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Introduction
Trichinellosis is a severe meat-borne parasitic disease. It is broadly spread around the world with considerable significance in several developing countries (Dyab, 2019). Human infection is caused by the ingestion of raw or insufficiently cooked meat from pigs or other Trichinella-infected animals. Despite the availability of effective and relatively safe drugs such as albendazole (ABZ) and mebendazole for the treatment of trichinellosis, these drugs have several drawbacks, such as the emergence of parasite drug resistance (Xiao, 2013; Abou-Shady et al., 2016), and cancer cells (Sharma et al., 2012; Liu et al., 2016).
To preserve the efficacy of current drugs and slow down the spread of resistance, several strategies were used, including the use of alternative drugs, rotation of drugs from different chemical groups, and use of drug combinations (Waller, 1997; Sangster, 2001; Singh & Yeh, 2017). The latter is considered the most effective approach to delaying resistance (Sangster, 2001). Therefore, the purpose of this study was to evaluate ABZ and MQ efficacy alone or in a low dose combination in T. spiralis infected mice, analyzing parasite burden and pathological changes in the intestine and muscles of these animals.
Materials and Methods
Mice
The study was carried out on one hundred and twenty male CD1 Swiss albino mice (20 ± 2 gm). The mice were bred and maintained at the Schistosome Biology Supply Center (SBSC) of Theodor Bilharz Research Institute, Giza, Egypt. Mice handling and treatment were conducted according to internationally valid guidelines and ethical conditions.
Parasite and Infection
T. spiralis larvae were originally isolated from the diaphragms of infected pigs obtained from El-Bassatine local Abattoir, Cairo, Egypt. The larvae were routinely maintained in our laboratory by a consecutive passage in mice. Muscles of mice heavily infected with T.spiralis were cut and digested in a solution formed of 1 % pepsin and 1 % concentrated hydrochloric acid in warm tap water as described by Dunn and Wright (1985). After overnight incubation at 37 °C, larvae were extracted using the sedimentation technique, washed several times with saline, and the number of larvae/ml was counted using a hemocytometer. The 12-hour fasting mice were infected orally with 200 larvae using a tuberculin syringe according to Wassom et al. (1988).
Experimental design
ABZ (Alzental) suspension was purchased from the Egyptian International Pharmaceutical Industries Co. (EIPICO) as 20mg/ml and was orally given in a dose of 50mg/kg/day. MQ (mephaquin) (Mepha Ltd., Aesch-Basel, Switzerland) was orally administered in one dose of 400 mg/kg/day which was freshly suspended in 7 % (v/v) Tween-80, 3 % (v/v) ethanol and distilled water (Keiser et al., 2009; Fakahany et al., 2014). A combination of ~ 1/3 doses of both drugs [ABZ (20 mg/kg/day) and MQ (140 mg/kg)] both were prepared separately, then orally administered one after the other. All mice were infected with 200 T. spiralis larvae and randomly divided into two main experimental groups according to the time of initiation of the treatment: group I, treated in the acute phase of infection (from day 1 post-infection); group II, treated in the chronic phase (from day 35 post-infection). Each group was further divided into 4 subgroups (n = 15), non-treated control (group C); ABZ-treated (50 mg/kg/day for 3 consecutive days) (group ABZ); MQ-treated (a single 400 mg/kg dose) (group MQ); ABZ (20 mg/kg/day for 3 consecutive days) and MQ (140 mg/kg)-treated (group ABZ+MQ). Mice from group I were sacrificed on day 7 post-infection to analyze intestinal worm burden (n = 10) and histopathological changes in the small intestine (n = 5). Mice from group II were sacrificed on day 48 post-infection to determine larval muscle burden (n = 10) and histopathological changes in the diaphragm (n = 5).
Isolation of adult worms and muscle larvae
Adult worms were collected using the method of Wranicz et al. (1998). The worms recovered from the small intestine were counted, and the intestinal worm load was expressed as the total number of intestinal worms per mouse. Muscle larvae were recovered from infected mice’s carcasses by artificial digestion according to standard procedures (Jiang et al., 2012). Muscle larval load was determined by counting all the larvae present in a carcass digest aliquot; it was expressed as a total number of encysted larvae per mouse.
Histopathological examination
Small intestine samples were obtained from mice sacrificed in the acute phase of infection (group I, subgroups C, ABZ, MQ and ABZ + MQ) according to the method described by Nasseff et al. (2018). The diaphragm was removed after euthanasia from mice treated in the chronic phase (group II, subgroups C, ABZ, MQ and ABZ + MQ). The specimens were fixed in 10 % buffered formalin solution, dehydrated, cleared, and embedded in paraffin blocks. Five μM-thickness paraffin sections were processed, mounted on glass slides, and stained with hematoxylin-eosin for histopathological examination.
Statistical analysis
Results are expressed as mean ± standard error. Test of normality, Kolmogorov-Smirnov test, was used to measure the distribution of data. So, a comparison between variables was performed using one way ANOVA followed by LSD test as a post-hoc test if the significant result was recorded. Statistical Package for Social Sciences (SPSS) computer program (version 19 windows) was used for data analysis. P-value ≤ 0.05 was considered significant.
Ethical Approval and/or Informed Consent
All the experiments on animals were carried out according to the internationally valid guidelines after the approval of the Institutional Ethical Committee of Theodor Bilharz Research Institute (TBRIREC), the serial number of the protocol: PT (527).
Results
Adult T. spiralis worm count in the small intestine of mice sacrificed at the 7thday post-infection
There is a statistically significant difference in the mean value of worm count between the four studied subgroups (F= 118.199; p= 0.001). The adult T. spiralis worm count is significantly decreased in all T. spiralis-treated groups (p= 0.001) compared with its corresponding value in (Group C). However, (group ABZ+MQ) recorded the highest percentage reduction in worm count (93.7 %) followed by (group ABZ) (88.7 %) and finally (group MQ) (84.7 %) (Fig. 1).
Fig. 1
Intestinal parasitic burden in CD1 Swiss albino male mice infected with T. spiralis L1 larvae and treated with Albendazol, Mefloquine, or both, in the acute phase of infection.
Description: The number of worms recovered from the intestine estimates the intestinal parasitic burden (see M&M). Each bar represents the mean ± SEM of 10 mice. Differences among treatment groups were analyzed with ANOVA; comparisons between treatments were done with LSD post-test. Differences were considered significant if P < 0.05. a: significantly different compared with the control group. #: percent change from the control group.
T. spiralis larvae count in the muscles of mice sacrificed 48 days post-infection
The mean value of larvae count had a statistically significant difference between the four studied subgroups (F= 333.881; p= 0.001). The T. spiralis larvae count significantly decreased in all infected-treated groups (p= 0.001), in comparison with its corresponding value in (group C). The total larval counts in (ABZ+MQ group) were significantly decreased in relation to its corresponding value in (ABZ group) (p=0.045). The combined treatment (ABZ+MQ) received on the 35th day post infection showed a drug efficacy of (86.2 %) on encysted T. spiralis larvae followed by (MQ group) (82.2 %) and finally (ABZ group) (79.6 %) (Fig. 2).
Fig. 2
Muscular parasitic load in CD1 Swiss albino male mice infected with T. spiralis L1 larvae and treated with Albendazol, Mefloquine, or both, in the chronic phase of infection.
Description: The number of encysted larvae recovered from the muscles estimates the muscular parasite load (see M&M). Each bar represents the mean ± SEM of 10 mice. Differences among treatment groups were analyzed with ANOVA; comparisons between treatments were done with LSD post-test. Differences were considered significant if P < 0.05. a: significantly different compared with the control group. b: significantly different compared with ABZ group. #: percent change from the control group.
Histopathological study
The histological analysis of small intestine sections from T. spiralis-infected non-treated mice showed inflammatory cells infiltrating both the mucosa and submucosa, in addition to, shedding of the epithelial lining, flattening of the villi and hyperplasia of the crypts of Lieberkühn (Fig. 3a). The infiltrate was composed mainly of lymphocytes and plasma cells, with few neutrophils, eosinophils, and fibroblasts. Intestinal sections of both ABZ- and MQ-treated groups showed a reduction in the inflammatory infiltrate, apparently intact epithelial lining and finger-like villi, and a similar recovery to its normal shape (Figs. 3b and 3c). In the ABZ-MQ-treated group, this reduction reached its maximum, accompanied by a marked decrease in all cellular infiltrates (Fig. 3d).
Fig.3
Representative micrographs illustrating the histology of the small intestine of mice infected with T. spiralis and treated with antiparasitics in the acute stage of infection.
Description: Tissue samples were obtained on day 7 post-infection. (a) non-treated control, showing heavy larvae embedded in the musculature of the diaphragm (black arrow) surrounded by intense inflammatory reaction (white arrow), atrophy, distancing, and tearing of muscle (M). H&E, X200 (b) ABZ-treated, showing larval deposition (black arrow) surrounded by mild muscle inflammation (white arrow). H&E, X200 (c) MQ-treated, showing minimal cellular inflammation (white arrow) and single larva deposition (black arrow). H&E, X400 (d) ABZ+MQ-treated, showing degenerated larvae (D) with broken down incomplete capsule which is completely invaded and surrounded by inflammatory cells (white arrow), inflamed skeletal muscle bundles (M), and single larval deposition (black arrow). H&E, X200.
The diaphragms of non-treated mice showed a massive presence of diffused T. spiralis larvae (Fig. 4a). Each larva was limited by a collagenous capsule and heavy inflammatory cellular infiltration with atrophy, distancing, and muscle tearing. In contrast, diaphragm tissue samples of treated animals showed fewer encysted larvae and minimal inflammatory cellular infiltration surrounding them (Figs. 4b and 4c). Furthermore, the diaphragm sections of the mice that received the combined treatment ABZ+MQ had much fewer encysted larvae, most showing degenerative changes in their contents and capsules (Fig 4d).
Fig.4
Representative micrographs illustrating the histology of the diaphragm of mice infected with T. spiralis and treated with antiparasitics in the chronic stage of infection.
Description: Tissue samples were obtained on day 7 post-infection. (a) non-treated control, showing dense inflammatory cellular infiltrate (white arrow), mainly in the core of the villi, shedding of the epithelial lining (E), and flattening and hyperplasia of the crypts of the villi (black arrow). H&E staining, magnification 200X. (b) ABZ-treated, showing mild inflammatory infiltrates, mainly within the core of the villi (white arrow) with apparently intact epithelial lining (E) with finger-like villi (black arrow). H&E, X400. (c) MQ-treated, showing intact epithelium lining (white arrow), intact epithelium lining (E), and finger-like villi (black arrow). H&E, X400. (d) ABZ+MQ-treated, showing more reduction in the intensity of the inflammatory infiltrate (line) with intact lining epithelium (white arrow) (E) and finger-like villi (black arrow). H&E, X400.
Discussion
To date, the drugs against T. spiralis are restricted all over the world. The broad-spectrum drug, ABZ, has many adverse drug responses such as encephalitis, epilepsy, extreme medicate eruptions, and even death (Shalaby et al., 2010; Yadav & Temjenmongla, 2012; Matadamas-Martínez et al., 2013). Moreover, it shows diminished effectiveness against T. spiralis- encysted larvae (Pozio et al., 2001; Siriyasatien et al., 2003; Kalaiselvan et al., 2007). Thus the search for novel, safe, and effective anthelminthic agent against the encapsulated muscle larvae of T. spiralis is a main objective in medical research.
Repurposing drugs to achieve antihelminthic efficacy is a matter of the utmost importance. This approach seems to result in lower costs, a lower hazard of failure, more safety, and speedier time during the drug development process (Andrews et al., 2014; Panic et al., 2014). MQ has been repurposed against a wide variety of infectious agents (Rodrigues-Junior et al., 2016; Aly et al., 2017; Balasubramanian et al., 2017) as well as helminths like Schistosomiasis (Keiser et al., 2009, 2010; Keiser & Utzinger, 2012) and Echinococcosis (Manneck et al., 2010; Fakahany et al., 2014; Rufener et al., 2018). The results of this study revealed a significant reduction in both T. spiralis intestinal worm and muscular larvae count in all treated groups compared to their corresponding T. spiralis infected control groups. This decrease was statistically comparable in the case of intestinal worms reaching its maximum peak in the combined ABZ-MQ-treatment (93.7 %). Moreover, the muscular larvae count recorded a significant maximal decline in the combined ABZ-MQ-treated group (86.2 %) compared with the ABZ-treated group (79.6 %) and at the same time MQ alone gave a better reduction (82.2 %) than ABZ, yet still insignificant. It was found that the efficacy of ABZ against Trichinella infection is more effective against the intestinal stage than against the muscular stage with a reduction percent extended from 62 % to 100 %, and its effectiveness against muscle larvae ranged from 26 to 91 reduction percent (Shoheib et al., 2006; Shalaby et al., 2010; Attia et al., 2015; Nada et al., 2018). The differences in the effectiveness of ABZ against both the intestinal and muscular stages depend on the dosage, time, and duration of treatment (Siriyasatien et al., 2003; Codina et al., 2015).
Herein, tissue damage and severe inflammations in the histopathological sections of both intestinal and skeletal muscles of T. spiralis infected mice was attributed to the increased levels of reactive oxygen species (ROS), superoxide dismutase (SOD), inducible nitric oxide synthase (iNOS), and the overexpression of COX-2, anti-apoptotic particles and inflammatory cells (Othman & Shoheib, 2016). These molecules have been produced not only by the parasite but also by the host cells, increasing tissue damage and reducing apoptotic processes during their defensive response. The parasite triggers angiogenesis through secretion of the panel of immunological molecules like vascular endothelial growth factor (VEGF) (Capo et al., 1998), fibroblast growth factor (FGF)-1, FGF-2, and insulin-like growth factor (IGF-1) (Kang et al., 2011) which in turn results in the development of blood vessels which are essential for larvae survival (Akiho et al., 2005; Serna et al., 2006; Romero et al., 2008; Othman et al., 2016).
Both the intestinal and skeletal muscles of the treated groups had mild inflammatory infiltrates and minimal cellular inflammation. The maximum reduction in these cells of the immune system was found in the combined ABZ-MQ-treated groups especially in muscles of the diaphragm surrounding the encysted larvae. As a result of the diminished number of both worms and larvae, restoration of the normal appearance of host cells was noted accompanied by an obvious diminishment in inflammatory cells (Attia et al., 2015; Abou Rayia et al., 2017; Elgendy et al., 2020) particularly in combined ABZ-MQ treated group. The confirmed parasiticidal effects of MQ in this study may be due to its involvement in ROS generation (Elmehy et al.,2021) in addition to its anti-angiogenic and apoptotic properties against the parasites in hosted tissues (Kamili et al., 2017).
In conclusion, the use of combined ABZ-MQ-treatment in reduced regimens has the maximum effect not only on reducing the two phases of parasite burden but also on the restoration of normal tissue architecture. MQ treatment is as successful as ABZ treatment in killing both phases of T. spiralis and consequently restoring the normal tissue architecture of the host. The synergistic activity of the two drugs overcomes their side effects and increases their biological activity. Besides, the chemical complexity of the mixtures reduces the risk of drug resistance.
