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Effect of Vitamin A on Listeria monocytogenes Infection in a Silkworm Model

View Article: PubMed Central - PubMed

ABSTRACT

Insect infection models have been used increasingly to study various pathogenic agents in evaluations of pathogenicity and drug efficacy. In this study, we demonstrated that larvae of the silkworm Bombyx mori are useful for studying Listeria monocytogenes infections in insects. Infection with the L. monocytogenes wild-type strain induced silkworm death. Infection by a listeriolysin O (LLO) deletion mutant also induced silkworm death, but the bacterial numbers in silkworms were lower than those of the wild-type strain. Intracellular growth was observed when the silkworm ovary-derived cell line BmN4 was infected with the wild-type strain. Explosive replication was not observed in BmN4 cells infected with the LLO mutant and the bacterial numbers of the LLO mutant were lower than those of the wild-type strain. Pretreatment with vitamin A did not affect silkworm mortality after bacterial infection, but the efficiency of infecting the hemocytes and BmN4 cells was decreased with vitamin A treatment. Our results indicate that silkworm larvae are a useful insect infection model for L. monocytogenes and that vitamin A has protective effects against bacterial infection in silkworms.

No MeSH data available.


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Infection of L. monocytogenes to silkworm pretreated with vitamin A.(A) Survival of infected silkworms after previous supplementation with vitamin A. Approximately 104 bacteria of the wild-type strain were used to infect silkworms pretreated with (open circles) or without vitamin A (black circles). Groups of five insects per group were checked daily for survival. Survival was observed until 8 days post infection. (B) Bacterial numbers of the wild-type strain in silkworms pretreated with (black bars) or without vitamin A (open bars) until 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the SEM (n = 9). Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (C) Hemocytes (red) containing L. monocytogenes strains (green, arrowheads) isolated from silkworm pretreated with or without vitamin A were observed by confocal laser scanning microscopy at 3 h and 24 h post inoculation. Scale bar represents 10 μm.
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pone.0163747.g003: Infection of L. monocytogenes to silkworm pretreated with vitamin A.(A) Survival of infected silkworms after previous supplementation with vitamin A. Approximately 104 bacteria of the wild-type strain were used to infect silkworms pretreated with (open circles) or without vitamin A (black circles). Groups of five insects per group were checked daily for survival. Survival was observed until 8 days post infection. (B) Bacterial numbers of the wild-type strain in silkworms pretreated with (black bars) or without vitamin A (open bars) until 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the SEM (n = 9). Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (C) Hemocytes (red) containing L. monocytogenes strains (green, arrowheads) isolated from silkworm pretreated with or without vitamin A were observed by confocal laser scanning microscopy at 3 h and 24 h post inoculation. Scale bar represents 10 μm.

Mentions: The innate immune system of the silkworm is considered to function at both the cellular and humoral levels. The role of vitamin A in the immune system extends to both innate and adaptive immune responses in vertebrates [14]. At first, we check the effects of vitamin A treatment in non-infected silkworms. No significant differences were observed in the health conditions, such as body weight, feeding activity and time to pupation, between the silkworms pretreated with vitamin A and control (data not shown). To examine the effects of vitamin A supplementation on L. monocytogenes infections in silkworms, we infected vitamin A-pretreated larvae with approximately 104 cells of the wild-type strain at 12 h after supplementation, and we then measured the survival rates of the supplemented silkworms and their CFU counts. There was no significant difference between the vitamin A-supplemented silkworms and the control (Fig 3A). There was a one-day delay in mortality between the two groups. Next, we determined the bacterial numbers in infected silkworms based on the CFU counts. To measure the bacterial numbers in infected silkworms, the larvae were infected with the wild-type strain and the bacterial counts were determined at 1 h, 24 h, and 48 h after infection. The number of bacteria was significantly lower in vitamin A-supplemented silkworms compared with the non-supplemented silkworms (Fig 3B). This effect was maintained throughout the course of infection. To investigate the effect of vitamin A supplementation on bacterial invasion into the hemocyte of silkworms, we examined hemocyte isolated from infected larvae using confocal microscopy. Intracellular bacteria were observed at 1 h and 24 h after infection, and there were no differences between vitamin A-supplemented and non-supplemented silkworms (Fig 3C).


Effect of Vitamin A on Listeria monocytogenes Infection in a Silkworm Model
Infection of L. monocytogenes to silkworm pretreated with vitamin A.(A) Survival of infected silkworms after previous supplementation with vitamin A. Approximately 104 bacteria of the wild-type strain were used to infect silkworms pretreated with (open circles) or without vitamin A (black circles). Groups of five insects per group were checked daily for survival. Survival was observed until 8 days post infection. (B) Bacterial numbers of the wild-type strain in silkworms pretreated with (black bars) or without vitamin A (open bars) until 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the SEM (n = 9). Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (C) Hemocytes (red) containing L. monocytogenes strains (green, arrowheads) isolated from silkworm pretreated with or without vitamin A were observed by confocal laser scanning microscopy at 3 h and 24 h post inoculation. Scale bar represents 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036829&req=5

pone.0163747.g003: Infection of L. monocytogenes to silkworm pretreated with vitamin A.(A) Survival of infected silkworms after previous supplementation with vitamin A. Approximately 104 bacteria of the wild-type strain were used to infect silkworms pretreated with (open circles) or without vitamin A (black circles). Groups of five insects per group were checked daily for survival. Survival was observed until 8 days post infection. (B) Bacterial numbers of the wild-type strain in silkworms pretreated with (black bars) or without vitamin A (open bars) until 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the SEM (n = 9). Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (C) Hemocytes (red) containing L. monocytogenes strains (green, arrowheads) isolated from silkworm pretreated with or without vitamin A were observed by confocal laser scanning microscopy at 3 h and 24 h post inoculation. Scale bar represents 10 μm.
Mentions: The innate immune system of the silkworm is considered to function at both the cellular and humoral levels. The role of vitamin A in the immune system extends to both innate and adaptive immune responses in vertebrates [14]. At first, we check the effects of vitamin A treatment in non-infected silkworms. No significant differences were observed in the health conditions, such as body weight, feeding activity and time to pupation, between the silkworms pretreated with vitamin A and control (data not shown). To examine the effects of vitamin A supplementation on L. monocytogenes infections in silkworms, we infected vitamin A-pretreated larvae with approximately 104 cells of the wild-type strain at 12 h after supplementation, and we then measured the survival rates of the supplemented silkworms and their CFU counts. There was no significant difference between the vitamin A-supplemented silkworms and the control (Fig 3A). There was a one-day delay in mortality between the two groups. Next, we determined the bacterial numbers in infected silkworms based on the CFU counts. To measure the bacterial numbers in infected silkworms, the larvae were infected with the wild-type strain and the bacterial counts were determined at 1 h, 24 h, and 48 h after infection. The number of bacteria was significantly lower in vitamin A-supplemented silkworms compared with the non-supplemented silkworms (Fig 3B). This effect was maintained throughout the course of infection. To investigate the effect of vitamin A supplementation on bacterial invasion into the hemocyte of silkworms, we examined hemocyte isolated from infected larvae using confocal microscopy. Intracellular bacteria were observed at 1 h and 24 h after infection, and there were no differences between vitamin A-supplemented and non-supplemented silkworms (Fig 3C).

View Article: PubMed Central - PubMed

ABSTRACT

Insect infection models have been used increasingly to study various pathogenic agents in evaluations of pathogenicity and drug efficacy. In this study, we demonstrated that larvae of the silkworm Bombyx mori are useful for studying Listeria monocytogenes infections in insects. Infection with the L. monocytogenes wild-type strain induced silkworm death. Infection by a listeriolysin O (LLO) deletion mutant also induced silkworm death, but the bacterial numbers in silkworms were lower than those of the wild-type strain. Intracellular growth was observed when the silkworm ovary-derived cell line BmN4 was infected with the wild-type strain. Explosive replication was not observed in BmN4 cells infected with the LLO mutant and the bacterial numbers of the LLO mutant were lower than those of the wild-type strain. Pretreatment with vitamin A did not affect silkworm mortality after bacterial infection, but the efficiency of infecting the hemocytes and BmN4 cells was decreased with vitamin A treatment. Our results indicate that silkworm larvae are a useful insect infection model for L. monocytogenes and that vitamin A has protective effects against bacterial infection in silkworms.

No MeSH data available.


Related in: MedlinePlus