In vivo Anticoccidial Activity of Salvadora persica Root Extracts

Intestinal eimeriosis caused by the parasite, Eimeria spp. are considered to be one of the major threats affecting animal health and production (Mehlhorn, 2014). Such parasite has a great economic importance as it causes severe reduction in meat and milk production and death of domestic agriculture animals (Schito and Barta, 1997). There are many anticoccidial agents used against eimeriosis. However these agents have many side effects (Mehlhorn, 2014; Wunderlich et al., 2014). Researchers efforts are now directed towards finding natural agents with reduced or with no side effects on the host infected with Eimeria. Extracts of pomegranate, neem, palm date, garlic and others were found to be good agents against E. papillata induced infection in mice jejunum (Wunderlich et al., 2014).

In this study, we used Salvadora persica, commonly known as miswak, and used most extensively against microbes (Elvin-Lewis, 1982). The propagation and use of miswaks is due to the spread of Islamic culture in many countries (Bos, 1993).

It was reported that, S. persica, has antimicrobial activity against numerous oral pathogens (Poureslami et al., 2007). Recently, Abdel-Baki et al. (2016) investigated the antiparasitic activity of S. persica against against Echinococcus granulosus.

S. persica extracts contain important phytochemicals like vitamin C, salvadorine, salvadourea, alkaloids, trimethylamine, cyanogenic glycosides, tannins, saponins and salts mostly as chlorides (Alali et al., 2005).

Miswak was harvested from the root of S. persica and methanolic extract was prepared for studying its potential against E. papillata infection in mice.

Fresh roots of Salvadora persica were harvested from Jizan region, Kingdom of Saudi Arabia. A botanist, Department of Botany, College of Science, King Saud University, Saudi Arabia authenticated the samples. S. persica root methanolic extracts were prepared according to Amer et al. (2013). Briefly, the roots were dried, grinded by an electronic blender into a powder that extracted by 70% methanol. The used collected residue was dissolved in water for the inoculation in mice.

Adult male C57BL/6 mice were obtained from the Department of Comparative Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. The mice were bred under specified pathogen-free conditions and fed a standard diet and water ad libitum. The experiments were approved by the state authorities and followed Saudi Arabian rules on animal protection.

To prepare Eimeria papillata oocysts for infection, the stored sporulated E. papillata were inoculated into mice using an epigastric tube. Every mouse received 100 µl saline contained 1000 sporulated oocysts.

Animals were divided into 5 groups of 5 mice per group. The first group was the non-infected control group. Mice of this group were daily inoculated with 100 µl saline for 5 days. The second, third, fourth and fifth groups group were infected with 1000 sporulated E. papillata oocysts. One hour later, the third, fourth and fifth groups were daily treated with 300, 600 and 900 mg/kg, respectively.

The faeces were daily collected from each mouse, weighed and prepared for counting using McMaster chamber (Dkhil et al., 2011). To investigate the change in weight, all mice were weighed at the beginning and at the end of the experiment.

Jejuna of mice were collected after 5 days postinfection with E. papillata. Each jejunum was washed and then fixed in 10% formalin. Tissue sections were prepared for histological examination (Adam and Caihak, 1964). To calculate the number of parasitic stages, the sections were stained with hematoxylin and eosin.

To calculate the number of goblet cells in non-infected, infected and infected-treated mice, jejunum tissue sections were stained with Alcian blue and the number of goblet cells was scored in 10 well orinted jejuna villi (Allen et al., 1986).

To determine the expression of MUC2-mRNA, we used a quantitative real-time PCR. In brief, the total RNA was isolated from mice jejuna using Trizol (Invitrogen). RNA samples were treated with DNase (Applied Biosystems, Darmstadt, Germany) and then converted into cDNA using the reverse transcription kit (Qiagen, Hilden, Germany). Quantitative real-time PCR (qRT-PCR) was performed using the ABI Prism 7500HT sequence detection system (Applied Biosystems, Darmstadt, Germany) with SYBR green PCR master mix from Qiagen (Hilden, Germany). MUC2 primer was obtained from Qiagen (Dkhil et al., 2013). mRNA levels were normalized to 18S rRNA. The fold induction of mRNA expression upon infection with E. papillata was determined using the 2−ΔΔCT method (Livak and Schmittgen, 2001).

Statistical analysis was carried out using one-way ANOVA, and Duncan’s test was used for comparison between groups.

On day 5 p.i. the expelled oocysts of E. papillata were at its maximum level being about 6242.7 ± 731.5 oocysts / g faeces in infected group. S. persica root extracts was significantly able to suppress the oocyst output by 56.8, 22.8 and 32.2 %, respectively (Table I).

Table 1.- S. persica induced reduction in oocyst output.

Values are means ± SD.

*Significance at P≤0.001.

It is clear now that the dose 300 mg/kg is the best dose for suppression of oocyst output. So, we used only 300 mg/kg root extracts to investigate the histological changes as well as the goblet cell response to infection and/or treatment.

Control non-infected mice grew normally resulting in an average weight gain of about 5.8 %. Whereas experimentally infected mice with E. papillata had lost their original weight by about 2.8 %. After treatment with 300 mg⁄kg S. persica root extracts, the loss of the original weight was reduced to -1.89 % (Fig. 1).

Table II shows the change in the number of parasitic stages on day 5 p.i. due to treatment of mice with 300 mg/kg S. persica root extracts. The developmental stages of E. papillata clearly appeared inside the intestinal epithelial of the villi and we found no parasites inside goblet cells.

Examination of Alcian blue stained sections showed that the infection induced a significant decrease in the number of goblet cells inside the jejuna villi (Table II).Treatment of mice with S. persica root extracts (Table II) could significantly increase the number of goblet cells compared to the infected group.

Table II.- S. persica induced reduction in the number of parasitic stages, goblet cells and weight change of mice infected with Eimeria papillata.

Values are means ± SD.

*Significance at P≤0.001.

The expression of the jejunal goblet cell gene, MUC2 was downregulated due to infection of mice with E. papillata (Fig. 2). S. persica root extracts could significantly upregulate MUC2 in the infected jejuna (Fig. 2).

Recently, several studies used plant extracts as antiparasitic agents (Klimpel et al., 2011; Metwaly et al., 2015; Alzahrani et al., 2016). The results of the current study indicated that, S. persica root extracts exhibit good anticoccidial properties. This effect may be due to the content of the extract. Khan et al. (2010) found that the root extracts contain flavonoids, alkaloids, carbohydrates, glycosides, saponins, steroids and tannins.

S. persica was able to improve the weight loss due to infection with E. papillata. This weight loss was previously studied by others (Anwar et al.,2008; Metwaly et al., 2013; Dkhil et al., 2014) and explained this loss due to decrease in water and food intake (Anwar et al., 2008). Also, Metwaly et al. (2013) related the weight loss due to the consumption of the parasitic stages to the carbohydrate content inside the intestinal villi and also led to structural alterations of the villi. The most structural change is the disruption of the host epithelium due to the discharge of the developed oocysts to the intestinal lumen.

Through the examination of the infected jejunum tissue sections, we observed that, different stages of E. papillata develop in the crypt region. Also, Cheng (1974) reported that, crypt region contains the stem cells producing goblet cells. These cells were invaded by the parasite leading to the decrease in the number of produced goblet cells in the jejunal villi. These observations were also documented by Dkhil (2013) during the investigation of the anticoccidial action of neem extract and its ability to increase the reduced goblet cell number due to E. papillata infection.

Dkhil et al. (2013) reported that, MUC2 gene was downregulated in the mice jejuna due to the infection with E. papillata. This gene is responsible for the immune response against pathogen-induced injury. S. persica root extract could significantly downregulate the MUC2 expression due to infection however, this mechanism of downregulation has not been studied before.

The high potential effect of S. persica is due to the antioxidant (Mohamed and Khan 2013) and anti-inflammatory (Ezmirly et al., 1979) activities of the plant extract constituents. The dose 300 mg/kg was also proved to have a significant anti-inflammatory activity on rat induced paw oedema (Ahmed et al., 2011) but until now there was no anti-coccidial study about this and our study was the first one dealing with the anti-eimerial properties of S. persica extract.

Based on these findings, we conclude that S. persica root extract exhibits antieimerial activities and could be used with the normal food of animals as an additive.

This research project was supported by a grant from the “Research Center of the Center for Female Scientific and Medical Colleges”, Deanship of Scientific Research, King Saud University.

We declare that we have no conflict of interest.