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Evaluation the efficacy of anthrax vaccine against challenge with a highly virulent strain of bacillus anthracis isolated from soil in sheep, goats and guinea pigs in iran

Archives of Razi Institute, Vol. 64, No. 1, June (2009) 45-50
Razi Vaccine & Serum Research Institute In vivo effect of albendazole and mebendazole on hydatid
cyst of mice
Hashemi Tabar 1, 2, G.R., Razmi1, G.R., maleki1, M.
1. Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad , Mashhad, Iran 2. The Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran Received 12 Oct 2008; accepted 28 Apr 2009 ABSTRACT
In current study, a few sheep cystic livers and lungs were obtained from Mashhad slaughter house and
protoscolex were separated aseptically. Thirty 6-week-old Swiss mice were divided into 5 groups and two
thousands of live protoscolices were inoculated intrapritoaneally in each mouse. In group 1 and 2
(prophylactic groups), mice were given 150 mg/kg of oral albendazole and mebendazole for 10 days. In
group 3 and 4 (treated groups), mice were treated orally with 300 mg/kg of albendazole and mebendazole
for 22 days with an interval of 2 days after every 4 days of treatment 6 months after inoculation. The control
group (group 5), was sham injected with normal saline. Mice were killed after 7 months and internal organs
were observed for hydatid cyst. In group 1, 2 cysts were observed in the liver of mice. In this group,
although albendazole did not prevent cyst formation, but the number and also the size of the cysts were
lower and smaller than the control group. In group 2, 3 cysts were observed in internal organs. In group 3,
there was no cyst in internal organs and so in this group it is concluded that albendazole prevented cyst
formation. In group 4, 1 cyst was observed on the liver of mice. In control group a lot of cysts were
observed in internal organs of mice and the average size of cysts were bigger than prophylactic and also
treatment groups.
Keywords: Hydatid cyst, Albendazole, Mebendazole, Mouse
INTRODUCTION
methylcarbamates derivatives (BZD) were proved to be effective against E. granulosus, and since Cystic echinococcosis (CE) caused by the then, many investigators have used albendazole metacestode (larval) stage of Echinococcus (ABZ) and mebendazole (MBZ) for the treatment granulosus (E. granulosus ) is still an important of human hydatidosis (Teggi et al 1993). public health concern in many countries of the Albendazole and mebendazole are considered to be world, such as Mediterranean and South American equally effective. When evaluated up to 12 months countries The disease affects humans as well as after initiation of benzimidazole treatment, 10%– domestic livestock including cattle, sheep, camels, 30% of patients exhibit cyst disappearance, 50%– pigs, horses and others (Torgerson & Budke 2003). 70% shows degeneration of cysts and 20%–30% In the early 1970s, benzimidazole show no morphological changes in the cyst appearance. Continuous or intermittent treatment *Author for correspondence. E-mail: hashemit@um.ac.ir
46
Hashemi Tabar, et al / Archives of Razi Institute, Vol. 64, No. 1, June (2009) 45-50
with albendazole is recommended for a period of 6 collected aseptically from cysts and were maintained in Hank’s Balanced Salt Solution benzimidazole methylcarbamate compounds such (HBSS) containing penicillin (500 IU/ml), as albendazole and mebendazole have been used to streptomycin (500 IU/ml). Prior to inoculation, the treat CE in humans (El-On. 2003). Mebendazole collected protoscolices were rinsed 2-3 times with was the first compound widely used (Bekhti et al HBSS containing antibiotics. Viability of the 1997). Although effective, the main disadvantage protoscolices was confirmed by visual inspection was the extremely high doses and prolonged through inverted microscope. The protoscolices administration period required to achieve a satisfactory clinical efficacy. Albendazole was protoscolices in 0.5 ml of HBSS were inoculated therefore a major breakthrough because, while intraperitoneally in each mouse. Male and female absorption of the parent compound remained poor, mice weighing 20–22 g were used in these studies. its metabolite, albendazole-sulphoxide (ABZSO) Thirty mice were inoculated with protoscolices was shown to be an active anthelmintic agent of E. granulosus and divided into 5 groups. Two (Horton. 2003). Both ABZ and ABZSO have been prophylactic groups, just after challenge with shown to be active against protoscolices of E. protoscolices, received 150 mg/kg BW /day granulosus in in vitro culture systems. However, albendazole (group 1) and/or mebendazole (group the in vitro protoscolicidal action of ABZ and 2) orally for 10 days. Two other groups, as ABZSO is very slow and requires a longer treatment groups, 6 months after challenge with incubation period compared to that observed for protoscolices were treated orally with 300 mg/kg of MBZ (Pérez-Serrano et al., 1994). In vitro and in albendazole (group 3) and mebendazole (group 4) vivo differences in the protoscolicidal activity of every 4 days with an interval of 2 days for totally of BZD anthelmintics have been observed. The 24 treatment days (six times). The fifth group significance of the effects of albendazole, animals were assigned as control and mice were sham injected with normal saline. All mice were researchers (Pérez-Serrano et al 2001, Rodríguez- sacrificed after 7 months and internal organs were Caabeiro et alet al 2004). The aim
observed for hydatid cyst. For histopathological of the present work was to determine in vivo study, tissue samples were taken from liver of protoscolicidal effect of albendazole and control and test mice. Samples were fixed in buffer mebendazole against E. granulosus in mouse. formalin 10% and then histopathologic section was prepared by routine method. Samples were stained by hematoxylin and eosin and finally were MATERIALS AND METHODS
E. granulosus hydatid cysts containing conditions from infected sheep with hydatid cysts presented for routine slaughter in abattoirs in All the mice were killed 7 months after the Mashhad. This study has been conducted in 2008. inoculation and internal organs were inspected for Briefly, the hydatid cysts (2–5 cm in diameter) hydatid cyst(s). In group 1, which received were cut open and vesicle fluid (containing albendazole for 10 days, 2 cysts were observed on protoscolices) was separated from the metacestode liver of mice. The size of cysts of this group was tissue and host adventitia. Protoscolices were smaller than the size of the cysts in internal organs Hashemi Tabar, et al / Archives of Razi Institute, Vol. 64, No. 1, December (2009) 45-50
47
of the control group. In group 2, which received the internal organs of all mice and the average size mebendazole for 10 days, 3 cysts were observed in of cysts were about 6.5mm and bigger than internal organs and the size of cysts was bigger than prophylactic and treated animals with both drugs. the group 1, but smaller than the control group. In Histopathological study of the cyst showed that group 3, which treated with albendazole for 22 typical double layered wall and in some cases with days, no cyst was found in the internal organs clear fluid inside (Figure 2). Some samples revealed a precystic structure, which consisted of connective tissue and scattered inflammatory cells. DISCUSSION
In the present study, the efficacy of albendazole and mebendazole in the prevention of hydatid cyst in mice was conducted. In a study by (Keshmiri et al., 2001) the effect of albendazole on hydatid cysts of human has been evaluated. Twenty-nine patients with 240 cysts received albendazole (400 mg twice a day, in 3 cycles of 6 weeks long with 2 weeks off between cycles). Some patients with liver cysts after treatment showed increasing heterogeneity and density suggestive of inactive cysts. Patients with larger cysts and those with pulmonary involvement were better responders. The observed results are encouraging, showing high efficacy of albendazole for the treatment of hydatidosis and should be offered to patients as an alternative before surgical treatment is considered. In comparison with the results of (Keshmiri et al 2001), our results showed that when mice were treated with albendazole, no cysts were found in internal organs and this result indicated that 2001, & 2004, et al 2006). It Figure 1. Treated group with albendazole. None of mice had
has been reported that continuous long term viable hydatid cysts in internal organs (Above). Hydatid cysts in internal organs of mice in control group (Below). Group 4, animals which were treated with parasiticidal against larval stage of E. multilocularis mebendazole for 24 days, just one cyst was especially in the early stages of infection (Liu et al observed on the liver of mice. In control group 1998). The effectiveness of albendazole (ABZ) and (Figure 1) a high number of cysts were observed in albendazole sulphoxide (ABZ.SO) and ABZ + 48
Hashemi Tabar, et al / Archives of Razi Institute, Vol. 64, No. 1, June (2009) 45-50
drugs (mebendazole, albendazole & ricobendazole) Echinococcus granulosus protoscolices has been were compared by analyzing their in vivo activity described. The results indicated that ABZ and ABZ against Echinococcus granulosus cysts in a mouse + ABZ.SO had an important effect upon larval growth in E. granulosus (Perez-Serrano et al., mebendazole-treated mice, at doses of 25-50 1997). The efficacy of Ivermectin (IVM) alone, albendazole (ABZ) alone and a combination of IVM plus ABZ against Echinococcus granulosus benzoimidazole used, has some beneficial effects protoscolices was studied by means of an in vitro on the disease in selected patients, it has also been incubation. The maximum protoscolicidal effect associated with treatment failure in some cases, was detected when combination of IVM+ABZ perhaps because of its poor absorption. It has were used (Casado et al 2002). It has been reported shown by et al 1998) that hydatid cysts that protoscolices of E. granulosus were incubated were severely damaged in mice treated with in vitro with praziquantel (PZ), albendazole (ABZ), mebendazole and new vesicles did not develop or a combination of both (PZ + ABZ). PZ and ABZ displayed slower protoscolicidal activity when applied separately than when used in combination. The PZ + ABZ treatment was effective only against small cysts, which had collapsed at 10 days postinoculation (Urrea-Paris et al 2000). It has been reported by (Dvoroznakova et al 2004), that the reduction of cyst growth after treatment with ABZ and liposome.ABZ was similar up to week 4 after last dose, but the parasitostatic effect of liposome. ABZ lasted 4 weeks longer than the effect of free drug. At the present time, albendazole is considered the treatment of choice Figure 2. Microscopic cyst in liver of mice. The arrows
indicate microscopic cyst and tissue reactions around cyst.
for echinococcosis in human (Shuhua et al 2002). Feng et al (1995) have shown that when infected Also, hydatid cysts reappeared after treatment with mebendazole, for 14 days, the glutathione S- transferase (GST) activity of both collapsed and full indicated that the postoperative prophylactic course cyst walls were inhibited by 30.1% and 26.8%, of mebendazole is reliable, safe and with minor side respectively. Albendazole (300 mg/kg/day) for 14 effects and the recurrence rate of the disease was days had no apparent effect on GST of E. granulosus cyst wall and it has been suggested that the inhibition of GST activity in the cyst wall induced by mebendazole might damage the defense Acknowledgment
In another experiment by (Daniel-Mwuambete et The authors thanks to Dr. Fallah-Rad, Mr. Azari al 2003), two different preparations, solution and and Mr. Mohammadnejad for their time and help. suspension, of three benzimidazole carbamate This research was supported by the Ferdowsi Hashemi Tabar, et al / Archives of Razi Institute, Vol. 64, No. 1, December (2009) 45-50
49
Liu, Y., Wang, X., Gao, J., Yao, Y. and Yu, D. (1998) Reevaluation of effect of albendazole on echinococcus References
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chemotherapy exhibits profound anti-parasitic activity. challenge. Research in Veterinary Science 74(3):191- International journal of Parasitology 34: 615–24. Teggi, A., Lastilla, G.M. and De Rosa, F. (1993) Urrea-Paris, MA., Moreno, M.J., Casado, N. and Therapy of human hydatid disease with mebendazole Rodriguez-Caabeiro, F. (2000) In vitro effect of and albendazole. Antimicrobial Agents and praziquantel and albendazole combination therapy on the larval stage of Echinococcus granulosus. Parasitology research 86(12):957-64. Echinococcosis--an international public health

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