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Bacterial biofilms are organized sessile communities of bacteria enclosed in extracellular polymeric substances (EPS). To analyze organization of bacteria and EPS in high resolution and high magnification by scanning electron microscopy (SEM), it is important to preserve the complex architecture of biofilms. Therefore, fixation abilities of formalin, glutaraldehyde, and Methacarn (methanol/chloroform/acetic acid-6:3:1) fixatives were evaluated to identify which fixative would best preserve the complex structure of bacterial biofilms. Economically important Gram-negative Mannheimia haemolytica, the major pathogen associated with bovine respiratory disease complex, and Gram-positive Staphylococcus aureus, the major cause of chronic mastitis in cattle, bacteria were selected since both form biofilms on solid-liquid interface. For SEM analysis, round glass coverslips were placed into the wells of 24-well plates and diluted M. haemolytica or S. aureus cultures were added, and incubated at 37°C for 48-72 h under static growth conditions. Culture media were aspirated and biofilms were fixed with an individual fixative for 48 h. SEM examination revealed that all three fixatives were effective preserving the bacterial cell morphology, however only Methacarn fixative could consistently preserve the complex structure of biofilms. EPS layers were clearly visible on the top, in the middle, and in the bottom of the biofilms with Methacarn fixative. Biomass and three-dimensional structure of the biofilms were further confirmed spectrophotometrically following crystal violet staining and by confocal microscopy after viability staining. These findings demonstrate that Methacarn fixative solution is superior to the other fixatives evaluated to preserve the complex architecture of biofilms grown on glass coverslips for SEM evaluation.

Background Milk exosomes are a rich source of microRNAs (miRNAs) that are protected from degradation. Ingestion of milk and subsequent absorption of miRNAs into recipient cells by endocytosis may play a role in the regulation of neonatal innate and adaptive immunity. In contrast, the miRNA content of milk exosomes may also be indicative of a lactating animal’s health; whereby, the presence or absence of specific miRNAs could serve as biomarkers for early detection of bacterial infection that can lead to mastitis. In the present study, we therefore analyzed and compared miRNA expression profiles of milk exosomes from four Holstein cows obtained during mid-lactation prior to and after infection (48 h) of the mammary gland with Staphylococcus aureus . Methods Milk exosomes, purified from control and S. aureus infected cows, were extracted for RNA. Following preparation indexed libraries from both groups the samples were subjected to next generation sequencing. Results Next generation sequencing of eight, unpooled small RNA libraries derived from milk exosomes produced about 60.5 million high-quality, bovine-specific sequence reads for comparison of miRNA expression between treatments. Sequence identity analysis showed the miRNAs make up about 13 % of the average RNA content of these exosomes. Although 417 known bovine miRNAs were identified, miRNAs represented the least diverse class of RNA accounting for only 1 % of all unique sequences. The 20 most prevalent unique sequences within this class accounted for about 90 % of the total miRNA-associated reads across samples. Non-annotated, unique reads provided evidence for another 303 previously unknown bovine miRNAs. Expression analyses found 14 known bovine microRNAs significantly differed in frequency between exosomes from infected and control animals. Conclusions Our survey of miRNA expression from uninfected milk exosomes and those produced in response to infection provides new and comprehensive information supporting a role for delivery into milk of specific miRNAs involved in immune response. In particular, bta-miR-142-5p, and −223 are potential biomarkers for early detection of bacterial infection of the mammary gland. Additionally, 22 mammary-expressed genes involved in regulation of host immune processes and response to inflammation were identified as potential binding targets of the differentially expressed miRNAs.

Mastitis remains the most prevalent and costly disease to dairy producers. Granulocytes are the primary host innate immune cell responders during infectious mastitis. Here we examine three mid-lactation Holsteins challenged with ~ 150 CFU of Staphylococcus aureus (Newbould) that developed chronic mastitis as assessed by bacteria and somatic cell counts in a single quarter. Single-cell RNA-sequencing (scRNA-seq) of blood and milk cells identified immune cell populations of interest from both tissues, and the proportion of cell types recovered via scRNA-seq were highly similar to those recovered via flow cytometry. Granulocytes were the predominating cell type in both blood and milk samples; however granulocytes identified via scRNA-seq revealed several clusters comprised primarily of milk-derived cells. Milk-enriched granulocyte clusters were further investigated to identify gene signatures indicative of the granulocyte-specific localized immune responses in the mammary gland during chronic mastitis infection. Biological process enrichment analysis of gene signatures further revealed relevant networks such as granulocyte migration, myeloid cell differentiation, and inflammatory responses. In total, the work describes the immune landscape occurring at both peripheral and local sites of cattle with mastitis and identified important granulocyte-specific features of the localized immune response occurring during chronic infection.

Objective Bile and its individual components, mainly bile acids, are important for digestion and drive bacterial community dynamics in the upper gastrointestinal tract of chickens. However, specific responses to bile acids have been characterized in only a few commensal bacteria, and it is unclear how other members of the microbiota respond to biliary stress. Here, we used label-free LC–MS/MS to assess the proteomic response of a common inhabitant of the chicken small intestine, Turicibacter bilis MMM721, to 24 h of growth in anaerobic growth media supplemented with 0.1% whole chicken bile, 0.1% taurochenodeoxycholic acid (TCDCA), or 0.1% taurocholic acid (TCA). Results Seventy, 46, and 10 differentially expressed proteins were identified in Turicibacter bilis MMM721 cultured with supplements of chicken bile, TCDCA, and TCA, respectively, when compared to unsupplemented controls. Many differentially expressed proteins were predicted to be involved in ribosomal processes, post-translational modifications and chaperones, and modifications to the cell surface. Ultimately, the T. bilis MMM721 response to whole bile and bile acids is complex and may relate to adaptations for small intestine colonization, with numerous proteins from a variety of functional categories being impacted.

Leptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C). L. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent differential expression of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203. Investigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets.

Deficiency of serum levels of 25-hydroxyvitamin D3 has been related to increased risk of lower respiratory tract infections in children. Respiratory syncytial virus (RSV) is a leading cause of low respiratory tract infections in infants and young children. The neonatal calf model of RSV infection shares many features in common with RSV infection in infants and children. In the present study, we hypothesized that calves with low circulating levels of 25-hydroxyvitamin D3 (25(OH)D3) would be more susceptible to RSV infection than calves with high circulating levels of 25(OH)D3. Calves were fed milk replacer diets with different levels of vitamin D for a 10 wk period to establish two treatment groups, one with high (177 ng/ml) and one with low (32.5 ng/ml) circulating 25(OH)D3. Animals were experimentally infected via aerosol challenge with RSV. Data on circulating 25(OH)D3 levels showed that high and low concentrations of 25(OH)D3 were maintained during infection. At necropsy, lung lesions due to RSV were similar in the two vitamin D treatment groups. We show for the first time that RSV infection activates the vitamin D intracrine pathway in the inflamed lung. Importantly, however, we observed that cytokines frequently inhibited by this pathway in vitro are, in fact, either significantly upregulated (IL-12p40) or unaffected (IFN-γ) in the lungs of RSV-infected calves with high circulating levels of 25(OH)D3. Our data indicate that while vitamin D does have an immunomodulatory role during RSV infection, there was no significant impact on pathogenesis during the early phases of RSV infection. Further examination of the potential effects of vitamin D status on RSV disease resolution will require longer-term studies with immunologically sufficient and deficient vitamin D levels.

In dairy cows, the period from the end of lactation through the dry period and into the transition period, requires vast physiological and immunological changes critical to mammary health. The dry period is important to the success of the next lactation and intramammary infections during the dry period will adversely alter mammary function, health and milk production for the subsequent lactation. MicroRNAs (miRNAs) are small non-coding RNAs that can post transcriptionally regulate gene expression. We sought to characterize the miRNA profile in dry secretions from the last day of lactation to 3, 10, and 21 days post dry-off. We identified 816 known and 80 novel miRNAs. We found 46 miRNAs whose expression significantly changed (q-value < 0.05) over the first three weeks of dry-off. Additionally, we examined the slopes of random regression models of log transformed normalized counts and cross analyzed the 46 significantly upregulated and downregulated miRNAs. These miRNAs were found to be associated with important components of pregnancy, lactation, as well as inflammation and disease. Detailing the miRNA profile of dry secretions through the dry-off period provides insight into the biology at work, possible means of regulation, components of resistance and/or susceptibility, and outlets for targeted therapy development.

Numerous in vitro studies have shown that toll-like receptor signaling induces 25-hydroxyvitamin D3 1α-hydroxylase (1α-OHase; CYP27B1) expression in macrophages from various species. 1α-OHase is the primary enzyme that converts 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Subsequently, synthesis of 1,25(OH)2D3 by 1α-OHase in macrophages has been shown to modulate innate immune responses of macrophages. Despite the numerous in vitro studies that have shown 1α-OHase expression is induced in macrophages, however, evidence that 1α-OHase expression is induced by pathogens in vivo is limited. The objective of this study was to evaluate 1α-OHase gene expression in macrophages and mammary tissue during an in vivo bacterial infection with Streptococcus uberis. In tissue and secreted cells from the infected mammary glands, 1α-OHase gene expression was significantly increased compared to expression in tissue and cells from the healthy mammary tissue. Separation of the cells by FACS9 revealed that 1α-OHase was predominantly expressed in the CD14+ cells isolated from the infected mammary tissue. The 24-hydroxylase gene, a gene that is highly upregulated by 1,25(OH)2D3, was significantly more expressed in tissue and cells from the infected mammary tissue than from the healthy uninfected mammary tissue thus indicating significant local 1,25(OH)2D3 production at the infection site. In conclusion, this study provides the first in vivo evidence that 1α-OHase expression is upregulated in macrophages in response to bacterial infection and that 1α-OHase at the site of infection provides 1,25(OH)2D3 for local regulation of vitamin D responsive genes.

Background Mastitis is the most common health concern plaguing the modern dairy cow and costs dairy producers estimates of two billion dollars annually. Staphylococcus aureus infections are prevalent, displaying varied disease presentation and markedly low cure rates. Neutrophils are considered the first line of defense against mastitis causing bacteria and are frequently targeted in the development of treatment and prevention technologies. We describe a case of naturally occurring, chronic mastitis in a Holstein cow (1428), caused by a novel strain of S. aureus that was not able to be cleared by antibiotic treatment. Case presentation The infection was identified in a single quarter, 2 months into the cow’s first lactation. The infection persisted for the following 20 months, including through dry off, and a second calving and lactation. This case of mastitis was associated with a consistently high somatic cell count, however presented with no other clinical signs. This cow was unsuccessfully treated with antibiotics commonly used to treat mastitis, consisting of two rounds of treatment during lactation and an additional round at the beginning of dry off. The chronic infection was also unchanged through an experimental mid-lactation treatment with pegylated granulocyte-colony stimulating factor (PEG-gCSF) and an additional periparturient treatment with PEG-gCSF. We isolated milk neutrophils from 1428 and compared them to two cows challenged with experimental S. aureus, strain Newbould 305. Neutrophils from 1428’s milk had higher surface expression of myeloperoxidase compared to experimental Newbould challenged animals, as well as increased presence of Neutrophil Extracellular Traps. This suggests a heightened activation state of neutrophils sourced from 1428’s naturally occurring infection. Upon postmortem examination, the affected quarter revealed multifocal abscesses separated by fibrous connective tissues. Abscesses were most common in the gland cistern and collecting duct region. Microscopically, the inflammatory reaction was pyogranulomatous to granulomatous and consistent with botryomycosis. Colonies of Gram-positive cocci were found within the eosinophilic matrix of the Splendore-Hoeppli reaction within granulomas and intracellularly within the acinar epithelium. Conclusions Collectively, we describe a unique case of chronic mastitis, the characterization of which provides valuable insight into the mechanics of S. aureus treatment resistance and immune escape.

Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain. Traditional production consists of floating culture incubation at 37°C, organism inactivation by autoclaving, coarse filtration, and protein precipitation. Three traditional production PPDs were used in this study including lot 9801, which served as a reference and has been used in the field for decades. Alternative production PPDs (0902A and 0902B), in which the autoclaving step was removed, were also analyzed in this study. SDS-PAGE analysis revealed protein smearing in traditional PPDs, but distinct bands were observed in the alternative PPD preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, whereas an immunoreactive smear was observed with the traditional PPDs. Mass spectrometry identified 194 proteins among three PPD lots representing the two different production methods, ten of which were present in all PPDs examined. Selected proteins identified by mass spectrometry were recombinantly expressed and purified from E. coli and evaluated by the guinea pig potency test. Seven recombinant proteins showed greater erythema as compared to the reference PPD lot 9801 in paired guinea pigs and were able to stimulate interferon-gamma production in blood from Johne's positive animals. These results suggest that autoclaving culture suspensions is not a necessary step in PPD production and specific proteins could supplant the PPD antigen for intradermal skin testing procedures and for use as in-vitro assay reagents.