Indian Journal of Forensic Medicine & Toxicology, April

Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2 2673

Comparison of Histological Changes in Mice Infected with the Cryptosporidium Parvum after Treatment with an Aquatic

Leaf Extract of Salvia Officinalis, Pimpinella Anisum and, Spiramycin Drug

Khadiji Khleaf Al-Dulaimi1, Karama Tahreer Ahmed Al-Taee2, Thaer Abdulqader Salih3

1Lecturer, Department of Basic Sciences, College of Dentistry, University of Anbar, 2Lecturer, Department of Basic Sciences, College of Dentistry, University of Anbar, 3Assistant Professor, College of Sciences/ Department of

Biology, University of Anbar

AbstractBackground: Cryptosporidium parvum was cause Cryptosporidiasis .In this study the therapeutic effect of two aquatic extracts of plants in infected white mice with the C. parvum were examined and the results were compare with the therapeutic effect of Spiramycin through the study of the histological changes in mouse intestine. Methods: Thirty male mice were divided into six groups: group 1(non infected, negative control ), group 2 (feeding with C. parvum oocysts, positive control ), group 3 (infected and treatment with leaf extract of Pimpinella anisum), group 4 (infected and treated with Salvia officinalis extract ,the group 5 and 6 (infected and treatment with 5mg and 10mg Spiramycin Respectively ) The existence of oocysts in the feces of animals was counted daily during the therapeutic period .After two weeks; the changes of cecum histological sections were examined. Conclusions: The oocysts counted in feces samples, showed significant differences between the treatments (P <0.05). The cecum histological changes in the group 3 and 4 indicated that leaf extract of plants have moderate efficacy in treatment of C.parvum infection. The cecum appears close to normal at the concentration of 5mg Spiramycin there is no abnormal tissue in the concentration of 10 mg.

Keywords: Cryptosporidium, plants extract, histological changes, Spiramycin.

Corresponding author: Karama Tahreer Ahmed Al-Taee, Lecturer Department of Basic Sciences, College of Dentistry, University of Anbar E-mail: [email protected].

Introduction

One of the important parasites that belong to phylum Apicomplexa is Cryptosporidium parvum, which causes infection to the epithelium of the intestine. These obligate enteric protozoa found in human and most mammalian. [1,2]. Cryptosporidium parvum causes five to ten million deaths per year [3] .

Cryptosporidium parvum was known as the cause of the diseases Cryptosporidiasis transmitted by Food

and water in humans and animals. The parasite is also a common pathogen related at self-limiting diarrhea in people with immune competence, but in low immune deficiency is life-threatening like AIDS patients [4,5].Globally, Cryptosporidiasis is considered the second disease after rotavirus causing death among children lower than 2 years due to diarrhea, which may be severe or moderate. [6] Cryptosporidium parvum causes acute diarrhea, particularly in children lower than five years old and small animals [7].

In neonates Cryptosporidiasis cases, naturally and experimentally C. parvum not only influenced the distal small intestine, but affected proximal small intestine too and several portions of the large intestine. [8,9] .In infected animals with parasite, animal’s intestine appear pathological changes like, villus atrophy, degeneration

2674 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2

in villus, crypt hyperplasia, infiltrations in inflammatory cells and degeneration in microvillus. As a result of these changes many intestinal functions would be affected such as digestion (maldigestion), absorption (malabsorption) and osmotic imbalance [10, 11].

The use of chemical therapy for the disposal of parasites is useful, but in this way there are many problems like resistance to drugs, residue and side effects associated. Therefore, it is important to find a replacement medication. [12].Herbal medicine was adopted centuries ago to treat parasites in most parts of the world. Plants have been used by humans to eradicate several diseases. Medicinal herbs are used in current years, largely for their reliability High efficiency and low cost [13, 14] Spiramycin (Rovamycine ) was examined and prove as an effective drug against cryptosporidiosis in patients infected with AIDS [15]. More than twenty years spiramycin was used in Europe and Canada in bacterial and protozoa disease, like Toxoplasma gondii [16].

Sage (Salvia officinalis L.) is a remarkable pharmaceutical herb to have aromatic characters used as a raw substance in different perfumery, medicinal uses, food, and in the cosmetics industry. S. officinalis is included in the family of Lamiaceae it is growing commercially for the production of essential oil [17]. Leaves used in extraction of essential oil are strongly aromatic, that have greater than 49 aromatic ingredients. Different pharmacological research proves that sage products hold a substance that inhibits oxidation, diabetic, inflammatory, microorganism causing disease and cancer. [18].

Pimpinella anisum called aniseed, belonged to Apiaceaeis family, a flowering plant. As a medicinal herb the uses of Anise are stimulating action of digestion and drug against parasites, bacteria and fungi. Aniseed is used in traditional European herbal medicine as treatment of diarrhea, colic and menstrual cramps [19]. Therefore, the purpose of this study was to know the impact of both Salvia officinalis and Pimpinella anisum on Cryptosporidium parvum infection and compare the result with treatment by spiramycin drug.

Materials and Methods

Cryptosporidium parvum oocysts:

Oocysts of Cryptosporidium parvum were obtained from a feces specimen that arrived to the (Laboratory of parasite), at the Teaching Hospital for Obstetrics and Gynecology to four year child. The feces specimen was examined by using modified Ziehl-Neelsen (ZN) staining to ensure the presence of oocysts. A drop of the sample was spread on a glass slide, left until dehydration after boiling it with Sheather’s sucrose solution fixed with ethyl alcohol, stained with the modified (ZN) and examined under compound light microscope on 40x objectives [20]. The infective specimen was maintained by 2.5% potassium dichromate as described by [21]

.The number of C. parvum oocysts was detected by haemocytometer in the stock inoculums [22].

Experimental animals: Thirty of laboratory Seviss Albino Mice (strain Balb/C type Mus musculus), four weeks old, male, weighing about 23 -28 g each were used in the current study which was obtained from animal house at the College of Science / University of Anbar. The mice were raised in special cages and multiplied under appropriate conditions in terms of temperature and provision of food consisting of a cereal fodder Soybeans and protein. In this study we followed all ethical protocols of animal’s treatment.

Determination of infected dose in experimental mice: 20 ml of oocysts were washed several times with distilled water and dosed 21 mice (for each dose three mice) with a suspensory of oocysts (10, 20, 30, 50, 100, 1000, and 10,000 oocysts / mouse). By using the Haemocytometer chamber, the highest dose and lowest dose were calculated to cause infection without fatality [22].

Collection of plants: The leave of Pimpinella anisum and Salvia officinalis were collected from different places from the College of Agriculture / Abu Ghraib, after being diagnosed by the national herbivore in the Department of Biology / College of Science / University of Baghdad, it was dried, ground and preserved at a temperature of 20 ° C.

Preparation of aquatic extracts: 100 grams of dry powder Leaves were infused in 1000 ml distilled water on magnetic mixer for 30 minutes then the solution was filtered and the filtrated liquid was placed in the centrifuge for 15 minutes, and then quit in electric oven at a temperature of 50 ° C, till gaining dry extract. The


stock solution was prepared by dissolving 1 g of each extract in 100 ml of distilled water. From this stock solution, a series of dilutions were prepared [23].

Design of Experiments: The mice, infection experiment with the parasite and treatment was designed as addressed in the table 1. All the animals were feeding orally by using the stomach tube, the animals in the second, third; fourth, fifth and sixth groups were infected by parasite by feeding the mice orally with 0.5 ml (103 oocysts). The mice feces were examined daily by direct

stool examination to confirm the appearance of the infection, after 2 to 4 days the infection occurrence was confirmed. The aquatic extract was given to the affected mice experimentally to the third and fourth group with a concentration of 5 mg / ml while, the fifth and sixth group was treated respectively by Spiramucin in concentration 5 mg and 10 mg for each 20 g of body weight. Spiramycin obtained from (Samarra Pharmaceutical Factory) was dissolved in distilled water. Each mouse was given a dose every day during the experiment that continues for two weeks.

Table 1. The treatments of experimental animals.

Dose of treatment No.of animalsType of treatmentNo.of Group

0.5 ml PBSFive animalsNegative control

Non infected Group 1

0.5 ml (103 oocysts )Five animalsPositive control

Infected Group 2

5 mg / mlFive animalsInfected and treated with the aqueous extract, Pimpinella anisum, Group 3

5 mg / mlFive animalsInfected and treatment with the aqueous extract of Salvia officinalisGroup 4

Spiramycin 5 mg / 20 g of body weightFive animalsInfected and treatment with

SpiramycinGroup 5

Spiramycin 10 mg / 20 g of body weightFive animalsInfected and treatment with

SpiramycinGroup 6

Oocysts counting: Haemocytometer chamber was used to counts the oocysts in feces of experimental mice daily during 14 days, table 2 [22].

Preparation of the Tissue : After two weeks the mice were dissection , the cecum was harvested and fixed in formalin in concentration 4% for 24 hours before processing and then used paraffin wax for embedded stage ,The sections that in thickness 3-5 μm were stained with H&E according to[24].

Statistical analysis: The experiment is designed as a simple one-factor experiment. The results obtained

from our current study were analyzed using SPSS Ver.22 statistical program, as a Randomize Complete Block Design (RCBD) was used. The significance of the averages was tested at a probability level (P <0.05), and the meanings of the coefficients were compared using the Least Significant Difference (LSD ) test using the Duncun test [25,26].

Rustles and Discussion

In this study, the therapeutic activity of sage leaf extract, anise, and spiramycin drug were examined in infected mice with Cryptosporidium parvum. The


comparison between the four treatments was adopted in terms of the effect of oocysts shedding number in the feces of infected mice, as well as the histological changes in the intestine of laboratory mice infected with the parasite.

To investigate the number of oocysts, the feces samples were examined in all groups of mice that infected

with the parasite. The examination revealed that infected and untreated mice (G2 positive control) shedding more oocysts than infected groups that were treated with aqueous extract of anise and sage (G3,G4) [27]. Also the Spiramycin drug (G5, G6) show high effective in the reducing oocysts count during the experiment that continue (14 days) with significant differences (P <0.05), this results identified with many studies [28].

Table 2. The number of oocysts during the period of experiment 14 days.

Spiramycin 10 mgSpiramycin 5 mg Salvia officinalis

Pimpinella anisum

Control positive

Control negative

Type of treatment

0 A0 A0 A0 A0 A0 A1st day

0 A0 A0 A0 A0 A0 A2nd day

0 A0 A0 A0 A15 B0 A3rd day

0 A0 A11 B42 B74 B0 A4th day

2 A21 B100 B100 B124 B0 A5th day

2 A22 AB 276 BC189 B378 C0 A6th day

0 A46 AB367 B387 B788 C0 A7th day

0 A2A476 B418 B2138 C0 A8th day

0 A 0 A588 B416 B6930 C0 A9th day

0 A0 A790 C321 C14678 B0 A10th day

0 A0 A700 C210 C24980 B0 A11th day

0 A0 A389 C103 C34529 B0 A12th day

0 A0 A378 C12 C55712 B0 A13th day

0 A0 A301 C0 A87645 B0 A14th day

0 A6.5312.5715716285.10Mean

The different letters within the same row indicate significant differences between the treatments with a significance level of 0.05.

In this study, after two weeks the cecum was harvested and the histological section were prepared and the changes in the tissue were examined under compound light microscope. Histological sections of the positive control group fig2 showed severe histological changes such as vocalization in the secretory glands and the presence of large numbers of parasites around the secretory glands, compared to the negative control

animals fig1 that revealed no changing in the histological section of cecum .

From the examination of the mice cecum tissue, that infected and treated with anise the study found that anise plant extract can assist to restore the tissue and secretory glands to normal state and minimize the number of oocysts fig 4 over time when compared with the positive


control group fi g 2, as it is noticed vacuolization in the secretory glands and the presence of huge numbers of oocysts around the secretory glands, meaning that the anise extract is get rid of the parasite. Similar results reveal its antiprotozoal action, such as the anticoccidial activity against infection of poultry with Emeria tenella [29]. In another study, the methanolic extract of Pimpinella anisum proven effective against infection with Amoeba [30].

Also, the extract of the Salvia offi cinalis helps in the return of the secretory glands to a normal state fi g 3 when compared to the positive control group fi g 2. Similar results were reported regarding the effect of

sage against worms, results showed signifi cant reduction in the number of eggs excreted by Hymenolepis nana, Aspiculuris tetraptera, Syphacia obvelata [31] Similar results about antiprotozoal effects of sage extracts were recorded against Cryptosporidium parvum [32].

In the case of treatment with Spiramycin at a concentration of 5 mg, there is an increase in the thickness of the intestine wall as a result of slight fi brosis with slight bleeding, but in the concentration of 10 mg, there is emergence of the normal state after treatment and the return of the secretory glands to their normal state.

Figure 1. Negative control .Histological section of the intestine mice, without infected parasite and without treatment, showed the cohesion of the secretory glands tissue H&E

stain (40x).Figure 2. Positive control. histological

section of the intestine mice, the occurrence of vacuolization in the secretory glands and the presence of large numbers of parasites s

around the secretory glands (H&E) stain 40x)Figure 2. Figure 1.

Figure 3. Section in Intestine mice was treated with Salvia offi cinalis, notes the secretory glands and their return to normal, H&E )

stain (40x). Figure 4. Intestinal histological section of

the treatment mice with Pimpinella anisum, noting the treatment attempt to restore the normal secretory tissue and glands, H&E

stain (40x)). Figure 4.

Figure 1.

Figure 3.


Figure 5. Histological section of the intestine mice that treated with Spiramycin at a

concentration of 5 mg notes the increase in thickness and thickening of the intestine walls

as a result of minor fi brosis ( ) with slight bleeding ( ), H&E (40x).

Figure 6. Histological section of the intestine mice that treated with Spiramycin at a

concentration of 10 mg. Normal condition appears after treatment, and the ejaculate

glands return to normal Figure 6. Figure 5.

As for the drug spiramycin, it is highly effective, as it led to a high decrease in oocysts shedding rates as shown in table (1) this is consistent with the studies [33,

34] that the drug spiramycin is used to treat diarrhea it is caused by the Cryptosporidium parasite and can be used as a high-dose treatment for a long time, Sparimycin affects the protein synthesis process in the ribosome. Other study concluded that spiramycin a high infl uence on the Cryptosporidium parvum especially when mixed with vitamin E. [35].

Competing Interest statement:

The authors advertise no confl ict of interest.

Funding statement: This study did not receive any specifi c grant from funding agencies in the public, commercial or not-for-profi t sectors.

Acknowledgements: The excellent technical assistance of Faculty of Sciences, Department of Biology that facilitated the achievement of this work is acknowledged.

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Indian Journal of Forensic Medicine & Toxicology, April