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Pharmacodynamic evaluation of a reference and a generic toltrazuril preparation in broilers experimentally infected with Eimeria tenella or E. acervulina a

b

c

d

Y. Alcala-Canto , E. Ramos-Martinez , G. Tapia-Perez , L. Gutierrez & H. Sumano

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Departamento de Parasitología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico b

Departamento de Medicina Experimental. Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico c

Departamento de Genética y Bioestadística,. Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico d

Departamento de Fisiologia y Farmacologia, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico Accepted author version posted online: 08 Jan 2014.Published online: 16 Apr 2014.

To cite this article: Y. Alcala-Canto, E. Ramos-Martinez, G. Tapia-Perez, L. Gutierrez & H. Sumano (2014) Pharmacodynamic evaluation of a reference and a generic toltrazuril preparation in broilers experimentally infected with Eimeria tenella or E. acervulina, British Poultry Science, 55:1, 44-53, DOI: 10.1080/00071668.2013.872770 To link to this article: http://dx.doi.org/10.1080/00071668.2013.872770

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British Poultry Science, 2014 Vol. 55, No. 1, 44–53, http://dx.doi.org/10.1080/00071668.2013.872770

Pharmacodynamic evaluation of a reference and a generic toltrazuril preparation in broilers experimentally infected with Eimeria tenella or E. acervulina Y. ALCALA-CANTO, E. RAMOS-MARTINEZ1, G. TAPIA-PEREZ2, L. GUTIERREZ3 H. SUMANO3

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Departamento de Parasitología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico, 1Departamento de Medicina Experimental. Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico, 2Departamento de Genética y Bioestadística,. Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico, and 3Departamento de Fisiologia y Farmacologia, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico

Abstract 1. The aim of this study was to investigate the effects on pigmentation, faecal oocyst output, immune responsiveness and reactive oxygen species (ROS) generation following treatment with either the reference toltrazuril (Baycox) or a generic preparation (gen-TTZ), during an experimental Eimeria tenella (Et) or E. acervulina (Ea) infection of 210 Ross broiler chickens. 2. Results showed a significant difference on the anticoccidial efficacy 6 d after treating infected animals with Baycox (Et: 99.69% and Ea: 99.52%) or gen-TTZ (Et:85.71% and Ea 81.81%). 3. Gen-TTZ-treated animals were less strongly carotenoid-pigmented than Baycox-treated broilers. Mean plasma carotenoid concentrations were significantly higher in groups treated with Baycox than in broilers given gen-TTZ. 4. Treatment of animals with Baycox led to a significant decrease in ability of the peripheral blood mononuclear cells to produce ROS in contrast to gen-TTZ-treated groups. Baycox, but not generic toltrazuril, increased IL-10 and decreased tumour necrosis factor alpha (TNF-α) concentrations in chickens infected with E. tenella and E. acervulina. 5. It is suggested that differences in anticoccidial efficacy may be observed when using a generic toltrazuril product. Hence, in addition to plasma profiles of drugs, standardised clinical control tests may be necessary for generic formulations, particularly if other parameters are important to achieve a better control of coccidiosis.

INTRODUCTION Eimeria spp. is a protozoan genus that causes the parasitic disease known as coccidiosis and produces severe intestinal damage in poultry due to disorders in nutrient absorption and food digestion. Economic losses are mainly due to reduced feed conversion efficiency, retarded chicken growth as well as depressed weight gain. This disease can produce bloody excreta and mortality

may range from 5% to 70% (Zhang et al., 2012). Several species of Eimeria that parasitise poultry have been described. Nevertheless, E. acervulina and E. tenella are two of the most frequently diagnosed pathogens. The former infects the duodenum whereas E. tenella is found in the caecum (Cornelissen et al., 2009). Published studies have described the general features of the immune response to Eimeria species (Haritova and Stanilova, 2012). The first line of defence against

Correspondence to: H. Sumano, Departamento de Fisiologia y Farmacologia, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico. E-mail: [email protected] Accepted for publication 15 October 2013.

© 2014 British Poultry Science Ltd

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these pathogens is represented by phagocytes such as macrophages that produce cytokines and consequently promote the development of cellmediated immunity (Chow et al., 2011). Previous reports have demonstrated that T helper 1/T helper 2 (Th1/Th2) (Casagrande et al., 2011) balance is necessary to protect the host against coccidiosis, and that the Th1 response is dominant during coccidiosis in birds older than 7 d (Cornelissen et al., 2009). Cytokines such as Interleukin-12 (IL-12), Interferon gamma (IFN-γ), IL-1b and tumour necrosis factor alpha (TNF-α) are associated with inflammatory responses, while Interleukin-10 (Yang et al., 2006) favours the development of Th2 responses and thus plays an important role in preventing the development of strong responses driven by Th1type cytokines and consequently reduces immunemediated damage (Rothwell et al., 2004). Reactive oxygen species (ROS) (Alonso-Alvarez et al., 2004) are produced upon macrophage activation and act against pathogens by activating the NADPH oxidase system to form the superoxide anion from molecular oxygen. Subsequently, biotoxic compounds such as peroxynitrite, hypochlorous acid and hydroxyl radicals are generated (Sild et al., 2011). However, ROS can damage the host as a result of overproduction (Chow et al., 2011), thereby compromising the integrity of tissues. The immune system is also sensitive to ROS because plasma membranes of immune cells contain large amounts of polyunsaturated fatty acids (Simons et al., 2012). Antioxidants prevent damage caused by this ROS, and when antioxidant systems do not quench free radicals, oxidative stress and tissue injury is induced (Kielland et al., 2009). A recent study indicated that carotenoids can buffer the pro-oxidant effect of the NADPH oxidase system activation (Kandeel, 2011) and therefore improve immune system responsiveness by protecting immune cells and proteins from the collateral damage induced by ROS and by serving a physiological role involved in tissue repair (Sild et al., 2011; Simons et al., 2012). Eimeria inhibits the gastrointestinal uptake of several essential and dietary components such as carotenoids, and one would expect that infection should negatively affect immune functioning and the state of oxidative stress. Furthermore, carotenoid-based pigmentation of skin and legs would also be depressed. In wild birds, carotenoid-based sexual traits are involved in the mate-choice process (Baeta et al., 2008). Nonetheless, in poultry, pigmentation depends upon several aspects such as health, breed, age or sex. In modern and traditional Mexican markets, consumers tend to favour broiler carcass pigmentation, which is based on the ingestion of xanthophylls. By adding these carotenoid-based pigments, broiler producers

45

obtain more profit from poultry products. Previous studies have demonstrated that infection with Eimeria and other parasites negatively affects pigmentation in wild and domestic birds as a consequence of malabsorption (Allen, 1997; Hõrak et al., 2004; Martinez et al., 2004; Zhao et al., 2006; Martinez-Padilla et al., 2007; Baeta et al., 2008; Butler and McGraw, 2010, 2011). Consequently, it would be expected that Eimeria control using anticoccidial drugs would improve the absorption and utilisation of carotenoids supplemented to experimentally infected broilers, in addition to improving immunocompetence and reducing tissue damage induced by ROS. Several anticoccidial drugs are used in Mexico. Toltrazuril is an antiprotozoan drug available as an oral formulation that is extensively used to control coccidiosis. This product acts against intracellular sexual and asexual stages of the pathogen, excluding oocysts (Kandeel, 2011). Several generic formulations of toltrazuril have been introduced into the pharmaceutical market in Mexico after the original patent of the first approved toltrazuril formulation expired (Baycox, Bayer). Regulatory standards require that a pharmaceutical equivalence be regarded as bioequivalent when plasma or serum bioavailability and key pharmacokinetic variables are not statistically different within a range of 80-125% as compared to the reference preparation. Yet, in spite of bioequivalence of generic compared with the reference preparation, different antiparasitic efficacies have been reported among preparations of antiparasitic agents such as ivermectin (Genchi et al., 2008; Suárez et al., 2013). Considering the above, an experiment was conducted to evaluate the pharmacodynamic properties of toltrazuril in order to better understand the relevance of substitutability among reference (Baycox, Bayer) and generic (TTZ-G) preparations, based on Eimeria oocyst output, absorption of carotenoids, pigmentation, cytokine production and the state of oxidative stress in experimentally infected Eimeria tenella and E. acervulina poultry.

MATERIALS AND METHODS Animals The experiments were conducted in accordance with the approved guidelines of the Institutional Committee for the Purpose of Control and Supervision of Experimentation on Animals of the National Autonomous University of Mexico (UNAM). A total of 210 ten-d-old ROSS broilers were purchased from a commercial poultry farm. All animals were raised on concrete floors covered with wood shavings and fed a standard diet

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Y. ALCALA-CANTO ET AL.

prepared as recommended by the National Research Council (1994). A xanthophyll-based pigment extracted from Tagetes erecta denominated Xamacol (IQF ENAMEX, S.A de C.V.) was added to the diet at 80 mg/kg (Martínez et al., 2004). Feed and water were provided ad libitum.

carotenoids in plasma were measured only when the broilers were 12 and 24 d old. The estimation of ROS concentrations was carried out before treatment (zero time), and at 6, 12 and 24 h after treatment. Interleukin (IL)-10 and TNF-α concentrations were measured on d 7 post-infection (17 d of age).

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Parasites For experimental infections, a stock of E. tenella and E. acervulina sporulated oocysts were used. Oocysts were obtained from a poultry farm and were kept separated by species at the Department of Parasitology, UNAM. Briefly, Eimeria oocysts were collected from faeces, small intestine and ceca of naturally infected chickens. Oocysts were sporulated and microscopically identified according to taxonomic keys. E. tenella and E. acervulina oocysts were isolated, washed with phosphate buffered saline (PBS, pH 7.4) and counted using a McMaster chamber. Experimental design A longitudinal study was carried out on the broilers. Birds were randomly distributed into 7 groups which were randomly allocated in three separate cages of 10 birds per cage, with enough room for growing animals. Groups 1, 3 and 5 (Et, Et-BAY and Et-TTZ-G) were orally inoculated with 0.4 ml of PBS containing 1.0 × 104 E. tenella sporulated oocysts; groups 2, 4 and 6 received 0.4 ml of PBS containing 1.0 × 104 E. acervulina sporulated oocysts orally (Kim et al., 2013). Previous studies carried out in our laboratory have shown that these doses lead to pathology, but are not lethal (unpublished data). Animals belonging to groups 1 and 2 (Et-BAY, Ea-BAY) were treated with 7 mg/ kg of a reference toltrazuril (Baycox Bayer Sanidad Animal México) at 11 and 12 d postinfection (Mathis et al., 2004); whereas groups 3 and 4 (Et-TTZ-G, Ea-TTZ-G) were provided with 7 mg/kg of a generic commercial brand of toltrazuril. To maintain confidentiality, this product was designated gen-TTZ. The labelling of the generic toltrazuril brand used in the current trial does not contain any indication of having a different vehicle composition or formulation as compared to the first approved toltrazuril product (Baycox). Animals in the positive control groups 5 (Et) and 6 (Ea) were orally administered 0.4 ml of PBS. Group 7 was neither infected nor treated at all (UC). Reference and generic commercial preparation of toltrazuril were added to the diet according to manufacturer’s instructions. Data of yellowness were measured individually in all broilers when they were 21, 28, 35 and 49 d old. The number of oocysts per g was recorded on d 7, 11 and 14 post-infection, that is when broilers were 17, 21 and 24 d old, whereas the concentrations of

Anticoccidial efficacy The number of oocysts per gram of litter was determined according to Conway and McKenzie (2007). Litter samples were collected from each cage on d 7, 11 and 14 after the experimental infection. Three pooled samples from 10 broilers in each were processed as follows: 10 g of the composite sample were immersed in 100 ml of water, filtered and centrifuged at 800 × g for 5 min. The supernatant liquid was discarded and the pellet re-suspended in a 15-ml volume saturated solution of sodium chloride. This suspension was shaken and a sample was placed in a McMaster counting chamber to calculate the number of oocysts per gram of litter. Anticoccidial efficacy was determined using the following formula (Holdsworth et al., 2004): E¼

XC  XT  100 XC

where: E = efficacy (%) XC = mean of excreted oocysts per gram of faeces in infected and untreated controls XT = mean of excreted oocysts per gram of faeces in treated animals. In vivo pigmentation evaluation The system colour profile of yellowness was measured using a reflectance colorimeter Minolta CR300 (Konica Minolta Business Solutions de Mexico S.A. de C.V., Tlanepantla, Edo. de Mexico, Mexico) on the right lateral apteria (Castaneda et al., 2005). Blood sampling and plasma carotenoid concentration Blood samples from each chicken were collected from the brachial vein at 28 and 42 d of age (Yang et al., 2006) in 4 birds per group using sterile needles and heparinised tubes. Blood was centrifuged (4000 × g for 15 min). Circulating carotenoid concentration was immediately assessed as previously published (Alonso-Alvarez et al., 2004). Briefly, 20 µl of plasma were diluted in 180 µl of absolute ethanol. The dilution was mixed in a vortex during 1 min and flocculent proteins were precipitated by centrifugation at 1500 × g

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for 10 min. The supernatant was examined in a spectrophotometer and the optical density of the carotenoid peak at 450 nm was determined. Carotenoid concentration was estimated from a standard curve of lutein (alpha-carotene-3,3′-diol, Sigma-Aldrich, México).

Statistical analysis

Isolation of peripheral blood mononuclear cells (PBMC)

Analysis

Heparinised blood was collected from all birds at 0, 6, 12 and 24 h after treatment with Baycox or gen-TTZ. PBMC were obtained by density gradient centrifugation using Ficoll–Hypaque. Cells were washed with PBS, suspended to a final concentration of 1 × 106 cells/ml in RPMI-1640 culture medium (Sigma-Aldrich México), supplemented with 10% heat-inactivated new born calf serum, 5% phyto-haemagglutinin, 50 IU/ml penicillin and 50 μg/ml streptomycin. Cell viability was determined by trypan blue dye exclusion (Kuwana et al., 2002).

Estimation of ROS concentrations To evaluate the generation of ROS at 6, 12 and 24 h after treatment with Baycox or gen-TTZ, the cell-permeant probe 6-carboxy-2,7-dichlorodihydrofluorescein diacetate (DCFDA) (Duranteau et al., 1998) was used. Briefly, 1 × 106/ml PBMC were resuspended in RPMI-1640 culture medium, H2DCFDA was added at 5 µM and the background was read at 530 nm on a VICTOR X4 multi-label plate reader (Perkin-Elmer, Norwalk, CT, USA); later phorbolmyristicacetate (PMA, Sigma-Aldrich, México) (Prowse et al., 1992) was added and the cultures were incubated for 30 min at 37°C in the dark. Finally, the ROS estimation was determined using the same conditions as in the background.

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Sample size

The sample size was obtained with the G-Power programme (Faul, 2007) for repeated measures with a small effect size = 0.11 for a test power (1 – β) = 0.82 (Cohen, 1992).

Longitudinal data, as yellowness, efficacy (%) of product, concentration of carotenoids in plasma and ROS, were analysed with a mixed model; the model incorporated random subject effects to accommodate between-subjects variability and autocorrelation for within-subject variability; quasi-binomial approximation to the binomial was used to convert continuous proportions into the ratio of successes over total number of trials (N). Residual maximum likelihood estimation (REML) was used to estimate the equations for the variance parameters, because those variables were not normally distributed (Gill, 2000). Group differences were analysed by Bonferroni’s multiple-range test. Cytokine concentrations were compared by ANOVA followed by Bonferroni’s multiple-range test. Data are presented as means ± SD. Differences between groups were considered significant when P < 0.05. The analysis was carried out using SPSS v19.

RESULTS Table 1 presents the P-values of fixed effects (group) for in vivo pigmentation, plasma carotenoid concentration, estimation of ROS concentrations and cytokine measurements of samples collected from broilers treated with a generic (gen-TTZ) or reference toltrazuril (Baycox) preparation. Anticoccidial efficacy

Cytokine measurement On d 7 post-infection, animals were bloodsampled via the brachial vein. Blood was centrifuged at 500 × g for 10 min at 4°C. The supernatant was collected and stored until analysis. IL10 and TNF-α concentrations were measured using a commercial Nitrite/nitrate assay kit (Kamiya Biomedical Company, Gateway Drive, Seattle WA, USA) (Chalhoub et al., 2011). The absorbance was measured at 405 nm with an automated ELISA reader. The cut-off point was determined as the mean of 10 uninfected controls plus three standard deviations. A sample was considered positive if the calculated absorbance value was equal to or greater than the cut-off point. Each sample was assayed in duplicate.

After experimental infection, it took approximately 6 d for the animals to show clinical signs such as diarrhoea and watery stools; however, no lethality was recorded. In untreated controls, an increase in oocyst shedding in faeces was observed. The group treated with Baycox on d 11 and 12 post infection showed the highest oocyst inhibitory effect (99.69%), statistically higher than that for the generic toltrazuril (85.71%) (P < 0.05). A statistically significant difference was also observed in E. acervulina-infected broilers treated with Baycox (99.52%) or gen-TTZ (81.81%). The level of oocyst excretion was significantly (P < 0.05) higher in gen-TTZ-treated animals throughout the faecal sampling and coprological testing period, as shown in Table 2.

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Table 1. Summary of results in the mixed model analysis for in vivo pigmentation, plasma carotenoid concentration, estimation of ROS concentrations (ROS) and cytokine measurement in broilers either treated with generic (gen-TTZ) or reference toltrazuril (Baycox) (n= 10) Random effects Time1

Fixed effects Group Variable

df2

F

P

Time

Concentrations

Variance3

6 3 6 5 5 5

1840.0 10.6 110.8 218.2 152.0 56.2

0.0001 0.001 0.001 0.0001 0.0001 0.0001

D D H H

3 4 3 3

1.1 1600.0 120.3 120.1

Yellowness, b* Efficacy, % Carotenoids, μg/ml ROS, Fluorescence Intensity Units TNF-α, pg/ml IL-10, pg/ml

Estimated variances for the repeated variable in linear non-normal models in which there exists a low-dimensional sufficient statistic for the fitted values analysed by REML. 2 The model included the intercept; 6 df indicates that the untreated control was added to model; 3 df indicates that the 4 treated groups were analysed; 5 df indicates that 4 treated groups and 2 positive controls were analysed. 3 P < 0.001 in all time random effects. 1

Table 2.

Anticoccidial efficacy (%) of generic (gen-TTZ) or reference toltrazuril (Baycox) in E. tenella or E. acervulina-infected broilers (n= 10, mean ± SD)

Days post-treatment

Efficacy of Baycox, %

7 11 14

E. acervulina

Efficacy of gen-TTZ, %

a

Efficacy of Baycox, %

b

53.44 ± 3.22 90.24 ± 0.39a 99.69 ± 0.33a

Efficacy of gen-TTZ, %

c

8.62 ± 3.85 43.90 ± 3.54b 85.71 ± 0.54b

36.73 ± 6.29 88.37 ± 0.74a 99.52 ± 0.30a

8.16 ± 0.07b 41.86 ± 5.56b 81.81 ± 2.04b

a,b,c

Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05 (Bonferroni’s multiple-range test).

In vivo pigmentation evaluation

less strongly coloured (P < 0.05) than Baycox-treated broilers as colorimetric measures corresponded to paler values in the former group.

As shown in Figure 1, the yellow component of hue (b*) significantly decreased over time in infected animals irrespective of the Eimeria species, compared with treated and uninfected birds (P < 0.05). In broilers infected with sporulated E. tenella or E. acervulina oocysts, toltrazuril addition prevented the de-pigmentation on d 21 after infection and remained as such until the end of the study. Yellowness (b*) was higher than 20 from d 28 post infection until the end of the experiment. The gen-TTZ-treated animals were

Plasma carotenoid concentration At d 28, circulating carotenoids tended to decrease with increased coccidian burden in untreated groups. Moreover, E. acervulina-infected animals had significantly less carotenoids than E. tenella-infected birds (P < 0.05). Carotenoid concentrations were significantly higher in treated

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Figure 1. In vivo measurement of yellow pigmentation (b*) in broilers infected with E. tenella (Et) or E. acervulina (Ea) and treated with Baycox (Et-Baycox or Ea-Baycox) or generic toltrazuril (Et-gen-TTZ or Ea-gen-TTZ) (n= 10). a,b,c,d Different superscript letters between groups indicate a statistically significant difference (P < 0.0001, Bonferroni’s multiple-range test).

Plasma carotenoid levels (μg/ml)

PHARMACODYNAMIC EVALUATION OF TWO TOLTRAZURIL PREPARATIONS 3.5 e 3.0

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PBMC ability to produce ROS, as compared to infected and untreated broilers (P < 0.05). However, changes in fluorescence also differed significantly (P < 0.05) between the Baycox and the gen-TTZ-treated groups, as ROS production was significantly higher in the latter experimental group at 6, 12 and 24 h after receiving anticoccidial treatment. Cytokine measurement

n Co

Figure 2. Measurement of plasma carotenoids in broilers infected with E. tenella (Et) or E. acervulina (Ea) and treated with Baycox (Et-Baycox or Ea-Baycox) or generic toltrazuril (Etgen-TTZ or Ea-gen-TTZ) (n= 10). Different superscript letters between groups indicate a statistically significant difference (P < 0.05, Bonferroni’s multiple-range test).

than in non-treated broilers (P < 0.05). However, the increase was higher in Baycox-treated birds than in animals that received the generic brand. On the other hand, uninfected control birds showed no significant change in plasma carotenoids. The same pattern was observed on 42-dold broilers, with treated groups having more carotenoids than on d 28. Overall, carotenoids did not reach the same concentration in groups treated with Baycox or gen-TTZ throughout the experiment, as samples collected from the former treated group showed higher pigment concentrations at all measured times (see Figure 2). Estimation of ROS concentration As shown in Figure 3, ROS generation in treated animals of both groups (generic and reference toltrazuril) led to a significant decrease in the

In uninfected control broilers, production of TNF-α and IL-10 remained below the limit of detection (data not shown). However, the mean concentration of TNF-α was significantly higher in birds infected with E. tenella (Et) or E. acervulina (Ea) (P < 0.05). Also, animals treated with generic toltrazuril showed greater values of TNF-α (E. tenella: 27.44 ± 1.37 pg/ml, E. acervulina: 29.18 ± 0.12 pg/ml, Et-gen-TTZ: 23.09 ± 1.83 pg/ ml, Ea-gen-TTZ: 21.42 ± 0.53 pg/ml) than the Baycox-treated broilers (Et-Baycox: 17. 15 ± 1.06 pg/ml, Ea-Baycox: 16.67 ± 1.32 pg/ml). In contrast, serum concentrations of IL-10 in broilers treated with Baycox were significantly higher than that of generic toltrazuril-treated animals and birds infected with Eimeria, irrespective of the species (P < 0.05) (see Figure 4).

DISCUSSION Results showed a statistically significant difference in the anticoccidial efficacy 6 d after treating infected animals with Baycox (Et: 99.69% and Ea: 99.52%) or gen-TTZ (Et:85.71% and Ea 81.81%). Anticoccidial efficacy was measured by estimating the reduction of faecal oocyst output after the administration of toltrazuril. Moreover, it was also assessed by demonstrating the decrease of

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Et-Baycox Ea-Baycox Et-gen-TTZ Ea-gen-TTZ E. tenella E. acervuling

Figure 3. Generation of ROS in broilers infected with E. tenella (Et) or E. acervulina (Ea) and treated with Baycox (Et-Baycox or EaBaycox) or generic toltrazuril (Et-gen-TTZ or Ea-gen-TTZ) (n= 10). Different superscript letters between groups indicate a statistically significant difference (P < 0.001, Bonferroni’s multiple-range test).

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TNF-αand IL-10 levels (pg/ml)

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Experimental groups

Figure 4. Serum concentrations of TNF-α and IL-10 in broilers infected with E. tenella (Et) or E. acervulina (Ea) and treated with Baycox (Et-Baycox or Ea-Baycox) or generic toltrazuril (Et-gen-TTZ or Ea-gen-TTZ) (n= 10). Different superscript letters between groups indicate a statistically significant difference (P < 0.001, Bonferroni’s multiple-range test).

unfavourable consequences of coccidiosis, such as the limitation on the availability of carotenoids, ROS generation and the induction of a protective immune response. This study was not designed to assess the mechanisms that link coccidian infection with circulating carotenoids, but it was possible to examine whether anticoccidial treatment may influence the outcome of the infection. There is extensive correlation evidence of the effect of infection and carotenoid availability in parasiteinfected birds (Allen, 1997; Zhao et al., 2006; Baeta et al., 2008). The present findings are in agreement with these statements because a decrease in pigmentation was observed in E. tenella or E. acervulina-infected broilers. The in vivo decline in colour, measured by a reflectance colorimeter in this study, is consistent with several studies that have reported decreased carotenoid concentrations to be among the most important clinical signs shown in the peak phase of coccidial infections in birds; and that have also demonstrated that parasites slow down the assimilation of carotenoids in the blood even in carotenoidsupplemented birds (Martinez-Padilla et al., 2007; Butler and McGraw, 2010). It is important to mention that in broilers, yellowness (b*) of over 20 is considered acceptable. Decreases in pigmentation might be due to the reduction of digestive and absorptive capacity of mucosa after the destruction of epithelial cells during the reproduction of the parasite. Experimental assessment of carotenoid access was warranted by measuring circulating carotenoid content. Interestingly, treatment with Baycox led to a faster increase in both circulating concentrations and colouration, whereas a significant reduction of pigments was observed in untreated and gen-TTZ-treated birds. It is tempting

to speculate that these findings might be related to the fact that the generic toltrazuril produced a delayed and significantly lower oocyst output reduction than the reference preparation of toltrazuril. At this stage, it is not known whether this is a direct lack of antiparasitic activity or a pharmacokinetic difference with the reference drug, which could allow a longer period of time for Eimeria to disturb the permeability of epithelial cells and affect absorption of nutrients, including carotenoids (Horak et al., 2004; Baeta et al., 2008). Decreased concentrations of carotenoids in peripheral blood as well as in visible tissues is consistent with their antioxidant functions (Baeta et al., 2008). Simons et al. (2012) showed that carotenoids increase resistance against free radicals. It was previously shown that during the E. tenella challenge, cytokines have the potential to activate cells to generate ROS that mediate parasite damage by killing them (Prowse et al., 1992; Allen, 1997). ROS production was significantly greater in untreated controls and in broilers that were given the generic toltrazuril compound. Inflammation and oxidative stress, such as that induced by coccidiosis, may contribute to tissue damage and hyperalgesia (Gao et al., 2007). In the present study, ROS generation was decreased in Baycox and gen-TTZ-treated broilers. Nonetheless, a significant difference in reduction of ROS production was shown in favour of Baycox. Previous studies have provided support for the idea that carotenoid supplementation allows infected individuals to cope with the costs associated with immune responses (Butler and McGraw, 2010). It is therefore likely that an anticoccidial treatment such as toltrazuril could limit infection more efficiently if it

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PHARMACODYNAMIC EVALUATION OF TWO TOLTRAZURIL PREPARATIONS

does not interfere with immunity (Peek and Landman, 2011). However, it has been shown that toltrazuril eliminates parasites at an early phase of infection and thus allows the proliferation of T cells that recognise early-phase antigens that are critical for protection in contrast to later antigens (Steinfelder et al., 2005). During an Eimeria infection, production of cytokines such as IL-12, IFN-γ, IL-1β and TNF-α induces the development of cell-mediated immunity against coccidiosis. Amongst these cytokines, TNF-α has been implicated in mucosal damage by increasing leukocyte adherence. It has been shown that ROS induce TNF-α as a defence mechanism and consequently, TNF-α recruits monocytes and macrophages to generate more ROS (Yadav et al., 2013). On the other hand, the role of IL-10 has been reported to be relevant in coccidiosis as it changes the T-helper bias during infection and thereby contributes to susceptibility of a specific line of chickens (Rothwell et al., 2004), promotes the development of humoral-mediated immunity and is implicated in anti-inflammatory responses (Chow et al., 2011). Therefore, in the present study, we measured concentrations of IL-10 and TNF-α in chickens treated with Baycox and gen-TTZ to assess the induction of two important cytokines released during coccidiosis in chickens. The results demonstrate that IL10 increases in toltrazuril-treated animals and the effect was paralleled by a decrease in TNF-α and ROS concentrations in agreement with previous studies (Chow et al., 2011; Huet et al., 2013). These authors showed that IL-10 decreases the level of inflammation induced TNF-α in cells by reducing the TNF-α-induced ROS. In fact, many aggressive situations, such as infection or trauma, have been associated with increased cytokine concentrations and consequent down-regulation of pro-inflammatory cytokine production (Chalhoub et al., 2011). Interestingly, in the present study, anticoccidial treatment with Baycox induced the production of higher concentrations of IL-10 and lower concentrations of TNF-α compared with gen-TTZ. On the whole, the results of the present study are in agreement with previous experiments that show that carotenoids sustain immune functions (Baeta et al., 2008; Sepp et al., 2011). Furthermore, it is postulated that the more effective and early elimination of intracellular stages of Eimeria by Baycox is associated with decreased inflammation, less damage to epithelial cells and reduced invasion by the parasite. Indeed, Baycox has been proven to reduce the release of inflammatory mediators and preserve intestinal integrity (Veronesi et al., 2013). The fact that Baycox only acts against intracellular stages earlier than the genTTZ product tempts us to speculate that the immune system is challenged by fewer antigens of destroyed parasites and would therefore be less likely to induce tissue damage in contrast to an exaggerated response

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elicited by a larger number of dead parasite-derived antigens. This hypothesis is supported by the current data, as lower concentrations of ROS and TNF-α contributed to reduced damage in the gastrointestinal tract of infected animals. To prevent parasite multiplication and reduce epithelial cell alteration, several drugs with anticoccidial activity have been approved for broilers. Some of them target only the gamonts, but as the life cycle is almost completed, most of the damage has already been done (Mundt et al., 2003a, 2003b, 2005a, 2005b). In contrast, toltrazuril has been shown to be effective against all species and intracellular stages of Eimeria in chickens (Mathis et al., 2004). It does not interfere with the development of natural immunity and can even enhance it (Greif, 2000). The reference brand of toltrazuril is Baycox (Bayer); however, several generic preparations are now available worldwide. Based on the regulatory specifications, a generic preparation is granted permission to enter the local market mainly based on pharmacokinetic data. To the best of our understanding, other pharmacodynamic data, except clinical efficacy, are rarely compulsory to register a new generic formulation. Thus, clinicians’ decisions on the brand of toltrazuril to be used are mainly based on direct comparative costs, with the underlying perception that local regulatory agencies endorse similar efficacy of all preparations of toltrazuril. The World Health Organization (1998) defines a generic drug as one with the same qualitative and quantitative composition of the active ingredient, the same pharmaceutical presentation and whose bioequivalence with the reference drug has been demonstrated. Yet, this definition allows variation among generic preparations. For example, Patel et al. (2012) consider a generic preparation to be of poor quality when it does not meet established standards in terms of identity, purity, bioavailability of the active pharmaceutical ingredient, as well as appropriate packaging and labelling of the finished product. However, recent studies have found increasing numbers of poor-quality medicines in some countries (Patel et al., 2012) and bioequivalence of a generic vs. a reference preparation is not always warranty of comparable clinical efficacy (Genchi et al., 2008; Suárez et al., 2013) Results obtained in this study supports these latter views, and considering the importance of this drug in veterinary medicine as well as the lack of reports of comparative anticoccidial efficacy of generic preparations prompt the suggestion that a more complete screening of key pharmacodynamic parameters may be required to fully declare certain drug preparations as generic.

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Y. ALCALA-CANTO ET AL.

This work was designed as a longitudinal study; the defining feature of a longitudinal data set is repeated observations on experimental units. Longitudinal data require special statistical methods because the set of observations on one subject tends to be intercorrelated. These correlations must be taken into account to draw valid scientific inferences. The method chosen here to estimate the covariance parameters in linear nonnormal models, in which there exists a low-dimensional sufficient statistic for the fitted values, was REML. REML estimators maximise only the portion of the likelihood that does not depend on the fixed effects, and orthogonally estimates that effect which in this work is the group of treatment (Blood and Cheng, 2011). The estimated variance component of time in the efficacy variable was the largest of all; this was expected since the response data was not count data and a quasi-binomial approximation to the binomial was used to convert continuous proportions to the ratio of successes over total number of trials, N, for a variety of possible N values for the percentage variable at each pool (three for each group) (Shannon, 2004). Summarising, taken the statistically significant differences observed in this trial, namely anticoccidial efficacy, immune responsiveness, carotenoid availability and generation of ROS, it is clear that important differences exists between the reference (Baycox) and the tested generic preparation of toltrazuril. In turn, these differences prompt the suggestion that, at least for some key veterinary products, customised tests are required if a given preparation is to be declared a truly generic one.

ACKNOWLEDGEMENTS The authors are thankful to Dr Elizabeth Posadas Hernández and Dr Carlos López Coello of the Centro de Enseñanza, Investigación y Extensión en Producción Avícola and Departamento de Medicina y Zootecnia de Aves (FMVZ/UNAM), respectively, for providing support in the use of the reflectance colorimeter and Dr Irene Cruz Mendoza of the Departamento de Parasitología (FMVZ/UNAM) for assistance in the isolation of Eimeria oocysts.

CONFLICT OF INTERESTS Bayer Animal Health did not provide financial support for this study. The authors declare that there were no competing interests and that the conceptual design, the conduct, the interpretation of results and all scientific aspects of the study were not influenced by Bayer.

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