MATERIALS AND METHODS

Cryptosporidium oocysts isolation and purification: The Cryptosporidium oocysts used in this study were originally isolated from fresh faeces of broilers, naturally infested in Morocco. The oocysts were purified and sieved by flotation in a saturated solution as described by Tumova et al.²⁴. Species identification was achieved by evaluation of morphometric characteristics of the oocysts (size), using the microscope and micrometer²⁵. The average measurements are of 6.3×5.2 μm and 8.3×6.3 μm for C. baileyi and C. galli respectively.

Counting of Cryptosporidium oocysts: The number of oocysts was determined by transferring 20 μL of the sample suspension of Cryptosporidium oocysts to a Malassez chamber for microscopic examination and counting. Cryptosporidium oocysts were counted in 10 fields of view by using standard techniques²⁶ and the mean number of oocysts per milliliter of the sample was calculated. The identification of Cryptosporidium species was done based on the microscopic observation thereby allowing the identification of the Cryptosporidium species according to the method described by Ng et al.²⁵. The percentage of each species in the mixed suspension was approximately 43.44% for C. galli and 56.56% for C. baileyi.

Effect of EOs and MCs on the number of Cryptosporidium oocysts: The EOs used in this study were: oregano oil (Oreganum compactum), thyme oil (Thymus vulgaris), Clove oil (Eugenia caryophylata), rosemary oil (Rosmarinus officinalis) and Artemisia oil (Artemisia absinthium). These EOs were purchased from Seema International (India). Carvacrol, thymol and eugenol used in this study were purchased from Sigma Aldrich (France).

All EOs and their MCs were dispersed in a liquid medium containing 0.2% agar in distilled water. This dispersion method was improved by Mzabi et al.²⁷. Each EO or EO component was tested in increasing concentrations (0, 0.25, 0.33, 0.5, 1 and 2 mg mL^–1). The activity of each EO or MC was determined in triplicate in Eppendorf tubes by incubation at room temperature (25°C) on rotary shaker for 24 h of an inoculum of 25 μL containing 1.22×10⁷ oocysts mL^–1 which was put in direct contact with the EO or MC at various concentrations for a final volume of 200 μL^28,29. The different curves were drawn, expressing the number of oocysts with respect to the concentration of EOs or MCs, after 3 and 24 h for each species of Cryptosporidium. The LC₅₀ was then inferred from this curve by identifying the concentration in which the number of oocysts was equal to half of the initial number.

Effect of anticoccidial agents on the decrease of the number of Cryptosporidium oocysts: The action of anticoccidials such as monensin, salinomycin and robenidine was tested using the previously described method on EOs with the following concentrations 0, 0.25, 0.33, 0.5, 1 and 2 mg mL^–1. These concentrations were prepared from a concentration of 100 mg mL^–1 of each of the three anticoccidial agents. All anticoccidials were purchased from Sigma Aldrich (France).

Decrease of the oocyst number in parallel with the release of substances absorbing at 273 nm after treatment with increasing concentrations of thymol and carvacrol: The release of cellular material absorbing at 273 nm from oocysts cells treated with increasing concentrations of carvacrol and thymol was determined. This experiment was performed on aliquots incubated for 24 h at ambient temperature with one milliliter suspension containing:

After incubation, the samples were centrifuged at 320 g for 5 min at 4°C. 500 μL of the supernatant was used to measure the UV absorption by a Beckman spectrophotometer^28,29. For each concentration, the oocyst number was counted as previously described by Ding et al.²⁶.

Statistical analysis: The results were expressed as mean values ±S.E.M (standard error of mean). Statistical analysis of the data was performed with one-way analysis of variance followed by Tukey’s Multiple Comparison Test (one way ANOVA followed by Tukey’s test) (Graph Pad Prism, version 5.03). Significant differences were indicated by values inferior to 0.05.

RESULTS

Effect of EOs concentrations on Cryptosporidium oocysts number: Figure 1 and 2 show that the number of C. baileyi (Fig. 1: a-b) and C. galli (Fig. 2a-b) oocysts decreases with the majority of EOs tested in a dose-dependent manner at a concentration ranging between 0 and 2 mg mL^–1, after the 3 and 24 h of treatment.

After the 3 h of exposure of Cryposptoridium oocysts to various concentrations of EOs, the number of oocysts of both species of Cryptosporidium noticeably decreased by very low concentrations (0.25-0.5 mg mL^–1) of thyme oil, oregano oil and clove oil.

Fig. 1(a-b):	The effect of different EOs on the viability of Cryptosporidium baileyi oocysts after (a) 3 h and (b) After 24 h of treatment

EOs of artemisia and rosemary require a high concentration (2 mg mL^–1) for a significant reduction of more than 50% in the number of oocysts.

After treatment with clove oil, oregano oil and thyme oil, 1 mg mL^–1 was sufficient to destroy all C. baileyi (Fig. 1b) and C. galli oocysts (Fig. 2b) for a period of 24 h. However, a concentration of 2 mg mL^–1 was required to obtain an oocysticidal action by the EOs of artemisia and rosemary. When expressed in terms of lethal concentration 50% (LC₅₀), our results show that the most efficacious EOs are thyme (0.31 mg mL^–1) followed by clove (0.33 mg mL^–1) and oregano oils (0.37 mg mL^–1) according to their LC₅₀ less than 0.4 mg mL^–1, the LC₅₀ of artemisia and rosemary is high, measuring 0.69 mg mL^–1 and 0.88 mg mL^–1 respectively. It was inferred from the curves expressing the number of oocysts with respect to the concentration of EOs.

Effect of MCs concentrations on Cryptosporidium oocysts number: Figure 3 and 4 show that the number of Cryptosporidium oocysts decreases after the 3 and 24 h of treatment with the three MCs used in a dose-dependent manner at a concentration that ranges between 0 and 2 mg mL^–1.

After 3 h of treatment, the MCs were able to reduce the number of C. baileyi (Fig. 3a) and C. galli (Fig. 4a) oocysts in a significant proportion (88%), at a concentration 0.5 mg mL^–1.

Fig. 2(a-b):	The effect of different EOs on the viability of Cryptosporidium galli oocysts after (a) 3 h and (b) After 24 h of treatment

Fig. 3(a-b):	The effect of different MCs on the viability of Cryptosporidium baileyi oocysts (a) After 3 h and (b) After 24 h of treatment

Eugenol can reduce the number of oocysts up to 100% at a concentration of 1 mg mL^–1, while no oocysts were visible using carvacrol and thymol at concentrations of mg mL^–1.

Fig. 4(a-b):	The effect of different MCs on the viability of Cryptosporidium galli oocysts (a) After 3 h and (b) After 24 h of treatment

After 24 h of treatment (Fig. 3b and Fig. 4b), all oocysts of both Cryptosporidium species are destroyed by thymol and eugenol at the concentration of 0.5 mg mL^–1. Carvacrol provokes an oocysticidal effect at the high dose of 1 mg mL^–1. The expression in terms of lethal concentration 50% (LC₅₀) shows that the three MCs tested, have a practically similar LC₅₀ in both Cryptosporidium species. Thymol and carvacrol have the same LC₅₀ (0.22 mg mL^–1), followed by eugenol with an LC₅₀ of 0.25 mg mL^–1.

Effect of anticoccidial drugs on Cryptosporidium oocysts number: Figure 5a and 6a show that the number of C. baileyi and C. galli oocysts decreased slightly after 3 h of treatment with increasing concentrations of the three antiparasitic drugs tested at the concentrations 0, 0.25, 0.33, 0.5, 1 and 2 mg mL^–1. No change in the viability of oocysts was observed even after 24 h of treatment (Fig. 5b and 6b).

Effect of carvacrol and thymol concentrations on the release of 273 nm absorbing material after 24 h of treatment from Cryptosporidium oocysts: Figure 7a and b show that after adding carvacrol (A), or thymol (B) in concentrations ranging from 0-2 mg mL^–1, the number of oocysts in a mixture of C. baileyi and C. galli noticeably decreases with very low concentrations (0.25-0,5 mg mL^–1).

Fig. 5(a-b):	The effect of different anticoccidials on the viability of Cryptosporidium baileyi oocysts (a) After 3 h and (b) After 24 h of treatment

Fig. 6(a-b):	The effect of different anticoccidials on the viability of Cryptosporidium galli oocysts (a) After 3 h and (b) After 24 h of treatment

This treatment causes a considerable release of 273 nm absorbing material that increases in a linear manner according to the concentration of the MC.

Fig. 7(a-b):	The Effect of (a) Carvacrol and (b) thymol concentrations on Cryptosporidium oocysts number and the release of 273 nm absorbing material

DISCUSSION

The Cryptosporidium parasite is at the origin of the gastroenteritis syndrome (infectious diarrhea) that affects humans and animals^4,7. The parasite’s ability to infect animals in poultry farms and the high mortality that results from it has made of it a major pathogenic agent³⁰. In the last decades, researchers have been investigating effective anti-parasitic drugs that could treat Cryptosporidiosis. Today, all solutions rely mainly on symptomatic treatments and preventive measures^18,23. However, in the case of human medicine, one drug has been approved by the FDA: the Nitazoxanide³¹.

Studies have tested the efficiency of natural substances such as curcuma, ginkgolic acids (Gingko biloba), onion, cinnamon on oocysts of C. parvum or C. andersoni, either in vitro on cell cultures³², or in vivo on mice^33,34. The last publication in this context is that of Gaur et al.³⁵, which showed that the EO of oregano and its MC carvacrol can inhibit up to 50% of the infectivity of C. parvum on adenocarcinoma ileo-caecal cells HTC-8 to the concentration of 30-60 μg mL^–1. However, in this experimental model, it is not possible to obtain more inhibition owing to the toxicity of this EO and its MC on culture cells. In that regard, we decided to test in vitro the anti-parasitic effect of five EOs and their MCs directly on the oocysts of C. baileyi and C. galli responsible of Cryptosporidiosis in poultry. We, therefore, tested their effectiveness on a mix of C. baileyi and C. galli oocysts containing approximately 1.22×10⁷ oocysts mL^–1. The results obtained show that the EOs of thyme, oregano and clove can cause a significant decrease in the number of oocysts of about 85% after 3 h and 98% after 24 h, to the concentration of 1 mg mL^–1. At this concentration, the EOs of rosemary and artemisia only cause a decrease of about 60% after 3 h and 80% after 24 h. The results obtained also show that the two species of Cryptosporidium used have the same sensitivity to the different EOs after 3 and 24 h.

Since the EOs of thyme, oregano and clove were more efficient and had a LC₅₀ inferior to 0.4 mg mL^–1, we decided to test the oocysticidic efficiency of their MC which are thymol, carvacrol and eugenol. The results showed that thymol, carvacrol and eugenol have practically the same efficiency from the concentration of 0.5 mg mL^–1. Below 0.5 mg mL^–1, thymol and carvacrol proved to be more efficient than eugenol.

EOs and their MC have demonstrated an oocysticidal activity in vitro on Eimeria species as described by Remmal et al.^28,29. These authors have noted the presence of deformed Eimeria oocysts with cracked walls and debris after treatment. These publications also identified the mechanism of action of EOs and their MC by measuring the release of the constituents of oocysts absorbing at 273 nm. For this reason, we also measured the liberation of substances absorbing at 273Nm (UV) present in the suspension of Cryptosporidium oocysts to gauge the destructive action of these MCs on oocysts. The results obtained show that at the concentration of 0.5 mg mL^–1, there is an important decrease in the number of oocysts along with an important increase of absorbing material at 273 nm (UV). This allows us to conclude that the MCs have an oocysticidal action.

Considering that we were working on protozoan oocysts and that Moroccan veterinarians use anticoccidials as a last recourse to fight cryptosporidiosis, or when in doubt in their diagnosis between cryptosporidiosis and coccidiosis. Their doubt can be due to the similarity of the clinical signs of the two diseases, or to the similar oocysts aspect of the two protozoans. Following this reasoning, we decided to test a few anticoccidial agents in order to verify if they have a destructive effect on Cryptosporidium oocysts. These agents are usually used to fight avian coccidiosis. They belong to the ionophores family such as salynomycin, monensin, or to the non-ionophores family such as robenidine. The results show that this oocysticide effect is very feeble. This weak sensitivity of Cryptosporidium to anticoccidials has already been described in many studies in vitro and in vivo on other Cryptosporidium species^19,36,37.

In order to ascertain the action of EOs and their MC on Cryptosporidium oocysts for animals, we have performed trials on 7-day-old broiler chicks infected on a daily basis with Cryptosporidium oocysts for 15 days. They were then kept under observation until they reached the age of 42 days. Significant improvements were observed in their zootechnical parameters: body weight, weight gain, feed gain ratio and mortality for the animals treated thymol or carvacrol compared to animals from the control batch. The injuries observed during autopsy were also significantly better (results not shown).

CONCLUSION

We can conclude from this in vitro study that the tested EOs and their MC have an oocysticidal activity with regards to Cryptosporidium spp for poultry. In parallel, a light decrease in the number of oocysts has been provoked by conventional anticoccidial drugs. To our knowledge, this is the first time that these EOs and their MCs to be tested on avian Cryptosporidium oocysts.

ACKNOWLEDGMENTS

This study is a partial fulfillment of Mariam Tanghort’s PhD thesis. The authors are deeply grateful to Miss Soukaina Remmal and Mrs Hélène Mock for their assistance in checking the English of the manuscript.