Application of ultrasound contra infection with

secondary hydatidosis in mice

B.B. Ramdan and A.A. Ali

Department of Biology, College of Education for Pure Sciences; University of Mosul, Mosul, Iraq

Email: ¹baraa631991@gmail.com, ²dr.asmaa_abdulaziz@uomosul.edu.iq

Abstract

The present research explained the impact of ultrasound on the viability of Echinococcus granulosus protoscolices in vitro application of fixed frequency waves at 20000 pulse/sec and 1.8 w/cm², with different exposing periods, thirty, twenty, fifteen, twelve, ten and five seconds respectively, subsequently prevention of mice against interjected with minor hydatid cysts, in comparison with the control animals infected with protoscolices without exposure to ultrasound waves, depending on many criteria including the numbers, weights and diameters of developed hydatid cysts. The results displayed an obvious impact of ultrasound waves on Echinococcus granulosus protoscolices viability number by increase of exposure period in vitro, in addition to a decrease of 100% of hydatid cysts numbers in the dealt ones with 17% fertility exposed for 20 seconds, the reduction rate was 99.23% in the group of fertility 35% which exposed for 15 seconds, after four and five months of infection.

Keywords: Echinococcus granulosus, Ultra Sonication, Hydatidosis, Mice

DOI: 10.33899/ijvs.2019.126132.1240, ©2020, College of Veterinary Medicine, University of Mosul.

This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).

تطبیق الأمواج فائقة الصوت ضد الإصابة بداء العُدارِی الثانوی فی الفئران

براء باسل رمضان و أسماء عبدالعزیز علی

فرع علم الحیاة، کلیة العلوم للعلوم الصرفة، جامعة الموصل، الموصل، العراق

الخلاصة                    

حددت الدراسة الحالیة تأثیر الأمواج فائقة الصوت فی عیوشیة الرؤیسات للدودة المشوکة الحبیبیة خارج الجسم بتطبیق تردد ثابت لکل المعاملات بمقدار 20 الف ذبذبة / ثانیة و 1.8 واط/سم² ولفترات تعریض مختلفة 5، 10، 12، 15، 20 و 30 ثانیة على التوالی، بالإضافة الى تمنیع الفئران ضد الإصابة بداء الأکیاس العدریة الثانوی بالمقارنة مع حیوانات السیطرة والمحقونة بالرؤیسات الأولیة غیر المعرضة للأمواج فائقة الصوت. اعتماداً على معاییر عدة تضمنت التغیرات الحاصلة فی أعداد وأوزان وأقطار الیرقات البعدیة المتکونة. بینت النتائج تأثیر الموجات فائقة الصوت فی عیوشیة الرؤیسات لدودة المشوکة الحبیبیة بزیادة فترة التعریض خارج جسم الحیوان، واختزال أعداد الیرقات البعدیة فی الفئران المعاملة بالأمواج فائقة الصوت بنسبة بلغت 100% فی المجموعات التی کانت نسبة عیوشیة الرؤیسات الأولیة فیها 17% والمعرضة لفترة 20 ثانیة للأمواج فائقة الصوت، بینما بلغت نسبة الاختزال 99.23% للمجموعة البالغة نسبة حیویتها 34% والمعرضة لفترة 15 ثانیة، بعد أربعة وخمسة أشهر من الإصابة.

Introduction

Hydatidosis is one of the most important parasitic infections on public and veterinary health worldwide. The disease has significant economic importance concerning mortality and morbidity (1-3) as estimated losses of global production of livestock are two billion dollars (4). The disease has a wide geographical scope and occurs in all continents, including the tropical and subtropical regions. Cystic echinococcosis (hydatid cyst) (CE) was diagnosed by the basic methods of clinical examination, imaging and serological tests (5-7). CE is treated by a variety of ways, including chemical therapy (drug therapy), percutaneous aspiration injection and respiration (PAIR), surgical treatment, or the option of observation without surgical intervention (8,9). Chemotherapy involves the use of albendazole, mebendazole or benzimidazole carbamates, chemotherapy is recommended as the only option in cases. where surgical treatment is not possible or when multiple cysts are available (9). This option includes aspiration of the fluid, injection of lethal agents and re-suction. The non-invasive control option is carried out with the CE4-CE5 stages of cyst development according to the world health organization (WHO) classification to avoid the possibility that these cysts remain fertile. Ultrasound and Serological tests are used for surveillance.

Ultrasound waves are waves with a frequency greater than 20 KHZ (human hearing threshold) (10,11). These are inaudible high-frequency mechanical vibrations produced by the electric generator and are converted to acoustic energy by mechanical deformation of a piezoelectric crystal placed in the power transformer (12). Ultrasonic frequency can be classified primarily into two sections: low-power high-frequency diagnostic ultrasound in the MHZ range, and the low-frequency high-power ultrasound is in the KHZ range. Ultrasound waves are a physical therapeutic method where non-ionizing radiation is applied in the form of mechanical sonicated on the tissues to produce heat. Ultrasound has been used to detect variety of diseases, including parasitic infections since 1970 (13), High Intensity Focused Ultrasound (HIFU) was initially used to treat cancer (14). It was used in the 1940s to treat brain diseases such as Parkinson's disease (15), uterine fibroids and prostate tumors (16). Ultrasound waves have also been used extensively in medical diagnostics and increasingly for therapeutic purposes as they have been applied to break up kidney stones, as a surgical tool for the removal of tumor tissue and as a tool for administration of drugs (17).

Hence the aims of this study were to show the impact of ultrasound in with the vitality of different concentration of protoscolices in vitro and in the prevention of albino mice from infection with hydatidosis. This study is considered as a first study in the country regarding application of ultrasound against hydatidosis.

Materials and methods

Segregation and testing hydatid cysts

The hydatid cysts were found (Figure 1) after being separated from liver of slaughtered sheep, which collected from butchers' shops in Mosul, and upon arrival in the laboratory, hydatid cysts were examined to ascertain the vitality.

Figure 1: Hydatid cysts (hydatid cysts) in the liver of sheep.

Collection and assessment of E. granulosus protoscolices

Sheep cysts containing protoscolices were obtained under sterilized conditions, a live protoscolices, 20 µl of protoscolices suspension were added to 20 µl of 0.1% of eosin pigment on a slide, then tested under light microscope. Bright green protoscolices were considered as alive because of eosin exclusion by protoscolices membranes, whereas red protoscolices were regarded as dead because of acceptance of the pigment. Gained and counted according to authenticated manner (18).

Experimental animals

120 Albino mice (four to five weeks old) were used and cared for in the Department of Biology, College of Education for Pure Sciences, University of Mosul, according to the conditions required for the experiments.

The device used in the experiment

The ultrasonic waves device was used, catalogue number 1/96/40/80/3/4 supplied by the Omni international (Figure 2).

Effect of the ultrasound waves on the vitality of sheep protoscolices in vitro

To determine the effect of ultrasound waves on the vitality of protoscolices in vitro within a certain time and frequency of 20.000 pulse/second, protoscolices with a 100% viability were taken at a concentration of 2000 protoscolex /ml, and then exposed to the ultrasonic waves, amount of ice was placed around the sample to avoid high temperature during exposure. Samples were exposed for 30, 20, 15, 12, 10 and 5 seconds, the post exposure vital percentages were 0, 17, 34, 50, 80 and 90% respectively. The protoscolices were examined under a light microscope for viability taking into consideration the flame cells movement, morphological abnormalities, and exclusion of eosin pigment.

Figure 2: Ultrasound waves device. (1) Operating power, (2) Main and power stand by for operation too when the sample is exposed, (3) the amplitude capacity starts from 0-28, the frequency is 20 thousand vibration/second.

Effect of ultrasound waves in protoscolices injected in vivo

protoscolices with vitality 90, 80, 50, 34, 17 and 0% were selected for mice injection in addition to the control with 100% vitality. A total of 30 male mice, aged 5-3 weeks, were divided into six group (five mice/ each group). The 1^st group was injected with 2000 protoscolices of vitality 0%. The 2^nd group was injected with 2000 protoscolices of vitality 17%. The 3^ed group was injected with 2000 protoscolices of vitality 34%. The 4^th group was injected with 2000 protoscolices of vitality 50%. The 5^th group was injected with 2000 protoscolices of vitality 80%. The 6^th group was injected with 2000 protoscolices of vitality 90%. The 7^th group (control) was injected with 2000 protoscolices of vitality 100%. All groups were all dissected after 2, 3, 4, 5 months.

Statistical analysis

Results of this study were analyzed statistically based on Completely Randomized Design (CRD) to study the effect of intervals and exposures and the integration between them, then the differences between the averages of each factor and the compatibility between factors were estimated depending on Duncan's Multiple Range Test (DMRT). Statistical Analysis System (SAS version 9) was utilized for applied for data analysis (19).

Results

Table 1 exhibited the changes in the numbers, diameters and weights of the hydatid cysts, and the of significance of F in the ANOVA analysis, table 2 DMRT for averages test explained a decrease in the average number of hydatid cysts in the treated groups (Figure 3, 5, 7 and 8) compared to control animals (Figure 4 and 6). The treated mice exhibited a reduction in the number of cysts 0.000 at 30 sec exposure, while control mice displayed the highest average of 9.650, 5 months post infection. Table 3 demonstrated the highest percentage reduction of hydatid cysts number in treated mice at 30 seconds exposure followed by 20, 15,12,10 and 5 seconds, respectively.

Table 1: Effect of ultrasound on numbers, diameters and weights of hydatid cysts in all experience animals

* mean significance at P<0.01, while ** mean significance of P<0.05.

Diameters are shown in table 1 illustrated F significance, table 4 (DMRT) for averages revealed significant differences between the exposure and non-exposed at 30 and 20 seconds. Significant differences were also observed between mean averages compatibility exposure and period, metacestode diameters in mice injected with protoscolices exposed with ultrasound decreased, with a minimum 5 months post injury 0,000 mm at 30 seconds exposure, followed by 20, 15, 12, 10 and 5 seconds respectively, contrast to the hydatid cysts control group was 0.890 mm. Related to the weight of the hydatid cysts table 1 showed F insignificancy for interference between the two factors (exposure and period).

However, the DMRT (Table 5) showed significant differences between the averages of compatibility regarding periods and exposures. The cysts weights in treated animals decreased to 0.00mg at 30 seconds and all months followed by 20, 15, 12, 10 and 5 seconds, respectively, in contrast to the control group (0.2760mg) 5-month post injury.

Table 2: Effect of ultrasound waves on the number of hydatid cysts in treated animals

The different letters indicate significance, while the similar letters indicate insignificancy.

Table 3: Percentage of protoscolices reduction in treated animals for all months

Table 4: Effect of ultrasound waves on diameters of hydatid cysts in treated animals

The different letters indicate significance, while the similar letters indicate insignificancy.

Table 5: Effect of ultrasound waves on weight of hydatid cysts in treated animals

The different letters indicate significance, while the similar letters indicate insignificancy.