Journal of Babylon University/Pure and Applied Sciences/ No.(4)/ Vol.(21): 2013




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Journal of Babylon University/Pure and Applied Sciences/ No.(4)/ Vol.(21): 2013

Prevalence Of Cryptosporidium Sp. And Treatment By Using Some Plants Extracts In Al-Hilla City\Babylon Province

Fadhil Hassan Alwan Al-Dulaimi

Local environmental research center Babylon Univ.

Ahmed Khudhair Aubbies Al-Hamairy

Science college of women Babylon Univ.

Abbass Hussain Mughier

Basic education Babylon Univ

.

Abstact :

The study was conducted to investigat the prevalence of Cryptosporidium sp. in children who were attended to laboratories maternity and pediatrics general hospital in Al-Hilla city of Babylon province for the period from Septmber 2010 to May 2011. The age groups of children was ranged from less than 6 months to 5 years old and to show the effect of six plants extracts on the parasite that injected in Balb\c mice. The study was carried out on 605 fecal samples of children, 318 males and 287 females. Detection of Cryptosporidium oocysts was done by modified Ziehl-Neelsen methods.The results show the rate of Cryptosporidium infection was (9.42 %), it did not vary between males and females. The highest rate of infection was among age group less than 6 months to less than one year old. The distribution of infection was highest in September (23.75 %) followed by March (13.75 %) and November (10.3 %) and the lowest was in January (4.54 %). The effect of six plant extracts on the shedding of oocysts in experimental mice, decrease the shedding of Cryptosporidium oocysts in mice by using 250 mg / kg body weight, route it was shown that the efficacy of Peganum harmala (65.9 %) followed by Artemesia herba alba (56.5 %), Ricinus communis (34.3 %).and lowest was Thymus vulgaris (19.1 %). It was also shown that the efficacy of plants extracts (500 mg / kg body weight) was higher than (250 mg / kg B.W.) in shedding of the parasite oocysts.



الخلاصة :

اجريت الدراسة الحالية للتحري عن البويغيات الخبيئة Cryptosporidium sp. في الأطفال المراجعين لمختبرات مستشفى الولادة والاطفال في مدينة الحلة / محافظة بابل للمدة من ايلول 2010 إلى ايار 2011 تراوحت مجاميع أعمارهم بين اقل من ستة اشهر الى خمس سنوات، وكذلك استخدام ستة مستخلصات نباتية وملاحظة تأثيرها على البويغات المحقونة في الفئران المختبرية . ونفذ التحري من خلال فحص 605 عينة غائط من الأطفال المراجعين( 318 من الذكور و 287 من الإناث). لقد استعمل لغرض الكشف عن أكياس البيضة لجنس Cryptosporidium طريقة زيل نلسن المحورة . أجريت الدراسة في الفئران البيضاء المختبرية لبيان تأثير فعالية مستخلصات نباتية مختلفة على طرح أكياس البيضة للطفيلي وهي : الزعتر Thymus vulgaris ،الشيحArtemisea herba-alba ، الخروع Ricinus communis ، الزيتون Olea europea ، الحرمل Peganum harmala ، الثوم Allium sativum بينت نتائج الدراسة ان معدل انتشار الطفيلي كان (9.42 %) مع عدم وجود فروق بين الذكور والإناث وكان أعلى معدل في الفئة العمرية( أصغر من ستة أشهر) إلى (أصغر من سنة واحدة). سجل اعلى نسبة للاصابة في أيلول (23.75 %) وتلاه (13.75 %) في آذار ثم ( 10.3%) في تشرين الثاني بينما اقل نسبة للاصابة كانت في كانون الثاني(4.54%) . وقد وجد أن تأثير المستخلصات النباتية على طرح أكياس البيضة في الفئران المختبرية المصابة تجريبيا بالطفيلي قد أدى إلى تقليل أعداد الأكياس المطروحة في براز الفئران. إن استعمال 250 ملغم / كغم من وزن الجسم من مستخلص الحرمل Peganum harmala هو أعلى فعالية في الكفاءة (65.9 %) وأعقبه الشيح Artemisea herba-alba(56.5%) ثم الخروع Ricinus communis

(34.3 %) وأقلها كان للزعتر Thymus vulgaris (19.1 %) . كما ظهر أن الجرعة 500 ملغم / كغم من وزن الجسم كان أكثر كفاءة علاجية في التأثير على أعداد الأكياس المطروحة.



Introduction :

Cryptosporidium zoonotic agents which frequently infected human as well as domestic and wild animals it is waterborne parasites , transmitted via fecal contamination (Oyibo et al. ,2011).

particularly as a result of comparative studies involving both medical and veterinary workers, Cryptosporidium was also recognized in man many of the early reports of the infection in humans were in immunocompromised subjects, particularly those suffering from AIDS (Hassanpour et al., 2011)

The disease is associated with self-limited, mild to sever diarrheas lasting for several days among healthy individuals and profuse-cholera like diarrhea often becomes irreversible among immune deficient ones, that may contrast sharply, causing prolonged life threatening disease a symptomatic form of Cryptosporidium infection is common and constitutes a source of infection for many hosts (Syed & Javid . 2011 ) . In symptomatic type of infection there may be excessive loss of fluid, varies from 3-6 to 17 liters of watery diarrhea/day in human (Mannheimer & Soave ,1994).

Feacal-oral mode of transmission may be very important between domestic animals and man (Mackenzie et al.,1994). In human, however, the contact between person to person spreads the infection that has been implicated in many outbreaks in all parts of the world (Mahammad et al., 2011) .

The parasite can be easily transmitted from one mammalian species to another, All these transmissions of the parasite cause contamination of surface and ground water (Mancassola ,1997). The first case of human cryptosporidiosis was reported from contamination of drinking water (Schmidt & Roberts, 2000). As cryptosporidiosis is self-limited illness in immunocompetent patients, therefore only supportive management is required while in immunocompromised patients, it is life threatening and may lead to death (Michael et al., 2011).

Subsequently, particularly as a result of collaborative studies involving both medical and veterinary workers, cryptosporidiosis was also recognized in man (Markell et al. ,1999). In the past, many reports assumed the parasite to be host specific and several species of Cryptosporidium were named on the basis of host occurrence. It has been demonstrated in preliminary cross-reaction studies that infections were readily transmittable between various hosts (Tzipori et al.,1980). Furthermore the several studies indicated that some degree of host specificity might occur between vertebrates (Abdel – Wahab & Abdel – Moagood , 2011) . Members of the genus Cryptosporidium are placed taxonomically within the phylum Apicomplexa the classification of the genus Cryptosporidium (Diego & Mirna, 2008).

The aim of the study are the prevalence of Cryptosporidium sp. in who were attended to laboratories maternity and pediatrics general hospital in Hilla city of Babylon province, and Isolating Cryptosporidium sp. from human and conducting trials of treatment of the parasitic infection and primary searching effective of six plants extracts on parasite .

Material & Methods :


The study was conducted on 605 random samples of feacal rectal smears of children attained to lab. Maternity & pediatrics general hospital in Hilla city, age ranged from less than 6 months to 5 years started from September 2010 to May 2011.

The information for each sample in this work were registered. The lab. examinations were conducted at lab. of researches in college science of women, department of biology.

The modified Ziehl- Neelseen stain M ZN-ST (acid fast) was used for examination of feacal smears as following (Henriksen & Pohlenz ,1981).

smear was fixed with Methanol alcohol for 5 minutes and allowed to dry at room temperature.Then the dried smears were stained for an hour in carbol fucshin prepared by dissolving 15% carbol fucshin in methanol (stock solution). ziehl fucshin 10 ml added to 90 ml 5% phenol.Then rinsed in tap water.Then differntiate in 2% H2SO4 solution for 20 seconds with agitating the slide.Rinsed in tap water.Then smear was stained with 5% malachite green solution for 5 minutes and washed in tap water and followed until dried.Then examined by using 40x and 100x oil immersion objectives.

Oocysts were obtained from naturally infected children with acute diarrhea and proved to be infected with Cryptosporidium by M ZN-ST (acid fast) .

Isolation of the oocysts from children faeces (Arrowood & Sterling ,1987): as follows: The faeces was suspended in PBS of pH 7.2 in ratio of 20-80 and centrifuged at 500g for 10 minutes. The supernatant was discarded and the sediment was suspended in 15ml centrifuge tubes of PBS pH 7.2, and 3-5ml of ether were added and mixed vigorously.Then centrifuged at 500 g for 1 minute was made and four layers were differentiated from bottom to the top:- 1-Sediment, 2-PBS, 3-Debris and 4-Solvent. Layers were discarded and the sediment layer that consisted of 75% of oocysts was used for purification of the oocysts. These oocysts were prepared in 2.5% K2Cr2O7 until used.

The mice were infected with Cryptosporidium sp. oocysts that isolated from human faeces using a dose of 103 oocysts. This dose was determined after trail infections of three doses: 102, 5x102, and 103 oocysts to each mouse per os, in order to achieve the infection that give the suitable number of oocysts sheds from infected mice. Collection of the oocysts of Cryptosporidium sp. from infected mice (Blagburn et al., 1998):

Intestine of infected mice were cut in small pieces, was added water and homogenized in plastic tube with sputa sol 25 and incubated at 4C˚. and homogenate was transferred in room temperature 90-120 minutes and homogenized thoroughly. The homogenate was centrifuged at 1000 g for 10 minutes.Then the sediment was washed two times with cold tween 20 (0.1%) in distilled water. The sediment was stored in 2.5% K2Cr2O7.

White mice of Balb\ c strain were reared in lab-animal room and managed in standard plastic cages bedding with wood shaving, and they were cleaned 1-2 times weekly according to the number of mice. They were fed with concentrated diet .

Six groups of mice each of 3 mice 2 weeks age were infected orally by stomach tube per os with 10 oocysts . The infected mice and control examined for 2 weeks by fecal floatation Sugar sheathers.Six species of plant were used in this stud were collected from Al-Hilla herbs shops:

Thymus vulgaris , Artemisea herba-alba , Ricinus communis , Peganum harmala , Olea europea , Allium sativum .

The leaves were the only part that was used in this study and collected parts of plants were dried in the air in shade and milled in mortar and pistol. The milled substances were sieved by using fine mesh sieve and the powder material adjusted in dissector containing CaCl2 in order to complete dryness. All the powders were extracted in ethyl alcohol 96% according to Harboren (1973). The dried plant powder was extracted in ethyl alcohol (96%) in ratio 1-10 (W/V) mixture was shacked continuously up to 24 hours under 4ْ C.

The solution filtrations under high pressure in buchner funnel for expression of the filtration for 15 minutes. The filtrate was collected and evaporated in vaccum. Rotary evaporator under less than 60ْ C. The rest of the extracted substance (chlorophylic extracts) and water was filtrated by using whatman filter paper no. 4. The filtrate was returned to evaporator to remove the water. The extracted substances (in dark bottle) were sent to college of medicine in Al-Qadesyia University for lyophilization. The lyophilized material was stored at 4ْ C until used.

Experimental studies for the lyophilized plants extracts:

The effect of lyophilized extracts on the oocysts of Cryptosporidium sp. was carry out in two lines in vivo.



A-Six groups of 3 white mice Balb\c each, 1 week old were infected with 103 oocysts of Cryptosporidium sp. collected from naturally infected human and purified in ether per os using stomach tube. Faeces of infected mice were examined daily for oocysts and counted. After 3 days post infection (P.I) when the maximum number of the oocysts was reached, the mice groups were treated with the Lyophilized extract in dose equivalent to 250 mg / kg body weight per os using stomach tube. at the sixth day post infection (P.I )the oocysts were numerated.

B- A group of 3 mice were infected with 104 oocysts and a group kept without treatment as a control group. The treated and control groups of mice were sacrificed and the intestine were separated for collection and enumeration of the total number of oocysts .

C-The lyophilized extract of higher effect on shedding of oocyst were chosen to another run of treatment with dose of 500 mg / kg body weight; as in A.

The efficiency of the extract was done according to Egerton (1963) by using the following formula:



Rate No. of the control group - Rate No. of the treated group
Efficacy %= _____________________________________________________ X 100
Rate No. of the control group


The different statistical tests were used for analysis the results according to the type of the data( F- test. ANOVA) The significance level p ˂ 0.05, 0.01 was regarded significant (Snedcor & Cochran, 1978 ).

Results :

Table(1) show the distribution of cryptosporidiosis was done according to age and sex. A total of 605 stool samples were examined from children, 318 were males and 287 were females. It was found that the rate of cryptosporidiosis in males (7.55 %) did not vary significantly from females (9.75%) .

Regarding the age distribution, statistically there was no significant difference between the age groups. It can be seen in table (1), that the rate of Cryptosporidium positive cases was highest among age groups from less than 6 months to more than one year (10.95 % & 10.76 %), followed by those below less than 6 months (6.87 %) and the lowest rate was among more than 5 years of age (2.5 %). Table (2) shows that the distribution of Cryptosporidium oocysts in children in rural areas (5.24 %) was significantly (P >0.05) lower than urban areas (12.72 %). Although statistically there was no significant difference in the rate of Cryptosporidium oocysts in children in different months of the year, but it was shown that the highest rate of oocysts infected occussed in September (23.75 %), followed by March (13.75 %), November (10.3%), December (5.20%), February (4.65 %), January (4.54%) and April (4.08%) respectively.

The effect of plants extracts on the shedding of oocysts in mice is indicated in (Table 3), that 250 mg / kg body weight of different plant extracts decrease the shedding of Cryptosporidium oocysts in infected mice, with significant difference in the number of oocysts between pre and post treatment (P> 0.05).

Table (4) indicates that the efficacy of 500 mg/ kg body weight of plants extract on the shedding of oocysts in mice. The efficacy of Peganum harmala was highest (69.7 %) followed by Allium sativum (65.6%) and the lowest was Thymus vulgaris (20 %).

It is clearly observed that the efficacy of 500 mg/ kg body weight of different plants extract on the total number of Cryptosporidium oocysts in the intestine of infected mice was greater than those treated with 250 mg / kg body weight of the plants extract


Table (1): Distribution of Cryptosporidium sp. oocysts in children According to


sex and age .

*Sex

Male

Female

Total




**Age Group

Exam.

No.


Infec.

No.


(%)

Exam.

No.


Infec.

No.


(%)

Exam.

No.


Infec.

No.


(%)

> 6 Months

51

3

5.88

65

6

9.23

131

4

6.87

<6 Months

80

9

11.25

60

4

6.6

146

16

10.95


141

11

7.80

143

18

12.6

288

31

10.76

>5 Years

46

1

2.17

19

-

-

40

1

2.5

Total

318

24

7.55

287

28

9.75

605

57

9.42

*Sex p< 0.05

**Age p< 0.05



Table (2): Distribution of Cryptosporidium sp. oocysts in children according to

Living area and months.


Region

*Rural

*Urban

Total

**Month

Exam.

No.


Infec.

No.


(%)

Exam.

No.


Infec.

No.


(%)

Exam.

No.


Infec.

No.


(%)

September


34

3

8.82

46

16

34.78

80

19

23.75

November

40

3

7.5

57

7

12.3

97

10

10.3

December

41

1

2.43

55

3

5.45

96

5

5.20

January

37

1

2.7

51

3

5.88

88

4

4.54

February

40

1

2.5

46

4

8.7

86

4

4.65

March

33

3

9.1

48

8

16.6

80

11

13.75

April

42

2

4.76

35

2

5.71

98

4

4.08

Total

267

14

5.24

338

43

12.72

605

57

9.42
*Living area p > 0.05

**Month p < 0.05


Table (3): Effect of plant extracts 250 mg / kg body weight on the shedding of

Cryptosporidium oocysts in infected mice.

Plant Extracts

No. Oocysts (X103)

*Prt 6th day **post



Efficacy %

Control group

Peganum harmala

3.5 1.1

65.9

3

Artemisea herba-alba

2.5 1.0

56.5




Ricinus communis

3.8 2.4

34.3




Olea europea

3.6 1.5

32.1




Allium sativum

4.3 3.5

31.6



Thymus vulgaris


3.4 2.7

19.1




*Prt= Pre-treatment

**Post= Post-treatment

P > 0.05
Table (4): Effect of some chosen plant extracts in dose 500 mg / kg body weight

on the shedding oocysts in infected mice.


Plant Extracts.

Oocysts No. (X103)

Prt. Pot.



Efficacy %

Control group

Peganum harmala


3.5 1.1

69.7

3.5

Allium sativum

3.3 1.2

65.6




Artemisea herba-alba

3.2 1.4

50.3




Ricinus communis

2.5 1.3

44.3




Olea europea

2.7 1.6

33.9




Thymus vulgaris

3.4 2.7

20




*Prt=Pre-treatment

**Post=Post-treatment

P > 0.05
Disscussion :

From 605 fecal smears from children examined 57 were positive with the rate of infection being (9.42%). This is high, which indicates that cryptosporidiosis is one of significant cause of diarrhea among children in Hilla city. The high rate of infection in this study might be related to poor hygiene, contamination of food or water supply and close contact with infected persons or animals specially dogs and cats (Radostitis et al. ,1994).

Comparing the finding of this study with that reported by other studies carried out in the country. It is lower than that found in Baghdad (14.6%), (Al-Gelany,1998) . in Mosul (14.3%), (Al-Alousi et al.,2003); (20.52%), (Khalil,2000) , and in Kirkuk (12.62%), (Al-Moula,1999). almost identical to that found in Basrah (8.82%), (Nadham,1996) but higher than in Diyala (2.8%), (Al-Taie,1997).

The difference between the finding of this study and other provinces might be related to period of study, environmental factors, sample size, and the techniques used for identification of the infection( Schmidt & Roberts ,2000).

It is also lower than that reported in other countries e.g. (12.7%), (Gerba et al., 1996) in Sudan; (67%), (Nimiri and Hijazi,1994) in Jordan; (14%), (Chen et al., 1991) in China.

The distribution of cryptosporidiosis does not differ significantly between sexes, this reflects that both male and female children are equally susceptible. This finding is not in agreement with that shown in Kirkuk, (Al-Moula,1999);in Baghdad, (Al-Gelany,1998); in Basrah, (Nadham,1996); and in China, Chen et al.,1991) who found that the distribution of infection in males was higher than females but not in agreement with that reported by Al-Moula (1999) in Kirkuk, who showed the rate of infection in females higher than males.

Concerning the age distribution, it seems from the result of this study that the distribution of cryptosporidiosis is not related with the age of children, as there is no significant difference in the rate of infection among different age groups. The highest rate of infection in the studied groups is among those below 6 months to below one year of age. This might be due to low care and attention given to children from their mothers such as using unsterilizing feeding bottles and unboiled water, (Ungar, 2000 ; Maryam et al ., 2010). This finding goes with finding of Al-Moula (1999) who found the highest rate of infection in infants aging from 1 month to 1 year, Iqbal et al. (2001) and Nimiri and Hijazi (1994), which reveals that cryptosporidiosis is common among infants and young children suffering from diarrhea, especially within the first two years of age (Mohammad et al ., 2011).

As far the residency concern, it is shown that children in rural areas have lower rate of infection than urban ones. This might be related to type of feeding, as most children in urban areas have fed on artificial diet, which have more chance to get infection them those fed on breast feeding similar finding by was reported in Kirkuk province (Al-Moula, 1999 ; Othman,2000), but against the finding in Korea (Chai et al., 2001) who reported higher rate of infection in rural (14%) than urban area (3.7%).

The lowest rate of infection in January might be due to oocysts of parasites destroyed in low temperature (4ْ C) statistically there was no significant difference in the rate of infection between different months. The present study was not carried out throughout a year; therefore seasonal comparison of rate of infection is impossible. It was found the highest rate of infection in September and the lowest in January ( Sultan et al ., 2011) .

Shedding light on the study of other workers, it is shown that rate of infection is hight summer and spring seasons and low in winter months (Othman,2000), in Kirkuk and (Al-Yassin,1999) in Tikrit. It has been shown that Cryptosporidium oocyst can be found during warm and humid months, (Syed and Javid,2011).

In the present study six plants extracts were tried (Peganum harmala, Artemisea herba-alba, Ricinus communis , Olea europea, Allium sativum , and Thymus vulgaris), as alternatives of chemical drugs due to their toxicity and unavailability in the country. The medical plant extracts efficacy was tried in mice as alternative of human and economically important live stocks (Maryam et al ., 2010) .

Using 250 mg/kg body weight of plant extracts, the highest efficient plant extract was Peganum harmala (65.9%), followed by Artemisea herba-alba (56.5%), Ricinus communis (34.3 %),Olea europea (32.1%) , Allium sativum (31.6%) and Thymus vulgaris (19.1%).



Peganum harmala is a medical plant, its chemical contents are glucosindate and prosporing and it is used as antidysentric (Majeed & Mahmood ,1988). The second effective drug was Artemisea herba- alba, its efficacy might be due to its contents of artemisin and santonin. It was used against Ascaris parasite (Al-Khazraji, 1991) and was found to be actives against hydatidosis in laboratory mice (Al-Nakeeb ,2004). The anti-parasitic action of Allium sativum, is also reported and suggested that might be due to its chemical contents of glucose and fructose. The anticryptosporidial activity of sugar has been found by Harp (1999), who found that sucrose and to a lesser extent isomaltose reduced intestinal colonization of C. parvum in neonatal mice (Syed & Javid . 2011) .

The efficacy of Ricinus communis was also high, which also included glucose in its content in addition coumarine, volatile oil, sulphur, isothiocyanate, and glucosinolate and it acted as vermifuge and intestinal carminative (PDR,1998).

Al-Musaui (2000) studied the effect of alcoholic extract of Artemisea herba- alba plant extract on Hymenolepis nana, and, she observed 10-14 compounds in both the dry and fresh parts of plants, among the compounds determined was Esculetin as a coumarine compound. He concluded that the dry and wet extracts of Artemisea herba- alba were effective on infertility, egg development, and average eggs output of H. nana. He also found the efficacy of alcoholic extract of Artemisea herba- alba on Hymenolepis nana was (100%), on days fifth and sixth post-infection using 500 and 250 mg/kg body weight respectively.

Regarding the efficacy of Olea europea, it was (32.1%), which contents include Mannite, glucose, oleosteral, resine, olivine, and resine . It has an active antiparasitic effect (Al-Aubaidy ,1999).

It is also reported that the efficacy of Thymus vulgaris was (19.1%), which is also reported to be parasite killer. Its chemical contents include beta caryophyllena, (Maryam et al., 2010).

Increasing the dose of plant extracts from 250 to 500 mg/kg body weight to show the effect of five plants ( Artemisea herba-alba, Olea europea, Peganum harmala, Allium sativum ) on Cryptosporidium oocysts. It was clearly indicated that the efficacy of plant extracts was increased with increasing the doses of plant extracts as shown in table (4).

from the results of this study, it is concluded the followings:

The prevalence of cryptosporidiosis is high among children at Hilla city and the plant extract of Peganum harmala have an effect on Cryptosporidium sp. infection in Balb/c mice.Freshly isolated oocysts from man are more infective to laboratory mice.




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