Remarkable Histopathological Improvement of Experimental Toxoplasmosis Treated with Spiramycin-loaded Chitosan Nanoparticles

The present study investigated the anti-Toxoplasma effect of chitosan nanoparticles [CS NPs], spiramycin, spiramycin co-administered with metronidazole and spiramycin-CS NPs formulation on the parasite burden and histopathological changes in the liver, spleen and brain in experimentally infected mice. Seventy male Swiss albino mice were classied into seven equal groups: healthy control (I), infected untreated control (II), infected group receiving CS NPs (III), spiramycin administered infected group (IV), infected group receiving spiramycin-metronidazole (V), infected receiving 400 mg/kg spiramycin-CS NPs (VI) and infected treated with spiramycin-loaded CS NPs 100 mg/kg (VII). All groups were inoculated intraperitoneally with 2500 T. gondii tachyzoites RH strain except the healthy control group. All groups were sacriced on the 8th day after infection. Density of the parasite and histopathological examination of the liver, spleen and brain of all treated mice revealed reduction in the mean tachyzoites count as well as decreased inammation, congestion and necrosis within tissue sections. Spiramycin-loaded NPs displayed the highest signicant reduction in the pathological insult tailed by spiramycin-metronidazole and CS NPs. In conclusion, spiramycin-loaded CS NPs showed a promising synergistic combination in the treatment of the histopathology caused by toxoplasmosis.


Introduction
Toxoplasmosis is a highly prevalent foodborne disease relating to the intracellular parasite Toxoplasma gondii [T. gondii]. It infects about one third of the population worldwide [Wohlfer et al. 2017]. The parasite exists in two distinct forms; a rapidly replicating invasive tachyzoite and a slowly replicating bradyzoite inside tissue cysts. T. gondii [RH strain] tachyzoites showed no potential for tissue cyst [bradyzoites] and oocysts formation in laboratory infected rats and mice [Dubey et al. 1999; Mordue et al. 2001; Asgari et al.2013]. The faster replication of the tachyzoites leads to many pathological changes ranging from mild congestion to severe degeneration involving mainly the liver, spleen and brain [Unno et al. 2013 Although many drugs are used for toxoplasmosis treatment, yet no de nitive "gold standard" treatment has been available. Some medicines have been commonly used such as pyrimethamine-sulfadiazine, trimethoprim-sulfamethoxazole, pyrimethamine-clindamycin, azithromycin and traditional Chinese medicine. They may be poorly tolerated, have severe side effects and/or have poor tissue penetration. Thus, the uncertainty of their e cacy is still not resolved [Valentini et al. 2015;Wei et al. 2015].
Spiramycin is less toxic than other drugs and it is used for the treatment of T. gondii infection among human population and in the prevention of vertical transmission from mother to foetus in the rst trimester of pregnancy. It is absorbed e ciently after oral intake and spreads quickly to blood and tissues [Shi et al. 2005]. Despite its signi cant tissue penetration, it reveals poor crossing of the blood brain barrier [BBB] owing to the presence of the e ux transporters for which spiramycin is a substrate [Grover and Benet, 2009]. For enhancement of its brain bypass, spiramycin could be co-administered with metronidazole to inactivate the e ux pumps present in the BBB [Chew et al. 2012].
In the search for new strategies that overcome the disadvantage of the used treatment regimens, it was found that application of nanomedicine improved the drug biodistribution, modi ed bioavailability and decreased toxicity [Assolini et al. 2017].Numerous types of nanoparticles [NPs] such as silver and gold are used, but their use may be hampered by their toxic side effects, adding to their relatively higher production cost in comparison to the natural based NPs [Cameron et al. 2016;Benelli, 2018].Chitosan[CS] is a normal non-toxic polysaccharide obtained from shells of crustaceans and molluscs by chitin deacetylation [Yong et al.2015]. The advantages of CS make it the material of choice for NPs preparation in various elds. Its biocompatible, biodegradable, nontoxic nature and relatively low cost encourage its suitable applicability especially in developing countries. CS has been extensively known as an antimicrobial agent due to its intrinsic positive charge density and chelating capacity [Kong et

VI. Histopathological study
Specimens from the liver, spleen and brain were taken from all the groups and xed in 10% formalin.
They were dehydrated in an ascending series of ethyl alcohol [70%, 80%, 95% and 100%] then cleared in xylene. The samples were embedded in molten para n at 60°C for 1-2 hrs to form para n blocks. Block sections were cut using microtome. Each section was 4 µm thick. Two sections were prepared from the different organs of each mouse, they were mounted on slides, then left in the oven at 40°C to dry and x on the slides. Slides were depara nized by dipping them in xylene followed by passing them in descending series of ethyl alcohol [100%, 95%, 80% and 70%] then rinsed with water for rehydration. Slides were stained with haematoxylin, rinsed under running water then counterstained with eosin. They were dehydrated using an ascending series of ethyl alcohol and then mounted in Canada balsam. Tissue

I. Parasite burden:
The infected untreated control group showed median parasite counts of 12, 8 and 3 / 30 oil immersion eld (OIF) in liver, spleen and brain respectively. There was a signi cant reduction in parasite burden in the groups treated with spiramycin-CS NPs formulation in comparison to other groups [ Figures 1-3].
II. Histopathological study: II.1. Liver (Fig. 4) Microscopic examination of sections of the liver from healthy control (group I) showed normal hepatic architecture divided into lobules. The hepatocellular cords were mostly one or two layers thick radiating from the central vein towards the periphery of the lobule. The normal hepatocytes were polygonal in shape with centrally placed round or oval dark violet nuclei and granulated eosinophilic cytoplasm. The cords of hepatocytes were separated by blood sinusoids lined by endothelial cells (EC). At the periphery of the lobule, the portal triads showed no in ammatory cellular in ltrates [ Figure 4.1].
In the infected untreated control (group II), microscopic examination of liver tissue revealed invasive tachyzoites aggregated among the hepatocytes. Markedly dilated and congested central veins and sinusoids with evident focal necrosis and in ammation. (Fig. 4.2) The liver sections of CS NPs treated group (III) showed pseudocysts with necrotic tachyzoites. Residual apoptotic bodies of hepatic nuclei and degenerative hepatocytes with marked dilated congested central veins and sinusoids were noticed. Moreover, there was evidence of lobular in ammation [ Figure 4 In CS NPs treated group (III), areas of macrophages loaded by tachyzoites were observed. Disorganized splenic architecture in addition to extramedullary hematopoiesis were evident in the red pulp [ Figure 5.4].
The microscopic examination of spiramycin infected treated group (IV) illustrated pseudocysts containing necrotic tachyzoites and numerous apoptotic cells. Severe disorganized splenic architecture with enhancement of the in ammatory cells and reduction in the lymphocytes were observed [ Figure 5.5].
The spleen of spiramycin-metronidazole treated group (V) demonstrated moderate disorganized architecture with extramedullary hematopoiesis and excess eosinophils [ Figure 5.6].
In spiramycin-CS NPs 400 mg/kg treated group (VI), necrotic tachyzoites in pseudocysts in the red pulp and at the margin of regenerative lymphocytes of the white pulp were observed. Mild disorganized spleen architecture with evident tissue recovery and extramedullary hematopoiesis were noticed [ Figure 5.7].
Moderate disorganized spleen architecture with regenerative white pulp forming well de ned germinal centres as well as frequent epithelioid histiocytes were evident [ Figure 5.8].
II.3. Brain (Fig. 6) Examination of the brain cortex of the healthy control [group I] revealed normal architecture composed of neuroglial cells with ill-de ned cytoplasm and pyramidal cells. Normal blood vessels were present [ Figure  6.1].
In infected untreated control (group II), the cerebral cortex showed gliosis, apoptosis as well as perivascular and parenchymal in ammation [ Figure 6.2].
The microscopic examination of CS NPs treated group (III) illustrated mild dilated blood vessels. Small pyramidal cells and others with vacuolated cytoplasm were noticed. Apoptosis and marked gliosis were observed [ Figure 6.3].
In the spiramycin treated group (IV), the brain revealed the occurrence of perivascular in ammatory in ltrate, gliotic nodules and necrotic neurons [ Figure 6.4].
In the brain of spiramycin-metronidazole treated group (V), no tachyzoites were detected. Mild congested blood vessels, moderate gliosis and ferrugination (mineralized bodies of necrotic neurons) were observed [ Figure 6.5].
Spiramycin-CS NPs 400 mg/kg treated group (VI) revealed absence of tachyzoites with mild dilation of blood vessels. Normal neuroglial cells with no evidence of in ammation were observed [ Figure 6.6].
In the brain of spiramycin-loaded CS NPs 100 mg/kg treated group (VII) spongiosis, moderate gliosis and apoptosis were seen. No in ammation was observed [ Figure 6.7].

Discussion
Toxoplasmosis treatment is a worthy challenge to achieve, particularly that the parasite is intracellular and crosses the blood brain barrier [Briones et al. 2008].
To ful l the present work, CS NPs, spiramycin, spiramycin co-administered with metronidazole and spiramycin-CS NPs were evaluated for treating acute toxoplasmosis regarding parasite load and histopathology of the liver, spleen and brain in Swiss albino mice.
The parasite burden decreased in liver, spleen and brain among all treated mice in comparison to the infected untreated control. The least parasite load was obtained after the treatment with spiramycinloaded NPs compared to the other groups regarding all the studied organs.
In the present work, histopathological study demonstrated severe tissue damage in the liver, spleen and brain of acute T. gondii infected untreated mice. Marked in ammation, congestion, areas of lytic necrosis and free tachyzoites within tissue sections were detected as compared to those of the healthy control and of the treated mice. Activated in ammatory cells that were attracted by the free tachyzoites, triggered the in ammatory response, causing cell lysis. Their effect incorporates with the mechanical damage produced by the tachyzoites. These ndings coincide with various reports of previous studies [Unno et al. 2013; Fuentes-Castroet al. 2017].
All the used treatments [CS NPs, spiramycin, spiramycin co-administered with metronidazole and spiramycin-CS NPs] modulated the pathological effects in the liver, spleen and brain of mice infected with T. gondii RH strain. The various degrees of response were characterized by disappearance of necrosis and reduction of in ammation.
Concerning the liver of infected untreated mice, it was characterized by congested veins and sinusoids accompanied by lobular necrosis, vacuolar degeneration of hepatocytes and in ammatory in ltrate in both lobular and portal areas with interphase hepatitis. Free tachyzoites were seen in the necrotic foci. The ndings agreed with those described by Unno et al.[2013] and Mady et al. [2016]. All treated groups showed reduction in the in ammatory in ltrate with brosis. Regeneration of hepatocytes was noted together with minimal mononuclear in ammatory in ltrate. In mice receiving spiramycin-metronidazole, residual portal and bridging brosis were noted. Interestingly, the groups of spiramycin-CS NPs 400 mg/kg treated and spiramycin-CS NPs 100 mg/kg showed nearly normal architecture with necrotic tachyzoites.
Regarding the spleen of the infected untreated group, there was disorganized architecture with areas of necrosis and absent germinal centers. Extramedullary hematopoiesis appeared with frequent megakaryocytes. This latter sign was previously described in the literature and was clari ed by the fact that T. gondii induces systemic levels of IL-12 which promotes high level of extramedullary hematopoiesis [EMH] [Zaretsky et al. 2012]. In the spleen of the treated groups, extramedullary hematopoiesis persisted. However, in the groups treated with spiramycin-CS NPs 400 mg/kg and spiramycin-loaded CS NPs 100 mg/kg, the necrosis disappeared completely and restoration of close normal architecture was identi ed. There was regeneration of white pulp, well-structured lymphoid follicles with reactive germinal centers and healthy demarcated red pulp.
In the brain of the infected untreated mice, the injury was evidenced by astrocytic proliferation [gliosis] and scattered necrotic neurons. Perivascular in ammation, spongiosis and congestion were observed. . Treatment greatly reduced the in ammation; dead neurons were mineralized or replaced by gliotic nodules. Best results were seen in the group treated with spiramycin-CS NPs 400 mg/kg; normal neuroglial cells with no sign of in ammation were detected.
Conclusion: The most signi cant tissue regenerative effect in liver, spleen and brain with absence of living tachyzoites was noticed in the mice treated with spiramycin-CS NPs 400 mg/kg. Thus, it could be a promising treatment for toxoplasmosis due to its capability to penetrate the tissues and particularly the BBB.

Con ict of interest
The authors declare that there is no con ict of interest regarding the publication of this article.