A paradox in bacterial pathogenesis: Activation of the local macrophage inflammasome is required for virulence of streptococcus uberis

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host–pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1β from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1β was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection

An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid

Background: Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis. Methodology/Principal Findings: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results. Conclusions/Significance: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.This work was supported by Regione Autonoma della Sardegna (http://www.regione.sardegna.it/)Pubblicat

Detection of cathelicidin-1 in the milk as an early indicator of mastitis in ewes

The objective of the study was the investigation of the behaviour of cathelicidin-1 in the milk after experimental infection with two prominent bacterial pathogens (experiment 1: Mannheimia haemolytica, experiment 2: M. haemolytica and Staphylococcus chromogenes) as a potential early indicator for diagnosis of mastitis in sheep. In two experiments, after bacterial inoculation into the udder of ewes, bacteriological and cytological examinations of milk samples as well as proteomics examinations [two-dimensional gel electrophoresis analysis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis] were performed sequentially. Cathelicidin-1 was detected and spot densities obtained from PDQuest v.8.0 were recorded. Associations were calculated between cell content and spot densities as well as between presence of mastitis in a mammary gland at a given time-point and detection of cathelicidin-1 in the respective milk sample. All inoculated mammary glands developed mastitis, confirmed by the consistent bacterial isolation from mammary secretion and increased leucocyte content therein. Spot density of cathelicidin-1 in samples from inoculated glands increased 3 h post-inoculation; spot density of cathelicidin-1 in samples from inoculated glands was higher than in samples from uninoculated controls. There was clear evidence of correlation between cell content and cathelicidin-1 spot densities in milk samples. There was significant association between presence of mastitis in the mammary gland and detection of cathelicidin-1 in the respective milk sample; overall accuracy was 0.818—this was significantly greater during the first 24 h post-challenge (0.903) than after the first day (0.704). In conclusion, detection of cathelicidin-1 in milk was significantly associated with presence of mastitis in ewes. The associations were stronger during the first 24 h post-infection than after the first day. Cathelicidin-1 has the advantage that it can be a non-specific biomarker, as simply a “positive” / “negative” assessment would be sufficient. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

What we have lost: Mastitis resistance in Holstein Friesians and in a local cattle breed

In Holstein Friesian dairy cows, selective pressure for increased milk production has led to a higher propensity to disease, including mastitis, when compared to less selected and lower producing dairy breeds. The biology underpinning the higher resistance to disease of such \ue2\u80\u9clocal breeds\ue2\u80\u9d is not fully understood. With the aim of investigating the factors associated to this phenomenon, we applied a multidisciplinary approach to compare innate immune response patterns, metabolic parameters, milk protein profiles and the milk microbiota in Holstein Friesian and Rendena cows reared in the same farm and under the same management conditions. Quarter milk samples and blood plasma were collected from all cows at dry-off, 1 day after calving, 7\ue2\u80\u9310 days after calving and 30 days after calving. Quarter milk samples were subjected to bacteriological culture, characterization of the milk microbiota by 16S metagenomics, milk protein profiling by electrophoresis and densitometry, somatic cell counting, measurement of the inflammation marker cathelicidin and assessment of different innate immune-related mediators such as lysozyme, CD45, IL-1\uce\ub2, TNF-\uce\ub1, PTX3, IL-1R8. In parallel, the main inflammometabolic parameters were measured in blood plasma samples. Despite having relatively few animals (6 moderate-yielding Holstein Friesian and 4 low-yielding Rendena) some important differences were apparent. Holstein Friesian cows showed a more severe fat mobilization and systemic inflammatory response postpartum in comparison with Rendena cows, which had a greater postpartum muscle mass and an increased amino acid mobilization compared to Holstein Friesians. Upon bacteriological analysis, contagious bacteria such as Staphylococcus aureus and Streptococcus agalactiae were absent, but significant differences were seen in the general composition of the milk microbiota of the two breeds. Concerning the milk protein abundance profile, pronounced differences were seen in colostrum, with significantly higher amounts of immunoglobulins and other immune-related proteins in Rendena. Added to this, the expression of innate immune related genes such as PTX-3, IL-1\uce\ub2, TNF-\uce\ub1, and KRT5 expression in milk epithelial and leukocyte cell components, respectively, was lower in Holstein Friesian colostrum compared with Rendena. In conclusion, several differences were observed in the two breeds, in spite of the same farming conditions. The observations reported in this work present numerous pointers to the factors that may provide autochthonous, more rustic breeds with a higher resistance to disease