Animals and parasite
A total of sixty parasite free, laboratory-bred, male Swiss Albino mice 5 weeks old, weighing about 20-25gm each, were used. Mice were obtained from animal house of Theodor Bilharz Research Institute (Giza, Egypt) and maintained in accordance with institutional and national guidelines. Mice were infected orally with 200-250 T. spiralis L1 larvae per mouse (Dunn and Wright 1985).
Experimental design
Animals were divided into 5 groups (12 mice each) as following: G (1) Non-infected control group (healthy control), G (2): Infected untreated (infected control), G (3): Infected then treated with albendazole and prednisolone, G (4): Infected then treated with Zingiber officinale extract and G (5): Infected then treated with Cinnamomum zeylanicum extract.
On the 7th day p.i., six mice from each group were sacrificed and blood samples were collected for determination of serum TNF-α. The abdominal skin was sterilized then incised and the peritoneum was opened to dissect the small intestine which is then preserved in formalin 10% for histopathological examination and immunohistochemical study. The rest of the intestine was used for T. spiralis adult worm counting. Adults were then preserved in fresh modified Karnovsky’s fixative to be examined later on under electron microscopy. On the 35th d.p.i., 6 mice from each group were sacrificed. Blood samples were collected for the determination of serum TNF-α. The peritoneum was opened and the diaphragm was carefully dissected for histopathological and ultrastructure studies. The rest of the muscle samples were digested for total larval count.
Drugs
Albendazole was used as Alzental suspension (EIPICO), 20 mg/ml. Each mouse received a dose of 50 mg/kg orally for 3 consecutive days starting from the 3rd day post infection according to Attia et al. (2015). Prednisolone was available as Predsol suspension, 5 mg/ml. It was given in a dose of 0.7 mg/kg orally for 3 successive days starting from the 3rd day post infection (Manzur et al. 2008).
Plant material and preparation of extracts
Briefly, 300 g of fresh ginger rhizome and 250 g of dried Cinnamomum zeylanicum bark were cut into small pieces, macerated into 90% ethyl alcohol and left for 10 days. Alcohol was replaced by fresh one every 3 days. The extract was filtrated and ethanol was removed via rotary evaporator at 50o C under reduced pressure to obtain viscous residues of crude plant extracts. These residues were then subjected to lyophilization to afford 40 gm of powdered extract of the plant. The suspension of the lyophilized extract was prepared for oral administration using 0.5% Tween-80 (ADWIC, Egypt) in normal saline. The concentration was adjusted that each 0.1 ml of the prepared suspension contains 0.3 mg of the plant extract (Mahmoud et al. 2014). Extracts were orally administered at a dose of 100 mg/kg (Hsiang et al. 2015) and 50 µg/g body weight (Kwon et al. 2011) once daily for Zingiber officinale and Cinnamomum zeylanicum respectively starting from the 1st day of infection till the day of sacrifice.
Parasitological study
Isolation and counting of adult worms
Mice were sacrificed and the small intestine was taken off. After the intestine was washed with physiological saline, it was split longitudinally along its entire length and divided into 2-cm portions, then placed in a beaker full of physiological saline for 3-4 hrs at 37°C. The intestine was shaken well in the saline, rinsed in a similar amount of saline and finally removed and discarded. Adults were allowed to sediment by standing in the container for half an hour. The supernatant was removed; the sediment was reconstituted in 3–5 drops of physiological saline and poured into a petri-dish. The number of adults was counted under the dissecting microscope at X 20 power (Shin et al. 2008).
Total larval burden in muscles
Mice were sacrificed on the 35th day p.i. Muscle larvae counts in whole carcasses were determined according to the method described by Martínez-Gómez et al (2009). Briefly, each mouse was skinned and eviscerated. Teeth, tail and ears were removed and the rest of the animal was cut into small pieces by scissors and digested in 1% pepsin-hydrochloride in 200 ml distilled water. Following incubation of the mixture at 37°C for 2 hrs with continuous stirring by an electromagnetic stirrer, encysted larvae were collected via the sedimentation technique and washed several times in distilled water. The number of larvae was counted microscopically using a McMaster counting chamber.
Histopathological study
The formol-saline preserved intestinal and muscle tissue specimens were processed in an automated tissue processor in two steps; fixation and dehydration. Initially, tissue samples were immersed in 10% buffered formalin for 48 hrs, after that the fixative was removed in distilled water for 30 minutes. Dehydration process was then carried out by running the tissues through a graded series of alcohol (70%, 90 %and 100%). Tissue specimens were then cleared in several changes of xylene, then the samples were impregnated with molten paraffin wax, then embedded and blocked out. Paraffin sections (4–5 µm) were stained with hematoxylin and eosin (Suvarna et al. 2013).
Immunohistochemistry
The standard immunohistochemical methods were adopted with Bancroft and Gamble (2008). Briefly, sections were mounted on positively charged glass slides (Biogenex, USA), and deparaffinized in xylene. After xylene was removed by absolute ethanol, slides were placed in an unsealed plastic container filled with sufficient antigen retrieval solution (Citrate buffer solution, pH 6) and microwaved for 5 minutes at power 10. The container was removed and allowed to cool for 15 minutes and the slides were washed in deionized water for several times then placed in phosphate buffer saline (PBS) for 5 minutes. Tissue sections were incubated with an endogenous peroxidase blocking reagent containing hydrogen peroxide and sodium azide (DAKO peroxidase blocking reagent, Cat. No.S 2001). Excess buffer was blotted off, and the slides were allowed to dry except for the tissue section. The supersensitive primary monoclonal antibody against transforming growth factor-beta 1 (TGF-β1) was added to the sections and incubated for 60 min at room temperature then, the slides were rinsed in PBS, incubated with biotin-streptavidin (BSA) system. After that, 1-2 drops of the ready-to-use DAKO EnVision + system were applied for 20 minutes at room temperature and rinsed again with PBS. Diaminobenzidine (DAB) was used as a chromogen. The slides were mixed for 10-20 min until a desirable brown color was obtained then counterstained with Mayer's hematoxylin.
Quantitative scoring method
TGF-β1 immuno-activity quantitation was made by digital image analysis through the image J analysis software on five fields from each slide. This software could measure the total positive stained brown color/areas throughout the unstained cells. From this data an index (positively stained cells per a total of 1000 cells) can be computed.
Determination of serum TNF-α
The concentrations of serum TNF-α were quantitatively determined in all groups, on days 7 and 35 p.i. using Quantikine®ELISA kits, Cat. No. MTA00B (R&D systems, USA). All ELISA techniques were performed according to the manufacturer’s instructions. The absorbance of the serum samples was read within 10 min at a wave length of 450 nm using an ELISA micro-titer plate reader.
Transmission electron microscopy
Adults
The collected adults were centrifuged at 7000 rpm for 1 min, and then the parasites were resuspended in fresh modified Karunovsky’s fixative and fixed at 4°C for 3 days. Fixative was then removed and the parasites were post-fixed for 1 hour in 2% osmium tetroxide. Adults were then dehydrated in ethanol followed by acetone series solutions of increasing concentration (Karunovsky 1965). The material was embedded in the epoxy resin Epon 812 according to Luft's standard method (Luft 1961). After hardening, blocks were removed and sections were cut using an ultramicrotome. Ultrathin sections were examined using a JEM 2100 transmission electron microscope (TEM).
Muscle samples
1 mm³ diaphragm muscle samples were cut then fixed within 5 minutes. Muscle samples were then prepared by the same steps as adults.
Ethical consideration
The study protocol was approved by the ethical committee of the Institutional Review Board (IRB) Unit, Faculty of Medicine, Zagazig University (approval number: 4002).
Statistical analysis
Quantitative values of the measured parameters were expressed as mean ± standard deviation (SD). Data were analyzed by one way-ANOVA to determine significance of differences between studied groups using Statistical Package for Social Sciences (SPSS), version 18.0. All statistical tests were considered significant at p < 0.05 and highly significant at p < 0.01.