Effect of the granulometric characteristics of monensin sodium on controlling experimental coccidiosis in broiler chickens

The aim of the present study was to investigate the efficacy of monensin sodium grain size on controlling coccidiosis in broiler chickens. Three hundred and fifty Cobb 700 chicks were infected experimentally with 8 × 104 sporulated oocysts of Eimeria spp. The birds were fed with diets supplemented with powdered (n = 150) or granulated (n = 150) monensin, with particle sizes of ≤100 μm and 450-650 μm, respectively. The remaining chicks (n = 50) were used as an unmedicated control group. The birds’ weights and number of oocysts per gram of litter in each group were recorded weekly. A total of 96 birds were randomly selected and culled during the trial. Their intestinal oocyst counts and lesion scores were determined. No significant differences in body weights were found between the groups at the end of the study. The percentages of infected animals, oocyst counts and lesion scores were significantly higher in the control group than in the other two groups; the two treated groups did not show any appreciable differences to each other. Enteritis was observed in 14 birds, all in the group supplemented with granulated monensin. These results suggest that powdered and granulated monensin have comparable efficacy in controlling coccidiosis in broiler chickens. However, treatments with monensin sodium of high grain size may be subject to contraindications relating to enteritis.

There are basically two means of preventing coccidiosis: chemoprophylaxis and vaccination.While vaccination is today less applied in broiler production, also because of its higher cost, chemoprophylaxis using anticoccidials in the feed is by far the most popular method, insofar as it is estimated that 95% of broilers produced receive anticoccidials (CHAPMAN, 1999).
In the European Union, 11 coccidiostatic substances are currently authorized for prevention of coccidiosis in one or more animal species.Of the 40.65 million tonnes of feed produced annually for chickens, turkeys and rabbits, approximately 18.33 million tonnes are manufactured with the addition of a coccidiostat (IFAH, 2007).Feed business operators generally produce a broad range of products within a single feed plant.Several studies have shown that completely contamination-free production of premixes and compound feeds in existing multi-product plants is impossible in practice (KENNEDY et al., 1998;STRAUCH 2002aSTRAUCH , b, 2003)), thus resulting in cross-contamination of subsequent feed-batches.
Cross-contamination is a recognized problem, to the point that European Commission Directive 2009/8/EC recently amended a previous Directive 2002/32/EC (EUROPEAN COMMISSION, 2002COMMISSION, , 2009) ) regarding the "[…] maximum levels of unavoidable carry-over of coccidiostats or histomonostats in non-target feed".
The physicochemical characteristics of coccidiostatic additives, such as their high dusting potential and adherence due to electrostatic charge, as well as the environmental conditions of production sites, can contribute towards cross-contamination (EFSA, 2008).Moreover, occupational exposure to monensin through accidental inhalation and/or dermal contact can represent potential negative health risks for workers (STORY; DOUBE, 2004).
Due to concerns regarding the use of anticoccidials, particularly about the potential for workers and non-target animals to be exposed to their residues, there is a constant search for innovative solutions.Granulated formulations of monensin sodium characterized by high particle size have been recently produced.Increasing the grain size can limit the dust emission from monensin sodium, consequently reducing the risks for handlers and those relating to cross-contamination throughout the feed chain.
The effect of drug particle size on dissolution rates and the corresponding drug absorption has been well established both theoretically and experimentally (FINCHER, 1968;KANENIWA;WATAKI, 1978;KANENIWA et al., 1978;HINTZ;JOHNSON, 1989;LU et al., 1993).For low to moderate-solubility drugs administered orally, like monensin sodium, a change in particle size can have a dramatic impact on their ability to be absorbed by the cell membranes (LIPINSKI, 2001(LIPINSKI, , 2002)).
The aim of this study was to investigate the efficacy of monensin sodium against coccidiosis in broiler chickens, according to its granulometric characteristics (powder vs. granules).

Animals, diets and housing system
The experiment lasted 42 days and was carried out at the experimental broiler farm of CISRA (Inter-Departmental Center for Animal Husbandry Services, University of Turin).
Three hundred and fifty one-day-old Cobb 700 male chicks were purchased from a local hatchery.On arrival at the experimental farm, they were divided into three groups, balanced according to body weight.The groups were allocated to different pens.The housing method was a litter system with a stocking density of eight birds per square meter.Before the birds were placed there, the pens were cleaned, washed, disinfected and provided with new wood shavings, in a layer of thickness 5 cm.The ambient temperature was gradually decreased from 32 °C on the first day to 20 °C at the end of the study.Ambient temperature and relative humidity were monitored at one-hour intervals.
Standard hygiene and prophylactic measures against infectious diseases were applied.All the broilers received a starter diet (0 to 2 weeks) and a finisher diet (3 to 5 weeks), and both were provided ad libitum (Table 1).Tap water was freely available.The diets were supplemented with 100 ppm of powdered (particle size: ≤100 µm) monensin sodium (group P, n = 150) or 100 ppm of granulated (particle size: 450-650 µm) monensin sodium (group G, n = 150).The remaining chicks (group C, n = 50) did not receive any coccidiostat and they were used as an untreated positive control.

Experimental infection
The birds were experimentally infected at 5 days of age.A mixed coccidial inoculum of 3 × 10 4 sporulated oocysts of Eimeria acervulina, 2.5 × 10 4 of E. maxima and 2.5 × 10 4 of E. tenella per bird was administered with 1 mL of tap water directly into the crop by using a self-calibrated 1 mL micropipette.The challenge oocysts were isolated from naturally infected chickens.The oocysts, separated by using sieving and sedimentation techniques, were allowed to sporulate at 25 °C in 2.5% potassium dichromate solution.The sporulated oocysts were cleared and counted using a modified McMaster technique (LONG; ROWELL, 1958).

Sampling and measurements
Separate working clothes, footwear and equipment were used for each pen throughout the experimental period in order to avoid cross-infection among groups.
During the trial, the broilers were individually weighed once a week in order to determine their weight gain.
Representative litter samples (a mixed sample of five handfuls taken in five sections per pen) were collected weekly from day 2 post-infection to the end of the experiment.The mean number of oocysts per gram (OPG) of litter was calculated in accordance with the standard procedures of the World Association of Animal Veterinary Parasitology (WAAVP) (HOLDSWORTH et al., 2004).
Ninety-six chickens were randomly selected and euthanized on day 10 (six in groups P and G; two in group C), on day 25 after inoculation (18 in groups P and G; five in group C) and on day 37 after inoculation (18 in groups P and G; five in group C), to evaluate pathological abnormalities.Intestinal lesions, if present, were investigated in accordance with the 0 to 4 scoring system proposed by Johnson and Reid (1970).The method covers a range of coccidial-specific lesions and is related to both the degree and the amount of intestine affected.The lesions were scored as follows: 0 = lesions absent; 1 = a few scattered lesions; 2 = a greater number of discrete lesions involving more of the affected zone of the intestine; 3 = lesions extensively developed with coalescence and some thickening of the intestinal walls; and 4 = extensive coalescence of lesions with thickening of the wall and bloody intestinal contents.A microscope was used to examine scrapings for coccidia whenever there was doubt about the cause of a lesion.
In addition, the number of oocysts per gram of intestinal contents was individually recorded with regard to the same euthanized chickens.
Necropsies were performed on four chicks (1 P, 1 G, and 2 C) that died during the experiment.Neither the euthanized birds nor the birds that died were replaced.
The statistical analysis on the data was performed using the SPSS 16.0 software (SPSS, 2007).Weighted data were subjected to one-way analysis of variance (ANOVA).The non-parametric Kruskal-Wallis test was performed on the number of intestinal oocysts per gram of content and lesion scores; related post-hoc comparisons were performed using Mann-Whitney U tests.Significance was declared at p < 0.05.

Results and Discussion
No significant differences in body weights were observed at the end of the trial among the three experimental groups (mean weight: group C, 1,720 g; group P, 1,800 g; and group G, 1740 g).
Figure 1 shows the temporal pattern of the number of Eimeria spp.oocysts detected in the litter for each experimental group.From day 9 onwards, the OPG value in the litter was higher in the control group than in the other two groups.
Eimeria spp.oocysts were found in the intestinal contents of all the euthanized broilers of group C.Among the euthanized birds, i.e. in 12 (29%) of group G and in 7 (17%) of group P, no intestinal oocysts were detected.
Regarding the intensity of infection, the oocysts counts in group C were always significantly higher than in group G, whereas differences between groups C and P were detected only on days 25 and 37.The intestinal OPG counts did not differ between groups P and G on day 25, but significantly lower levels in G than in P were detected on both day 10 and day 37 (Figure 2).
The coccidial lesion scores were always significantly higher in group C than in the other two groups.Comparable intestinal lesion scores in groups P and G were observed on both day 10 and day 37, whereas these two groups differed significantly on day 25 with lower levels detected among birds in group G (Figure 3).
In 14 birds (two on day 10, four on day 25 and eight at the end of the trial), all belonging to group G, enteritis was observed in the necropsies.
Coccidiostats for poultry are exclusively used as feed additives, and are mixed with compounded feeds in the producing feed mills upon request.Since 1971, with the introduction of monensin, contamination.As previously stated, the physical characteristics of additives, such as high dusting potential and small particle size, are a well-recognized problem in relation to cross-contamination of feed batches during the technical process of commercial feed production.Moreover, emission of monensin dust has to be taken into consideration in relation to handlers throughout the feed chain.
Although the dissolution rate of drugs is slower with larger particles, which is likely to result in lower bioavailability, our results showed that the efficacy of the granulated formulation against coccidia was comparable to that of the powdered formulation.As reported by Sigfridsson et al. (2011), since monensin is evenly absorbed over the entire intestinal tract, the rate of dissolution does not influence the extent of absorption.
Since enteritis was observed as a postmortem finding in some birds in group G, further studies will be needed in order to assess whether a correlation exists between the increased particle size of the drug and the enteric damage.

Conclusion
The granulated monensin sodium tested in this study was found to be very efficacious and comparable to the powdered formulation in controlling coccidiosis in broiler chickens.However, further investigations will be needed to assess whether the increased grain size of the drug might be the cause of the observed intestinal damage.most commercial broilers have been medicated with this drug.The main reason for the popularity of monensin, apart from its high efficacy, is the relatively limited risk of complete resistance, at least compared with the risk of resistance towards synthetically produced drugs (PEEK; LANDMAN, 2003;WILLIAMS, 2006;JENKINS et al., 2010).
The granulated monensin formulation tested in this study was characterized by a particle size of 450-650 µm.In comparison with powdered monensin formulations that are commonly used in commercial feed production, such increased particle sizes may present many advantages with regard to dispersion in cross-

Figure 1 .
Figure 1.Number of Eimeria spp.oocysts per gram (OPG) of litter as a function of the number of days post-infection (DPI) for the two groups treated with monensin sodium (powdered, P, and granulated, G), and the unmedicated control group (C).

Table 1 .
Experimental basal starter and finisher diets for broiler chickens.