Male Swiss mice (Mus musculus) as a most suitable experimental model for the study of Giardia duodenalis BIV

In this study, we proposed to verify the most suitable murine experimental model for studying human giardiasis. In total 150 animals were used. Fifty mice (Mus musculus) from each lineage (Swiss, Balb/c and C57BL/6), 25 females and 25 males, were divided into 5 groups with 5 animals each, according to the lineage/sex. Three groups were infected with 10 cysts of Giardia duodenalis of assemblage BIV and 2 negative control groups. The animals were followed and evaluated for 15 days after receiving the inoculum. The clinical parameters evaluated were body weight, water and feed intake, excretion, appearance of fur and feces, elimination of Giardia spp cysts and behavioral assessment. The clinical parameters of the groups infected with G. duodenalis were compared with the non-infected groups within their own lineage/sex. In the 15 days of monitoring, only the male Swiss mice presented differences in these parameters. The infected animals consumed more feed, water and eliminated more excreta than the non-infected group. There was no difference in the general average of the weight of the animals or in the behavioral assessment in any group. Only the infected male Swiss mice eliminated G. duodenalis cysts in the feces, which was confirmed by the molecular diagnosis and by observing the presence of trophozoites in the intestinal mucosa. The results demonstrate that the most suitable animal model for the study of human giardiasis is the male Swiss mice, since it was the only one capable of developing infection by G. duodenalis cysts.


Introduction
The etiologic agent of giardiasis, Giardia duodenalis (synonyms: Giardia lamblia and Giardia intestinalis) is one of the most prevalent protozoa in the human gastrointestinal tract (Colli et al., 2015;Hooshyar et al., 2019), and the most associated with cases of infectious diarrhea (Cock et al., 2020), which are an important cause of morbimortality in children under five years old (Baker & Alonso, 2018). Transmission is via the fecal-oral route and occurs mainly by accidental ingestion of cysts in contaminated water and food, being favored by the absence of water and sewage treatment systems, and by the conglomerate of people (Fantinatti et al., 2016).
Giardiasis has a global distribution, reaching an average of between 2 and 7% of the population in developed countries, and might reach 30% in developing or underdeveloped countries (Fantinatti et al., 2016), totaling more than 200 million cases diagnosed by year (Hooshyar et al., 2019). It can be asymptomatic or symptomatic with chronic diarrhea and intestinal malabsorption, leading to disorders in growth and intelectual/cognitive development in children (Lima et al., 2019).
Until now, eight genetic assemblages of G. duodenalis (A-H) have been described (Thompson et al., 2000;Lasek-Nesselquist et al., 2010;Feng & Xiao, 2011;Colli et al., 2015, Qi et al., 2020. Assemblages A and B are considered zoonotic and are responsible for the majority of known human cases of the disease (Matsuchita et al., 2017). The assemblage A can be subdivided into subassemblages AI and AII, and assemblage B can be subdivided into BIII and BIV. In human infection, some authors report that assemblage B is the most commom (Cacciò & Ryan, 2008;Feng & Xiao, 2011). In the southern region of Brazil, there is a predominance of the assemblage BIV (Colli et al., 2015).
Due to its unique and remarkable cellular characteristics, G. duodenalis has been intensively studied (Mayol et al., 2019); however, it is still unclear which is the most suitable experimental murine model for the study of this pathology.
Rodents are the most used experimental models in scientific research (Chorilli et al., 2007;Guénet, 2011). In general, for the study of human pathologies, mice of the species Mus musculus are predominant (Ehret et al., 2017), due to their great genotypic homology and physiological similarities to human beings. These animals are susceptible to infections (Pavanelli et al., 2018), are easy to maintain and observe, have a high reproduction rate and have a large amount of basic information available (Chorilli, et al., 2007).
Due to their different genotypes, the lineages of experimental animals can behave differently when infected by the same pathogen (Ehret et al., 2017). Among the lineages of Mus musculus species, the following stand out: Swiss, heterogeneous animals, resulting from random mating, in which each animal from the same colony responds in a variable way when subjected to the same experiment; Balb/c and C57BL/6, isogenic animals, resulting from inbreeding crosses with a homozygosity index of 99%, which makes the animals from the same colony to respond similarly to the same experiment (Massinori, 2009). In this context, the aim of our study was to suggest, among Swiss, Balb/c and C57BL/6 mice, the most suitable animal model for the study of human giardiasis, based on the evaluation of parasitological, molecular and clinical parameters.

Methodology
An unprecedented experimental animal study was carried out. In total 150 animals were used. Fifty mice (Mus musculus) from each lineage (Swiss, Balb/c and C57BL/6), 25 females and 25 males, were divided into 5 groups with 5 animals each, according to the lineage/sex. The experiment consisted of groups of control animals and groups of infected animals. Three groups were infected with 10 4 cysts of Giardia duodenalis of assemblage BIV and 2 negative control groups.
The variables observed were: initial parasitological and molecular evaluation, to ensure that the animals were free of previous infections, and monitoring of the pre-patency period -period ranging from inoculation of the cysts to the development of infection with elimination of the cysts in the feces, in which quantitative parameters (body weight, water and feed, intake and elimination of excreta) and qualitative (appearance of fur and feces), elimination of cysts in feces and behavioral testing were evaluated. All variables from the groups of infected animals were compared with the negative control groups according to their lineage/sex. The experimental groups, the steps and chronology of the experiments are described in the following sub-items.

Ethical aspects
This study was approved by the Ethics Committee on the Use of Animals (CEUA/UEM) of the State University of Maringá -Brazil (process number 9375170816) and all the guidelines of the Brazilian Society of Sciences in Laboratory Animals were followed.

Animals
Were used 150 mice (Mus musculus), 50 from each lineage (Swiss, Balb/c and C57BL/6), being 25 females and 25 males, 21 days old, with no statistical difference in the average weight of the animals in each group. The Swiss and Balb/c animals were from the Central Vivarium of the State University of Maringá (Universidade Estadual de Maringá -UEM) and the C57BL/6 mice were from the Central Vivarium of the State University of Western Paraná (Universidade Estadual do Oeste do Paraná -Unioeste).
The animals were maintained in the vivarium of the Environmental and Food Parasitology Laboratory at the State University of Maringá (LPAA/UEM) and placed into micro-acclimated cages (Alesco®; polysulfone cages of 20 cm width x 32 cm length x 21 cm height). During the experiments, there was control of temperature (22.7 ± 1.2 ºC), 12h/12h light/dark cycle, with filtered/boiled water and feed (Nuvital® Nuvilab Cr-1) offered ad libitum.

Experimental groups
The mice were distributed into 30 groups with five animals each, according to the lineage and sex. The experiments were conducted in three different periods, as described in Table 1.

Parasitological evaluation and molecular diagnosis
Feces from each animal were collected individually. The samples were processed by the method of Faust et al. (1938), in order to analiser the presence of pathogens in the intestinal tract, such as, cysts of Giardia muris. The analysis were performed for three consecutive days taking into account the intermittent elimination of cysts in the feces, to verify the absence or presence of Giardia.
A pool of fecal samples (1 g) from each group was processed using the modified Ritchie method (Bezagio et al. 2020). The recovered Giardia cysts were quantified in a Neubauer chamber, in triplicate, and the results were obtained by calculating the simple average. The samples were classified as low, medium or high parasitic load according to the standards established by Uda-Shimoda et al. (2014).
The presence of G. muris was also investigated through Polymerase Chain Reaction (PCR) using primers G18S2 and G18S3, which amplify a fragment of 470 base of pairs (bp) of the 18S ribosomal RNA gene, according to Monis et al. (1999): 94°C for 2 minutes; 35 cycles 94°C for 30 seconds; 55°C for 30 seconds; 72°C for 1 minute; ending with 55°C for 7 minutes.
The amplified product was visualized in polyacrylamide gel (4.5%) and revealed by silver salts.
The animals with positive parasitological/molecular results for G. muris (including negatives control groups) were etiologically treated according to the protocol established by Bezagio et al. (2017): Fembendazole 50 mg/kg -1× only on the first day and metronidazole 500 mg/kg -1×/day/7 days. The treatment was administered intragastrically using a gavage probe.
Research, Society and Development, v. 10, n. 10, e493101019250, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10. 33448/rsd-v10i10.19250 5 The materials which came into contact with the animals were sterilized daily and the animals were maintained in microacclimated cages on an acrylic support with holes that avoided contact with their own feces.
After the end of treatment, feces samples were individually collected for analysis during three consecutive days. Animals with negative results by the method of Faust et al. (1938) and by the molecular method were considered cured.

Inoculum and infection with cysts of G. duodenalis
The cysts of G. duodenalis used in this study were isolated from recently eliminated human fecal samples, with approval from the Research Ethics Committee Involving Human Beings (COPEP / UEM-439/2009), which were genotyped by PCR-RFLP as assemblage BIV and preserved at -20 ºC in the LPAA/UEM.
In order to determine the viability of G. duodenalis cysts, the Trypan Blue test was performed. This dye at 0.4% was added to a concentrate of cysts previously quantified in a 1:1 ratio (Strober, 2015).
The inoculum was prepared with 0.85% saline solution so that the concentration was 10 4 viable G. duodenalis cysts (Nakada et al. 2018) in a volume of 300 µl/animal. The animals in the negative-control group received the same volume (300 µl/animal) of saline solution. Administration was performed intragastrically, using a gavage probe.

Assessment of clinical signs
During the 15 days following the infection, the animals were monitored and evaluated daily for the parameters: quantitative (body weight, water and feed intake and elimination of excreta) and qualitative (appearance of fur and feces).

Elimination of cysts
A pool of feces samples from each group of infected animals were daily collected and processed by the method of Faust et al. (1938), until cysts of Giardia spp. were observed by microscopy and positive molecular method, or during the 15 days following infection.
DNA was extracted from the cysts, using the fragment kit PureLink PCR Purification® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations and Uda-Shimoda et al. (2014). The glutamate dehydrogenase gene (GDH) was amplified by the Semi-Nested PCR method with modifications proposed by Colli et al. (2015), for identification of G. duodenalis. The external forward primer GDHeF, internal forward primer GDHiF and reverse primer GDHiR were used to amplify a fragment of approximately 432 bp. The amplified product was visualized in polyacrylamide gel (4.5%) and revealed with silver salts.

Behavioral testing -Open Field Test (OFT)
It was performed on the 7th day postinfection (dpi) (Pavanelli et al., 2018). The open field consisted of a circular arena of 30 cm in diameter surrounded with opaque walls of 30 cm high, composed of a central area of 15 cm in diameter divided into four quadrants and a peripheral area divided into eight quadrants. Individually, the animals were placed in the center of the arena and freely explored the open field for a period of 5 minutes.
The sessions were recorded on video (Webcam LifeCam Cinema HD 720p Microsoft® using Microsoft LifeCam software version 3.22) and blindly analyzed using Open Field software. The analysis included central locomotion, peripheral locomotion, total locomotion and time spent in the central region and time spent in the peripheral region.

Necropsy and assessment of intestinal mucosa
After evaluating the pre-patent period, all mice were anesthetized with inhalable isuflurane and euthanized by craniocervical dislocation. The small intestine of two infected animals from each group, randomly chosen, was removed by opening the peritoneal cavity with a median insertion and washed with 0.85% saline. The intestines were longitudinally sectioned, dipped into 25 ml of 0.85% saline and scraped using a glass slide. A portion of fresh sample was examined by Direct Microscopy for searching of trophozoites in the intestinal mucosa.

Statistical analysis
The data were tabulated and analyzed using the GraphPad Prism software version 7.0 (GraphPad Software Inc., La Jolla, CA, USA). The analysis was performed by bidirectional ANOVA. When statistical differences were identified by twoway ANOVA, Bonferroni's post hoc test was performed to assess specific differences between the experimental groups. A pvalue was considered significant when p < 0.05.

Results
In the experiment #01, all 50 Swiss mice (100%), male and female, presented positive results for G. muris at the beginning, classified as high parasite load (more than 2 cysts/field on the 20x objective) as confirmed by amplification the minor ribosomal subunit gene (G18S). After the etiological treatment, only the male Swiss animals achieved the cure criteria, in which G. muris cysts were not observed in the parasitological examination and amplification of the parasite's DNA by the molecular method did not occur. In the experiments #02 and #03, the Balb/c and C57BL/6 mice had negative parasitological results for the three days of analysis. In the three experiments, the results obtained by PCR confirm 100% of the results obtained by the parasitological exams performed initially, as shown in Table 2. Female Swiss The averages of feed (Table 3) and water (Table 4) consumption, elimination of excreta (Table 5) and body weight, per animal, were calculated on days 1, 3, 6, 9, 12 and 15. For the general average of days, there was a difference only for the male Swiss mice, in which the infected animals ate more (p < 0.0001), and eliminated a greater amount of excreta (p < 0.0001) when compared with the non-infected animals. The overall mean water consumption of infected male Swiss was not significantly higher, but a significant increase was noted on days 9,12 and 15. Regarding the average body weight, the male Swiss mice were the only groups with a significant difference, in which the non-infected animals had an average weight greater than the infected ones on the 15th day (p = 0.0004). No diarrheal feces were observed. Some male and female Swiss mice, both infected and non-infected, had bristly hair in the first 4 days after infection. *p-value is considered significant when p < 0.05. All p-value were adjusted according to Bonferroni's multiple comparisons test. **The difference in means between the negative control and infected groups is significant The average feed consumption corresponds to the overall average consumption of each animal according to the day, individually, expressed in grams of feed/animal. It is important to note that on a few isolated days, the feed consumption of the infected and non-infected groups, within the lineage/sex itself, showed a significant difference, in which on some days the infected group consumed more and on others the group negative control, however, the difference in the overall average consumption of all days was not significant. In the group of male Swiss animals, the feed consumption of the infected group was significantly higher than the negative control both in general average and in the days 1, 3, 6, 9 and 15. Source: Authors. *p-value is considered significant when p < 0.05. All p-value were adjusted according to Bonferroni's multiple comparisons test. **The difference in means between the negative control and infected groups is significant The average water consumption corresponds to the general average consumption of each animal according to the day, individually, expressed in milliliters of water/animal. It is important to note that on a few isolated days, the water consumption of the infected and non-infected groups, within the lineage/sex itself, showed a significant difference, in which on some days the infected group consumed more and on others the group negative control, however, the difference in the overall average consumption of all days was not significant. In the group of male Swiss animals, the water consumption of the infected group was significantly higher than the negative control both in general average and in the days 9, 12 and 15. Source: Authors. *p-value is considered significant when p < 0.05. All p-value were adjusted according to Bonferroni's multiple comparisons test. **The difference in means between the negative control and infected groups is significant The average excreta elimination corresponds to the general average of excretion of each animal according to the day, individually, expressed in grams of excreta/animal. It is important to note that on a few isolated days, the elimination of excreta of the infected and non-infected groups, within the lineage/sex itself, showed a significant difference, in which on some days the infected group eliminated more and on others the group negative control, however, the difference in the overall average excreta elimination of all days was not significant. In the group of male Swiss animals, the elimination of excreta of the infected group was significantly higher than the negative control both in general average and in the days 1, 3, 9, 12 and 15. Source: Authors.
The OFT performed on the 7th dpi revealed that there was no difference in the behavioral parameters between the groups of infected animals when compared with the non-infected ones, for the studied lineages.
At the end of the experiments, the analysis of the intestinal mucosa of the animals evidenced trophozoites of Giardia spp. in both male and female Swiss mice, with more than 10 trophozoites/field, being classified as high parasitic load. In both male and female Balb/c and C57BL/6 mice, no trophozoites were observed in the intestinal mucosa.

Discussion
As far as we know, this is the first work that aimed to investigate which is the most suitable murine experimental model for studying human giardiasis, by comparing parasitological, molecular and clinical parameters and the pre-patent period of infection with G. duodenalis BIV in the lineages of Mus musculus (Swiss, Balb/c and C57BL/6). Only male Swiss mice acquired the infection, which was confirmed by parasitological and molecular methods, with a pre-patency period that ranged from the 6th to the 8th dpi, with significantly greater consumption of feed, water and excreta elimination when compared with non-infected animals. This indicates that infection with G. duodenalis alters the animal's physiology, and is consistent with what can also occur in humans infected with this parasite, especially children.
The elimination of the first cysts of G. duodenalis in MS1 and MS2 groups occurred on the 6th dpi. This result is in agreement with Buret et al. (1991), who also reported this pre-patency period; however, it differs from other studies that report elimination of cysts from the 3rd dpi (Pavanelli et al., 2018;Ware & Villegas, 2019). Some studies show that this period can vary from one to two weeks (Astiazarán-García et al., 2000;Soares et al., 2008). In MS3 group, the elimination of the first cysts occurred on the 8th dpi. This difference in the pre-patent period may be associated with host factors, such as immune response (Scott et al., 2000).
The alterations in clinical parameters regarding the consumption of feed, water, and greater elimination of excreta found in groups of animals that developed infection with G. duodenalis, are in agreement with the findings of Bezagio et al. (2017), when Swiss mice infected with G. muris were evaluated. Animals from groups that did not develop the infection also presented alterations in these parameters, raising the hypothesis that the animals' physiology and immune response may also be involved in this process. Since there was no specific pattern of differences between the groups with infected and noninfected animals, and as in most cases the alterations in these animals were not significant and when they were, they occurred on isolated days, it makes suggesting that the simple presence of the antigen could have led to these alterations.
Changes in these parameters, more intensely in animals that developed the infection, are probably related to the pathogenic action of the parasite. The trophozoites attached to the mucosa form a carpet and act as a physical barrier that hinders the absorption of water and nutrients. It can be aggravated by injury or atrophy of intestinal microvilli (Pavanelli et al., 2018), as it leads to a significant decrease in enzyme activities (Scott et al., 2000;Halliez & Buret, 2013). In an attempt to supply the nutritional deficiency caused by the difficulty of absorption, the animals increase the intake of water and feed, and due to the reduced absorption, the amount of excreta becomes greater. Change in the composition of the intestinal microbiota, lesions in the intestinal epithelium of the host and increase in the mucus production of male Swiss mice infected with assemblages AII and BIV of G. duodenalis (Pavanelli et al., 2018) reinforce a pathogenic action of the presence of the parasite and confirm the idea of using this sex and lineage as an experimental model for human giardiasis.
Cysts of G. duodenalis BIV did not infect Balb/c and C57BL/6 mice, even with inoculum of 10 4 parasites. Infection with cysts in these two lineages was reported in malnourished C57BL/6 ( Bartelt et al., 2013), in BALB/cAnUnib Specified Pathogen Free (SPF) (Nakada et al., 2018), and also in immunosuppressed CF1 heterogeneous mice (Ware & Villegas, 2019), indicating that nutritional status and other environmental factors during the maintenance of mice directly influence their capacity to resist to pathogens (Mizutani et al., 2017;Vaidya, 2017). The host's immune response is also a determining factor for infection. The intestinal microbiota is an efficient defense mechanism of the mice's innate immune system and commensal bacteria have an anti-Giardia effect through competition, direct toxicity or modulation of the immune response (Goyal et al., 2013). Infection can be favored if, for example, SPF mice have absence or deficiency in their first line of defense. These Infection with G. duodenalis in C57BL/6 mice was also reported when 10 6 trophozoites were administered by gavage (Li et al., 2014), or when these animals had concomitant infection with other parasite (Von-Allmen et al., 2006). Some authors report that cysts of G. duodenalis of assemblages A and B are not able to naturally infect mice and rats (Lebbad et al., 2010;Zhao, 2015), but the inoculation of trophozoites can lead to infection (Lemée et al., 2000). In this work, we opted for inoculation of cysts because this is the infectious form in humans, and according to Pavanelli et al. (2018), the conditions for testing the parasite in an animal model are different from those that occur in culture.
For the behavioral parameters, there was no difference between the groups of infected animals and the non-infected ones. Increased exploratory behavior in animals can occur in response to pain (Pavanelli et al., 2018). Baker (2006) describes that when the parasitic load in the animal is very high, some degree of inappetence and lethargy may occur, indicating the need for further studies with regard to animal behavior throughout the infection.
One difficulty and limitation of our study is that all Swiss mice, both male and female, came from the vivarium of the institution of origin naturally infected with G. muris, presenting a high parasitic load according to the classification of Uda-Shimoda et al. (2014). High infection rate with this parasite has also been reported in mice and rats produced in vivariums of Brazilian institutions (Bicalho et al., 2007;Bezagio et al., 2017;Almeida et al., 2021). As this parasite can cause an inflammatory response in the intestinal mucosa with increased mucus production and alterations in the animal's imune response, as well as alterations in clinical signs (Bezagio et al., 2017), the animals were etiologically treated before being infected with G. duodenalis. On the other hand, this treatment increases the experimentation time, making the animals to come into contact with the anti parasitic drugs, previously to the experimental infection, but this seems to be a reality in the vivariums of Brazilian institutions. However, it was possible to obtain cure and infection with G. duodenalis in the male Swiss animals, indicating that they are the most adequate experimental model for studies of human giardiasis. On the other hand, the Balb/c mice, which had the same origin as the Swiss mice, and the C57BL/6 mice from another institution, were not infected with G. muris, and did not get infected with G. duodenalis, presenting a lower susceptibility to infection, which varies according to sex, age, weight, lineage, among other factors inherent to the host.

Conclusion
It is concluded that male Swiss (Mus musculus) is the most suitable experimental model for the study of human giardiasis assemblage BIV, as it is the only lineage, compared with the other lineages studied, capable of becoming infected and eliminating G. duodenalis cysts in the feces. In this model, significant alterations in clinical parameters were observed in infected animals when compared with uninfected animals, signaling that important mucosal lesions, similar to the lesions present in human infection, may be occurring.
Once we were able to observe the resistance of Balb/c and C57BL/6 mice to develop infection by G. duodenalis from the intragastric inoculation of the cysts, new research and study possibilities arise, aiming to elucidate the resistance mechanisms presented by these animals. Knowing the mechanisms by which these mice destroy the parasite and prevent the infection from taking hold can be of great use, especially for researching new drugs for the treatment of human giardiasis.

Financing
This work was carried out with the support of the Coordination for the Improvement of Higher Education Personnel -(CAPES) -Brazil -Financing Code 001.