Uruguay and South America (1,2). The use of acaricides in Uruguay has been the main tool for tick control (3). In control programs of ticks, drugs administration requires a strategic dosage plan with established times according to the residuality of the drugs or depending if the establishment is in a tick control or eradication program (4). Dip acaricides used are organophosphates, pyrethroids and amidines. While injectable and pour-on applications are presented for macrocyclic lactones, pronil and uazuron (5). Resistance has been reported for all the aforementioned, except for uazuron in which there is no practical in-vitro tests for its detection (6).
Fluazuron is responsible for inhibiting chitin synthesis on immature ticks, preventing them from achieving adult stages. Insect growth regulators, such as uazuron, could be responsible for the long-term response given the delayed effect that this type of compounds have on insect´s life cycle (7), reducing the number of immature life stages of Rhipicephalus microplus (R. microplus).
The application of uazuron pour-on formulation in the initial period of lactation of the cow, generates uncertainty about the levels reached in cattle or their lactating calves. Likewise, the persistence levels or the elimination rate of uazuron in lactating cow beefs is not clear (8). Concentration levels due to digestive absorption in lactating calves are also not reported. Furthermore, differences in the practical application technique at the eld level, using different empirical recommendations in the topical administration of the product, varying the extension on the application surface, but maintaining the dose, remains as an uncertainty.
Therefore, the e cacy and associated bene t of the uazuron poured preparation as a tick control alternative in lactating cows needs to be documented. The objective of this study was to determine in endemic tick infestation the incidence of the application pattern of uazuron pour-on on the plasma levels of this acaricide in cows and calves in the lactation period of the beef cow.

Results
The prevalence of ticks at the beginning of the experiment (0 day) was 1% (3/313), as expected by the epidemiological model described for Uruguay. In the following evaluations (32, 77 and 117 days), no animals with presence of ticks were identi ed, except three animals (in short group 0.7 [1/145] and in long group 1.3 [2/147], P > 0.05) and one calf at the time of the third application (77 day). The infestation rate was not different (p > 0.05) between the two application surfaces groups throughout the evaluation periods.
The individual levels of uazuron in cattle determined prior to the third and fourth repeated application for each subgroup (n = 10) are presented in Fig. 1. There were no differences after 40 days post-treatment of the second application (30 ± 5 ppb vs 28.5 ± 12 ppb, P > 0.05) and 45 days after the third application (147 ± 55 ppb vs 140 ± 46 ppb, P > 0.05) between the groups of cows treated with the long or short pouron application, respectively. In view that the cattle groups (long vs short) did not present statistical differences, it was considered as a global cattle group. Forty days after the second administration, the average concentration (± standard deviation) for global cattle group and calves were 29.3 ± 8.9 a ppb and 147 ± 43 b ppb (P < 0.05), respectively (Fig. 2). The application of the third repeated treatment and the determination of the concentrations 45 days later increased the average concentration reaching 143 ± 49 a ppb and 417 ± 87 b ppb (P < 0.05) in the treatments of global cattle and calf groups, respectively (Fig. 2). Plasma concentration of uazuron prior to the third and fourth application increased 3.3 and 2.9 times (respectively) in calves under free suckling vs. cows (Fig. 2). Nevertheless, the global cattle group (4.9 times) and the calves (2.8 times) showed a signi cant increase in plasma concentration of uazuron, between 77 at 117 days (P < 0.05) (Fig. 2).

Discussions
The bioavailability of a drug is directly dependent on the rate and degree of absorption of the drug at the site of administration. Factors that affect the absorption of the drug, including patterns for the pour-on application, will directly affect the bioavailability of the drug. Our results suggest that the drug dispersion pattern long vs short, does not represent a relevant variable when the correct dose is respected. The topical absorption of pour-on formulation can be explained through the Fick law of diffusion in which the drug molecule moves according to the concentration gradient from a higher drug concentration to a lower one until equilibrium is reached (9). Although in our study we doubled the absorption surface, we did not modify the dose, therefore the concentration was decreased per contact surface, which possibly determined that the passive diffusion of the drug was not signi cantly modi ed.
When redosing lipophilic drugs such as uazuron at established times, not only the pharmacokinetic pro le on the dam is affected, but also the plasma levels on lactating calves. After pour-on treatment at 1.5 mg/kg, the mean plasma levels remained quite stable between 9 and 35 days after treatment, ranging from 35 to 41 ppb and declined at about 7 ppb at 16 weeks (10). Using the same dose and formulation, but from a single administration and during a short period of time, Ferreira et al., (2019) reported lower plasma concentration values. In our study, the dosing interval was set at 4-6 weeks following the manufacturer recommendations and the governmental guidelines on tick control for Uruguay, which could explain the higher concentration levels of uazuron found in cattle and calves. The Committee for Medical Products for Veterinary Use (11) issued a Summary report on uazuron pharmacokinetic behavior, with uazuron being excreted via cow's milk to calves, resulting in higher plasma and fat residue levels in calves compared to the ones in cows.
Accumulation is usually considered with regard to plasma, overlooking accumulation of the drug in tissues with poor blood perfusion (12), leaving the accumulation of drug concentration on lactating animals relegated when it comes to explaining whether accumulation occurs and the possible impacts of such process. According to (11), a steady state between absorption and elimination was observed for three to four weeks after treatment. When a single dose of uazuron is administered topically to cattle, the depletion from plasma is slow, with an elimination half-life of 10.5 weeks. Therefore, multiple doses could lead to drug accumulation both in the cow and the calves. As reported by (11) multiple treatments with 12-week intervals did not lead to accumulation of uazuron residues.
The greater accumulation of this acaricide in lactating calves in relation to cows and the increase between subsequent administrations, could be explained by the continuous intake via the digestive tract through milk. Different authors document the importance of licking in the topical administrations and the relative importance of the dermal and oral routes in the removal of drugs from the skin (13,14). We ruled out the licking effect since at the time of application we made a quick visual inspection and determined a null licking behavior in the rst hours after applying all treatments (Unpublished Data). As stated by (15), excretion of parent compounds and/or their metabolites from plasma into milk is a complex process related with physico-chemical properties and membrane interactions. Fat content is one of the main factors that contribute to the concentration of hydrophobic drugs into milk (16,17). Given the lipophilic characteristic of uazuron, fat and milk residue levels are expected to be higher than the plasma levels.
In this study, the prevalence and burden of R. microplus found in the animals is consistent with the data provided by the conceptual epidemiological model described by (1) for Uruguay. Under ideal conditions of temperature and relative humidity (27°C and over 80% RH), the rst generation of ticks takes place between August and October while the second generation appears from December to February. The tick populations parasitizing cattle are higher in relation to the rst generation, with an average of 25 ticks per animal. Considering that the study was carried out in a farm with a history of presence of ticks in previous autumn months, and that the prevalence was lower than expected by the epidemiological model, we can consider that the concentration levels of uazuron in the animals were su cient to prevent the development of the parasitic cycle of the tick.
The rational use of drugs is crucial in food-producing animals because of the potential adverse effects of drugs that appear as residues in edible tissues (18)(19)(20). Milk excretion needs to be quanti ed in order to understand the pharmacokinetic changes that could occur during the suckling period and the impact on parasites control and drug residue levels, especially on parasites with high health impact such as R. microplus. Further studies are needed to quantify milk excretion of uazuron under pour-on administration on lactating cows. Achieving a pharmacokinetic model for this excretion route is the rst step on understanding the use of this chemical tool for tick control around parturition.

Conclusions
The main conclusions of this study were the following: 1) Different administration patterns (long vs short) did not differ in plasma levels of uazuron; 2) Given that only cattle were treated, and lactating calves presented higher plasma levels of uazuron than cows, passage through milk appears to be relevant and possibly due to a cumulative effect and continued drug intake.

Study location
The study was conducted from August to December 2019 in a farm located in Artigas, Uruguay, South America (30°06′ South, 57°04'West). The farm has a history of tick and use of all approved drugs (Ivermectin, Fipronil, Amitraz or Fluazuron) for the control of R. microplus. Animals were not treated with acaricides in the 60 days previous to the uazuron treatment. Tick infestations were present in a very low number of animals in the farm prior to the study. The prevalence was the expected for the period selected for the trial, which corresponds to the rst generation of ticks within the epidemiological model of the country (1). Rainfall during 2019 (January to December) was 1440 mm and the minimum and maximum temperatures were 4.6°C (July) and 30.9°C (October), respectively. Experimental design Three hundred and thirteen beef cows (Hereford) around parturition were treated with commercial pour-on formulation of uazuron ( uazuron 2.5%, pour-on, Acatak Pour-on®, Novartis, Brasil) at a rate of 2.5 mg/kg of body weight. A total of 4 treatments were carried out with a dosing period set in a range of 32 to 45 days. At the administration time two different patterns for the pour-on application were randomly tested and used as a grouping variable: long (~ 60 cm pour-on application surface, n = 163) and short (~ 30 cm pour-on application surface, n = 150). The calves remained free of antiparasitic treatment. Throughout the study, cow-calf pairs were kept in the same paddocks and under eld conditions. Both treatment schemes were designed to prevent the annual persistence of ticks. A control group was not included because the farm was in a global tick control strategy. Data recovery Quantitative analysis of tick infestation: All the cattle and calves in the study were qualitatively examined for tick infestation the day of each treatment (0, 32, 77 and 117 day). Each animal was examined for presence of ticks (adult engorged female ticks) (4.5-5 mm of diameter)) on the head, ears, neck, back, perineum and tail (21). A careful examination was carried out in 10 percent of the animals (randomly selected), which included the examination of the belly and legs. Presence of one or more ticks on an animal was recorded as a positive case.

Fluazuron Analytical procedures
Before the third and fourth application, blood was taken from 10 animals from each group of cows (long and short) and 10 calves randomly (using heparinized tubes).
Fluazuron concentration in plasma was determined by HPLCMS/MS, based on the procedure validated by our group obtaining acceptable accuracy according to the guidance document on analytical quality control and method validation procedures for pesticide residues analysis in food and feed from the European Commission (22). Matrix-matched calibration curves in the range from 5 to 1000 ppb (µg/kg) were established using least squares linear regression analysis and correlation coe cients (r = 0.99) and back calculated concentration residuals below 20%. A limit of quanti cation (LOQ) of 5 ppb was de ned as the lowest measured concentration with relative standard deviation below 20% and an absolute recovery ≥ 70%. Concentration values below the LOQ were not considered in the analysis of experimental data.

Statistical analysis
The prevalence (%) of ticks was determined as the ratio of the number of existing cases during a certain period of time and the population at risk during that speci ed period (23). The plasma concentration data are reported as arithmetic mean ± SD. Differences between cattle groups (long or short) at each time were analyzed for statistical signi cance using the independent samples t test. Due to the fact that the long and short groups did not present statistical differences, they were considered a single group of cattle (global cattle group). The differences between calves and global cattle groups within and between times were analyzed for statistical signi cance using the independent samples t test.
Statistical analyses of differences in the prevalence rates between groups were performed with Fisher's exact test. A p value < 0.05 was considered statistically signi cant. Informed consent was obtained from farm owners for including animals in the study.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Figure 1 Fluazuron adult cattle plasma concentration (ppb) before dose repetition (77 and 117 days from rst dose) of a uazuron pour-on formulation (2.5 mg/kg, pour-on, 2.5%, Acatak®), following two different application schemes: short (30 cm) and long (60 cm). Long_77d = long application at 77 day; Short_77d = short application at 77 day; Long_117d = long application at 117 day; Short_117d = short application at 117 day. P Value Pairwise (T-test) indicates the signi cant difference between groups.