Protective effect of the probiotic Lactobacillus acidophilus ATCC 4356 in BALB/c mice infected with Toxocara canis

ABSTRACT

Human toxocariasis consists of chronic tissue parasitosis that is difficult to treat and control. This study aimed to evaluate the action of the probiotic Lactobacillus acidophilus ATCC 4356 on larvae of Toxocara canis and the effect of IFN-γ cytokine on parasite-host in vivo (1.10⁹ CFU) and in vitro (1.10⁶, 1.10⁷, 1.10⁸, 1.10⁹ CFU) interactions. Four groups of six BALB/c mice were formed: G1 - L. acidophilus supplementation and T. canis infection; G2 - T. canis infection; G3 - L. acidophilus supplementation; and G4 - PBS administration. Mice were intragastrically suplemented with probiotics for 15 days before inoculation and 48 h after inoculation with 100 T. canis eggs. The inoculation of T. canis was also perfomed intragastrically. The recovery of larvae took place through digestion of liver and lung tissues; the evaluation of IFN-γ gene transcription in leukocytes was performed by qPCR. The in vitro test consisted of incubating the probiotic with T. canis larvae. The supplementation of probiotics produced a reduction of 57.7% (p = 0.025) in the intensity of infection of T. canis larvae in mice, whereas in the in vitro test, there was no larvicidal effect. In addition, a decrease in the IFN-γ gene transcription was observed in both, T. canis-infected and uninfected mice, regardless of whether or not they received supplementation. The probiotic L. acidophilus ATCC 4356 reduced T. canis infection intensity in mice, however, the probiotic did not have a direct effect on larvae, demonstrating the need of interaction with the host for the beneficial effect of the probiotic to occur. Yet, the proinflammatory cytokine IFN-γ did not apparently contributed to the observed beneficial effect of probiotics.

Toxocariasis; Probiotic; Innate immunity; Cytokine; INF-γ

INTRODUCTION

Human toxocariasis involves chronic tissue parasitosis, is globally distributed and is most prevalent in countries with tropical climate¹. The main etiological agents are the nematodes Toxocara canis and Toxocara cati, intestinal parasites of dogs and cats, respectively. In humans, larvae of these nematodes are incapable of reaching maturity they undergo tissue migration affecting various organs and causing lesions of varied degrees of severity. The main form of infection in humans is through the ingestion of embryonic eggs of the parasite, but it can also occur through the ingestion of larvae present in meat or viscera of paratenic hosts, ingested raw or undercooked²,³. This parasitosis is considered a neglected disease that is prevalent in some developed⁴ and developing countries⁵.

In the host immune response triggered by T. canis, the parasite switches the Th1 to Th2-type response⁶. Studies using murine models have shown that in the early phase of T. canis infection, there is an increase in IFN-γ, TNF-α and IL-12 cytokine levels⁷,⁸. During infection, T. canis modulates the immune response by suppressing proinflammatory cytokines production and stimulating the production of cytokines by Th2 and Treg cells⁶,⁹. This modulation is a form of parasite evasion from the host immune response as proinflammatory cytokines, such as IFN-γ and IL-12, are important macrophages activators that act in the defense against the nematode⁸,¹⁰-¹².

Human toxocariasis is a parasitosis that is difficult to treat due to the moderate effectiveness of drugs on the encysted larvae of Toxocara spp. in tissues, making it necessary to investigate other forms of control¹³-¹⁵. An alternative treatment is the use of probiotics, which have demonstrated potential to control murine visceral toxocariasis¹⁶-²⁰. One of the modes of action of probiotics is through their capacity to counterbalance the Th1-, Th2- and Treg-type immune responses²¹ and by their ability to inhibit the adhesion of pathogenic microorganisms to the intestinal epithelium through the production of antimicrobial substances such as short chain fatty acids, ammonia, hydrogen peroxide and bacteriocins²².

Probiotics of the genus Lactobacillus contribute to reduce the luminal pH by stimulating the growth of epithelial cells and increasing the blood flow, modifying the intestinal motility, improving the absorption of water and minerals and increasing mucus production²³. In addition, in vivo studies with Lactobacillus spp. have shown that these probiotics are capable of stimulating the Th1-type immune response, with increased IFN-γ, IL-12 and TNF-α cytokines production²⁴-²⁹, which may also be beneficial for the control of visceral toxocariasis⁸. This study aimed to evaluate the action of the probiotic Lactobacillus acidophilus ATCC 4356 on the infection intensity of T. canis in BALB/c mice and the effect of the proinflammatory cytokine IFN-γ on the parasite-host interactions.

MATERIALS AND METHODS

Mice

Male BALB/c mice of five to seven weeks of age were maintained under controlled environmental conditions at 22 °C (± 1 °C), with a 12 h light and 12 h dark cycle, and access to food and water ad libitum. This project was approved by the Animal Use Ethics Committee of the Federal University of Rio Grande (P039/2016, P078/2016).

Probiotic Lactobacillus acidophilus

The probiotic L. acidophilus ATCC 4356 obtained from the Fundacao Oswaldo Cruz in Rio de Janeiro/RJ - Brazil (FIOCRUZ) was cultivated in Man, Rogosa and Sharpe (MRS) broth for 48 h at 37 °C. It was then centrifuged at 4000 x g for 10 min and resuspended in sterile phosphate-buffered saline (PBS). Next, the colony-forming units (CFU) were determined. Probiotic production, quality control, culture viability, colony counts and purity were evaluated according to the methodology employed by Walcher et al.²⁰.

Collection and incubation of the eggs and larvae of Toxocara canis

Five to eight weeks-old dogs naturally infected with T. canis were treated orally with pyrantel pamoate (15 mg/kg) to recover adult specimens of T. canis. The adult forms of T. canis were sexed and the females were submitted to hysterectomy to obtain eggs, which were incubated in 2% formalin at 28 °C, with humidity greater than 80% and oxygenation for 30 days¹⁹. Following the eggs embryonation, T. canis larvae were extracted and incubated¹²,³⁰.

In vitro evaluation of the probiotic Lactobacillus acidophilus

The probiotic L. acidophilus was tested at concentrations of 1.10⁶, 1.10⁷, 1.10⁸ and 1.10⁹ CFU and incubated at concentrations of 100 larvae/well (microculture plate TPP^®) in RPMI-1640 medium supplemented with 25 mM HEPES, 1% glucose, 100 IU/mL penicillin, and 50 μg/mL streptomycin) at 37 °C with 5% CO₂ for 48 h, in triplicate³¹.

After the plate incubation, 0.2% Trypan blue cell viability indicator (Vetec^®) was added to all wells in the plate, and the material was incubated for an additional 30 min. Control RPMI-1640 medium with live T. canis larvae and control dead larvae (frozen at -20 °C/ 10 days, followed by heat shock at 60 °C) were also used. The viability evaluation was based on the following criteria: morphological integrity, motility and absence of Trypan blue staining. The evaluation was performed using an optical microscope at 100 x and 400 x magnification.

In vivo evaluation of the infection intensity

Four groups of six BALB/c mice were formed: G1 – mice were supplemented with the probiotic (1.10⁹ CFU) intragastrically (IG) for 15 days before the inoculation of 100 T. canis eggs (IG), and the supplementation condinued for 2 days post-inoculation (P.I); G2 – IG administered with sterile PBS, for 15 days preinoculation and 48 h post-IG inoculation of 100 T. canis eggs; G3 – IG supplemented with probiotic for 17 days; and G4 – IG-administered sterile PBS for 17 days²⁰. Mice were euthanized 2 days P.I and the experiment lasted 17 days. Euthanasia was perfomed with thiopental by intraperitoneal injections (75 mg/kg). Next, the digestion of the liver and lung tissues from animals of groups G1 and G2 was performed in 1% pepsin solution and 1% hydrochloric acid with constant shaking overnight at 37 °C³². Afterwards, T. canis larvae were recovered and quantified by optical microscopy at 100 x and 400 x magnification.

IFN-γ gene transcription

Whole-blood samples were obtained from the mice belonging to the four groups (G1, G2, G3, and G4) on the day of euthanasia using a pool of blood samples per group. Leukocytes were isolated from whole blood samples after cell lysis to eliminate red blood cells followed by washingin Hanks solution and storage in TRI Reagent^® (Sigma Aldrich) at -70 °C. Subsequently, RNA extraction and cDNA synthesis were performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) as instructed by the manufacturer. Quantitative polymerase chain reactions (qPCRs) were performed in a Step One Plus apparatus (Applied Biosystems, Foster City, CA, USA) using specific primers for IFN-γ and GAPDH, the latter was included as a reference gene⁸. The qPCRs were performed with 1 µL of cDNA (synthesized from 300 ng/µL of RNA), 6.25 µL of Go Taq^® qPCR Master Mix (Promega Corporation, Madison, Wisconsin, USA), 0.5 µM of each primer and 4.25 µL of RNAse-free water (Sigma Aldrich, Brasil Ltda) in a final volume of 12.5 µL. The temperatures used were as follows: an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 60 s, extension at 72 °C for 60 s and a final extension step at 72 °C for 5 min. All samples were tested in duplicate. The cycle threshold (Ct) values were used to calculate the variation in gene expression relative to GAPDH (control) expression.

Statistical analysis

Data regarding the infection intensity by T. canis and the IFN-γ gene transcription were compared by the Student’s t-test, with a significance level of 0.05 (BioEstat version 5.0, AnalystSoft Inc., Walnut, CA, USA). The viability of T. canis larvae (in vitro test) was evaluated by analysis of variance (ANOVA), and the means were compared by Tukey’s test, with a significance level of 0.05 (BioEstat version 5.0, AnalystSoft Inc., Walnut, CA, USA).

RESULTS

Compared to control mice (G2), animals supplemented with the probiotic L. acidophilus ATCC 4356 (G1) presented a 57.7% (p=0.025) reduction in the total number of larvae recovered from the liver (Table 1). Larval positivity in the liver was observed in all G1 and G2 mice, confirming the acute T. canis infection. No larvae were recovered from the lungs.

Table 1
Mean number of Toxocara canis larvae recovered from the liver 48 h post inoculation of 100 embryonated eggs in BALB/c mice supplemented with the probiotic Lactobacillus acidophilus (1.109 CFU; n = six mice/group).

Groups	Mean (± standard deviation)	p
Control	5.2 (± 2.16)
Lactobacillus acidophilus	2.2 (± 1.09)	0.025
Reduction (%)	57.7%

• (Student’s t-test)

In the in vitro test, compared to the control, the different concentrations of L. acidophilus probiotic had no direct effect on T. canis larvae during the 48 h incubation period with T. canis larvae (p > 0.05). The mean percentage of larvae with morphological integrity, positive motility and no Trypan blue staining ranged from 93.8% to 95.3%. These means were similar to those of the control live larvae, of 94.6%. In the control group of dead larvae, all larvae were not viable, that is, they showed no motility and Trypan blue staining.

IFN-γ gene transcription

Compared to that in control mice supplemented with PBS (G4), IFN-γ gene transcription was reduced by three times in mice supplemented with probiotics and infected with T. canis (G1) and six times in mice supplemented with probiotics (G3). IFN-γ gene expression in mice infected with only T. canis (G2) was not significantly different from that of mice in the PBS control group (G4; Figure 1). After 48 h of T. canis egg inoculation, mice without supplementation had consistent IFN-γ (G2) gene transcription levels.

Figure 1
IFN-γ gene transcription in Balb/c mice leukocytes (samples analyzed in one pool/group). G1 (Tox + L. ac) - infected by Toxocara canis and supplemented with Lactobacillus acidophilus; G2 (Tox) - infected by Toxocara canis; G3 (L. ac) - supplemented with Lactobacillus acidophilus; G4 (Cont) - not supplemented and not infected.

DISCUSSION

Several studies have already shown the potential of different Lactobacillus species probiotics to prevent and treat parasitosis, such as Trichinella spiralis, Giardia lamblia and Toxocara canis²⁰,³³-³⁵. The results obtained in this study have shown that supplementation with L. acidophilus ATCC 4356 has significantly reduced the infection intensity of Toxocara canis larvae in experimentally infected mice. The rate of reduction in the intensity of infection induced by this probiotic was similar to that observed by Walcher et al.²⁰, who used the probiotic L. rhamnosus ATCC 7469 (53.3%). These results confirm the potential of Lactobacillus spp. to control toxocariasis under the tested conditions.

Despite the in vivo action of L. acidophilus ATCC 4356 on reducing the infection intensity, no in vitro direct effect on T. canis larvae was observed. Similar results have been shown with the probiotics S. boulardii³¹ and L. rhamnosus ATCC 4356²⁰, which reduced the intensity of infection in mice and showed no in vitro effect on the larvae. In addition, our results are corroborated by those of other studies suggesting that the interaction between S. boulardii and the intestinal mucosa of T. canis-infected mice is necessary for the development of the beneficial effect of this probiotic³¹,³⁶.

To understand the mechanism underlying the action of this probiotic, the IFN-γ gene transcription was evaluated, as this is an important cytokine involved in the recruitment of macrophages during the acute phase of toxocariasis¹⁰. However, in this study, mice supplemented with L. acidophilus ATCC 4356, both infected and uninfected, had reduced IFN-γ gene transcription levels, similar to those of mice supplemented with L. casei ATCC 7469 and infected with Trichinella spiralis³³. According to these authors, IFN-γ gene transcription levels were reduced in comparison with those of the control as IFN-γ production was poorly induced due to the small number of infective larvae, and the same phenomenon may have occurred in this study. However, animals supplemented with other species of Lactobacillus have shown increased levels of this proinflammatory cytokine²⁵,²⁷,²⁹, indicating that the effect obtained with one species of microorganism used as a probiotic cannot be extrapolated to other species, since closely related species can produce distinct effects or even opposite effects. Therefore, it is important that probiotics are characterized at the strain level³⁷.

Other hypotheses that may explain the variation in the results of different studies that evaluated the protective effect of probiotics against T. canis infections include the use of different probiotics (Enterococcus faecalis CECT 7121, S. boulardii and L. rhamnosus ATCC 7469), the dose of the administered probiotic, the species and lineage of the host and the evaluated organ⁸,¹⁷-²⁰,³⁶.

Although no increment in the transcription of IFN-γ was observed in animals supplemented with L. acidophilus, the obtained results suggest that the effect of this probiotic against T. canis involves the innate immunity participation, as the reduction in the number of larvae occurred within 48 h, an insufficient period for the development of an adaptive immune response⁸,¹⁰. In addition, in this study, IFN-γ transcription levels were evaluated at the systemic level (blood) and the action of the probiotic can occur at the local level (intestinal mucosa)³⁶, and this aspect, like other pro-inflammatory and anti-inflammatory interleukins need to be evaluated in future studies.

CONCLUSION

The probiotic Lactobacillus acidophilus ATCC 4356 reduced the intensity of T. canis infection. However, the proinflammatory cytokine IFN-γ was not involved in this reduction and in the establishment of infection by T. canis larvae, and the probiotic did not have a direct effect on larvae, demonstrating the need of the interaction with the host for the probiotic beneficial effect to occur. Thus, it is important to evaluate other possible mechanisms of action of the probiotic, as well as other cytokines, to elucidate its protective effect.

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Publication Dates

• Publication in this collection
  29 Jan 2021
• Date of issue
  2021

History

• Received
  30 Aug 2020
• Accepted
  30 Nov 2020