Assessing the effect of diclazuril on the intestinal absorptive capacity of broilers infected with experimental coccidiosis, using d-xylose absorption test

Coccidiosis is among the most important diseases of poultry worldwide caused by a protozoan parasite, Eimeria. The bird develops a disability to absorb dietary nutrients through the disruption of the integrity of the intestinal mucosae (Ruff & Wilkins, 1980; Adams et al., 1996; Greif, 2000; Persia et al., 2006; Zhao et al., 2006). Although coccidiostats are able to prevent or control the disease, reports are scarce on the influence of coccidiostats on the absorption capacity of intestinal epithelium in infected chickens.

d-Xylose absorption test has been successfully used to evaluate the malabsorption syndrome in chickens (Goodwin et al., 1985; Hill et al., 1985; Doerfler et al., 2000; Mansoori et al., 2007). Any change in plasma concentration of d-xylose, over 3-h period is quite indicative of its absorption from the intestinal tract of the bird (Doerfler et al., 2000). The present study aimed to assess the influence of Diclazuril, the active substance of commercially available coccidiostats, on the intestinal absorptive capacity of broiler chickens during experimental coccidiosis, using d-xylose absorption test.

The experiment contained four groups of 20 1-day old broiler cockerels (Ross 308). Birds were placed on wire-floored starter batteries and given ad libitum access to water and un-medicated starter feed (except Groups 3 and 4), which met or exceeded NRC (National Research Council) (1994) requirements. At 14 days of age, 10 chicks from each group were weighed, individually identified, and randomly assigned to nonchallenged (Group 1) or challenged groups (Groups 2–4).

The birds were kept individually in raised floor wire cages to adapt to the new environmental conditions for further 9 days. The experimental groups were as follows: Group 1, negative control received no Eimeria oocysts; Group 2, positive control challenged with mixed Eimeria oocysts; Group 3, positive control plus Clinacox 0.5% (diclazuril 1 mg/kg of medicated feed; Kimiafaam Pharmaceutical Co., Jomhuri Ave, Tehran, Iran) from day 1; Group 4, positive control plus Dilacox 0.5% (diclazuril 1 mg/kg of medicated feed; Roshd Daneh Gorgan Pharmaceutical Co., Agh-ghala Industrial Park, Gorgan, Iran) from day 1. All experimental birds survived during the experiment. The experiment was carried out at the Poultry Station, Veterinary Research Institute, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. The experimental procedure was approved by the Animal Research Committee of the University of Tehran.

Oocysts (mixed Eimeria species of local isolates, mainly E. acrervulina, E. maxima, E. necatrix, and E. tenella) maintained in the Parasitology Laboratory, Faculty of Veterinary Medicine, University of Tehran were processed for sporulation in 2.5% potassium dichromate solution (Ryley et al., 1976). Sporulated oocysts were given two washings with phosphate-buffered saline (PBS; pH 7.2), and stored in PBS at 4 °C for further use.

Chicks of Groups 2, 3, and 4 were inoculated by 0.5 mL crop intubations of water containing 3 × 10⁵E. acervulina, 1.2 × 10⁵E. maxima, 1.2 × 10⁵E. necatrix, and 6 × 10⁴E. tenella oocysts to create an experimental coccidial infection on day 24 of the experiment. Uninfected chicks (Group 1) received a sham inoculation of tap water.

Five days after coccidial inoculation, food and water were removed from each group of the bird 12 h prior to the first collection of blood. The birds were given d-xylose solution (50 mg/mL deionized water; Sigma Chemical Co., St Louis, MO, USA) at a dose of 500 mg/kg BW, via oral gavage. One blood sample before, and five others after the oral administration of test material, were collected by ulnar vein puncture on 30 min basis for 150 min, using heparinized micro-hematocrit capillary tubes (Code No. 9100260; Hirschmann Laborgerate Techcolor, Eberstadt, Germany). The tubes were centrifuged and plasma was collected. The d-xylose absorption test was carried out by the method of Eberts et al. (1979), as modified by Goodwin et al. (1985).

Analysis of data was carried out using one-way analysis of variance of Minitab system (Minitab 13.2 statistical package, Minitab Inc., State College, PA, USA 2000). Polynomial regression analysis was used to investigate the relationship between d-xylose level and time (Kaps & Lamberson, 2004).

Plasma d-xylose level of Group 1 (negative control) had a quadratic correlation with time (P < 0.001, r² = 0.93) and reached to its peak (54.2 mg/dL) at 30–60 min after the administration of the solution (Table 1). The administration of Eimeria oocysts severely suppressed the intestinal absorption of d-xylose in Group 2 to the level that the concentration of plasma d-xylose reached to its peak (38 mg/dL) at 90 min after the administration of d-xylose. However, the concentration of d-xylose followed a quadratic correlation with time (P < 0.001, r² = 0.87). In Group 3, the concentration of plasma d-xylose reached to its peak (45.3 mg/dL) at 60–90 min after the d-xylose administration. The concentration of plasma d-xylose of Group 3 followed a quadratic correlation with time (P < 0.001, r² = 0.76). In Group 4, the concentration of plasma d-xylose reached to its peak (47.7 mg/dL) at 60–90 min after the administration of d-xylose solution. The plasma d-xylose concentration of Group 4 also followed a quadratic correlation with time (P < 0.001, r² = 0.79).

A decrease in intestinal absorption of d-xylose in response to experimental coccidiosis in this experiment is in line with other reports on the influence of coccidiosis on the intestinal absorption of nutrients (Hill et al., 1985; Adams et al., 1996; Greif, 2000; Kettunen et al., 2001; Persia et al., 2006; Zhao et al., 2006). Hill et al. (1985) reported that the intestinal d-xylose absorption was a sensitive method for evaluating the effects of coccidial infection in broilers and correlated well with body weight gain and feed conversion ratio.

A decrease in absorption capacity of d-xylose at the intestinal mucosae may be mediated directly by the parasite or by the host’s immuno-inflammatory response, which may result in changes in the intestinal mucosal morphology (Barker, 1993). Coccidiosis changes the gut normal homeostasis as a result of an increase in total mucosal thickness, a decrease in villus height, an increase in crypt length, an increase in the rate of replacement and turnover of intestinal epithelial cells, and/or in metabolism of mucosal cells, an increase in tissue moisture (edema), a decrease in pH of the infected area, an increase in feed passage time, and finally, an alteration in mechanisms responsible for transport of nutrients across the intestinal wall particularly Na⁺-dependent active transport systems in the duodenum and jejunum might reduce the absorption capacity of intestine (McKenzie et al., 1987; Kettunen et al., 2001).

Both coccidiostats resulted an improvement in the trend of d-xylose absorption from the intestine of the challenged birds to a similar extent. Clinacox 0.5% and Dilacox 0.05% contain diclazuril as the active substance. Diclazuril, a benzeneacetonitrile anticoccidial, has potent activity against various stages of Eimeria life cycle. Maes et al. (1988) showed that a single treatment of experimentally infected chickens with diclazuril resulted in a complete interruption of the life cycle and oocyst shedding of E. tenella. The first- and second-generation schizonts showed extensive degenerative changes including loss of internal structure, the appearance of intracytoplasmic vacuoles, and incomplete merogony. Diclazuril was also effective against merozoites and both the microgametocytes and the macrogametocytes. In the macrogametocytes, wall-forming bodies either did not develop or disappeared rapidly. The authors concluded that diclazuril at the level of 1 ppm in the feed was lethal against both the asexual and the sexual stages of E. tenella. Vanparijs et al. (1990) reported that feeding broilers diclazuril, at dose levels of 0.5, 0.75, 1, and 2 ppm prevented mortality, suppressed or reduced lesion scores, and allowed for normal weight gains as well as productivity.

McDougald et al. (1990) measured the anticoccidial efficacy of diclazuril in broilers, using field isolates of E. acervulina, E. brunetti, E. maxima, E. necatrix, E. mitis, and E. tenella. Results showed that Diclazuril at 0.5–1.5 ppm was almost completely effective against all Eimeria species.

Lower correlation coefficients of Groups 3 and 4 compared to Groups 1 and 2 in this experiment, was likely due to a broader variation in response of individual birds in Groups 3 and 4 to the infection and/or diclazuril. In conclusion, d-xylose absorption test is a sensitive test for measuring the absorption capacity of small intestine in normal birds as well as in birds infected with coccidiosis. This test showed that diclazuril, the active substance of Clinacox and Dilacox, was successful in improving the intestinal absorption capacity of infected chicken with coccidiosis is likely because of the anticoccidial effect of diclazuril.