Forty male albino rats weighing 120-160 g were obtained from the Laboratory Animal Unit of Beni-Suef University's Faculty of Pharmacy. The rats were divided into four groups of ten rats each, and they were housed in plastic cages. Rats were kept in the Laboratory of Parasitology, Faculty of Veterinary Medicine, Beni-Suef University, Egypt, at an ambient temperature of 20-25 °C, with a relative humidity of 55.0%, on a 12 h light-dark cycle, with the lights turned off at 7 p.m. The animals were fed standard rodent pellets and were given unlimited access to water.
The purified thymol used in the present study was purchased from Sigma- Aldrich (CAS Number: 89-83-8; St. Louis MO, USA). The commercial anthelmintic ivermectin was brought from Pharma Swede Ph. Comp. (Egypt).
Embryonation of Toxocara eggs
Adult female T. vitulorum were obtained from naturally infected buffalo calves brought to veterinary clinics. Gravid female worms were collected and thoroughly washed in 0.85% normal saline before gravid uteri were removed with fine scissors. The eggs were sieved, washed, and precipitated several times with 1.0% formal-saline before being stored in sufficient solution in the refrigerator as an egg stock (Amerasinghe et al. 1992). In addition, the collected eggs were placed in clean Petri dishes (90 mm in diameter) containing formal saline (1.0%). The dishes were incubated at 28 oC for 14-18 days, with the solution changed every two days, aerated, and examined under a microscope to see how the embryonic cells developed. After about 10 days of incubation, the second-stage larvae began to emerge from these eggs.
In vitro effect of thymol and ivermectin on Toxocara eggs
Using dimethyl sulfoxide (DMSO) as an emulsifier, different concentrations of thymol (10, 5, 2.5, 1.25, and 0.625 mg/mL) were prepared. To make the emulsifier, 120 mg of purified thymol was dissolved in 600 µl DMSO (10.0%). In a microtiter plate (96 well), double-fold serial dilutions in saline solution (NaCl 0.9%) were performed to a final concentration of 0.625 mg/100 µl. To each well, one hundred T. vitulorum eggs in formol saline were added to achieve a final concentration ranging from 10.0% to 0.625% (Arafa et al. 2020). Ivermectin was diluted in distilled water to obtain five concentrations of 0.08, 0.04, 0.02, 0.01 mg/mL, and 0.005 mg/mL. As a negative control, 1.0% formol saline was used. The plates were incubated at 28 °C for 10 days before being examined under a light microscope to determine the percentage of larval development. All treatments were carried out in triplicate, and experiments were independently repeated thrice.
In vivo effect of thymol and ivermectin on Toxocara migration in liver and lungs
The experiment was divided into four groups of ten rats each. Throughout the experiment, a negative control group was given phosphate buffer saline orally. A positive control group of rats was infected but not treated. The rats in the thymol-treated group received thymol in the drinking water at a dose of 40.00 mg/kg body weight for seven days prior to challenge (via stomach tube) (Arafa et al. 2020). For seven days prior to infection, the final group (ivermectin-treated group) received ivermectin (0.2 mg/kg) in the drinking water. All groups (except the negative control group) were inoculated orally with 2500 embryonated eggs per rat via a gastric tube (Barriga and Omar 1992). The experiment was continued until day 7 post-infection, at which point all rats were euthanized via intraperitoneal barbiturate injection (Dutton 2019). Immediately prior to this, blood samples from the medial canthus of the eye were taken for serum biochemical and cytokine analyses. Macro- and microscopically, the liver and lung were examined for larval migration. The ethical standards for animal regulations were followed and approved by Beni-Suef University's Faculty of Veterinary Medicine (BSU/0256/2019).
The protective effect of thymol and ivermectin against Toxocara larvae in vivo
Postmortem inspection of internal organs
The liver and lung were minced separately and digested in a pepsin-HCl solution (20 times their volume) (0.7 g pepsin and 0.7 ml HCl in normal saline) for 24 h at 37 °C (Horiuchi et al. 2005). A sieve was used to filter the digested suspension. Centrifuge the filtrate for 2 min at 1,500 rpm. The pellets were then collected and examined for the presence of T. vitulorum larvae. At a magnification of 10X, the larvae were counted using a light microscope. The efficacy percentages of thymol and ivermectin treatments were determined by the number of recovered larvae counted (Amerasinghe et al. 1992), with the following formula:
Efficacy (%) of either thymol or ivermectin treatments = 100X (the mean number of recovered larvae in the negative control – the mean number of recovered larvae in the treated group) / (the mean number of recovered larvae in the negative control).
Biochemical and cytokine parameters
On the seventh day after infection, five rats were bled, and blood was collected. Serum samples were used to determine biochemical parameters. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using the method described by Elissondo et al. (2008). Protein and albumin levels were estimated as previously denoted by Bergmeyer et al. (1985). Serum nitric oxide (NO) value was determined using the colorimetric method as stated by Bories and Bories (1995). Moreover, cytokines IL-4 and IFN-γ levels were measured in the serum of rats using Sandwich ELISA with anti-cytokine antibodies (Stenken JA, Poschenrieder 2014) and following the manufacturer’s instructions (Phar Mingen, San Diego, USA).
At day 7 after infection, all groups had their liver and lungs removed. The isolated organs were cut into small pieces with a thickness of 2-3 mm and fixed in Bouin's fluid for 72 h. The fixed samples were dehydrated in ethyl alcohol in increasing concentrations, cleared in xylene, and embedded in Paraplast®. Hematoxylin and eosin (H&E) for general histological examination (El-Ashram et al. 2017; El-Ashram et al. 2020), Periodic-acid Schiff (PAS) for mucin detection, and Crossmon's trichrom stain for collagen fiber detection were used to cut and mount μm 4-5 m thick sections on clean and dry glass slides (Suvarna et al. 2019). In addition, the size and number of granulomas in each section of the rat liver and lung were measured in five fields (i.e., fifteen fields for each group). The Image J analysis software program (NIH, Bethesda, Maryland) and LEICA (DFC290 HD system digital camera, Heerbrug, Switzerland) connected to the light microscopy (10X objective lens) were used to score histopathological lesions in the liver and lung stained with H&E in terms of the degree of cell damage (Gibson-Corley et al. 2013).
One-way analysis of variance (ANOVA) was used for the statistical analysis, followed by the Tukey multiple comparison post-hoc test. P values less than 0.05 were considered significant when the obtained data were expressed as a mean ± SD. Statistical Package for the Social Sciences 22 was used to perform all calculations (SPSS, Chicago, Illinois, USA).