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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 silkworms with L. monocytogenes.(A) Survival rate of silkworms. Fifteen insects per group were infected and monitored to determine the time required to kill 50% of the silkworms. Approximately 104 bacteria belonging to the wild-type strain (WT, circles), LLO deletion mutant (Δhly, triangles), and LLO-complemented strain (Δhly::hly, squares) were sufficient to kill 50% (LD50) of the silkworms within 4 and 5 days. Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (B) The bacterial numbers of each strain detected in silkworms at 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the standard error of the mean (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) were observed by confocal laser scanning microscopy at 1 h and 24 h post inoculation. Scale bar represents 10 μm. (D) Immunoblot analysis of LLO expression. Samples were prepared from the wild-type strain, LLO deletion mutant, and LLO-complemented strain cultivated at 37°C or 25°C in BHI broth.
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pone.0163747.g001: Infection of silkworms with L. monocytogenes.(A) Survival rate of silkworms. Fifteen insects per group were infected and monitored to determine the time required to kill 50% of the silkworms. Approximately 104 bacteria belonging to the wild-type strain (WT, circles), LLO deletion mutant (Δhly, triangles), and LLO-complemented strain (Δhly::hly, squares) were sufficient to kill 50% (LD50) of the silkworms within 4 and 5 days. Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (B) The bacterial numbers of each strain detected in silkworms at 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the standard error of the mean (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) were observed by confocal laser scanning microscopy at 1 h and 24 h post inoculation. Scale bar represents 10 μm. (D) Immunoblot analysis of LLO expression. Samples were prepared from the wild-type strain, LLO deletion mutant, and LLO-complemented strain cultivated at 37°C or 25°C in BHI broth.

Mentions: In order to determine whether the silkworm B. mori can be used as a model system for L. monocytogenes infection, we first examined the infectivity of wild-type L. monocytogenes EGD, an LLO deletion mutant, and an LLO-complemented strain at room temperature. L. monocytogenes was serially diluted and then injected into 15 individual silkworms per group, before monitoring the time required to kill 50% of the silkworms (LT50). The PBS-injected larvae survived for at least six days and subsequently matured into pupae (data not shown). The LT50 was determined when approximately 104 cells of the wild-type strain, LLO deletion mutant, or LLO-complemented strain were injected at room temperature. The wild-type strain and LLO-complemented strain obtained LT50 values of 120 h and 96 h (five and four days), respectively (Fig 1A). A delay in the LT50 (approximately six days) was observed for the LLO deletion mutant. All of the infected larvae looked normal for at least two days and the activity then decreased, before they stopped eating and finally died. Their skin color became dark only after death.


Effect of Vitamin A on Listeria monocytogenes Infection in a Silkworm Model
Infection of silkworms with L. monocytogenes.(A) Survival rate of silkworms. Fifteen insects per group were infected and monitored to determine the time required to kill 50% of the silkworms. Approximately 104 bacteria belonging to the wild-type strain (WT, circles), LLO deletion mutant (Δhly, triangles), and LLO-complemented strain (Δhly::hly, squares) were sufficient to kill 50% (LD50) of the silkworms within 4 and 5 days. Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (B) The bacterial numbers of each strain detected in silkworms at 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the standard error of the mean (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) were observed by confocal laser scanning microscopy at 1 h and 24 h post inoculation. Scale bar represents 10 μm. (D) Immunoblot analysis of LLO expression. Samples were prepared from the wild-type strain, LLO deletion mutant, and LLO-complemented strain cultivated at 37°C or 25°C in BHI broth.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036829&req=5

pone.0163747.g001: Infection of silkworms with L. monocytogenes.(A) Survival rate of silkworms. Fifteen insects per group were infected and monitored to determine the time required to kill 50% of the silkworms. Approximately 104 bacteria belonging to the wild-type strain (WT, circles), LLO deletion mutant (Δhly, triangles), and LLO-complemented strain (Δhly::hly, squares) were sufficient to kill 50% (LD50) of the silkworms within 4 and 5 days. Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (B) The bacterial numbers of each strain detected in silkworms at 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the standard error of the mean (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) were observed by confocal laser scanning microscopy at 1 h and 24 h post inoculation. Scale bar represents 10 μm. (D) Immunoblot analysis of LLO expression. Samples were prepared from the wild-type strain, LLO deletion mutant, and LLO-complemented strain cultivated at 37°C or 25°C in BHI broth.
Mentions: In order to determine whether the silkworm B. mori can be used as a model system for L. monocytogenes infection, we first examined the infectivity of wild-type L. monocytogenes EGD, an LLO deletion mutant, and an LLO-complemented strain at room temperature. L. monocytogenes was serially diluted and then injected into 15 individual silkworms per group, before monitoring the time required to kill 50% of the silkworms (LT50). The PBS-injected larvae survived for at least six days and subsequently matured into pupae (data not shown). The LT50 was determined when approximately 104 cells of the wild-type strain, LLO deletion mutant, or LLO-complemented strain were injected at room temperature. The wild-type strain and LLO-complemented strain obtained LT50 values of 120 h and 96 h (five and four days), respectively (Fig 1A). A delay in the LT50 (approximately six days) was observed for the LLO deletion mutant. All of the infected larvae looked normal for at least two days and the activity then decreased, before they stopped eating and finally died. Their skin color became dark only after death.

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