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Detection and characterisation of Lactobacillus spp. in the bovine uterus and their influence on bovine endometrial epithelial cells in vitro.

ABSTRACT: Bacterial infections and inflammation of the uterus are common in dairy cattle after parturition. In particular, pathogenic bacteria that cause endometritis have been the focus of research in cattle reproduction in the last ten years. The aim of the present study was to identify commensal lactobacilli in the bovine uterus and to examine their influence on the synthesis of pro-inflammatory factors in bovine endometrial epithelial cells in vitro. Lactobacillus species were isolated from healthy bovine uteri and further characterised. Bovine endometrial epithelial cells in the second passage (n = 5 animals) were co-cultured with the autochthonous isolates L. buchneri, L. ruminis and L. amylovorus as well as with a commercially available L. vaginalis in different multiplicities of infection (MOI = 1, 5 and 10, respectively). Endometrial epithelial cells cultured without bacteria served as controls. At distinct points in time (2, 4 and 6 h) total RNA was extracted from co-cultured epithelial cells and subjected to reverse transcription quantitative PCR of pro-inflammatory factors. Furthermore, the release of such factors by co-cultured epithelial cells was measured by ELISA or EIA after 24 and 48 h. L. ruminis and L. amylovorus induced increased interleukin (IL) IL1A, IL6, IL8 and prostaglandin-endoperoxide synthase 2 mRNA levels and the release of IL8 and prostaglandin F(2?) in endometrial epithelial cells compared with control cells. In contrast, L. buchneri did not significantly influence the expression and release of these factors. Toll-like receptors 2 and 6 transcripts were found unchanged in co-cultured and untreated epithelial cells in vitro. However, endometrial epithelial cells of each animal showed individual differences in the response to bacterial load. These results suggest that Lactobacillus species are present in the bovine uterus, revealing immunomodulatory properties.

SUBMITTER: Gartner MA

PROVIDER: S-EPMC4372290 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Detection and characterisation of Lactobacillus spp. in the bovine uterus and their influence on bovine endometrial epithelial cells in vitro.

Gärtner Martina A MA Bondzio Angelika A Braun Nicole N Jung Markus M Einspanier Ralf R Gabler Christoph C

PloS one 20150324 3

Bacterial infections and inflammation of the uterus are common in dairy cattle after parturition. In particular, pathogenic bacteria that cause endometritis have been the focus of research in cattle reproduction in the last ten years. The aim of the present study was to identify commensal lactobacilli in the bovine uterus and to examine their influence on the synthesis of pro-inflammatory factors in bovine endometrial epithelial cells in vitro. Lactobacillus species were isolated from healthy bo ...[more]

PMID: 25803719

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Project description:Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.

Project description:The presence of pathogenic bacteria in ejaculates has been a topic in boar semen preservation over the last decades. Since little information is available on commensal bacteria in boar semen, the aim of the present study was to identify commensal lactobacilli in fresh cryopreserved boar semen and to examine their influence on boar semen quality. Therefore, 111 boar ejaculates were investigated for the presence of Lactobacillus species. Thirty samples (27%) contained viable Lactobacillus species (e.g. L. amylovorus, L. animalis, L. reuteri and Weisella minor). L. animalis and L. buchneri DSM 32407 (isolated from the bovine uterus) qualified for further examinations based on their growth rate in six antibiotic-free boar semen extenders. After a 120 min short-term incubation with an antibiotic-free BTS-extender, progressive motility was diminished (P = 0.001) upon addition of 105 and 106 colony forming units (CFU/mL) L. animalis. The supplementation with L. buchneri DSM 32407 had no significant (P > 0.05) influence on sperm quality during short-term co-incubation. After 168 h long-term co-incubation, motility analysis revealed a negative (P = 0.026) impact of 105 CFU/mL L. buchneri DSM 32407. A concentration- and storage-dependent effect is particularly obvious (P < 0.001) using 106 CFU/mL L. buchneri DSM 32407. Most notably, the thermo-resistance (TRT) for 106 CFU/mL L. buchneri DSM 32407 (P = 0.001) was inferior to BTS with and without gentamicin after 72 and 168 h of semen co-incubation. The supplementation of 105 CFU/mL L. buchneri DSM 32407 impaired progressive motility to a lesser extent. The percentage of mitochondrially active spermatozoa after 96 h (P = 0.009) and membrane-intact spermatozoa after 168 h (P < 0.001) was lower when 106 CFU/mL L. buchneri DSM 32407 were suspended compared with all other groups. Finally, the addition of L. buchneri DSM 32407 to BTS-extended boar semen had no competitive effect on the total amount of bacteria 48 h after co-incubation. In summary, the present study demonstrated that there are Lactobacillus species present in the porcine seminal plasma, which can be cultivated using standard procedures. However, long-term co-incubation of lactic acid bacteria with spermatozoa had a negative influence on spermatozoa.

Project description:BackgroundCryptosporidium is a genus of apicomplexan parasites that cause enteric disease in vertebrates. In pigs, infections are most often asymptomatic, but may result in diarrhoea and poor growth. The most common species detected in pigs are C. suis and C. scrofarum with low zoonotic potential. C. parvum, with higher zoonotic potential, may also be found. As previous knowledge on the occurrence of Cryptosporidium in Swedish pigs is scarce, this was investigated in our study. Faecal samples from 13 pig herds were collected and a total of 222 pooled pen samples, from suckling piglets (n = 48), growers, aged 6-12 weeks (n = 57), fatteners, aged 13-24 weeks (n = 67) and adult animals (n = 50) were included. Samples were analysed using microscopy and positive samples were further analysed using polymerase chain reaction and sequencing of the 18S rRNA gene and the 28S rRNA gene to determine species.ResultsCryptosporidium spp. were detected in all sampled herds and in 25% (56/222) of the individual pen samples. Infections were most common in growers and fatteners with 51% (29/57) and 35% (20/67) positive samples in each group, respectively. The piglets had 8% (4/48) positive samples and adults had 6% (3/50). Species determination showed C. suis and C. scrofarum in piglets and growers, C. scrofarum in the fatteners, and C. suis and C. parvum in the adults. Although no mixed infections could be confirmed we saw signs of double peaks in the 28S rRNA gene chromatograms, possibly indicating more than one species present per sample.ConclusionCryptosporidium spp. were detected on every sampled farm and in 25% of the individual pen samples in our study. We therefore conclude that Cryptosporidium spp. are present and likely common in Swedish pig herds, where pigs are loose and reared on solid floors. However, none of the farms reported any problems with poor weight gain, diarrhoea, or reduced appetite in their pig herds. The pig adapted C. suis and C. scrofarum were the predominant species identified. Two samples were positive for the more zoonotic C. parvum, and pigs should hence not be disregarded as a possible source of zoonotic cryptosporidiosis.

Dataset Information

Detection and characterisation of Lactobacillus spp. in the bovine uterus and their influence on bovine endometrial epithelial cells in vitro.

Publications

Detection and characterisation of Lactobacillus spp. in the bovine uterus and their influence on bovine endometrial epithelial cells in vitro.

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