Introduction
Toxocariasis is a worldwide parasitic zoonosis that occurs in both developing and developed countries (Hotez & Wilkins, Reference Hotez and Wilkins2009). However, the prevalence and impact of toxocariasis on public health are underestimated due to the difficultly of diagnosis (Smith et al., Reference Smith, Holland, Taylor, Magnaval, Schantz and Maizels2009). Toxocariasis is a tissue parasitosis; in humans, Toxocara canis larvae migrate in the tissues for long periods, which can cause syndromes ranging from asymptomatic to systemic forms (Macpherson, Reference Macpherson2013). The clinical presentations of toxocariasis are visceral toxocariasis, ocular toxocariasis and neurological toxocariasis, and subclinical manifestations may occur as a hidden toxocariasis (Magnaval et al., Reference Magnaval, Glickman, Dorchies and Morassin2001). The current pharmacological treatments have moderate efficiency (Magnaval et al., Reference Magnaval, Glickman, Dorchies and Morassin2001; Othman, Reference Othman2012). Therefore, it is necessary to develop treatments to prevent and avoid infection or reinfection (Magnaval & Glickman, Reference Magnaval, Glickman, Holland and Smith2006).
Since the first registered cases (Beaver et al., Reference Beaver, Snyder, Carrera, Dent and Lafferty1952), the nematode T. canis has been the etiological agent most frequently associated with human toxocariasis (Quattrocchi et al., Reference Quattrocchi, Nicoletti, Marin, Bruno, Druet-Cabanac and Preux2012). This geohelminth presents a complex biological cycle that involves multiple forms of transmission. The definitive hosts of T. canis are canids, mostly dogs; all mammals can play the role of paratenic hosts. Humans may be considered as accidental paratenic hosts (Glickman & Schantz, Reference Glickman and Schantz1981). Thus, the main prophylactic measures for human toxocariasis are based on avoiding the accidental intake of T. canis embryonated eggs (Epe, Reference Epe2009; Amaral et al., Reference Amaral, Rassier, Pepe, Gallina, Villela, Nobre, Scaini and Berne2010). There are also specific measures to prevent infection with larvae present in the meat or viscera of bird and mammal species that have roles as paratenic hosts (Dutra et al., Reference Dutra, Pinto, da Costa de Avila, de Lima Telmo, da Hora, Martins, Berne and Scaini2013; Cardoso et al., Reference Cardoso, Walcher and da Silva Cadore2020).
However, despite the importance of the route of vertical transmission in dogs, control of this infection route is difficult, in part, because the recommended doses of anthelmintic are not highly effective against dormant somatic larvae (Macpherson, Reference Macpherson2013; Overgaauw & Knapen, Reference Overgaauw and Knapen2013). This type of transmission has also been observed in paratenic hosts, such as mice (Lee et al., Reference Lee, Min and Soh1976; Reiterová et al., Reference Reiterová, Tomašovicová and Dubinský2003; De Souza Aguiar et al., Reference De Souza Aguiar, Furtado, de Avila, de Lima Telmo, Martins, Berne, da Silva and Scaini2015).
The intestines are a highly complex ecosystem in which nutrients, microbiota and host cells interact in a balance that determines health maintenance. Thus, there is interest in using probiotics to modulate the intestinal microbiota and prevent or treat diseases (Butel, Reference Butel2014). Trials with probiotics have shown promising results, including tissue parasite control of Trichinella spiralis (Bautista-Garfias et al., Reference Bautista-Garfias, Ixta-Rodríguez, Martínez-Gómez, López and Aguilar-Figureueroa2001) and T. canis (Basualdo et al., Reference Basualdo, Sparo, Chiodo, Ciarmela and Minvielle2007; Chiodo et al., Reference Chiodo, Sparo, Pezzani, Minvielle and Basualdo2010). Furthermore, the yeast Saccharomyces boulardii has been found to reduce the number of T. canis larvae in tissues during the initial and chronic toxocariasis phases (Avila et al., Reference Avila, Conceição, Telmo, Dutra, de Los Santos, Martins, Berne, da Silva and Scaini2012, Reference Avila, Leon, Moura, Berne, Scaini and Leivas Leite2016) and to reduce the intensity of infection in mice caused by the consumption of ser infected with T. canis (Cardoso et al., Reference Cardoso, Walcher and da Silva Cadore2020). However, no studies have evaluated the effects of probiotics on larvae vertical transmission.
In consideration of the complexity of the T. canis biological cycle and the possibilities of probiotic biotherapeutics (Avila et al., Reference Avila, Conceição, Telmo, Dutra, de Los Santos, Martins, Berne, da Silva and Scaini2012, Reference Avila, Leon, Moura, Berne, Scaini and Leivas Leite2016; Cardoso et al., Reference Cardoso, Walcher and da Silva Cadore2020), this study evaluated the effects of S. boulardii probiotic on the vertical transmission of T. canis in mice.
Materials and methods
Probiotic S. boulardii and food administration
The probiotic S. boulardii CNCM I-745 was cultivated in yeast peptone dextrose medium and incubated in a shaker (150 rpm) at 37°C for 72 h. The culture was then centrifuged and washed with sterile phosphate-buffered saline. To add the probiotic supplement to the food, the food was triturated, manipulated and remoulded into pellets. The pellets were then dried in a greenhouse with forced air circulation (Avila et al., Reference Avila, Leon, Moura, Berne, Scaini and Leivas Leite2016).
Collection and incubation of the eggs and larvae of T. canis
Young dogs naturally infected with T. canis were treated orally with pyrantel pamoate (12.5 mg/kg) for the recovery of adult worms. Eggs were obtained directly from the uterine tubes of female adults T. canis worms and incubated for 30 days in 2% formalin under a humidity greater than 80% and a temperature of 28°C with oxygenation (Avila et al., Reference Avila, Conceição, Telmo, Dutra, de Los Santos, Martins, Berne, da Silva and Scaini2012).
Experimental design
Two groups (G1 and G2) of eight female Swiss mice (outbred) aged five and seven weeks were established, and four males aged ten weeks were used for mating. The animals were housed in a controlled environment at 24°C (±1°C). The light/dark cycle was 12/12 h, and the animals had access to feed and water ad libitum. The mice were obtained from the central vivarium of the Federal University of Rio Grande.
The G1 mice received food containing 107 colony-forming units of S. boulardii per gram of food for 15 days before mating, during pregnancy and through 21 days of lactation. The G2 females received food without probiotics during the same periods.
Each G1 and G2 female was mated with a male mouse, and mating was confirmed by the presence of a vaginal plug. The pregnant females were housed in individual cages and inoculated with 300 T. canis embryonated eggs through an intragastric probe on the 14th day of pregnancy. After birth, the females were housed with their offspring for 21 days of lactation. The lactating females and their offspring were then euthanized, and brain, liver, lungs, heart, kidneys, spleen, eyes and carcasses were collected. The mammary glands from the lactating females were also collected. Each organ was macerated, and the tissue was digested with 1% pepsin and hydrochloric acid to recover T. canis larvae according to the methodology described by Xi & Jin (Reference Xi and Jin1998).
The following data were recorded: (1) larvae transmitted, defined as the number of larvae recovered in the offspring; (2) larvae retained in females, defined as the number of larvae recovered in the females; and (3) total larvae, defined as the number of larvae present in the females plus the number of larvae recovered from the offspring. To determine the transmission rate of T. canis larvae to offspring, the total number of larvae was considered 100%. Then, the number of larvae present in the offspring was used to determine the proportion of larvae transmitted – that is, the transmission rate (%).
The Shapiro–Wilk test was initially applied to all data to assess normality. The Mann–Whitney test was used to analyse the non-Gaussian data (recovery of larvae in lactating females and their offspring), and Test T was used to analyse the transmission rate (Gaussian samples). A significance level of 95% was used in both tests performed using GraphPad Prism version 7.0 (GraphPad Software, La Jolla, CA, USA).
Results
The addition of S. boulardii probiotic to the food of G1 mice reduced the transmission rate of larvae to offspring by 42% (P = 0.041) (table 1). However, there was no significant difference between the groups in the total number of larvae (P = 0.817) or the number of non-transmitted larvae (retained larvae) (P = 0.837). The examination of T. canis larvae distribution in the offspring revealed larval tropism for the brain and carcass. A 50% reduction (P = 0.023) in the number of larvae in the brain were observed in G1 offspring relative to G2 offspring (fig. 1).
Table 1. Distribution of larvae recovered from lactating females (retained larvae), offspring (transmitted larvae), total larvae recovered and percentage of larvae transmission from lactating to offspring.
G1, group supplemented with probiotic Saccharomyces boulardii; G2, control group; SD, standard deviation.
Different letters in the same column indicate a significant difference (P < 0.05).
Fig. 1. Toxocara canis larvae distribution in the offspring after vertical transmission in the G1 (Saccharomyces boulardii, n = 62) and G2 (control, n = 65) groups. SD, standard deviation. Different letters indicate significant differences.
The distribution of retained larvae in lactating females was evaluated, and tropism for brain was observed. The same average number (6.25) was observed in both groups. There was no statistically significant difference in the number of somatic larvae recovered in lactating females in the G1 and G2 groups (P = 0.088). In G1 females, there were larvae in the brain, liver, lungs, heart, kidneys, mammary glands and carcass. In G2, there was larval retention in the brain, liver, kidneys, mammary glands and carcass (fig. 2).
Fig. 2. Toxocara canis larvae distribution in G1 (Saccharomyces boulardii) and G2 (control) lactating females (n = 8). To investigate potential tropism and the difference in somatic larvae distribution, liver, lungs, heart, kidneys, mammary glands and skeletal muscle (carcass) were analysed. SD, standard deviation.
There was no significant difference between the sizes of the offspring between groups G1 and G2 (P = 0.621), which presented an average of 7.7 (±2.6) and 8.1 (±1.7) offspring generated per female in the G1 and G2 groups, respectively, yielding a total of 62 offspring in G1 and 65 in G2. The percentage of positive offspring for T. canis larvae was 46.77% and 56.96%, respectively, in groups G1 and G2 (P = 0.492).
Discussion
This study is the first to demonstrate the potential of probiotics to reduce the vertical transmission rate of T. canis larvae. A reduction of 42% (P = 0.041) in the transmission rate of T. canis larvae to offspring was observed in G1 group relative to G2 group, and 50% reduction in the number of larvae in the brains of the offspring was observed in G1 relative to G2 (P = 0.023). These results corroborate previous studies demonstrating promising results of probiotics Enterococcus faecalis CECT7121 (Basualdo et al., Reference Basualdo, Sparo, Chiodo, Ciarmela and Minvielle2007; Chiodo et al., Reference Chiodo, Sparo, Pezzani, Minvielle and Basualdo2010), Lactobacillus rhamnosus (Walcher et al., Reference Walcher, Cruz, Telmo, Martins, Avila, Berne and Scaini2018) and S. boulardii (Avila et al., Reference Avila, Conceição, Telmo, Dutra, de Los Santos, Martins, Berne, da Silva and Scaini2012, Reference Avila, Leon, Moura, Berne, Scaini and Leivas Leite2016; Cardoso et al., Reference Cardoso, Walcher and da Silva Cadore2020) in altering T. canis larvae infection intensity.
Despite a reduction in the transmission rate observed in offspring in the probiotic-treated females, no significant difference in the total number of larvae recovered was observed between the S. boulardii-treated and control females. Similarly, there was no significant difference between groups in the total number of larvae, which represents the number of larvae expected in females in the absence of pregnancy or lactation. These results are inconsistent with the findings of previous studies, which demonstrated a reduction in the number of T. canis larvae in mice S. boulardii supplementation (Avila et al., Reference Avila, Conceição, Telmo, Dutra, de Los Santos, Martins, Berne, da Silva and Scaini2012, Reference Avila, Leon, Moura, Berne, Scaini and Leivas Leite2016; Cardoso et al., Reference Cardoso, Walcher and da Silva Cadore2020). However, in this study, unlike previous ones, the females were in the reproductive period, which can alter the immune system (Mor & Cardenas, Reference Mor and Cardenas2010; Nauta et al., Reference Nauta, Ben Amor, Knol, Garssen and Van der Beek2013; Alijotas-Reig et al., Reference Alijotas-Reig, Llurba and Gris2014). In this study, inoculation of embryonated eggs occurred at a time (the final third of pregnancy) favourable for nematode survival (Oshima, Reference Oshima1961; Glickman & Schantz, Reference Glickman and Schantz1981; Strube et al., Reference Strube, Heuer and Janecek2013). Therefore, the physiological and immunological conditions during pregnancy and lactation and the parasite characteristics might have influenced the difference in infection intensity between G1 and G2 females (P > 0.05).
However, we revealed an influence of S. boulardii probiotic use on the vertical transmission of T. canis larvae in mice. The decrease in the transmission rate of larvae in offspring, despite the absence of a larval reduction in G1 females, indicates that there may have been a delay or decrease in larval migration in the G1 group, resulting in a lower level of larval transmission to offspring. However, there was no significant difference between the number of somatic larvae recovered in females in groups G1 (25 larvae) and G2 (eight larvae) (P = 0.088).
This microorganism has been revealed capable of modulating the mucosal response in mice (Moura et al., Reference Moura, Terto, Jeske, de Castro, Pinto, Avila, Leivas Leite and Berne2017). When present in the intestine, probiotics can stimulate specialized cells of the epithelium associated with lymphoid follicles (M cells) and dendritic cells located in Peyer's patches. These cells are important components of the gut-associated lymphoid tissue that functions as a trigger to activate immunological systemic responses (Jung et al., Reference Jung, Hugot and Barreau2010). This capacity may allow the probiotics to function as immunomodulators in distant anatomic sites and induce responses beyond the gut mucosa (Clancy, Reference Clancy2003). Although these systemic effects are probably insufficient to eliminate T. canis larvae in pregnant females, they can interfere with larval migratory capacity and reduce their transmission to offspring. These effects may derive from the ability of S. boulardii to increase the levels of pro-inflammatory interleukins IL-12 and IFN-γ (Avila et al., Reference Avila, Leon, Moura, Berne, Scaini and Leivas Leite2016; Moura et al, Reference Moura, Terto, Jeske, de Castro, Pinto, Avila, Leivas Leite and Berne2017), which can favour the formation of granulomas and thus decrease larval migration capacity; the increase of IL-12 is pointed out as the basis for the protective mechanism of this probiotic against T. canis (Avila et al., Reference Avila, Leon, Moura, Berne, Scaini and Leivas Leite2016).
The two groups of lactating females exhibited the same average number of larvae in brain (6.25), which is an anatomic site with immunological weakness. In the brain, T. canis larvae are protected from host immune system action, and larvae in the chronic phase of infection are commonly present in the brain (Oshima, Reference Oshima1961; Glickman & Schantz, Reference Glickman and Schantz1981; Jin et al., Reference Jin, Akao & and Ohta2008; Strube et al., Reference Strube, Heuer and Janecek2013), likely promoting their persistence until their transfer to the next generation (Dunsmore et al., Reference Dunsmore, Thompson and Bates1983).
Our results highlight the importance of somatic larvae to vertical transmission and revealed high heterogeneity in larval distributions in different tissues between the two groups. In both groups, larvae were found in the mammary glands, with slightly higher numbers being observed in G1 than G2. This finding can be attributed to the ability of the probiotic to interfere with larval migration. This result has significance, given the importance of transmammary transmission observed in BALB/c mice with chronic toxocariasis (De Souza Aguiar et al., Reference De Souza Aguiar, Furtado, de Avila, de Lima Telmo, Martins, Berne, da Silva and Scaini2015). Saccharomyces boulardii yeast exhibits potential for decreasing the vertical transmission of visceral toxocariasis. The use of this probiotic reduced the transmission rate of T. canis larvae transmitted to the offspring of females infected during pregnancy and the number in the brains of offspring. Additional studies should be conducted to elucidate the mechanisms and the impacts of probiotic use on the biological cycles and survival strategies of T. canis.
Financial support
This work was supported by the National Council for Scientific and Technological Development (CNPq), Brazil
Conflicts of interest
None.
Ethical standards
This study was approved by the Ethics Committee on Animal Use of Federal University of Rio Grande (report number 055/2011), and all experiments were performed according to Brazilian legislation on animal care.
