Evaluation of intracellular survival of Campylobacter fetus subsp. fetus in bovine endometrial cells by qPCR

Document Type : Full paper (Original article)

Authors

1 Department of Genetics and Virology, Faculty of Chemical Sciences, Autonomous University of Coahuila, Saltillo, Mexico

2 Laboratory of Agricultural Microbiology, Department of Agricultural and Animal Production, Metropolitan Autonomous University-Xochimilco, Mexico City, Mexico

3 Biological and Health Sciences Division, Department of Agricultural and Animal Production, Metropolitan Autonomous University-Xochimilco, Mexico City, Mexico

4 Laboratory of Molecular Biology, Department of Agricultural and Animal Production, Metropolitan Autonomous University-Xochimilco, Mexico City, Mexico

10.22099/ijvr.2021.38693.5632

Abstract

Background: Campylobacter fetus subsp. fetus is the causal agent of sporadic abortion and infertility in bovines that produces economic losses in livestock. Aims: This study evaluates the capability of C. fetus subsp. fetus to invade and survive in bovine endometrial epithelial cells and attempts to describe a pathogenic mechanism of this microorganism. Methods: Primary culture of bovine endometrial epithelial cells was challenged with C. fetus subsp. fetus. Intracellular bacteria, represented by the number of genomic copies (g.c.) were quantified at 0, 2, 4, 10, and 24 hours post-infection (h.p.i.), by quantitative polymerase chain reaction (qPCR). The presence of intracellular bacteria was evaluated by immunofluorescence and immunohistochemistry. Results: The results showed that only viable C. fetus subsp. fetus could invade endometrial cells. The g.c. number in assays with viable C. fetus subsp. fetus reached an average value of 656 g.c., remained constant until 4 h.p.i., then decreased to 100 g.c, at 24 h.p.i. In assays with non-viable microorganisms, the average value of g.c. was less than 1 g.c. and never changed. The intracellular presence of this bacteria was confirmed at 2 h.p.i. by immunofluorescence and immunohistochemistry. Conclusion: The results suggest that only C. fetus subsp. fetus viable can invade bovine endometrial epithelial cells but will not replicate in them, indicating that the endometrial cells do not represent a replication niche for this pathogen. Nonetheless, this invasion capability suggests that this type of cell could be employed by the pathogen to spread to other tissues.

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Agostinis, C; Mangogna, A; Bossi, F; Ricci, G and Kishore, U (2019). Uterine immunity and microbiota: A shifting paradigm. Front Immunol., 10: 1-11.
Alge, CS; Hauck, SM; Priglinger, SG; Kampik, A and Ueffing, M (2006). Differential protein profiling of primary versus immortalized human RPE cells identifies expression patterns associated with cytoskeletal remodeling and cell survival. J. Proteome Res., 5: 862-878.
Baker, NT and Graham, LL (2010). Campylobacter fetus translocation across Caco-2 cell monolayers. Microb. Pathog., 49: 260-272.
Blakes, A; Day, NPJ; Atwal, S; Giengkam, S; Blacksell, SD and Paris, DH (2015). Improved quantification, propagation, purification, and storage of the obligate intracellular human pathogen Orientia tsutsugamushi. PLoS Negl. Trop. Dis., 9: 1-20.
Boumart, Z; Velge, P and Wiedemann, A (2014). Multiple invasion mechanisms and different intracellular behaviors: a new vision of Salmonella–host cell interaction. FEMS Microbiol. Lett., 361: 1-7.
Brumell, JH; Steele-Mortimer, O and Finlay, BB (1999). Bacterial invasion: force feeding by Salmonella. Curr. Biol., 9: 277-280.
Bücker, R; Krug, SM; Fromm, A; Nielsen, HL; Fromm, M and Nielsen, H (2017). Campylobacter fetus impairs barrier function in HT-29/B6 cells through focal tight junction alterations and leaks. Ann. N. Y. Acad. Sci., 1405: 189-201.
Cagnoli, CI; Chiapparrone, ML; Cacciato, CS; Rodríguez, MG; Aller, JF and Catena, MDC (2020). Effects of Campylobacter fetus on bull sperm quality. Microb. Pathog., 149: 1-5.
Day, AS; Nahidi, L; Kaakoush, NO; Mitchell, HM; Zhang, L and Leach, ST (2010). Host Attachment, invasion, and stimulation of proinflammatory cytokines by Campylobacter concisus and other non-Campylobacter jejuni Campylobacter species. J. Infect. Dis., 202: 1855-1865.
Dersch, P (2003). Molecular and cellular mechanisms of bacterial entry into host cells. In: Herwald, H (Ed.), Host response mechanisms in infectious diseases. (1st Edn.), Karger, Switzerland. PP: 183-209.
Elsinghorst, EA (1994). Measurement of invasion by gentamicin resistance. Methods Enzymol., 236: 405-420.
Eritja, N; Llobet, D; Domingo, M; Santacana, M; Yeramian, A and Matias-Guiu, X (2010). A novel three-dimensional culture system of polarized epithelial cells to study endometrial carcinogenesis. Am. J. Pathol., 176: 2722-2731.
Feng, X; Cao, Y; Liu, Z; Fu, Y; Liu, B and Li, D (2012). Lipopolysaccharide increases Toll-like receptor 4 and downstream Toll-like receptor signaling molecules expression in bovine endometrial epithelial cells. Vet. Immunol. Immunopathol. 151: 20-27.
Gard, J (2016). Bovine genital campylobacteriosis-A review. Int. J. Vet. Sci. Res., 2: 29-31.
Graham, LL (2003). Campylobacter fetus adheres to and enters INT 407 cells. Can. J. Microbiol., 48: 995-1007.
González-Escalona, N; Fey, A; Höe, MG; Espejo, RT and Guzmán, CA (2006). Quantitative reverse transcription polymerase chain reaction analysis of Vibrio cholerae cells entering the viable but non-culturable state and starvation in response to cold shock. Environ. Microbiol., 8: 658-666.
Haeger, JD; Hambruch, N; Dantzer, V; Hoelker, M; Schellander, K and Klisch, K (2015). Changes in endometrial ezrin and cytokeratin 18 expression during bovine implantation and in caruncular endometrial spheroids in vitro. Placenta. 36: 821-831.
Hall, GA and Jones, PW (1977). A study of the pathogenesis of experimental Salmonella dublin abortion in cattle. J. Comp. Pathol., 87: 53-65.
Iraola, G; Forster, SC; Kumar, N; Lehours, P; Bekal, S and García-Peña, FJ (2017). Distinct Campylobacter fetus lineages adapted as livestock pathogens and human pathobionts in the intestinal microbiota. Nat. Commun., 8: 1367-1370.
Iraola, G; Hernández, M; Calleros, L; Paolicchi, F; Silveyra, S; Velilla, A; Carretto, L; Rodríguez, E and Pérez, R (2012). Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses. J. Vet. Sci., 13: 371-376.
Kaakoush, NO; Castaño-Rodríguez, N; Mitchell, HM and Man, SM (2015). Global epidemiology of Campylobacter infection. Clin. Microbiol. Rev., 28: 687-720.
Kienesberger, S; Sprenger, H; Wolfgruber, S; Halwachs, B and Thallinger, GG (2014). Comparative genome analysis of Campylobacter fetus subspecies revealed horizontally acquired genetic elements important for virulence and niche specificity. PLOS One. 9: 1-13.
Kisselbach, L; Merges, M; Bossie, A and Boyd, A (2009). CD90 expression on human primary cells and elimination of contaminating fibroblasts from cell cultures. Cytotechnology. 59: 31-44.
Konkel, ME and Joens, LA (1989). Adhesion to and invasion of HEp-2 cells by Campylobacter spp. Infect. Immun., 57: 2984-2990.
Louwen, R; Nieuwenhuis, EES; van Marrewijk, L; Horst-Kreft, D and de Ruiter, L (2012). Campylobacter jejuni translocation across intestinal epithelial cells is facilitated by ganglioside-like lipooligosaccharide structures. Infect. Immun., 80: 3307-3318.
Luangtongkum, T; Morishita, TY; El-Tayeb, AB; Ison, AJ and Zhang, Q (2007). Comparison of antimicrobial susceptibility testing of Campylobacter spp. by the agar dilution and the agar disk diffusion methods. J. Clin. Microbiol., 45: 590-594.
Michi, AN; Favetto, PH; Kastelic, J and Cobo, ER (2016). A review of sexually transmitted bovine trichomoniasis and campylobacteriosis affecting cattle reproductive health. Theriogenology. 85: 781-791.
Mshelia, GD; Amin, JD; Woldehiwet, Z; Murray, RD and Egwu, GO (2010). Epidemiology of bovine venereal campylobacteriosis: geographic distribution and recent advances in molecular diagnostic techniques. Reprod. Domest. Anim., 45: 221-230.
Oliveira, LM; Resende, DM; Dorneles, EM; Horácio, EC; Alves, FL; Gonçalves, LO; Tavares, GS; Stynen, AP; Lage, AP and Ruiz, JC (2016). Complete genome sequence of type strain Campylobacter fetus subsp. fetus ATCC 27374. Genome Announc., 4: e01344-16.
Paukszto, L; Mikolajczyk, A; Jastrzebski, JP; Majewska, M; Dobrzyn, K; Kiezun, M; Smolinska, N and Kaminski, T (2020). Transcriptome, spliceosome and editome expression patterns of the porcine endometrium in response to a single subclinical dose of Salmonella Enteritidis lipopolysaccharide. Int. J. Mol. Sci., 2: 1-25.
Ricchi, M; Bertasio, C; Boniotti, MB; Vicari, N; Russo, S and Tilola, M (2017). Comparison among the quantification of bacterial pathogens by qPCR, dPCR, and cultural methods. Front Microbiol., 8: 1-15.
Viejo, G; Gomez, B; De Miguel, D; Del Valle, A; Otero, L and De La Iglesia, P (2001). Campylobacter fetus subspecies fetus bacteremia associated with chorioamnionitis and intact fetal membranes. Scand. J. Infect. Dis., 33: 126-127.
Wang, HB; Lü, SH; Lin, QX; Feng, LX; Li, DX and Duan, CM (2010). Reconstruction of endometrium in vitro via rabbit uterine endometrial cells expanded by sex steroid. Fertil. Steril., 93: 2385-2395.
Watson, RO and Galán, JE (2008). Campylobacter jejuni survives within epithelial cells by avoiding delivery to lysosomes. PLOS Pathog., 4: 1-15.
Wattanaphansak, S; Gebhart, CJ; Anderson, JM and Singer, RS (2010). Development of a polymerase chain reaction assay for quantification of Lawsonia intracellularis. J. Vet. Diagn. Invest., 22: 598-602.
Yoon, H; Gros, P and Heffron, F (2011). Quantitative PCR-based competitive index for high-throughput screening of Salmonella virulence factors. Infect. Immun., 79: 360-368.