Separation of CWP and Preparation of TWH

Camel milk (4.23% fat and 3.47% total protein) was obtained from the Centre of Scientific Research in Marsa Matruh governorate. CWP was separated at the Dairy Science and Technology Department, Faculty of Agriculture, Alexandria, Egypt, according to Wang et al. [25]. Milk protein hydrolysate was prepared according to Otte et al. [26]. The hydrolysis process was initiated by adding 2 ml of trypsin solution to the protein solution and vortexed for 30 s. The mixture was incubated at 40 °C, and a 1 ml sample was withdrawn after 24 h of hydrolysis. Then, enzymatic inactivation was done by heating at 90°C for 15 min. Afterward, the mixture solution was cooled in an ice bath for 20 min, centrifuged at 10,000xg for 10 min, and the supernatant was used for further analyses [27].

Characterization of CWP and TWHFree amino acids analysis

The amino acid composition of CWP and TWH samples was analyzed using high-performance liquid chromatography (HPLC) Pico-Tag method (Waters Associates, Millipore Cooperative, USA). Pre-column derivatization of the samples’ amino acids was done using phenylisothiocarbamyl. The phenylthiocarbamyl derivatives were separated by reversed-phase gradient elution HPLC and detected by their ultraviolet (UV) absorbance at a fixed wavelength of 254 nm (2489 UV/Vis Detector) [28, 29].

Antioxidant activity assay

Free radical scavenging activity of CWP and TWH was measured by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) [30]. Using a spectrophotometer (UV-VIS Milton Roy), the absorbance (A) was measured at 517 nm. Ascorbic acid was used as a reference standard compound, and the experiment was performed in triplicate. Using the log dose inhibition curve, the inhibitory concentration of 50% (IC50%) of DPPH free radical, was calculated. The percentage of DPPH scavenging effect was determined using the following equation:

$$\:\begin &\text\:\text\:\text\:\%\\& \quad =\:\frac\:-\:\text\:}\text}\times100 \end$$

Animals and ethics statement

Laboratory-bred parasite-free Swiss albino male mice, aged 6–8 weeks and weighed 20–25 g, were obtained and retained in the animal house of Medical Parasitology, Faculty of Medicine, Alexandria University, Egypt, under standard housing conditions. Animals were housed, treated, and sacrificed according to the Egyptian national animal ethics guidelines. They were anesthetized by intraperitoneally injecting 40 mg/kg of sodium pentobarbital, and blood was collected from the jugular veins. Subsequently, the unconscious mice were immediately euthanized by cervical dislocation. The protocol was approved by the Ethical Committee of the Faculty of Medicine, Alexandria University, Egypt (protocol approval number: 0306391).

Parasite and animal infection

T. spiralis strain was initially purchased from Theodore Bilharz Research Institute (Giza, Egypt) and maintained in the Medical Parasitology laboratory for sequential infection. On the 28th day post-infection (dpi), the whole eviscerated skinned carcass was minced, and infective ML were retrieved by the artificial digestion method. The minced muscles of the infected mouse were digested in 1% pepsin and 1% concentrated hydrochloric acid in 200 ml distilled water and swirled at 37 °C using a magnetic stirrer. After two hours, the mixture was sieved in a conical flask. Thereafter, the supernatant was discarded after allowing ML to sediment and they were collected and microscopically counted. For infection, 250 T. spiralis ML were adjusted in 0.1 ml and orally inoculated into the mouse using a gastric tube [31].

Animal grouping and experimental design

Ninety-six mice were randomly allocated into 5 main groups. Group I included 6 non-infected non-treated mice. Group II, 18 non-infected treated mice were subdivided into 3 equal subgroups as follows: Subgroup IIa, non-infected CWP-treated; Subgroup IIb, non-infected TWH-treated; and Subgroup IIc, non-infected ALB-treated. Group III consisted of 12 T. spiralis-infected non-treated control mice. Group IV (Prophylactic group), 24 mice were subdivided equally as follows: Subgroup IVa, CWP-pretreated infected; and Subgroup IVb, TWH-pretreated infected. Group V (Therapeutic group) included 36 infected treated mice that were subdivided equally into 3 subgroups as follows: Subgroup Va, infected CWP-treated; Subgroup Vb, infected TWH-treated; and Subgroup Vc, infected ALB-treated.

According to Maghraby et al. 2005, the calculated daily oral dose of CWP and TWH was 200 µl per mouse [14], while ALB was administered to mice at a dose of 50 mg/kg [32]. The treatments were administered for five consecutive days by gastric tube. In the prophylactic subgroups (IVa and IVb), all mice started treatments five days before infection. From each therapeutic subgroup (Va, Vb, and Vc), six mice initiated the five-day treatment on the same day of infection, corresponding to the adult stage of T. spiralis [33]. While the remaining six mice started the treatment course on 23rd dpi, corresponding to the timing of nurse cell formation in the muscular phase [34]. From each infected Group (III, IV, and V), six mice were euthanized at two different time points, namely on the 5th and 28th dpi, corresponding to the adult and ML stages of T. spiralis, respectively, to undergo the assessment criteria [35]. The antitrichinellosis efficacy was assessed via parasitological, ultrastructural, biochemical, and histopathological studies (Fig. 1).

Fig. 1

Infographic representation of the experimental study design, animal grouping, and treatment timelines

Prophylactic and therapeutic activity against experimental trichinellosisParasitological studyAdult count

On the 5th dpi, the small intestine of the infected mice was longitudinally opened, washed with phosphate buffer saline, and fragmented into small pieces. After 2 h of incubation at 37°C, the adult worms were collected and microscopically counted [35].

Larval count

On the 28th dpi, encysted ML were retrieved by artificial digestion and counted microscopically as previously described [35].

Parasite burden reduction was calculated according to the following equation:

$$\begin & \text\left( \%\text\right) \\ &\quad= 100 - \left[ \frac}treated\:control} \times 100\right] \end$$

Ultrastructural study

The harvested adult worms and ML from infected non-treated mice (Group III) and infected therapeutic subgroups (Va, Vb, and Vc) were fixed in cold 2.5% buffered glutaraldehyde phosphate, dehydrated, and examined under scanning electron microscopy (SEM) (JEOL JSM, IT200, Japan) [32].

Biochemical study

Colorimetric measurement of Malondialdehyde (MDA) and reduced glutathione (GSH) were assayed in sera of mice in all studied groups using commercial kits (CAT. No. MD2529 and GR2511, Bio-Diagnostic, Egypt) according to the manufacturer´s instructions.

Histopathological study

One cm segments of jejunal tissue from infected groups (III, IV, and V) were taken on the 5th dpi, fixed in 10% formalin, processed, and embedded in paraffin blocks. Paraffinized sections were stained with hematoxylin and eosin (H&E) and examined microscopically. Ten low-power fields (LPFs) were examined per mouse for the detection of intestinal inflammatory cellular infiltrate, damage, and goblet cell density [31]. Jejunal inflammatory cellular infiltration was determined using a score system from 0 to 4 (0, normal cellular pattern; 1, scattered lamina propria inflammatory cellular infiltrate; 2, numerous lamina propria inflammatory cellular infiltrate; 3, confluent inflammatory cellular infiltrate extending to submucosa; and 4, transmural inflammatory cellular infiltrate). Jejunal tissue damage was evaluated by a scoring system ranging from 0 to 4 (0, normal architectural pattern with normal villous to crypt ratio; 1, minimal villous shortening and broadening with minimal crypt hyperplasia; 2, mild villous shortening and broadening with mild crypt hyperplasia; 3, obvious villous shortening and broadening with obvious crypt hyperplasia; and 4, extensive mucosal damage and extension through deeper structures of bowel wall. Jejunal goblet cell density was determined as mean goblet cell number per 10–15 enterocytes as follows: goblet cell depletion (< 1), average goblet cells (= 1), and goblet cell hyperplasia (> 1) [31, 35]. On the 28th dpi, diaphragms retrieved from infected groups (III, IV, and V) were fixed, processed, embedded in paraffin blocks, sectioned, and stained with H&E. Thedensity of ML and inflammatory cell infiltration were microscopically determined in ten LPFs. ML density was scored as follows: score 1, less than 5 ML per LPF; score 2, 5–10 ML per LPF; and score 3, more than 10 ML per LPF. Inflammatory cell infiltration surrounding the encysted ML was recorded as follows: 1, mild; 2, moderate; and 3, intense infiltrate [35, 36].

Statistical analysis of the data

Result values were analyzed using the IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). The normality of continuous data was assessed by the Shapiro-Wilk test. One-way ANOVA test was used for comparing normally distributed quantitative variables of different studied subgroups. Post Hoc test (Tukey) was used for pairwise comparison between each subgroup. The level of significance was judged at 5%.