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Campylobacter fetus is Internalized by Bovine Endometrial Epithelial Cells

LIZETH GUADALUPE CAMPOS-MÚZQUIZ

LIZETH GUADALUPE CAMPOS-MÚZQUIZ

Doctorado en Ciencias Agropecuarias, Universidad Autónoma Metropolitana XochimilcoCiudad de México, México
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CAMPOS-MÚZQUIZ, LIZETH GUADALUPE
, 
ESTELA TERESITA MÉNDEZ-OLVERA

ESTELA TERESITA MÉNDEZ-OLVERA

Doctorado en Ciencias Agropecuarias, Universidad Autónoma Metropolitana XochimilcoCiudad de México, México
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MÉNDEZ-OLVERA, ESTELA TERESITA
, 
BEATRIZ ARELLANO-REYNOSO

BEATRIZ ARELLANO-REYNOSO

Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Ciudad UniversitariaCiudad de México, México
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ARELLANO-REYNOSO, BEATRIZ
 and 
DANIEL MARTÍNEZ-GÓMEZ

DANIEL MARTÍNEZ-GÓMEZ

Doctorado en Ciencias Agropecuarias, Universidad Autónoma Metropolitana XochimilcoCiudad de México, México
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MARTÍNEZ-GÓMEZ, DANIEL
  
Jun 28, 2019
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Article Category: original-paper
Published Online: Jun 28, 2019
Page range: 217 - 224
Received: Dec 05, 2018
Accepted: Feb 14, 2019
DOI: https://doi.org/10.33073/pjm-2019-022
Keywords
© 2019 Lizeth Guadalupe Campos-Múzquiz et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Epithelial cells from bovine endometrium maintained in DMEM supplemented with 10% fetal bovine serum.A) Normal appearance of the epithelial-like cells (20×). B) Immunofluorescence. Cytokeratin 18 of epithelial cells of bovine endometrium stained with Alexa 488 (green) (40×). C) The results of RT-PCR for keratin 8 on 2% agarose gel after staining with ethidium bromide. In left lane: DNA ladder (Thermo Fisher Scientific); right lane: the amplicon of keratin 8 (215 pb).
Epithelial cells from bovine endometrium maintained in DMEM supplemented with 10% fetal bovine serum.A) Normal appearance of the epithelial-like cells (20×). B) Immunofluorescence. Cytokeratin 18 of epithelial cells of bovine endometrium stained with Alexa 488 (green) (40×). C) The results of RT-PCR for keratin 8 on 2% agarose gel after staining with ethidium bromide. In left lane: DNA ladder (Thermo Fisher Scientific); right lane: the amplicon of keratin 8 (215 pb).

Fig. 2.

The adherence assay. Epithelial cells from bovine endometrium were challenged with C. fetus (A) or E. coli (B) for 1 h, fixed with methanol and stained with Giemsa (100×). The arrows show the adhered bacteria.
The adherence assay. Epithelial cells from bovine endometrium were challenged with C. fetus (A) or E. coli (B) for 1 h, fixed with methanol and stained with Giemsa (100×). The arrows show the adhered bacteria.

Fig. 3.

The intracellular survival assay. Epithelial cells of endometrium of bovine were infected with C. fetus ATCC 27374 (a MOI of 1000:1). The intracellular bacteria were recovered and plated on the Campylobacter selective agar supplemented with 5% of blood. Average log CFU are shown at 0, 2, 4, 10 and 24 h p.i.
The intracellular survival assay. Epithelial cells of endometrium of bovine were infected with C. fetus ATCC 27374 (a MOI of 1000:1). The intracellular bacteria were recovered and plated on the Campylobacter selective agar supplemented with 5% of blood. Average log CFU are shown at 0, 2, 4, 10 and 24 h p.i.

Fig. 4.

Confocal differential fluorescent staining of internal C. fetus ATCC 27374 on the infected epithelial cells of bovine endometrium. Epithelial cells were grown on coverslips and infected with C. fetus ATCC 27374 at a MOI of 1000:1. Cytoskeleton was stained with phalloidin-FITC (green), and the bacteria with Alexa 594 (red) 2 h p.i. White arrows show intracellular C. fetus (70×).
Confocal differential fluorescent staining of internal C. fetus ATCC 27374 on the infected epithelial cells of bovine endometrium. Epithelial cells were grown on coverslips and infected with C. fetus ATCC 27374 at a MOI of 1000:1. Cytoskeleton was stained with phalloidin-FITC (green), and the bacteria with Alexa 594 (red) 2 h p.i. White arrows show intracellular C. fetus (70×).

Fig. 5.

The cytoskeleton inhibition assay. Epithelial cells of endometrium of bovine were treated with cytocalasin D or nocodazole before and during infection. The cells were infected with C. fetus ATCC 27374 (a MOI of 1000:1). The intracellular bacteria were recovered and plated on the Campylobacter selective agar supplemented with 5% of blood. Average log CFU are shown at 0 and 2 p.i. T-test was performed. All treatments were compared to the not-treatment control, *(p < 0.001).
The cytoskeleton inhibition assay. Epithelial cells of endometrium of bovine were treated with cytocalasin D or nocodazole before and during infection. The cells were infected with C. fetus ATCC 27374 (a MOI of 1000:1). The intracellular bacteria were recovered and plated on the Campylobacter selective agar supplemented with 5% of blood. Average log CFU are shown at 0 and 2 p.i. T-test was performed. All treatments were compared to the not-treatment control, *(p < 0.001).
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