| Abstract|| |
Viable protoscolices of Echinococcus granulosus injected into Swiss-albino mice by two different routes, viz. intraperitoneal and subcutaneous and in two different concentrations of 2000 and 1000 scolices, to observe the development of cysts have shown that subcutaneous route of infecting a mouse model is superior because of its low infecting dose and longer survival period of mouse. This finding should be helpful in experiments to observe the effect of drugs for the treatment of hydatidosis, in vivo, without the need of killing the mouse.
Keywords: Echinococcus granulosus, experimental model, subcutaneous infection
|How to cite this article:|
Kakru DK, Sofi BA, Assadullah S. Novel route of infection in experimental model of hydatid disease. Indian J Pathol Microbiol 2008;51:373-5
| Introduction|| |
Hydatid disease, caused by Echinococcus granulosus , is worldwide in distribution and is widely prevalent in India. , The treatment of the disease is mainly surgical  though mebendazole is being used for treatment of in-operable hydatid cysts ,, and so is albendazole, , though with limited success. Experimental model - Swiss-albino mouse - developed earlier  and subsequently used to find in vivo efficacy of mebendazole in containment of cyst mass  paved the way for experimenting with newer options of nonsurgical treatment, but is riddled with the problem of killing a large number of mice. This study was attempted to find a safer route of inducing infection in mouse to have a model in which in vivo efficacy of various newer drugs could be checked without the need of killing the mice.
| Materials and Methods|| |
Swiss-albino mice maintained in the animal house of a tertiary care hospital were used for the study. The mice were divided into four groups, with each group having forty (40) mice. Every single mouse in all the four groups was injected with scolices as under:
Group A: 2000 scolices, intaperitoneal
Group B: 1000 scolices, intraperitoneal
Group C: 2000 scolices, subcutaneous
Group D: 1000 scolices, subcutaneous
Protoscolices were obtained from a hydatid cyst, removed surgically from a patient in a tertiary care hospital and were washed five times with sterile normal saline. Viability of these scolices was checked with 5% eosin dye [Figure 1].  Two different concentrations of these scolices, viz. 2000 scolices/0.5 ml and 1000 scolices/0.5 ml in physiological saline containing 100 units and 200 µg/ml of penicillin and streptomycin were prepared for injection into mice as above. These mice were maintained in the animal house and observed regularly. Four mice from each group were killed every month and examined for the presence of cysts, if any, postmortem. The cysts were aspirated and cyst fluid examined microscopically for scolices and hooklets. The cyst wall was examined histologically after staining sections with hematoxylin and eosin stain. Blood samples of these killed mice were collected for detection of IgM and IgG antibodies by ELISA. 
Animal ethical committee approval has been obtained by the author before the study was conducted.
| Results|| |
Upto 80% of mice in Group A developed cysts, measuring 1-2 mm, in 2-3 months and had a distended abdomen after 6-7 months. None of the mice in this group survived beyond 7 months. In Group B, none of the mice developed any cysts. All the mice in Group C developed cysts in the abdominal wall that could be seen and palpated beneath the skin by the end of 3 months and there after with gradual increase in the size. Most of the mice in this group survived upto 8 months and some even for 9 months. In Group D also the cysts could be seen beneath the skin by the end of 3 months with gradual increase in the size there after, attaining a size of 2-3 cm in some mice. All the mice in this group survived 10 months.
Viable protoscolices were seen in the fluid aspirated from the cysts and histological examination of the cyst showed laminations. Antibody titers of both IgM and IgG antibodies recorded at different time intervals were shown in [Table 1]. Mice in Group B had an IgM antibody titer of 1:20 after 1 month, increasing to 1:40 at the end of 2 months and then fell back to 1:20 at 3 months, remaining at that level all through the period of study; mice in this group were negative for IgG antibodies. Antibody titers for both IgM and IgG antibodies in other three groups showed a rising pattern with peak titers of 1:640 (IgM) and 1:2560 (IgG) after 7 months in Group A; 1:640 (IgM) after 6 months and 1:2560 (IgG) after 9 months in Group C; and 1:640 (IgM) and 1:2560 (IgG) at the end of 10 months.
| Discussion|| |
Intraperitoneal induction of infection in mice has been compared in this study with induction of infection by subcutaneous route. Though both the routes have been successful in inducing infection, the subcutaneous route holds an advantage by way of longer survival of mice, i.e., 10 months or more as compared to less than 7 months in mice with intraperitoneal induction of infection. Subcutaneous route of induction is achieved with a smaller dose of 1000 scolices of Echinococcus granulosus of human origin while as an infetive dose of 1000 scolices could not induce infection in mice when injected itraperitoneally which is evident by absence in rise of IgG and low IgM titers in Group B and also by absence of cysts (postmortem) in these mice. On postmortem, cysts in the mice in which infection was successfully developed and also antemortem in Group D, aspiration of cyst fluid showed viable protoscolices. This viability of protoscolices can be taken advantage of, in finding efficacy of conventional as well as newer and experimental chemotherupatic agents. These agents can be tested, in vivo and also immune response monitored in the living model itself. Effect of these drugs can be observed without any further invasive procedures or need to kill the animal as these cysts can be palpated and even visualized and their size monitored in living animal models [Figure 2]. In the present day world, where prevention of cruelty to animals and limiting the use of animals in experimental work is, very rightly, the order of the day, this study should go a long way in helping those involved in medical research.
| Acknowledgment|| |
The authors are highly appreciative of S. Nissar Hussain, B.Sc., SGT, for his skilled technical support and care of animals, without which this experiment would not have been possible.
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Dalip K Kakru
Department of Microbiology, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar - 190 011, Kashmir (J and K)
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]