research article prevalence of cryptosporidium sp ...

Egypt. J. Exp. Biol. (Zool.), 8(2): 287 – 293 (2012)

© The Egyptian Society of Experimental Biology

RESEARCH ARTICLE A hm e d K h. A l - H am ai r y* A b b as H . M u g h e er * * F a d hi l H . A . A l - D ul ai m i * * *

PR EVALEN CE OF CR YPTO SPOR ID IU M SP. AN D TR E ATM ENT BY U SING SOME PL AN T EXT R AC T S IN AL -H IL L A C IT Y, BAB Y LON PR OVIN C E OF IR AQ

ABSTRACT: The present study was conducted to investigate the prevalence of Cryptosporidium sp. in children attending the laboratories of maternity and pediatrics general hospital in Al-Hilla City of Babylon province for the period from September 2010 to May 2011. The age groups of children ranged from less than 6 months to 5 years old. The study was also extended to show the in vivo effect of six plant extracts on the parasite in experimentally infected Balb\c mice. The study was carried out on 605 fecal samples (318 males and 287 females). Detection of Cryptosporidium oocysts was done by modified Ziehl-Neelsen methods. The rate of Cryptosporidium infection (9.42%) does not vary with host gender. The highest rates of infection were among age groups more than six months to more than one year old. The infection rate 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 was well illustrated where there was a marked decrease of the shedding of Cryptosporidium oocysts in mice by using 250 mg / kg body weight. It was shown that Peganum harmala (68.6%) showed highest anthelmintic activity followed by Artemesia herba alba (60.0%), Ricinus communis (36.8%), while Allium sativum and Thymus vulgaris show the lowest activity 18.6% and 20.5%, respectively. It was also shown that the efficacy of plant extracts at a dose of 500 mg/kg body weight was higher than 250 mg / kg BW in shedding of the parasite oocysts.

KEY WORDS: Cryptosporidium sp., Treatment, Plant extracts. CORRESPONDENCE: Ahmed Khudhair Al-Hamairy* *Science College for W omen-Biology, Babylon University E-mail: [email protected] ISSN: 2090 - 0511

Abbas Hussien Mugheer** **Basic science, Babylon University Fadhil H. Alwan Al-Dulaimi*** ***Local environmental research center, Babylon Univer sity

ARTICLE CODE: 36.01.12

INTRODUCTION: Cryptosporidium sp. is a zoonotic coccidian sporozoa frequently infecting human as well as domestic and wild animals. It is waterborne parasite transmitted via fecal contamination (Oyibo et al., 2011). The parasite inhabits the brush border of the small intestinal mucosa of a variety of mammals (Hassanpour et al., 2011) The disease is associated with selflimited mild to sever diarrheas lasting for several days among healthy individuals and profuse-cholera like diarrhea that often becomes irreversible among immune deficient patients that may contrast sharply. It is a prolonged life threatening disease and the asymptomatic form of Cryptosporidium infection is common and constitutes a source of infection for many hosts (Mirza-Qavami and Sadraei, 2011). In symptomatic type of infection there may be excessive loss of fluid varies from 3-6 to 17 liters of watery diarrhea per day in humans (Mannheimer and Soave, 1994). Fecal-oral mode of transmission may be very important between domestic animals and man (Mackenzie et al., 1994). In humans, however, the contact between peoples spreads the infection that has been implicated in many outbreaks in all parts of the world (Radfar et al., 2011). The parasite can be easily transmitted from one mammalian species to another. Transmission of the parasite causes contamination of surface and ground water (Mancassola, 1997). The first case of human cryptosporidiosis was reported from contamination of drinking water (Schmidt &

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Roberts, 2000). As cryptosporidiosis is a selflimited illness in immunocompetent patients, therefore only supportive management is required while in immunocompromised patients, it is life threatening and may lead to death (Brumlik et al., 2011). As a result of collaborative studies between medical and veterinary scientists, cryptosporidiosis was recognized in man (Markell et al., 1999). In the past 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 crossreaction studies that infections were readily transmittable between various hosts (Tzipori et al., 1980). Furthermore, studies indicated that some degree of host specificity might occur between vertebrates (Guiguet et al., 2008; Abdel-W ahab and Abdel-Moagood, 2011). The aim of the study is to record the prevalence of Cryptosporidium sp. in children attending the laboratories of the Maternity and Pediatrics General Hospital in Hilla City of Babylon province . The study also aims to isolate Cryptosporidium sp. from humans and evaluate the efficacy of six plant extracts on the parasite. MATERIAL AND METHODS: The study was conducted on 605 random samples of fecal smears of children from the laboratories of the Maternity and Pediatrics General Hospital in Hilla City. The age of children ranged from less than 6 months to 5 years. The study started from September 2010 to May 2011. Examinations were conducted at the laboratory of the College of Science of W omen, Department of Biolog y. The modified Ziehl- Neelseen stain M. ZN-ST. (acid fast) used for examination of fecal smears follows Henriksen and Pohlenz (1981). Smears were fixed with methanol alcohol for 5 minutes and allowed to dry at room temperature. Dried smears were stained for an hour in carbol fucshin prepared by dissolving 15% carbol fucshin in methanol (stock solution). Ten ml of Ziehl fucshin were added to 90 ml of 5% phenol. Smears were rinsed in tap water and differentiated in 2 % H 2 SO 4 solution for 20 seconds while agitating the slide and rinsed in tap water. Smears were counter stained with 5 % malachite green solution for 5 minutes, washed in tap water and left until dried. Stained smears were examined by using 40 X and 100 X oil immersion objectives. Oocysts were obtained from naturally infected children with acute diarrhea and proved to be infected with Cryptosporidium as confirmed by M. ZN-ST. (acid fast). ISSN: 2090 - 0511

Isolation of the oocysts from children feces (Arrow ood and Sterling, 1987): The feces were suspended in PBS (pH 7.2) at a ratio of 1: 4 and centrifuged at 500 g for 10 minutes. The supernatant was discarded and the sediment was suspended in 15 ml centrifuge tubes of PBS (pH 7.2) and 35 ml of ether were added and mixed vigorously, and then were centrifuged at 500 g for 1 minute. Four layers were differentiated from bottom to top: - 1- Sediment, 2- PBS, 3Debris and 4- Solvent. The upper three layers were discarded and the sediment layer that consisted of 75% of oocysts was used for purification of the oocysts. These oocysts were kept in 2.5% K 2 Cr 2 O 7 until used. Mice were infected with Cryptosporidium sp. oocysts isolated from human feces using a dose of 10 3 oocysts per ml. This dose was determined after trail infections of three doses: 10 2 , 5x10 2 , and 10 3 oocysts to each mouse, in order to achieve the best infective dose that gives the suitable number of oocysts shed from infected mice. Collection of the ooc ysts of Cryptosporidium sp. from infected mice (Blagburn et al., 1998): Intestine of infected mice were cut in small pieces, water was added and homogenized in plastic tube and incubated at 4ºC. The homogenate was kept in room temperature for 90-120 minutes and homogenized thoroughly. The homogenate was centrifuged at 1000 g for 10 minutes and the sediment was washed twice with cold tween 20 (0.1%) in distilled water. The sediment was stored in 2.5 % K 2 Cr 2 O 7 . W hite of Balb\ c mice were reared in lab-animal room in standard plastic cages and fed concentrated diet (seeds mixture, soya bean with animal protein in 1%). Six groups of mice, 3 mice each, of two weeks age were infected orally by stomach tube with 10 3 oocysts per os. The infected mice and control were examined for 2 weeks by fecal floatation sugar sheathers. The six species of plants tested in this study and purchased from AlHilla herbs shops are Thymus vulgaris, Artemisea herba-alba, Ricinus communis, Peganum harmala, Olea europea, Allium sativum. The study was done on the leaves of these plants. Leaves 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 was completely dried in a dissector containing CaCl 2 . Extraction was done according to Harboren (1973) where the dried plant powder was extracted in ethyl alcohol (96%) in ratio 1-10 (W /V). The mixture was shaken continuously up to 24 hours at 4ْ C. Filtration was done under high pressure in Büchner funnel for 15 minutes. The filtrate



Al-Hamairy et al., Prevalence of Cryptosporidium Sp. and Treatment by Some Plant Extracts in Al-Hilla City, Iraq

was collected and evaporated in vacuum, using rotary evaporator at temperature lower than 60ْ C. The rest of the extracted substance (chlorophyllic extracts) and water was filtrated by using W hatman filter paper (No. 4). The filtrate was returned to the evaporator to remove the excess 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 on plant extracts:

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RESULTS: Table1 shows the prevalence of cryptosporidiosis in relation to the age and sex of infected children. 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, there were no statistically significant differences 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 older than one year (10.95% and 10.76%, respectively), followed by those less than 6 months (6.87 %) and the lowest rate was among children younger than 5 years of age (2.5%).

The effect of lyophilized extracts on the oocysts of Cryptosporidium sp. was carried out in two lines in vivo. For each dose, six groups, each of 3 one week- old white Balb\c mice were infected with 10 3 oocysts of Cryptosporidium sp. collected from naturally infected humans and purified in ether using a stomach tube. Feces of infected mice were examined daily for oocysts and counted. After 3 days post infection (P.I), when the maximum number of oocysts was reached, the mice in all groups were treated with the lyophilized extract in a dose equivalent to 250 mg/kg or 500 mg/kg ody weight using a stomach tube. At the sixth day post infection the oocysts were counted. For each dose another six groups, 3 mice each were kept without treatment as control ones. On the sixth day post treatment the treated and control groups of mice were sacrificed and the intestines were separated for collection and enumeration of the total number of oocysts. The efficiency of the extract was done according to Egerton (1963) by using the following formula:

Table 1. Distribution of Cryptosporidium sp. oocysts in children in relation to sex and age *Sex

Male

**Age Group

Female

Total (%)

Exam. Infec. Exam. Infec. Exam. Infec. (%) (%) No. No. No. No. No. No.

6 months

51

3

5.88

65

6

9.23 131

4

6.87

6 months 1 year

80

9

11.25

60

4

6.6

146

16

10.95

One Year 5 years

141

11

7.80

143

18

12.6 288

31

10.76

> 5 Years

46

1

2.17

19

-

Total

318

24

7.55

287

28

40

1

2.5

9.75 605

-

57

9.42

*Sex p> 0.05 **Age p> 0.05

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%). Statisticall y there was no significant differences in the rate of Cryptosporidium oocysts in children in different months of the year, however the highest rate of oocysts occurred 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%).

Efficacy % = [(No. of oocysts from the control group - No. of oocysts from the treated group) / No. of oocysts fr om the control group] X 100

Statistical tests were done for analysis of the significance of the results according to the type of the data (F- test ANOVA) p > 0.05, 0.01 was regarded as significant (Snedcor and Cochran, 1978).

Table 2. Distribution of Cryptosporidium sp. oocysts in children in relation to living areas and months Region

*Rural

*Urban

Total

Exam. No.

Infec. No.

(%)

Exam. No.

Infec. No.

(%)

Exam. No.

Infec. No.

(%)

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

**Month

September

*Living area p > 0.05 **Month p > 0.05 ISSN: 2090 - 0511



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The effect of plant extracts on the shedding of oocysts in mice as represented in table 3 shows that 250 mg/kg body weight of different plant extracts decreased significantly (P≤ 0.05) the shedding of Cryptosporidium oocysts in infected mice(Table 3). Table 3. Effect of plant extracts at a dose of 250 mg/kg body weight on the shedding of Cryptosporidium oocysts in infected mice No. of oocysts (X103) at the 6th DPI

Plant Extracts

Efficacy (%)

Control group

Treated group

Peganum harmala

3.5

1.1

Artemisea herba-alba

2.5

1.0

60

Olea europea

3.6

1.5

58.3

Ricinus communis

3.8

2.4

36.8

Thymus vulgaris

3.4

2.7

20.5

Allium sativum

4.3

3.5

18.6

68.6

Table 4 shows the efficacy of administration 500 mg/ kg body weight of plant extracts on the shedding of oocysts in mice. The efficacy of Peganum harmala was highest (68.6%) followed by Allium sativum (63.6%) and the lowest was Thymus vulgaris (20.5%). It is clearly observed that the efficacy of 500 mg/ kg body weight of different plant extracts on the total number of Cryptosporidium oocysts in the intestine of infected mice was higher than that of 250 mg/kg body weight. Table 4. Effect of plant extracts at a dose of 500 mg / kg body weight on the shedding oocysts in infected mice No. of oocysts (X103) at the 6th DPI

Plant Extracts

Efficacy (%)

Control group

Treated group

Peganum harmala

3.5

1.1

68.6

Allium sativum

3.3

2.7

63.6

Artemisea herbaalba

3.2

1.4

56.3

Ricinus communis

2.5

1.3

48

Olea europea

2.7

1.6

40.7

Thymus vulgaris

3.4

2.7

20.5

DISCUSSION: From 605 fecal smears of the examined children, 57 were positive at a rate of infection (9.42%). This rate is high, which indicates that cryptosporidiosis is one of significant cause of diarrhea among children in Al-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 ISSN: 2090 - 0511

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; rate of infection (14.6%) is lower than that found in Baghdad (Al-Gelany, 1998), it was14.3% in Mosul (Al-Alousi et al., 2003) and 20.52% (Khalil, 2000), and rate of infection in Kirkuk was 12.62% (Al-Moula, 1999). Almost identical result (8.82%) was reported in Basrah (Nadham et al., 1996) but higher than hat recorded in Diyala which was 2.8% (Al-Taie, 1997). The difference between the finding of this study and other provinces might be related to the period of study, environmental factors, sample size, and the techniques used for identification of the infection (Schmidt and Roberts, 2000). The present finding are lower than that reported in other countries where Gerba et al. (1996) reported 12.7% in Sudan while Nimiri and Hijazi (1994) reported 67% in Jordan and Chen et al. (1993) reported a 14% 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 disagrees with that recorded in Kirkuk (AlMoula, 1999) in Baghdad, (Al-Gelany, 1998) in Basrah Nadham et al., 1996); and in China (Chen et al., 1993). These studies reported that the distribution of infection in males was higher than females. On the other hand, AlMoula (1999) found that the the rate of infection in females were higher than males in Kirkuk. Concerning the age distribution, it seems from the results of this study that the distribution of cryptosporidiosis is not related to 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 less than 6 months to one year of age. This might be due to low care and attention given to children from their mothers such as using unsterilized feeding bottles and unboiled water, (Ungar, 2000; Fathi et al., 2010). This finding goes with that of Nimiri and Hijazi (1994), Al-Moula (1999) and Iqbal et al. (2001) who found the highest rate of infection in infants aging from one month to one year which reveals that cryptosporidiosis is common among infants and young children suffering from diarrhea, especially within the first two years of age (Radfar et al., 2011). As regards the residency of the subjects understudy, it was shown that children in rural areas have lower rates of infection than those in urban areas. This might be related to the type of feeding, as most likely that children in urban areas feed on bottled milk, which is more subjected to contamination than breast milk. Similar finding were reported in Kirkuk




province (Al-Moula, 1999; Othman, 2000), but these finding disagrees with the finding in Korea (Chai et al., 1996) where higher rate of infection was reported in rural (14%) than urban area (3.7%). The lowest rate of infection was observed in January and the highest rate of infection was noted in September. This might be due to that the oocysts of the parasites destroyed in low temperature 4ْC (Ayaz et al., 2011). There were no significant differences in the rate of infection between different months. The present study was not carried out throughout a whole year; therefore seasonal comparison of rate of infection is impossible. In comparison with other studies, it is shown that the rate of infection was high in summer and spring seasons and low in winter months in Kirkuk (Othman, 2000) and in Tikrit (Al-Yassin, 1999). It has also been shown that Cryptosporidium oocysts can be found during warm and humid months (MirzaQavami and Sadraei, 2011). In the present study six plant extracts (Peganum harmala, Artemisea herba-alba, Ricinus communis , Olea europea, Alliu m sativum and Thymus vulgaris) were evaluated as alternatives for chemical drugs which are sometimes toxic and unavailabe in the country. The efficacy of the medicinal plant extracts was evaluated in mice as alternative for humans and economically important live stocks (Fathi et al., 2010). Using 250 mg/kg body weight of plant extracts, revealed that the highest efficient plant extract was that of Peganum harmala (68.6%) followed by Artemisea herba-alba (60%), Olea europea (58.3%), Ricinus communis (36.8%),Thymus vulgaris (20.5%), and Allium sativum (18.6%). Peganum harmala is a medicinal plant, used as antidysentric, its chemical contents are glucosindate and prosporing (Majeed and Mahmood, 1998). 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 active against hydatidosis in laboratory mice (Al-

291

Nakeeb, 2004). The anti-parasitic action of Allium sativum, is also reported and it is suggested that its activity 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 (Mirza-Qavami and Sadraei, 2011). The efficacy of Ricinus communis was also high. This plant includes glucose, coumarine, volatile oil, sulphur, isothiocyanate, and glucosinolate and was reported as vermifuge and intestinal carminative (PDR, 1998). Al-Musaui (2000) studied the effect of alcoholic extract of Artemisea herba- alba extract on Hymenolepis nana, and detected 10-14 compounds in both the dry and fresh parts of the 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 fertility, egg development, and eggs output of H. nana. He also found that the efficacy of alcoholic extract of Artemisea herba- alba on Hymenolepis nana was 100% on the fifth and sixth days postinfection using 250 or 500 mg/kg body weight. The efficacy of Olea europea was 58.3%. Its contents include mannite, glucose, oleosteral, resine, olivine, and resine that have an antiparasitic effect (Al-Aubaidy, 1999). The study also reported that the efficacy of Thymus vulgaris was 20.5%. Its chemical contents include beta caryophyllena (Fathi et al., 2010). Increasing the dose of the six plant extracts under study from 250 to 500 mg/kg body weight on Cryptosporidium oocysts, increased their efficacy. From the results of the present study, it is concluded that the prevalence of cryptosporidiosis is high among children at Al-Hilla City, freshly isolated oocysts from man are more infective to laboratory mice, and the ethanolic extract of Peganum harmala has the highest effect on Cryptosporidium sp. infection in Balb/c mice.

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Tzipori S, Angus KW, Campbell I, Gray EW. 1980. Cryptosporidium: Evidence for a singlespeacies genus. Infect. Immun., 30(3): 884-886. Ungar BL. 2000. Infectious diseases and their etiologic agents. In: “Principles and practice of infectious diseases. (Mandell GL, Bennett JE, Dolin R. Eds.)”. Churchill Livingstone, Philadelphia. p 2903-2914.

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