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Comparison of Bacterial Burden and Cytokine Gene Expression in Golden Hamsters in Early Phase of Infection with Two Different Strains of Leptospira interrogans.

Fujita R, Koizumi N, Sugiyama H, Tomizawa R, Sato R, Ohnishi M - PLoS ONE (2015)

Bottom Line: In addition, infection with serovar Manilae resulted in a significantly larger number of hamsters with tnfalpha upregulation (p = 0.04).These results demonstrate that serovar Manilae multiplied more efficiently in liver tissues and induced significantly higher expression of genes encoding pro- and anti-inflammatory cytokines than serovar Hebdomadis even in tissues for which a significant difference in leptospiral load was not observed.In addition, our results suggest a serovar Manilae-specific mechanism responsible for inducing severe damage in kidneys and hemorrhage in lung.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bio-Applications & Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan; Department of Bacteriology I, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

ABSTRACT
Leptospirosis, a zoonotic infection with worldwide prevalence, is caused by pathogenic spirochaetes of Leptospira spp., and exhibits an extremely broad clinical spectrum in human patients. Although previous studies indicated that specific serovars or genotypes of Leptospira spp. were associated with severe leptospirosis or its outbreak, the mechanism underlying the difference in virulence of the various Leptospira serotypes or genotypes remains unclear. The present study addresses this question by measuring and comparing bacterial burden and cytokine gene expression in hamsters infected with strains of two L. interrogans serovars Manilae (highly virulent) and Hebdomadis (less virulent). The histopathology of kidney, liver, and lung tissues was also investigated in infected hamsters. A significantly higher bacterial burden was observed in liver tissues of hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.01). The average copy number of the leptospiral genome was 1,302 and 20,559 in blood and liver, respectively, of hamsters infected with serovar Manilae and 1,340 and 4,896, respectively, in hamsters infected with serovar Hebdomadis. The expression levels of mip1alpha in blood; tgfbeta, il1beta, mip1alpha, il10, tnfalpha and cox2 in liver; and tgfbeta, il6, tnfalpha and cox2 in lung tissue were significantly higher in hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.05). In addition, infection with serovar Manilae resulted in a significantly larger number of hamsters with tnfalpha upregulation (p = 0.04). Severe distortion of tubular cell arrangement and disruption of renal tubules in kidney tissues and hemorrhage in lung tissues were observed in Manilae-infected hamsters. These results demonstrate that serovar Manilae multiplied more efficiently in liver tissues and induced significantly higher expression of genes encoding pro- and anti-inflammatory cytokines than serovar Hebdomadis even in tissues for which a significant difference in leptospiral load was not observed. In addition, our results suggest a serovar Manilae-specific mechanism responsible for inducing severe damage in kidneys and hemorrhage in lung.

No MeSH data available.


Related in: MedlinePlus

Quantification of expression of cytokine genes in the blood of hamsters infected with L. interrogans serovars Manilae or Hebdomadis at 96 h pi.Cytokine gene expression in the blood of hamsters infected with strains of L. interrogans serovars Manilae (filled boxes) or Hebdomadis (open boxes) was quantified at 96 h pi using real-time PCR (2−ΔΔCt method). The bottom, median, and top lines of the box indicate the 25th, 50th, and 75th percentiles, respectively. The vertical line with whiskers shows the range of values. Experiments were performed in duplicate using two independently extracted RNA samples for each hamster; each dot indicates the average of two experiments and gene expression relative to control hamsters. The dotted line indicates the expression level in control hamsters (for calibration). Data out of the range of values were excluded from statistical analysis. *, p < 0.05.
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pone.0132694.g002: Quantification of expression of cytokine genes in the blood of hamsters infected with L. interrogans serovars Manilae or Hebdomadis at 96 h pi.Cytokine gene expression in the blood of hamsters infected with strains of L. interrogans serovars Manilae (filled boxes) or Hebdomadis (open boxes) was quantified at 96 h pi using real-time PCR (2−ΔΔCt method). The bottom, median, and top lines of the box indicate the 25th, 50th, and 75th percentiles, respectively. The vertical line with whiskers shows the range of values. Experiments were performed in duplicate using two independently extracted RNA samples for each hamster; each dot indicates the average of two experiments and gene expression relative to control hamsters. The dotted line indicates the expression level in control hamsters (for calibration). Data out of the range of values were excluded from statistical analysis. *, p < 0.05.

Mentions: Of the 12 cytokine genes tested, the expression of three genes il1beta, mip1alpha, and tgfbeta was upregulated in the blood of infected hamsters (Fig 2). Moreover, the expression of mip1alpha was significantly higher in hamsters infected with serovar Manilae as opposed to serovar Hebdomadis (p < 0.05; Fig 2). In addition, the number of hamsters with detectable expression of tnfalpha was significantly higher in the group infected with serovar Manilae (5/7 animals) as opposed to serovar Hebdomadis (1/8 animals; p = 0.04, as determined by Fischer’s Exact test). The expression of eight of the 12 genes tested was upregulated in kidney tissues upon leptospiral infection, although significant differences were not detected between the two serovars Manilae and Hebdomadis (Fig 3). The expression of eight of the 12 genes tested was enhanced in both liver and lung tissues upon leptospiral infection (Figs 4 and 5); significantly higher expression levels of tgfbeta, il1beta, mip1alpha, il10, tnfalpha, and cox2 in liver (cox2, p < 0.05; other genes, p < 0.01; Fig 4) and of tgfbeta, il6, tnfalpha, and cox2 in lung (il6 and tnfalpha, p < 0.05; tgfbeta and cox2, p < 0.01; Fig 5) tissues were observed in the group infected with serovar Manilae as opposed to serovar Hebdomadis. The expression levels of il2, il4, and ifngamma were below the detection limit in all tissues in this round of infection experiments.


Comparison of Bacterial Burden and Cytokine Gene Expression in Golden Hamsters in Early Phase of Infection with Two Different Strains of Leptospira interrogans.

Fujita R, Koizumi N, Sugiyama H, Tomizawa R, Sato R, Ohnishi M - PLoS ONE (2015)

Quantification of expression of cytokine genes in the blood of hamsters infected with L. interrogans serovars Manilae or Hebdomadis at 96 h pi.Cytokine gene expression in the blood of hamsters infected with strains of L. interrogans serovars Manilae (filled boxes) or Hebdomadis (open boxes) was quantified at 96 h pi using real-time PCR (2−ΔΔCt method). The bottom, median, and top lines of the box indicate the 25th, 50th, and 75th percentiles, respectively. The vertical line with whiskers shows the range of values. Experiments were performed in duplicate using two independently extracted RNA samples for each hamster; each dot indicates the average of two experiments and gene expression relative to control hamsters. The dotted line indicates the expression level in control hamsters (for calibration). Data out of the range of values were excluded from statistical analysis. *, p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492770&req=5

pone.0132694.g002: Quantification of expression of cytokine genes in the blood of hamsters infected with L. interrogans serovars Manilae or Hebdomadis at 96 h pi.Cytokine gene expression in the blood of hamsters infected with strains of L. interrogans serovars Manilae (filled boxes) or Hebdomadis (open boxes) was quantified at 96 h pi using real-time PCR (2−ΔΔCt method). The bottom, median, and top lines of the box indicate the 25th, 50th, and 75th percentiles, respectively. The vertical line with whiskers shows the range of values. Experiments were performed in duplicate using two independently extracted RNA samples for each hamster; each dot indicates the average of two experiments and gene expression relative to control hamsters. The dotted line indicates the expression level in control hamsters (for calibration). Data out of the range of values were excluded from statistical analysis. *, p < 0.05.
Mentions: Of the 12 cytokine genes tested, the expression of three genes il1beta, mip1alpha, and tgfbeta was upregulated in the blood of infected hamsters (Fig 2). Moreover, the expression of mip1alpha was significantly higher in hamsters infected with serovar Manilae as opposed to serovar Hebdomadis (p < 0.05; Fig 2). In addition, the number of hamsters with detectable expression of tnfalpha was significantly higher in the group infected with serovar Manilae (5/7 animals) as opposed to serovar Hebdomadis (1/8 animals; p = 0.04, as determined by Fischer’s Exact test). The expression of eight of the 12 genes tested was upregulated in kidney tissues upon leptospiral infection, although significant differences were not detected between the two serovars Manilae and Hebdomadis (Fig 3). The expression of eight of the 12 genes tested was enhanced in both liver and lung tissues upon leptospiral infection (Figs 4 and 5); significantly higher expression levels of tgfbeta, il1beta, mip1alpha, il10, tnfalpha, and cox2 in liver (cox2, p < 0.05; other genes, p < 0.01; Fig 4) and of tgfbeta, il6, tnfalpha, and cox2 in lung (il6 and tnfalpha, p < 0.05; tgfbeta and cox2, p < 0.01; Fig 5) tissues were observed in the group infected with serovar Manilae as opposed to serovar Hebdomadis. The expression levels of il2, il4, and ifngamma were below the detection limit in all tissues in this round of infection experiments.

Bottom Line: In addition, infection with serovar Manilae resulted in a significantly larger number of hamsters with tnfalpha upregulation (p = 0.04).These results demonstrate that serovar Manilae multiplied more efficiently in liver tissues and induced significantly higher expression of genes encoding pro- and anti-inflammatory cytokines than serovar Hebdomadis even in tissues for which a significant difference in leptospiral load was not observed.In addition, our results suggest a serovar Manilae-specific mechanism responsible for inducing severe damage in kidneys and hemorrhage in lung.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bio-Applications & Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan; Department of Bacteriology I, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

ABSTRACT
Leptospirosis, a zoonotic infection with worldwide prevalence, is caused by pathogenic spirochaetes of Leptospira spp., and exhibits an extremely broad clinical spectrum in human patients. Although previous studies indicated that specific serovars or genotypes of Leptospira spp. were associated with severe leptospirosis or its outbreak, the mechanism underlying the difference in virulence of the various Leptospira serotypes or genotypes remains unclear. The present study addresses this question by measuring and comparing bacterial burden and cytokine gene expression in hamsters infected with strains of two L. interrogans serovars Manilae (highly virulent) and Hebdomadis (less virulent). The histopathology of kidney, liver, and lung tissues was also investigated in infected hamsters. A significantly higher bacterial burden was observed in liver tissues of hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.01). The average copy number of the leptospiral genome was 1,302 and 20,559 in blood and liver, respectively, of hamsters infected with serovar Manilae and 1,340 and 4,896, respectively, in hamsters infected with serovar Hebdomadis. The expression levels of mip1alpha in blood; tgfbeta, il1beta, mip1alpha, il10, tnfalpha and cox2 in liver; and tgfbeta, il6, tnfalpha and cox2 in lung tissue were significantly higher in hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.05). In addition, infection with serovar Manilae resulted in a significantly larger number of hamsters with tnfalpha upregulation (p = 0.04). Severe distortion of tubular cell arrangement and disruption of renal tubules in kidney tissues and hemorrhage in lung tissues were observed in Manilae-infected hamsters. These results demonstrate that serovar Manilae multiplied more efficiently in liver tissues and induced significantly higher expression of genes encoding pro- and anti-inflammatory cytokines than serovar Hebdomadis even in tissues for which a significant difference in leptospiral load was not observed. In addition, our results suggest a serovar Manilae-specific mechanism responsible for inducing severe damage in kidneys and hemorrhage in lung.

No MeSH data available.


Related in: MedlinePlus