Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc Avermectin toxicity in bovines less than thirty days old Daniel de Castro Rodriguesa, Carolina Buzullinib, Tiago Arantes Pereiraa, Breno Cayeiro Curzb, Lucas Vinicius Costa Gomesb, Vando Edésio Soaresc, Thiago Souza Azeredo Bastosd, Luiz Fellipe Monteiro Coutod, Welber Daniel Zanetti Lopesb,d,⁎, Gilson Pereira de Oliveirab, Alvimar José da Costab aMSD Animal Health, São Paulo, Brazil b CPPAR - Animal Health Research Center, Faculdade de Ciências Agrárias e Veterinárias, UNESP, Campus de Jaboticabal. Via de acesso Prof. Paulo Donatto Castellane, s/n°, CEP: 14884-900 Jaboticabal, São Paulo, Brazil cUniversidade Brasil, Campus de Descalvado, CEP 13690-970 São Paulo, SP, Brazil d Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás (IPTSP/UFG), Goiânia, Goiás 74605050, Brazil A R T I C L E I N F O Keywords: Abamectin Calves Central nervous system Ivermectin + abamectin A B S T R A C T The present study was designed to evaluate possible adverse effects of different dosages of avermectins (aba- mectin and a combination of ivermectin + abamectin) administered subcutaneously in calves less than one month of age. Clinical as well as biochemical parameters of blood and cerebrospinal fluid (CSF) were evaluated for animals subjected to various treatments. Thirty-five calves were divided into seven groups of five animals each: T01 (control), T02 (abamectin 200 μg/kg), T03 (abamectin 400 μg/kg), T04 (abamectin 600 μg/kg), T05 (ivermectin 450 μg/kg+ abamectin 250 μg/kg), T06 (ivermectin 900 μg/kg+ abamectin 500 μg/kg) and T07 (ivermectin 1350 μg/kg+ abamectin 750 μg/kg). Cerebrospinal fluid and blood samples were collected on day zero (treatment day) and on days 1, 7 and 14 following treatment. The biochemical parameters measured in blood samples were total protein and the AST, GGT and alkaline phosphatase enzymes. In CSF samples, color, aspects, pH, density, cellularity, total proteins, glucose and the CK, ALT and LDH enzymes were evaluated. No neurological toxicity or biochemical changes in the CSF or blood that could be correlated to administration of tested products were observed in this study. Though it is well known that abamectin may lead to intoxication in bovines less than four months of age, based on data from the present study, no adverse clinical effects to the CSF or blood were observed in calves< 30 days old that had received up to 600 μg/kg abamectin formulations and up to 1350 μg/kg ivermectin +750 μg/kg abamectin associations. It is possible that the level and activity of the multiple drug resistance gene (MDR1 - ABCB1) present in the calves may have influenced the results obtained in this study. Future studies should be carried out. 1. Introduction With an increased relevance to parasitology, avermectins and mil- bemycins appeared in the 1980s. Belonging to the macrocyclic lactone (ML) group, these endectocides act against arthropods and nematodes (Burg et al., 1979). As part of their mechanism, there is a hypothetical agonist action of gamma amino butyric acid (GABA) that increases the permeability of chlorine ions (Clˉ), resulting in muscular paralysis (Mellin et al., 1983; Albert et al., 1986). GABA is a known neurotransmitter found in ne- matodes and arthropods and is also present in the central nervous system (CNS) of mammals. Avermectin concentrations in the CNS of mammals subjected to normal therapeutic dosages presented in- expressible levels of the drug. However, elevated drug levels were verified in the brain tissue of affected bovines and dogs even after ap- plication of therapeutic dose (Prichard et al., 2012). Geyer and Janko (2012) describe that the therapeutic safety of macrocyclic lactones in animals is dependent of the expression level and activity of the multi- drug efflux transporter MDR1 (ABCB1) gene and P glycoprotein in the blood-brain barrier. Fromm (2004), Marzolini et al. (2004) and Mealey (2004) determined that mutation of the MDR1 (ABCB1) gene alters the pharmacokinetic properties of the P-glycoprotein transported drugs, leading to enhanced bioavailability and reduced drug elimination through the liver, kidney and gut. In othe words, MDR1 mutant dogs, https://doi.org/10.1016/j.rvsc.2018.04.003 Received 2 October 2017; Received in revised form 13 March 2018; Accepted 13 April 2018 ⁎ Corresponding author at: CPPAR - Animal Health Research Center, Faculdade de Ciências Agrárias e Veterinárias, UNESP, Campus de Jaboticabal. Via de acesso Prof. Paulo Donatto Castellane, s/n°, CEP: 14884-900 Jaboticabal, São Paulo, Brazil. E-mail address: wdzlopes@hotmail.com (W.D.Z. Lopes). Research in Veterinary Science 118 (2018) 403–412 0034-5288/ © 2018 Elsevier Ltd. All rights reserved. T http://www.sciencedirect.com/science/journal/00345288 https://www.elsevier.com/locate/rvsc https://doi.org/10.1016/j.rvsc.2018.04.003 https://doi.org/10.1016/j.rvsc.2018.04.003 mailto:wdzlopes@hotmail.com https://doi.org/10.1016/j.rvsc.2018.04.003 http://crossmark.crossref.org/dialog/?doi=10.1016/j.rvsc.2018.04.003&domain=pdf such as Collie, do not express P-glycoprotein at the blood-brain barrier, and for this reason may present severe neurological toxicity after ap- plication of therapeutic doses (Geyer et al., 2005) Despite this understanding reported above, Chrisman (1985), Feldman (1989), Braund (1994) and Wright (1978) describe the hy- pothesis of analyzing the constituents of the cerebrospinhal fluid (CSF), such the biochemical parameters, in animals that were administered avermectin may reflect physiologic pathological changes in brain functions. This specific research study was motivated by that need. The present study was designed to evaluate possible adverse effects of varying dosages and formulations, administered subcutaneously, containing different avermectins (abamectin -Duotin® - Merial Animal Health and an association of ivermectin + abamectin- Solution® - MSD Animal Health) in calves< 30 days old. To accomplish this goal, neu- rological and clinical parameters, as well the constituents of cere- brospinal fluid (CSF) from animals subjected to these treatments, were analyzed. Secondarily, the biochemical parameter of blood were ana- lyzed in accordance with recommends studies of target animal safety (VICH, 43). 2. Material and methods 2.1. Location, animal selection, animal feeding and allocation of animals into groups Prior to implementation, this study was approved by the Ethics Committee for Animal Use (Comissão de Ética no Uso de Animais - CEUA) of the School of Agricultural and Veterinarian Sciences (Faculdade de Ciências Agrárias e Veterinárias – FCAV), Jaboticabal campus of the São Paulo State University (Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP). It was approved only after being considered in agreement with ethical principles in animal ex- perimentation adopted by the National Council for the Control of Animal Experimentation (Conselho Nacional de Controle de Experimentação Animal - CONCEA). This experiment was conducted entirely at the Center for Research in Animal Health (Centro de Pesquisas em Sanidade Animal – CPPAR), located on the aforementioned campus. Thirty-five male Holstein newborn calves were used. The animals were obtained from a commercial farm located in the city of Descalvado, São Paulo State. The calves were separated from their mothers at the moment of birth. At the farm, these animals received colostrum in the first hours of life and were then transported to the CPPAR/FCAV/UNESP where they were kept in pens during the period of the experiment. During the entire experiment, the calves were arti- ficially fed twice per day with four liters of a milk substitute (Amamenta Standard®) prepared in the proportions recommended by the manufacturer (225 g of the substitute diluted in two liters of water, heated to a temperature of 39 °C). During this period, animals had ac- cess to granulated commercial ration (Fri-bezerros®), coarsely chopped Tifton (Cynodon sp.) hay and ad libitum fresh water. Animals were fed with 70% Tifton hay and 30% commercial ration. The amount of food provided was based on 3% of each animal's body weight. The broma- tological composition of this diet was evaluated at the Animal Nutrition Laboratory (Laboratório de Nutrição Animal - FCAV/UNESP). Each animal was individually weighed, and seven groups were then established with five calves of similar ages and body weights (Table 1). Experimental day zero was defined as the day when the animals were treated. Different formulations containing abamectin (Duotin® - Merial Animal Health) and ivermectin + abamectin (Solution® - MSD Animal Health) were used in this study (Table 1). The animals received the recommended normal dose (200 μg/kg abamectin; 450 μg/kg iver- mectin +250 μg/kg abamectin), 2x (400 μg/kg abamectin; 900 μg/kg ivermectin +500 μg/kg abamectin) and 3x (600μg/kg abamectin; 1350 μg/kg ivermectin +750 μg/kg abamectin) as recognized by VICH - 43 (2008). All treatments were administered subcutaneously. There are no neurologic studies with both formulations, but in ac- cordance with the manufacturer's information's, these products cannot be administered in calves< 16weeks (4months) old. 2.2. Clinical and neurological evaluation Identification records were created for each animal, containing the calf's id number, age and body weight. Clinical observations were conducted on day zero (prior to treatment) and on days 1, 7 and 14 post treatment (DPT). On these dates, clinical parameters (cardiac and re- spiratory frequencies and rectal temperature), eye abnormalities and the cardiovascular, respiratory, muscular-skeletal, integumentary (skin, hair and hooves), gastrointestinal, nervous, lymphatic and urinary systems were examined. During the experiment, all animals were also evaluated for the presence of clinical signs of systemic poisoning, such as ataxia, paresis, dysmetria, ocular changes such as mydriasis, sialor- rhea, apathy, prostration, somnolence, excitement, convulsions, dys- phonia, diarrhea, dyspnea, jaundice, cough, head tremors, skin tremors and hyperesthesia. If any of these animals had any signs, they would be classified as accented (+++), moderate (++), mild (+) and absent (−). 2.3. Cerebrospinal fluid evaluations 2.3.1. CSF collection CSF was obtained through sub-occipital puncture on the cisterna magna, using 20 G (40×0.9mm) needles. The liquid was transferred to 1.5 mL silicone tubes (Eppendorf®) for analysis of the cytological and biochemical aspects. The animals were immobilized using physical re- straint without the use of chemicals (Stober, 1993). Collections were performed before treatments (day zero) and on the 1st, 7th and 14th DPT. 2.3.2. Physical-chemical evaluation Color and aspects of CSF were evaluated in contrast with tubes containing distilled water, both placed against a white background with printed letters. Density was determined using refractometry (re- fractometer ATAGO Co. LTD., Tokyo, Japan), and indicator strips (Merck do Brasil, Rio de Janeiro, RJ) were used to determine the pH (Gama, 2003). 2.3.3. Cytological evaluation Global cell counts were conducted in Neubauer chambers, and dif- ferential counts for pleocytosis were performed using microscopic preparations obtained through cytocentrifugation (cytocentrifuge FANEM, São Paulo, SP). Afterwards, samples were stained with a me- thanol May-Grünwald-Giemsa mixture. Global and differential cell counts were performed immediately after obtaining the samples in order to avoid cellular degeneration (Gama, 2003). 2.3.4. Glycorrhachia analysis Glycorrhachia was determined using HK reagent sets (Labtest Diagnóstica, Belo Horizonte, MG) and a biochemical analyzer apparatus (Labquest - Labtest Diagnóstica, Belo Horizonte, MG) by means of colorimetric methods (Gama, 2003). 2.3.5. Enzymatic analysis CSF samples were analyzed for aspartate amino transferase (AST, optimized UV method - IFCC),1 creatine kinase (CK, kinetic UV method - IFCC)2 and total protein (using the total protein reagents1). Readings were performed in an automatic spectrophotometer 2. Determinations of CK, AST and lactate dehydrogenase activities 1 Wiener Lab. – Rosario, Argentina. 2 Automatic biochemical analyzer, Cobas Mira S, Roche do Brasil - São Paulo, SP. D. de Castro Rodrigues et al. Research in Veterinary Science 118 (2018) 403–412 404 were performed using a reagent set (Labtest) and the kinetic method, with readings obtained using a biochemical analyzer apparatus (Labquest - Labtest Diagnóstica, Belo Horizonte, MG) (Gama, 2003). 2.3.6. Protein analysis Determination of total protein values was performed using Sensi Prot reagent sets (Labtest Diagnóstica, Belo Horizonte, MG) and the pyrogallol red method, with readings obtained using a biochemical analyzer apparatus (Labquest - Labtest Diagnóstica, Belo Horizonte, MG) (Gama, 2003). 2.4. Blood enzyme and protein evaluations To separate the blood serum for biochemical examination, 10mL of blood was collected in vials without additives. This procedure was performed based on the recommendations of the Target Animal for Veterinary Pharmaceutical Products (VICH 43 –, 2008). The samples were analyzed for alkaline phosphatase (AP, Bowers & McComb mod- ified method),3 aspartate amino transferase (AST, optimized UV method - IFCC)4 and gamma glutamyl transferase (GGT kinetic method)1. Readings were obtained using an automatic spectro- photometer. Determination of total protein values was conducted using total protein reagents1. 2.5. Data analysis A design with parcels subdivided in time (“Split Plot in Time”) was adopted, considering the treated and control groups, each with five repetitions, as the main treatments and observation dates as the sec- ondary treatments. Data were analyzed using a methodology proposed by Little and Hills (1978), which required logarithmic transformation (log [x+ 1]). Analyses were performed by applying the F-Test, and averages were compared using the Tukey test (SAS, 1996). 3. Results and discussion 3.1. Clinical examinations Clinical symptoms due to different treatment formulations or do- sages were not observed in any of the experimental animals. Cases of lactone intoxication in bovine animals are expected to demonstrate neurological toxicity in animals; however, in the present Table 1 Experimental design. Group N° of bovines Mean age (days) Treatment Dose I 5 22.1 Saline solution saline 0,9% II 5 22.3 Abamectin 200 μg/kg (recommended dose) III 5 22.0 Abamectin 400 μg/kg IV 5 22.4 Abamectin 600 μg/kg V 5 22.3 Ivermectin 2.25%+Abamectin 1.25% 450 μg/kg-ivermectin 250 μg/kg-abamectin (recommended dose) VI 5 22.6 Ivermectin 2.25%+Abamectin 1.25% 900 μg/kg-ivermectin 500 μg/kg-abamectin VII 5 22.2 Ivermectin 2.25%+Abamectin 1.25% 1350 μg/kg-ivermectin 750 μg/kg-abamectin Table 2 Variation of the mean values of the clinical parameters of bovine animals under 30 days submitted to different concentrations of avermectin. Group Treatment Experimental day/Rectal temperature °C ZERO 1 7 14 I Saline solution 38.26 B 38.76 A 38.60 A 39.06 A II Abamectin (200 μg) - recommended dose 39.38 AB 38.38 A 39.20 A 38.46 A III Abamectin (400 μg) 38.40 B 38.66 A 38.34 A 38.78 A IV Abamectin (600 μg) 38.18 B 38.62 A 38.50 A 38.82 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 39.66 A 39.24 A 38.96 A 39.06 A VI Ivermectin (900 μg)+Abamectin (500 μg) 38.18 B 38.70 A 38.32 A 39.28 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 38.64 AB 38.76 A 38.44 A 39.68 A Group Treatment Experimental day/Heart frequency (BPM) ZERO 1 7 14 I Saline solution 89.60 A 82.40 A 77.60 AB 85.60 A II Abamectin (200 μg) - recommended dose 68.80 B 80.00 A 68.80 B 77.60 A III Abamectin (400 μg) 77.60 AB 82.40 A 83.20 AB 85.60 A IV Abamectin (600 μg) 75.20 B 82.40 A 83.20 AB 84.80 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 67.20 B 83.20 A 70.00 AB 70.40 A VI Ivermectin (900 μg)+Abamectin (500 μg) 63.20 B 85.60 A 81.60 AB 85.60 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 89.60 A 80.80 A 84.80 A 84.00 A Group Treatment Experimental day/Respiratory frequency (MPM) ZERO 1 7 14 I Saline solution 20.80 AB 23.20 A 23.20 A 25.60 A II Abamectin (200 μg) - recommended dosel 21.60 AB 25.60 A 20.80 A 27.20 A III Abamectin (400 μg) 20.00 AB 22.40 A 24.00 A 25.60 A IV Abamectin (600 μg) 21.60 AB 20.00 A 20.80 A 24.00 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 24.80 A 24.00 A 22.40 A 25.60 A VI Ivermectin (900 μg)+Abamectin (500 μg) 18.40 B 23.20 A 23.20 A 25.60 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 22.40 AB 22.40 A 22.40 A 24.00 A Means values followed by the same letter on the same colun do not differ significantly at a 95% reliability level. 3 Labtest Diagnóstica S.A. - Lagoa Santa, MG. 4 Wiener Lab. – Rosario, Argentina. D. de Castro Rodrigues et al. Research in Veterinary Science 118 (2018) 403–412 405 Mean Mean±SE Mean±2*SD s enila S noitulo nit ce ma b A g k/gµ002 nit ce ma b A gk/ gµ 004 nitce mab A gk/gµ006 054 ni tce mrev I tce mab A + gk/gµ gk/gµ052 ni 009 nitce mrevI tce ma b A + gk/gµ gk/ gµ 00 5 ni 3 1 nitce mr e vI mab A + gk /gµ05 gk/g µ057 nitce 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 Treatment Experimental Day 10 12 14 16 18 20 22 24 26 28 30 32 34 ) M P M( ycn euqerf yrotarip se R 30 40 50 60 70 80 90 100 110 120 ) M P B( ycneuqerf trae H 35 36 37 38 39 40 41 42 Cº erutarep met latce R Fig. 1. Boxplot of clinical parameters of bovine animals under 30 days submitted to different concentrations of avermectin. D. de Castro Rodrigues et al. Research in Veterinary Science 118 (2018) 403–412 406 Table 3 Data analysis of the cerebrospinal fluid parametres of bovine animals under 30 days submitted to different concentrations of avermectin. Group Treatment Experimental day/Density value (Cerebrospinal fluid) ZERO 1 7 14 I Saline solution 1.005 A 1.005 A 1.005 A 1.005 A II Abamectin (200 μg) - recommended dose 1.005 A 1.005 A 1.006 A 1.005 A III Abamectin (400 μg) 1.006 A 1.005 A 1.005 A 1.005 A IV Abamectin (600 μg) 1.006 A 1.005 A 1.005 A 1.005 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 1.006 A 1.007 A 1.006 A 1.005 A VI Ivermectin (900 μg)+Abamectin (500 μg) 1.005 A 1.005 A 1.005 A 1.005 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 1.005 A 1.005 A 1.005 A 1.005 A Group Treatment Experimental day/pH (Cerebrospinal fluid) ZERO 1 7 14 I Saline solution 7.10 AB 8.00 A 7.90 A 8.00 A II Abamectin (200 μg) - recommended dosel 7.80 A 7.70 A 7.80 A 8.00 A III Abamectin (400 μg) 7.40 AB 8.00 A 7.70 A 8.00 A IV Abamectin (600 μg) 7.00 B 7.80 A 8.00 A 7.80 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 7.40 AB 6.80 B 7.80 A 7.60 A VI Ivermectin (900 μg)+Abamectin (500 μg) 7.20 AB 7.90 A 7.70 A 7.80 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 7.30 AB 8.00 A 8.00 A 8.00 A Group Treatment Experimental day/Glucose (Cerebrospinal fluid) ZERO 1 7 14 I Saline solution 33.20 A 56.20 A 52.40 A 46.40 A II Abamectin (200 μg) - recommended dose 54.20 A 43.40 A 56.80 A 41.14 A III Abamectin (400 μg) 55.60 A 59.60 A 57.20 A 56.80 A IV Abamectin (600 μg) 45.00 A 54.20 A 47.00 A 46.40 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 57.20 A 43.40 A 51.00 A 37.20 A VI Ivermectin (900 μg)+Abamectin (500 μg) 46.80 A 48.80 A 49.20 A 45.00 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 48.60 A 51.40 A 50.80 A 43.20 A Group Treatment Experimental day/Leukocytes (Cerebrospinal fluid) ZERO 1 7 14 I Saline solution 2.00 A 2.00 B 2.90 A 2.40 A II Abamectin (200 μg) - recommended dose 0.80 A 5.00 B 2.80 A 2.40 A III Abamectin (400 μg) 0.50 A 0.50 B 0.00 A 1.00 A IV Abamectin (600 μg) 0.50 A 0.00 B 1.00 A 0.50 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 4.40 A 17.50 A 1.00 A 7.80 A VI Ivermectin (900 μg)+Abamectin (500 μg) 2.00 A 2.50 B 2.00 A 1.00 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 0.50 A 0.50 B 0.00 A 0.60 A Group Treatment Experimental day/LDH (Cerebrospinal fluid) ZERO 1 7 14 I Saline solution 0.6417 A 1.1572 A 0.2461 A 0.5498 A II Abamectin (200 μg) - recommended dose 0.8535 A 1.0043 A 1.2721 A 1.4033 A III Abamectin (400 μg) 1.0219 A 0.8167 A 0.5333 A 0.8527 A IV Abamectin (600 μg) 0.4922 A 1.0172 A 0.2644 A 0.8596 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 1.0763 A 1.2365 A 1.5898 A 0.6663 A VI Ivermectin (900 μg)+Abamectin (500 μg) 0.2461 A 1.0748 A 0.6113 A 0.8189 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 0.9844 A 0.6391 A 0.2910 A 0.6379 A Group Treatment Experimental day/AST (Cerebrospinal fluid) ZERO 1 7 14 I Saline solution 23.05 A 34.92 A 34.06 AB 9.42 A II Abamectin (200 μg) - recommended dose 17.51 A 26.19 A 40.94 A 17.81 A III Abamectin (400 μg) 24.15 A 29.14 A 14.27 B 16.37 A IV Abamectin (600 μg) 23.36 A 24.55 A 11.78 B 19.82 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 18.41 A 37.55 A 54.90 A 27.44 A VI Ivermectin (900 μg)+Abamectin (500 μg) 26.76 A 37.13 A 14.92 B 17.38 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 21.77 A 29.15 A 13.43 B 14.83 A Group Treatment Experimental day/CK (Cerebrospinal fluid) ZERO 1 7 14 I Saline solution 43.65 A 33.94 A 52.91 A 29.12 A II Abamectin (200 μg) - recommended dose 43.37 A 38.86 A 43.20 A 39.86 A III Abamectin (400 μg) 58.28 A 29.09 A 53.03 A 33.96 A IV Abamectin (600 μg) 48.57 A 24.23 A 48.23 A 38.76 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 53.14 A 43.54 A 38.40 A 29.15 A VI Ivermectin (900 μg)+Abamectin (500 μg) 38.84 A 43.60 A 33.83 A 38.94 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 43.70 A 53.26 A 46.80 A 33.95 A Group Treatment Experimental day/Total protein (Cerebrospinal fluid) ZERO 1 7 14 I Saline solution 32.46 A 26.08 A 23.14 A 23.18 A II Abamectin (200 μg) - recommended dose 22.38 A 9.10 A 27.48 A 20.22 A (continued on next page) D. de Castro Rodrigues et al. Research in Veterinary Science 118 (2018) 403–412 407 study, no neurological abnormalities were observed in calves. In ex- periments which induced acute and chronic abamectin intoxication in mice, rabbits, dogs and monkeys, animals presented mainly mydriasis, vomiting, tremors, ataxia, depression, seizures, coma and death (Lankas and Gordon, 1989). Evaluating tolerance of calves to different doses of abamectin (0.3 to 8mg/kg), Button et al. (1988) mostly observed signs of depression and ataxia. In this case, moderate intoxication symptoms were detected in animals after receiving dosages starting at 0.727mg/ kg. When the same researchers administered volumes between 2 and 8mg/kg, toxicity signs such as ataxia, progressive paresis, mydriasis, decubitus, sialorrhea and death occurred. On the present study, eval- uating clinical safety of different formulations containing abamectin (Duotin®,MerialSaúde Animal,and Solution®, MSD Saúde Animal), it was possible to observe that the highest dosages for each formulation containing this active component were 0.6 mg/kg and 0.75mg/kg, re- spectively. Independent of administered dosages, it was not possible to verify any signs of neurologic toxicity in animals. A possible explanation for calves on the present study not demon- strating signs of neurologic toxicity by different abamectin dosages may be found in more advanced studies with the MDR1 gene and P-glyco- protein, conducted in dogs. Dogs which showed toxicity signs to some macrocyclic lactones administered in therapeutic dosages may present a mutation on the MDR1 gene and become more sensible to such active principles. This gene (MDR1) codifies P-glycoprotein, which in turn functions as a cellular efflux pump for various medications in several species of animals (Geyer and Janko, 2012). Table 3 (continued) Group Treatment Experimental day/Density value (Cerebrospinal fluid) ZERO 1 7 14 III Abamectin (400 μg) 18.20 A 18.86 A 19.60 A 28.04 A IV Abamectin (600 μg) 17.42 A 19.44 A 18.44 A 26.68 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 41.24 A 28.95 A 19.66 A 22.52 A VI Ivermectin (900 μg)+Abamectin (500 μg) 21.94 A 47.50 A 27.86 A 24.88 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 17.40 A 23.40 A 25.72 A 17.30 A Means values followed by the same letter on the same colun do not differ significantly at a 95% reliability level. 1,002 1,003 1,004 1,005 1,006 1,007 1,008 1,009 1,010 1,011 1,012 e ul av ytis n e D s e ni l a S n oit ul o nitc e m a b A gk/ g µ 0 0 2 nit c e m a b A g k/ g µ 0 0 4 nit c e m a b A g k/ g µ 0 0 6 0 5 4 nitc e mr evI tc e m a b A + gk/ g µ gk/ g µ 0 5 2 ni 0 0 9 ni tc e mr evI t c e m a b A + gk/ g µ gk/ g µ 0 0 5 ni 3 1 nit c e m r evI m a b A + gk / g µ 0 5 gk/ g µ 0 5 7 nitc e 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 H p -40 -20 0 20 40 60 80 100 120 140 160 ) L d/ g m( es oc ul G Mean Mean±SE Mean±2*SD s e nil a S n oit ul o nit c e m a b A gk/ g µ 0 0 2 nit c e m a b A gk/ g µ 0 0 4 nit c e m a b A g k/ g µ 0 0 6 0 5 4 nitc e mr evI t c e m a b A + g k/ g µ gk/ g µ 0 5 2 ni 0 0 9 n itc e mr evI t c e m a b A + gk/ g µ gk/ g µ 0 0 5 ni 3 1 n it c e mr e vI m a b A + gk / g µ 0 5 gk / g µ 0 5 7 nitc e 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 Treatment Day -3000 -2000 -1000 0 1000 2000 3000 4000 s etyc ok u e L Fig. 2. (a) and (b). Boxplot of the cerebrospinal fluid parametres of bovine animals under 30 days submitted to different concentrations of avermectin. D. de Castro Rodrigues et al. Research in Veterinary Science 118 (2018) 403–412 408 According to Fecht et al. (2007), the gene is located on chromosome 14 and is composed by 28 exons. Mutation consists in deletion of the fourth exon, which results in synthesis of defective and nonfunctional P- glycoprotein, making the bearer of this mutation susceptible to in- toxication, even when receiving an active component in its therapeutic dosages (Bissonnette et al., 2008). The animal may be homozygous (MDR1 +/+) for the MDR1 gene, and in this case it is considered normal (or wild) and do not present the same risk as homozygous mutants. Homozygous mutant animals (MDR1 −/−) present extreme sensibility and predisposition to intoxication. Heterozygous individuals (MDR1 +/−) are bearing the mutation, being either susceptible to intoxication or not (Bissonnette et al., 2008). It is known from animal models (Schinkel et al., 1997) that MDR1 is up-regulated in the first weeks of life. Therefore, a hypothesis that could justify the results found in the present study is that if the MDR1 ex- pression level in young calves is already fully regulated, there is suffi- cient protection against brain penetration by ML in these animals. However, further studies should be conducted to confirm this hypoth- esis. Based on results expressed in Table 2 and Fig. 1, it is possible to verify that at no time during this study were significant differences observed for respiratory frequencies and rectal temperatures in ex- perimental calves; these clinical parameters remained within normal ranges as preconized by Feitosa (2008). Concerning the average values of cardiac frequencies measured in these animals, significant differ- ences (p≤ 0.05) were observed between groups on days zero (before treatment) and 7 (7th DPT). Groups of calves with mean cardiac fre- quency values above normal levels for this species were observed on all days post-treatment. However, this condition also occurred occasion- ally in animals from the untreated control group (T01 – saline solution, Table 2 ad Fig. 1). Another aspect that must be considered is the stress of animals submitted to physical restraint for sample collection. In addition, rectal temperatures and other physiological variables, such as age, may alter the vagal state, intensifying activity of the cardio-ac- celerator and vasoconstrictor centers and therefore elevating cardiac rates. Seixas et al. (2006) reported the occurrence of several intoxication outbreaks caused by abamectin, totaling at least 57 deaths over four years (including adult animals), reported from just three diagnostic services contacted in Brazil. This research study emphasized the pos- sibility that avermectin intoxications are frequent and that the numbers of iatrogenic intoxications are probably being underestimated. On the other hand, it is essential to reinforce that these cases re- ported by Seixas et al. (2006) were diagnosed based on administration records and observation of clinical symptoms after treatment. In most cases, exact information regarding animal ages, administered dosages and clinical characteristics were not available. Those researchers highlighted the importance of performing in-depth biochemical and neurological studies in order to better understand possible adverse ef- fects of abamectin in bovines. Benz and Cox (1989) and Courtney and Roberson (1995) considered the safety margin of avermectins to be elevated, even in animals highly -80 -60 -40 -20 0 20 40 60 80 100 120 140 L/ u H D L s e nil a S n oit ul o nit c e m a b A gk/ g µ 0 0 2 nit c e m a b A gk/ g µ 0 0 4 nitc e m a b A g k/ g µ 0 0 6 0 5 4 nitc e mr e vI tc e m a b A + gk/ g µ gk/ g µ 0 5 2 n i 0 0 9 nitc e mr evI tc e m a b A + gk/ g µ g k/ g µ 0 0 5 ni 3 1 nitc e mr evI m a b A + gk / g µ 0 5 gk/ g µ 0 5 7 nitc e 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 -40 -20 0 20 40 60 80 100 ) R O U QÍ L( L/ U T S A -40 -20 0 20 40 60 80 100 120 L/ u K C Mean Mean±SE Mean±2*SD s e nil a S n oit ul o nitc e m a b A g k/ g µ 0 0 2 nitc e m a b A g k/ g µ 0 0 4 nit c e m a b A g k/ g µ 0 0 6 0 5 4 nitc e mr evI tc e m a b A + gk/ g µ g k/ g µ 0 5 2 ni 0 0 9 ni tc e mr evI t c e m a b A + g k/ g µ gk/ g µ 0 0 5 ni 3 1 nit c e mr e vI m a b A + gk/ g µ 0 5 gk/ g µ 0 5 7 nitc e 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 Treatment Day -200 -150 -100 -50 0 50 100 150 200 250 300 )l d/ g m( ni et or p l at o T Fig. 2. (continued) D. de Castro Rodrigues et al. Research in Veterinary Science 118 (2018) 403–412 409 susceptible to intoxication, such as calves under four years old. They affirmed that dosages up to five times the recommended dose would not be lethal, even though severe signs of intoxication, and even deaths, occur when abamectin is used in dosages much greater (five to 10 times) than what is recommended or administered to animals pre- senting higher sensitivities. The results of the present study are similar to those obtained by the aforementioned authors, confirming their findings regarding safety of avermectins, even in susceptible animals. However, it is essential to reinforce that these results do not allow for the recommendation of abamectin use in calves under 30 days old. For these young calves, new studies must be performed. Nonetheless, intoxication does not seem to occur in the majority of susceptible ani- mals that received abamectin. It is important to note that other hy- potheses must be considered in clinical cases suggestive of intoxication diagnosed in the field, similar to those reported by Seixas et al. (2006). 3.2. Cerebrospinal fluid All samples collected, from both treated and control groups, were clear and colorless and within normal ranges as described by Kay et al. (1974), Wright (1978) and Bailey and Vernau (1997). For the re- maining parameters evaluated in CSF samples, it is important to re- inforce that no statistically significant differences (p > 0.05) were observed, across all experimental dates, in density, glucose, LDH, CK and total protein. For pH, it was possible to verify that values varied between 6.8 and 8.0, with significant changes observed before treatment (day zero) and on the first DPT for groups T02 (abamectin 200 μg/kg) and T04 (aba- mectin 600 μg/kg), as well as for group T05 (ivermectin 450 μg/ kg+ abamectin 250 μg/kg) and the remaining groups (Table 3 and Figs. 2a and b). Still, it is essential to highlight that pH results did not exceed normal physiological values given by Jean et al. (1997). Concerning the cellular composition in CSF samples, a significant difference (p≤ .05) was observed only on 1 DPT for average leukocytes of animals that received ivermectin (450 μg/kg)+ abamectin (250 μg/ kg) compared to the remaining groups (Table 3 and Figs. 2a and b). These CSF values can be considered normal and are similar to ob- servations by Welles et al. (1994) and Jean et al. (1997). Elevated average levels (p≤ .05) of CSF aspartate aminotransferase (u/L) were observed in calves from groups T02 (abamectin 200 μg/kg) and T05 (ivermectin 450 μg/kg+ abamectin 250 μg/kg) in comparison to average levels observed in animals treated with abamectin (400 and 600 μg/kg) and the ivermectin + abamectin (900 μg/kg+ 500 μg/kg, 1350 μg/kg+ 750 μg/kg) association. According to Feldman (1989), Chrisman (1985), Osuna (1992) and Lutsar (1994), increases in the activity of the AST enzyme in this location can constitute an important indicator of active necrosis of the CNS of vertebrates. However, despite the significant difference (p≤ 0.05) found be- tween groups T02 (abamectin 200 μg/kg) and T05 (ivermectin 450 μg/ kg+ abamectin 250 μg/kg) for AST, it is essential to reinforce that values quantified for the aforementioned groups did not differ sig- nificantly (p > 0.05) regarding average levels of CSF aspartate ami- notransferase (u/L) present in calves belonging to the control group (T01 – saline solution). Therefore, this result does not support the Table 4 Data analysis of the biochemical parameters performed to bovine animals under 30 days submitted to different concentrations of avermectin. Group Treatment Experimental day/Total serum protein (g/dL) ZERO 1 7 14 I Saline solution 6.86 A 6.99 A 6.88 A 7.09 A II Abamectin (200 μg) - recommended dosel 7.41 A 7.15 A 7.30 A 7.22 A III Abamectin (400 μg) 6.52 A 6.88 A 6.73 A 6.72 A IV Abamectin (600 μg) 6.96 A 7.04 A 6.90 A 7.16 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 7.39 A 7.01 A 7.14 A 6.32 A VI Ivermectin (900 μg)+Abamectin (500 μg) 7.18 A 7.11 A 6.80 A 7.18 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 7.39 A 7.74 A 7.47 A 7.58 A Group Treatment Experimental day/AST (g/dL) ZERO 1 7 14 I Saline solution 55.57 A 52.83 A 55.47 A 81.16 A II Abamectin (200 μg) - recommended dose 58.67 A 86.81 A 71.23 A 63.87 A III Abamectin (400 μg) 54.94 A 49.25 A 52.40 A 44.71 A IV Abamectin (600 μg) 49.70 A 53.39 A 55.48 A 55.04 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 88.95 A 143.69 A 44.00 A 53.89 A VI Ivermectin (900 μg)+Abamectin (500 μg) 59.83 A 48.15 A 54.50 A 61.21 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 55.39 A 48.89 A 65.72 A 52.94 A Group Treatment Experimental day/GGT (g/dL) ZERO 1 7 14 I Saline solution 36.40 A 36.40 A 28.80 A 33.80 A II Abamectin (200 μg) - recommended dose 32.60 A 42.60 A 57.00 A 54.40 A III Abamectin (400 μg) 47.80 A 36.40 A 28.00 A 29.80 A IV Abamectin (600 μg) 39.00 A 37.78 A 32.60 A 32.40 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose of label 58.00 A 60.40 A 41.40 A 40.20 A VI Ivermectin (900 μg)+Abamectin (500 μg) 25.80 A 56.80 A 41.60 A 41.40 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 29.80 A 32.40 A 30.00 A 38.80 A Group Treatment Experimental day/FA (g/dL) ZERO 1 7 14 I Saline solution 131.25 BC 96.01 B 107.20 A 109.45 A II Abamectin (200 μg) - recommended dose 104.64 C 134.32 AB 137.66 A 131.02 A III Abamectin (400 μg) 163.58 ABC 184.36 A 178.80 A 186.46 A IV Abamectin (600 μg) 226.70 A 195.22 A 164.12 A 172.16 A V Ivermectin (450 μg)+Abamectin (250 μg) - recommended dose 118.42 C 169.16 AB 145.92 A 145.96 A VI Ivermectin (900 μg)+Abamectin (500 μg) 201.80 AB 154.74 AB 139.12 A 141.34 A VII Ivermectin (1350 μg)+Abamectin (750 μg) 167.84 ABC 135.96 AB 122.52 A 129.40 A Means values followed by the same letter on the same colun do not differ significantly at a 95% reliability level. D. de Castro Rodrigues et al. Research in Veterinary Science 118 (2018) 403–412 410 inference that increases observed in groups T02 and T05 occurred as a consequence of synthetic chemical treatments performed on these an- imals. 3.3. Biochemical blood tests Total proteins analyzed stayed within normal limits for all experi- mental dates (Kaneko et al., 1997). Regarding AST, the majority of values were within normal ranges, while some were above normal. For GGT, all values were above those cited as normal for bovines (Kaneko et al., 1997). Nonetheless, it is possible to verify that no significant differences (p > 0.05) occurred among biochemical values of total proteins, AST and GGT obtained from animals treated with different formulations containing abamectin and/or ivermectin compared to the control group across all observational dates (Table 4 and Fig. 3). As for alkaline phosphatase, significantly greater (p≤ 0.05) average values were found for groups of calves treated with 400 and 600 μg/kg abamectin than in the other groups. However, it is essential to reinforce that significant differences (p≤ 0.05) between groups regarding mean alkaline phosphatase values were already observed before the start of treatments (Table 4 and Fig. 3). In addition, alkaline phosphatase va- lues observed in experimental calves (treated and control) throughout the experiment remained within normal parameters for the target species (Kaneko et al., 1997), supporting the finding that reported significant differences had no clinical significance. 4. Conclusion Even though it is well known that abamectin may lead to cases of intoxication in bovines less than four months old, based on results obtained in the present study, it was not possible to observe adverse effects, including neurological toxicity, biochemical and cerebrospinal fluid related, in calves< 30 days old that had received up to 600 μg/kg of an abamectin-based formulation, as well as an association containing up to 1350 μg/kg and 750 μg/kg ivermectin + abamectin, respec- tively,. The level and activity of the multiple drug resistance gene (MDR1 - ABCB1) present in the calves may have influenced the results found in this study. Future studies should be carried out. References Albert, J., Lingle, D.H., Marder, E., O'Neil, M.B., 1986. A GABA-activated chloride con- dutance not blocked by picrotoxin on spiny lobster neuromuscular preparetions. Br. J. Pharmacol. 87, 771–779. Bailey, C.S., Vernau, W., 1997. Cerebrospinal fluid. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. 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(Ed.), Clínical syndromes in -400 -200 0 200 400 600 800 ) L d/ g( T S A -100 -50 0 50 100 150 200 250 ) L d/ g( T G G s e nil a S n oit ul o nitc e m a b A g k/ g µ 0 0 2 nitc e m a b A g k/ g µ 0 0 4 nit c e m a b A gk/ g µ 0 0 6 0 5 4 nitc e m r evI tc e m a b A + gk/ g µ g k/ g µ 0 5 2 ni 0 0 9 nitc e mr evI tc e m a b A + gk/ g µ gk/ g µ 0 0 5 ni 3 1 nitc e mr evI m a b A + gk/ g µ 0 5 gk/ g µ 0 5 7 nitc e 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 50 100 150 200 250 300 350 400 ) L/ u( es at a h ps o h p e n il akl A Mean Mean±SE Mean±2*SD s e nil a S n oit ul o nitc e m a b A gk/ g µ 0 0 2 nit c e m a b A gk/ g µ 0 0 4 nit c e m a b A gk/ g µ 0 0 6 0 5 4 ni tc e mr evI tc e m a b A + gk/ g µ gk / g µ 0 5 2 ni 0 0 9 nitc e mr evI tc e m a b A + gk/ g µ gk / g µ 0 0 5 ni 3 1 nit c e mr evI m a b A + gk/ g µ 0 5 g k/ g µ 0 5 7 nitc e 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 0 1 7 14 Treatment Day 2 3 4 5 6 7 8 9 10 11 ) L d/ g( ni et or p m ur es l at o T Fig. 3. 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