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Mycobacterium Avium subsp. paratuberculosis isolates induce in vitro granuloma formation and show successful survival phenotype, common anti-inflammatory and antiapoptotic responses within ovine macrophages regardless of genotype or host of origin.

Abendaño N, Tyukalova L, Barandika JF, Balseiro A, Sevilla IA, Garrido JM, Juste RA, Alonso-Hearn M - PLoS ONE (2014)

Bottom Line: After 7 d of infection, variations in the estimated log10 CFUs between all the tested isolates were not statistically significant.In addition, ovine MDMs exhibited enhanced anti-inflammatory, antiapoptotic and antidestructive responses when infected with two ovine isolates of distinct genotype (C and S) or with two C-type isolates from distinct hosts (cattle and sheep); which correlated with the successful survival of these isolates within ovine MDMs. A second objective was to study, based on an in vitro granuloma model, latter stages of the infection by investigating the capacity of two Map isolates from cattle and sheep to trigger formation of microgranulomas.In summary, our results demonstrated that Map isolates from cattle, sheep, goats, deer, fallow-deer and wild boar were able not only to initiate but also to establish a successful infection in ovine macrophages regardless of genotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Health, Basque Institute for Agricultural Research and Development, NEIKER-Tecnalia, Technological Park of Bizkaia, Bizkaia, Spain.

ABSTRACT
The analysis of the early macrophage responses, including bacterial growth within macrophages, represents a powerful tool to characterize the virulence of clinical isolates of Mycobcaterium avium susbp. paratuberculosis (Map). The present study represents the first assessment of the intracellular behaviour in ovine monocyte-derived macrophages (MDMs) of Map isolates representing distinct genotypes (C, S and B), and isolated from cattle, sheep, goat, fallow deer, deer, and wild boar. Intracellular growth and survival of the selected isolates in ovine MDMs was assessed by quantification of CFUs inside of the host cells at 2 h p.i. (day 0) and 7 d p. i. using an automatic liquid culture system (Bactec MGIT 960). Variations in bacterial counts over 7 days from the baseline were small, in a range between 1.63 to 1.05-fold. After 7 d of infection, variations in the estimated log10 CFUs between all the tested isolates were not statistically significant. In addition, ovine MDMs exhibited enhanced anti-inflammatory, antiapoptotic and antidestructive responses when infected with two ovine isolates of distinct genotype (C and S) or with two C-type isolates from distinct hosts (cattle and sheep); which correlated with the successful survival of these isolates within ovine MDMs. A second objective was to study, based on an in vitro granuloma model, latter stages of the infection by investigating the capacity of two Map isolates from cattle and sheep to trigger formation of microgranulomas. Upon 10 d p.i., both Map isolates were able to induce the formation of granulomas comparable to the granulomas observed in clinical specimens with respect to the cellular components involved. In summary, our results demonstrated that Map isolates from cattle, sheep, goats, deer, fallow-deer and wild boar were able not only to initiate but also to establish a successful infection in ovine macrophages regardless of genotype.

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Morphological characterization of the cell populations recruited within in vitro- ovine granulomas.Primary ovine PBMCs (5x105) were seeded on an extracellular matrix and subsequently infected with the K10 reference strain (A, B and C) or with an ovine isolate of Map (2349/06-1) (D, E and F) at MOI (Bacteria:cells) 1:8. At 10 days p.i., the granuloma-like aggregates were harvested, processed for histopathology and stained with HE (A, B, D and E) and ZN stains (C and F). Original magnification in A and D  =  200X and in B, C, E and F  =  1000X. As shown in image B, macrophages (asterisk) and lymphocytes (arrows) were present in the granulomas. In images C and F, acid-fast bacilli (arrows) were observed within macrophages by ZN staining.
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pone-0104238-g004: Morphological characterization of the cell populations recruited within in vitro- ovine granulomas.Primary ovine PBMCs (5x105) were seeded on an extracellular matrix and subsequently infected with the K10 reference strain (A, B and C) or with an ovine isolate of Map (2349/06-1) (D, E and F) at MOI (Bacteria:cells) 1:8. At 10 days p.i., the granuloma-like aggregates were harvested, processed for histopathology and stained with HE (A, B, D and E) and ZN stains (C and F). Original magnification in A and D  =  200X and in B, C, E and F  =  1000X. As shown in image B, macrophages (asterisk) and lymphocytes (arrows) were present in the granulomas. In images C and F, acid-fast bacilli (arrows) were observed within macrophages by ZN staining.

Mentions: After 10 days of culture, the morphological characterization of the granuloma-like aggregates was confirmed by histological staining. Figure 4 shows the morphological characterization of the aggregates formed by primary ovine PBMCs infected with the K10 Map reference strain and with the ovine isolate of Map (2349/06-1) at MOI 1∶8. As shown in figures 4B and 4E, granulomas exhibited aggregation of lymphocytes around infected macrophages. When granuloma sections were stained with ZN, Map cells could be observed residing within the granulomas.


Mycobacterium Avium subsp. paratuberculosis isolates induce in vitro granuloma formation and show successful survival phenotype, common anti-inflammatory and antiapoptotic responses within ovine macrophages regardless of genotype or host of origin.

Abendaño N, Tyukalova L, Barandika JF, Balseiro A, Sevilla IA, Garrido JM, Juste RA, Alonso-Hearn M - PLoS ONE (2014)

Morphological characterization of the cell populations recruited within in vitro- ovine granulomas.Primary ovine PBMCs (5x105) were seeded on an extracellular matrix and subsequently infected with the K10 reference strain (A, B and C) or with an ovine isolate of Map (2349/06-1) (D, E and F) at MOI (Bacteria:cells) 1:8. At 10 days p.i., the granuloma-like aggregates were harvested, processed for histopathology and stained with HE (A, B, D and E) and ZN stains (C and F). Original magnification in A and D  =  200X and in B, C, E and F  =  1000X. As shown in image B, macrophages (asterisk) and lymphocytes (arrows) were present in the granulomas. In images C and F, acid-fast bacilli (arrows) were observed within macrophages by ZN staining.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128652&req=5

pone-0104238-g004: Morphological characterization of the cell populations recruited within in vitro- ovine granulomas.Primary ovine PBMCs (5x105) were seeded on an extracellular matrix and subsequently infected with the K10 reference strain (A, B and C) or with an ovine isolate of Map (2349/06-1) (D, E and F) at MOI (Bacteria:cells) 1:8. At 10 days p.i., the granuloma-like aggregates were harvested, processed for histopathology and stained with HE (A, B, D and E) and ZN stains (C and F). Original magnification in A and D  =  200X and in B, C, E and F  =  1000X. As shown in image B, macrophages (asterisk) and lymphocytes (arrows) were present in the granulomas. In images C and F, acid-fast bacilli (arrows) were observed within macrophages by ZN staining.
Mentions: After 10 days of culture, the morphological characterization of the granuloma-like aggregates was confirmed by histological staining. Figure 4 shows the morphological characterization of the aggregates formed by primary ovine PBMCs infected with the K10 Map reference strain and with the ovine isolate of Map (2349/06-1) at MOI 1∶8. As shown in figures 4B and 4E, granulomas exhibited aggregation of lymphocytes around infected macrophages. When granuloma sections were stained with ZN, Map cells could be observed residing within the granulomas.

Bottom Line: After 7 d of infection, variations in the estimated log10 CFUs between all the tested isolates were not statistically significant.In addition, ovine MDMs exhibited enhanced anti-inflammatory, antiapoptotic and antidestructive responses when infected with two ovine isolates of distinct genotype (C and S) or with two C-type isolates from distinct hosts (cattle and sheep); which correlated with the successful survival of these isolates within ovine MDMs. A second objective was to study, based on an in vitro granuloma model, latter stages of the infection by investigating the capacity of two Map isolates from cattle and sheep to trigger formation of microgranulomas.In summary, our results demonstrated that Map isolates from cattle, sheep, goats, deer, fallow-deer and wild boar were able not only to initiate but also to establish a successful infection in ovine macrophages regardless of genotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Health, Basque Institute for Agricultural Research and Development, NEIKER-Tecnalia, Technological Park of Bizkaia, Bizkaia, Spain.

ABSTRACT
The analysis of the early macrophage responses, including bacterial growth within macrophages, represents a powerful tool to characterize the virulence of clinical isolates of Mycobcaterium avium susbp. paratuberculosis (Map). The present study represents the first assessment of the intracellular behaviour in ovine monocyte-derived macrophages (MDMs) of Map isolates representing distinct genotypes (C, S and B), and isolated from cattle, sheep, goat, fallow deer, deer, and wild boar. Intracellular growth and survival of the selected isolates in ovine MDMs was assessed by quantification of CFUs inside of the host cells at 2 h p.i. (day 0) and 7 d p. i. using an automatic liquid culture system (Bactec MGIT 960). Variations in bacterial counts over 7 days from the baseline were small, in a range between 1.63 to 1.05-fold. After 7 d of infection, variations in the estimated log10 CFUs between all the tested isolates were not statistically significant. In addition, ovine MDMs exhibited enhanced anti-inflammatory, antiapoptotic and antidestructive responses when infected with two ovine isolates of distinct genotype (C and S) or with two C-type isolates from distinct hosts (cattle and sheep); which correlated with the successful survival of these isolates within ovine MDMs. A second objective was to study, based on an in vitro granuloma model, latter stages of the infection by investigating the capacity of two Map isolates from cattle and sheep to trigger formation of microgranulomas. Upon 10 d p.i., both Map isolates were able to induce the formation of granulomas comparable to the granulomas observed in clinical specimens with respect to the cellular components involved. In summary, our results demonstrated that Map isolates from cattle, sheep, goats, deer, fallow-deer and wild boar were able not only to initiate but also to establish a successful infection in ovine macrophages regardless of genotype.

Show MeSH
Related in: MedlinePlus