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Pathogenesis of Candida albicans infections in the alternative chorio-allantoic membrane chicken embryo model resembles systemic murine infections.

Jacobsen ID, Grosse K, Berndt A, Hube B - PLoS ONE (2011)

Bottom Line: While many aspects of the chicken embryo response resembled murine infections, we also observed significant differences: In contrast to systemic infections in mice, IL-10 had a beneficial effect in chicken embryos.Pathogenicity of the majority of 15 tested C. albicans deletion strains was comparable to the virulence in mouse models and reduced virulence was associated with significantly lower transcription of proinflammatory cytokines.C. albicans strains locked in the yeast stage disseminated significantly more often from the CAM into the embryo, supporting the hypothesis that the yeast morphology is responsible for dissemination in systemic infections.

View Article: PubMed Central - PubMed

Affiliation: Department for Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany. ilse.jacobsen@hki-jena.de

ABSTRACT
Alternative models of microbial infections are increasingly used to screen virulence determinants of pathogens. In this study, we investigated the pathogenesis of Candida albicans and C. glabrata infections in chicken embryos infected via the chorio-allantoic membrane (CAM) and analyzed the virulence of deletion mutants. The developing immune system of the host significantly influenced susceptibility: With increasing age, embryos became more resistant and mounted a more balanced immune response, characterized by lower induction of proinflammatory cytokines and increased transcription of regulatory cytokines, suggesting that immunopathology contributes to pathogenesis. While many aspects of the chicken embryo response resembled murine infections, we also observed significant differences: In contrast to systemic infections in mice, IL-10 had a beneficial effect in chicken embryos. IL-22 and IL-17A were only upregulated after the peak mortality in the chicken embryo model occurred; thus, the role of the Th17 response in this model remains unclear. Abscess formation occurs frequently in murine models, whereas the avian response was dominated by granuloma formation. Pathogenicity of the majority of 15 tested C. albicans deletion strains was comparable to the virulence in mouse models and reduced virulence was associated with significantly lower transcription of proinflammatory cytokines. However, fungal burden did not correlate with virulence and for few mutants like bcr1Δ and tec1Δ different outcomes in survival compared to murine infections were observed. C. albicans strains locked in the yeast stage disseminated significantly more often from the CAM into the embryo, supporting the hypothesis that the yeast morphology is responsible for dissemination in systemic infections. These data suggest that the pathogenesis of C. albicans infections in the chicken embryo model resembles systemic murine infections but also differs in some aspects. Despite its limitations, it presents a useful alternative tool to pre-screen C. albicans strains to select strains for subsequent testing in murine models.

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Characterization of the course of infection in chicken embryos infected with C. albicans SC5314 at different developmental days.(A) Mortality after infection on the CAM (Kaplan-Meyer curve). N = 20 per group per experiment, two independent experiments. Significant mortality (compared to age-matched PBS control, log rank test) was only observed for embryos infected on developmental day (DD) 8 (P<0.01). The logrank test for trend was significant for comparison of infected groups (P<0.0001). (B) Comparison of fungal burden in CAM in embryos infected at different developmental days (DD) as mean and SD, n = 10. (C) Frequency of positive isolation of C. albicans from liver (n = 10 per DD and time point). Dark: positve isolation; white: no fungi isolated. (D and E) Histology of embryos infected on developmental day 10. Periodic acid-Schiff stain (fungal elements: pink). (D) Hyphae invading into and penetrating the full thickness of the CAM 24 h after infection. Arrow: blood vessel penetrated by C. albicans. (E) Histology of macroscopically visible plaque 3 days after infection. Arrows indicate fungal cells.
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pone-0019741-g001: Characterization of the course of infection in chicken embryos infected with C. albicans SC5314 at different developmental days.(A) Mortality after infection on the CAM (Kaplan-Meyer curve). N = 20 per group per experiment, two independent experiments. Significant mortality (compared to age-matched PBS control, log rank test) was only observed for embryos infected on developmental day (DD) 8 (P<0.01). The logrank test for trend was significant for comparison of infected groups (P<0.0001). (B) Comparison of fungal burden in CAM in embryos infected at different developmental days (DD) as mean and SD, n = 10. (C) Frequency of positive isolation of C. albicans from liver (n = 10 per DD and time point). Dark: positve isolation; white: no fungi isolated. (D and E) Histology of embryos infected on developmental day 10. Periodic acid-Schiff stain (fungal elements: pink). (D) Hyphae invading into and penetrating the full thickness of the CAM 24 h after infection. Arrow: blood vessel penetrated by C. albicans. (E) Histology of macroscopically visible plaque 3 days after infection. Arrows indicate fungal cells.

Mentions: The outcome of infection with various pathogens, including C. albicans, in chick embryos has been described to depend on the developmental stage (age) at infection [17], [23]. In agreement with these observations, we found a clear correlation of age at infection and survival. At both high (107 cfu, data not shown) and low infectious doses (Fig. 1A), infection on developmental day 8 led to rapid mortality significantly higher than in embryos infected on developmental day 10 (P<0.001), while embryos infected on developmental day 12 were highly resistant and showed survival rates undistinguishable from the PBS control. It has been suggested that the increased resistance of older embryos to infection with pathogens might reflect maturation and increasing competence of the embryonic immune system [17]. Increased immune competence could enable the host to limit fungal burden and prevent dissemination of the pathogen, thus increasing survival of the host. To test this hypothesis, we determined fungal burden in the CAM and dissemination into the liver during the course of infection (day 1–5 post infection (p.i.)) in embryos infected on developmental day 8, 10 and 12 (n = 10 per day and age). C. albicans could be isolated from the CAM of all infected embryos (Fig. 1B). However, isolation from the liver, indicating systemic dissemination of C. albicans, was only sporadically successful. Although we observed a tendency towards lower dissemination rates in oldest embryos, this trend was not statistically significant (Fig. 1C). To determine whether dissemination might be an event associated with imminent death of an embryo, we additionally performed isolation from livers and complete abdomens of freshly deceased embryos. Similarly to living embryos, however, isolation was only infrequently positive (data not shown). Surprisingly, the embryonic age at infection did not have a significant impact on the number of cfu isolated from the CAM (Fig. 1B). Furthermore, cfu did not change over the course of the experiment (Fig. 1B). Neither fungi nor bacteria could be cultured from CAM and embryonic tissue of PBS-mock infected controls (n = 15), confirming the absence of contaminations.


Pathogenesis of Candida albicans infections in the alternative chorio-allantoic membrane chicken embryo model resembles systemic murine infections.

Jacobsen ID, Grosse K, Berndt A, Hube B - PLoS ONE (2011)

Characterization of the course of infection in chicken embryos infected with C. albicans SC5314 at different developmental days.(A) Mortality after infection on the CAM (Kaplan-Meyer curve). N = 20 per group per experiment, two independent experiments. Significant mortality (compared to age-matched PBS control, log rank test) was only observed for embryos infected on developmental day (DD) 8 (P<0.01). The logrank test for trend was significant for comparison of infected groups (P<0.0001). (B) Comparison of fungal burden in CAM in embryos infected at different developmental days (DD) as mean and SD, n = 10. (C) Frequency of positive isolation of C. albicans from liver (n = 10 per DD and time point). Dark: positve isolation; white: no fungi isolated. (D and E) Histology of embryos infected on developmental day 10. Periodic acid-Schiff stain (fungal elements: pink). (D) Hyphae invading into and penetrating the full thickness of the CAM 24 h after infection. Arrow: blood vessel penetrated by C. albicans. (E) Histology of macroscopically visible plaque 3 days after infection. Arrows indicate fungal cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3094387&req=5

pone-0019741-g001: Characterization of the course of infection in chicken embryos infected with C. albicans SC5314 at different developmental days.(A) Mortality after infection on the CAM (Kaplan-Meyer curve). N = 20 per group per experiment, two independent experiments. Significant mortality (compared to age-matched PBS control, log rank test) was only observed for embryos infected on developmental day (DD) 8 (P<0.01). The logrank test for trend was significant for comparison of infected groups (P<0.0001). (B) Comparison of fungal burden in CAM in embryos infected at different developmental days (DD) as mean and SD, n = 10. (C) Frequency of positive isolation of C. albicans from liver (n = 10 per DD and time point). Dark: positve isolation; white: no fungi isolated. (D and E) Histology of embryos infected on developmental day 10. Periodic acid-Schiff stain (fungal elements: pink). (D) Hyphae invading into and penetrating the full thickness of the CAM 24 h after infection. Arrow: blood vessel penetrated by C. albicans. (E) Histology of macroscopically visible plaque 3 days after infection. Arrows indicate fungal cells.
Mentions: The outcome of infection with various pathogens, including C. albicans, in chick embryos has been described to depend on the developmental stage (age) at infection [17], [23]. In agreement with these observations, we found a clear correlation of age at infection and survival. At both high (107 cfu, data not shown) and low infectious doses (Fig. 1A), infection on developmental day 8 led to rapid mortality significantly higher than in embryos infected on developmental day 10 (P<0.001), while embryos infected on developmental day 12 were highly resistant and showed survival rates undistinguishable from the PBS control. It has been suggested that the increased resistance of older embryos to infection with pathogens might reflect maturation and increasing competence of the embryonic immune system [17]. Increased immune competence could enable the host to limit fungal burden and prevent dissemination of the pathogen, thus increasing survival of the host. To test this hypothesis, we determined fungal burden in the CAM and dissemination into the liver during the course of infection (day 1–5 post infection (p.i.)) in embryos infected on developmental day 8, 10 and 12 (n = 10 per day and age). C. albicans could be isolated from the CAM of all infected embryos (Fig. 1B). However, isolation from the liver, indicating systemic dissemination of C. albicans, was only sporadically successful. Although we observed a tendency towards lower dissemination rates in oldest embryos, this trend was not statistically significant (Fig. 1C). To determine whether dissemination might be an event associated with imminent death of an embryo, we additionally performed isolation from livers and complete abdomens of freshly deceased embryos. Similarly to living embryos, however, isolation was only infrequently positive (data not shown). Surprisingly, the embryonic age at infection did not have a significant impact on the number of cfu isolated from the CAM (Fig. 1B). Furthermore, cfu did not change over the course of the experiment (Fig. 1B). Neither fungi nor bacteria could be cultured from CAM and embryonic tissue of PBS-mock infected controls (n = 15), confirming the absence of contaminations.

Bottom Line: While many aspects of the chicken embryo response resembled murine infections, we also observed significant differences: In contrast to systemic infections in mice, IL-10 had a beneficial effect in chicken embryos.Pathogenicity of the majority of 15 tested C. albicans deletion strains was comparable to the virulence in mouse models and reduced virulence was associated with significantly lower transcription of proinflammatory cytokines.C. albicans strains locked in the yeast stage disseminated significantly more often from the CAM into the embryo, supporting the hypothesis that the yeast morphology is responsible for dissemination in systemic infections.

View Article: PubMed Central - PubMed

Affiliation: Department for Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany. ilse.jacobsen@hki-jena.de

ABSTRACT
Alternative models of microbial infections are increasingly used to screen virulence determinants of pathogens. In this study, we investigated the pathogenesis of Candida albicans and C. glabrata infections in chicken embryos infected via the chorio-allantoic membrane (CAM) and analyzed the virulence of deletion mutants. The developing immune system of the host significantly influenced susceptibility: With increasing age, embryos became more resistant and mounted a more balanced immune response, characterized by lower induction of proinflammatory cytokines and increased transcription of regulatory cytokines, suggesting that immunopathology contributes to pathogenesis. While many aspects of the chicken embryo response resembled murine infections, we also observed significant differences: In contrast to systemic infections in mice, IL-10 had a beneficial effect in chicken embryos. IL-22 and IL-17A were only upregulated after the peak mortality in the chicken embryo model occurred; thus, the role of the Th17 response in this model remains unclear. Abscess formation occurs frequently in murine models, whereas the avian response was dominated by granuloma formation. Pathogenicity of the majority of 15 tested C. albicans deletion strains was comparable to the virulence in mouse models and reduced virulence was associated with significantly lower transcription of proinflammatory cytokines. However, fungal burden did not correlate with virulence and for few mutants like bcr1Δ and tec1Δ different outcomes in survival compared to murine infections were observed. C. albicans strains locked in the yeast stage disseminated significantly more often from the CAM into the embryo, supporting the hypothesis that the yeast morphology is responsible for dissemination in systemic infections. These data suggest that the pathogenesis of C. albicans infections in the chicken embryo model resembles systemic murine infections but also differs in some aspects. Despite its limitations, it presents a useful alternative tool to pre-screen C. albicans strains to select strains for subsequent testing in murine models.

Show MeSH
Related in: MedlinePlus