The anti-cryptosporidium activity of thymol was tested in vitro in different water samples used as drinking water in poultry farms. Samples were obtained from Tank (T) and water tower (WT) coming from a river and four different groundwater points (W1, W2, W3, and W4). They were collected in sterile bottles, stored in a cooler at 4°C and transported directly to the laboratory. They were analyzed within 24 hours of arrival.

The antiparasitic agent used was thymol. It’s the active principle of NP (15% of thymol), produced by the Industrial Laboratory of Veterinary Alternatives (LIAV, LLC) in Morocco. Thymol was obtained from Origanum compactum. In addition to thymol, other excipients have been added to provide stability and solubility. Different concentrations of the thymol (1, 2 and 4 g L^-1 of NP) were added to each water sample. A negative control was also prepared.

The anti-cryptosporidium test was carried out using microscopy counting. 10 µL of each sample was transfered to a Malassez chamber. Number of parasites was counted in 10 fields of view using standard techniques (Ryley, Meade, Hazelhurst, & Robinson, 1976), and the mean number of parasites per milliliter of sample was calculated.

The main goal of the in vivo test was to assess the efficiency of the consumption of the treated water with thymol on the parasitic intestinal load of chicken and their performances. One-day-old chicks in a weight range of 37 g were used in this study. The present work was approved by the Committee on Ethics and guidelines for the use of animals, University of Sidi Mohamed ben Abdelah. The photoperiod was adjusted on a daily basis to 12 hours of light and 12 hours of darkness. A fan was used to ensure the aeration. At the beginning of the experiment, the ambient temperature was 32°C. It was reduced by 2 to 3°C each week to reach 23°C at the end of the experiment. Water and feed were provided for ad libitum consumption. They were fed with maize-based food, free of antibiotics and antiparasitics. For sanitation, drinkers were cleaned daily.

The drinking water was obtained from the water supply system on a poultry farm (sample T). Before any treatment, the parasitic load of the water (sample T) was 1.7 10⁴Cells mL^-1. After treatment with thymol, it was reduced to 1.8 10³ Cells mL^-1 with 1 g L^-1 and 10² Cells mL^-1 with 2 g L^-1 of NP.

Animals were divided by random selection into groups of twenty and housed separately:

Group 1 (control group): Animals that consumed untreated drinking water.

Group 2: Animals that consumed treated drinking water with thymol (1 g L^-1 of NP).

Group 3: Animals that consumed treated drinking water with thymol (2 g L^-1 of NP).

The effect of the consumption of untreated water compared with treated water with thymol on the assessment of the parasitic intestinal load of animals was tested during the whole rearing period. One day a week (Day 1, Day 7, Day 14 and Day 21), 1 g of fresh fecal specimens from each group were collected and placed into sterile tubes containing 9 ml of physiological saline. From this stock solution, dilutions were performed. The intestinal load was calculated using microscopy counting. 10 µL of each sample was transferred to a Malassez chamber. Number of parasites was counted in 10 fields of view using standard techniques (Ryley et al., 1976), and the mean number of parasites per milliliter of sample was calculated.

In that same experiment, the thymol effect on drinking water was evaluated by body weight, body weight gain, food intake and consumption index of animals.

All values were expressed as means and standard error. SigmaStat 4.0 was used to analyze the data. The significance was verified for parasitic analysis of drinking water, intestinal load of animals and zootechnical parameters. p values less than 0.05 were considered as significant.

The variation of the Cryptosporidium load of the different tested samples is shown in Table 1. Before any treatment, the parasitic tests showed an important burden for all tested samples. After treatment with thymol, a significant reduction was observed for samples W2, W3, W4 and T (p <0.05; p <0.01; p <0.001) with the concentration 1 g L^-1 of NP. For all samples, a significant reduction (p <0.05; p <0.01; p <0.001) of the Cryptosporidium burden was obtained thanks to the increase of the treatment concentration.

Table 1. Variation of the Cryptosporidium load in different samples depending on the thymol concentration.

GP (Groundwater point); Values are means (n=6) ± SEM (Standard error of the mean); Comparison with control: * p <0.05; ** p <0.01; *** p <0.001.

The variation of the Cryptosporidium intestinal load for different groups of animals is shown in Table 2. At the beginning of the experiment (Day 1), the Cryptosporidium burden of all groups was 4.15 1012 Cells g^-1. Throughout the experience, the group 3 treated with thymol (2 g L^-1 of NP) was distinguished by a higher reduction load compared to the group treated with 1 g L^-1 and to the control.

Table 2. Evolution of the Cryptosporidium intestinal load.

Group 1 (untreated water); Group 2 (treated water with thymol (1 g L^-1 of NP)); Group 3 (treated water with thymol (2 g L^-1 of NP)). Values are means (n=6) ± SEM (Standard error of the mean); Comparison with control: * p <0.05; ** p <0.01.

Temporal evolution of body weights, body weights gain, food intake and consumption index for each group is shown in Table 3. During the whole rearing period, results show a significant increase (p <0.05; p <0.01) in the weight of the group 2 compared to the control. This increase is more significant (p <0.05; p <0.001) after treatment with the concentration 2 g L^-1. The body weight gain of animals in both treated groups was significantly (p <0.05; p <0.01; p <0.001) higher than the control. Concerning the consumption index, the control represents a higher consumption index compared to treated groups.

Table 3. The effect of water treatment on body weight, body weight gain, food intake and consumption Index.

Group 1 (untreated water); Group 2 (treated water with thymol (1 g L^-1 of NP)); Group 3 (treated water with thymol (2 g L^-1 of NP)). Values are means (n=20) ± SEM (Standard error of the mean); Comparison with control: * p <0.05; ** p <0.01; *** p <0.001.

Protozoa are difficult to treat and control because of their biological characteristics as cysts/oocysts resistant stage, very thick wall and small size of cellular body .They can survive in cold water for several months (Omarova, Tussupova, Berndtsson, Kalishev, & Sharapatova, 2018). They are responsible for a number of diseases such as severe gastro enteritis, chronic damages and high morbidity (Fletcher, Stark, Harkness, & Ellis, 2012).

Parasitic analysis revealed the contamination of both groundwater and surface water samples by parasites with dominance of Cryptosporidium. The Cryptosporidium load varies between 10⁴ and 10⁵ Cells mL^-1. Samples were treated with increasing concentrations of thymol, which are 1, 2 and 4 g L^-1 of NP. The treatment with thymol at 1 g L^-1 significantly reduced this load, and with the concentration 2 g L^-1 of NP, reduction was more important. This inhibitory effect is explained by the fact that thymol is one of the most effective antiparasitic agent. Tanghort et al. (2019) tested the oocysticidic efficiency of thymol and found that it decreases the number of Cryptosporidium baileyi and Cryptosporidium galli oocysts in a significant proportion (88%) at a concentration 0.5 mg mL^-1 after only 3 hours of treatment, while no oocysts were visible at the concentration of 1 mg mL^-1. After 24 hours of treatment, all oocysts of both Cryptosporidium species are destroyed at the concentration of 0.5 mg mL^-1.

The intestinal Cryptosporidium load was significantly lower in thymol-treated groups (group 2 and group 3). The effect is more important after treatment with 2 g L^-1. Most researchers have reported the efficiency of essential oils, against intestinal parasites. Studies conducted by Silva et al. (2009); Tsinas, Giannenas, Voidarou, Tzora, & Skoufos (2011) and Alp et al. (2012) investigated that the supplementation of broilers with oregano essential oils had a protective effect against avian coccidiosis. Oocyst shedding in excreta was reduced compared to control birds.

In the same experiment, even if animals of the control group consumed the same amount of food as treated groups, they showed worse zootechnical performances. This difference in growth between animals groups could be due to thymol which reduces the pathogen parasitic load and allows an intestinal microflora balance. This reduction affects intestinal integrity, therefore food may be more easily absorbed. A study conducted by Greathead and Kamel (2006) reported a positive effect of thymol and carvacrol supplementation to experimentally infected birds with sporulated oocysts of E. acervuline. It was concluded that this supplementation enhanced intestinal integrity by reducing expression of coccidiosis. To this end, thymol could represent a natural alternative to chemical biocides frequently used in poultry farming.