Relationship of Late Lactation Milk Somatic Cell Count and Cathelicidin with Intramammary Infection in Small Ruminants.

Late lactation is a critical moment for making mastitis management decisions, but in small ruminants the reliability of diagnostic tests is typically lower at this stage. We evaluated somatic cell counts (SCC) and cathelicidins (CATH) in late lactation sheep and goat milk for their relationship with intramammary infections (IMI), as diagnosed by bacteriological culture (BC). A total of 315 sheep and 223 goat half-udder milk samples collected in the last month of lactation were included in the study. IMI prevalence was 10.79% and 15.25%, respectively, and non-aureus staphylococci were the most common finding. Taking BC as a reference, the diagnostic performance of SCC and CATH was quite different in the two species. In sheep, receiver operating characteristic (ROC) analysis produced a higher area under the curve (AUC) value for CATH than SCC (0.9041 versus 0.8829, respectively). Accordingly, CATH demonstrated a higher specificity than SCC (82.92% versus 73.67%, respectively) at comparable sensitivity (91.18%). Therefore, CATH showed a markedly superior diagnostic performance than SCC in late lactation sheep milk. In goats, AUC was <0.67 for both parameters, and CATH was less specific than SCC (61.90% versus 65.08%) at comparable sensitivity (64.71%). Therefore, both CATH and SCC performed poorly in late lactation goats. In conclusion, sheep can be screened for mastitis at the end of lactation, while goats should preferably be tested at peak lactation. In late lactation sheep, CATH should be preferred over SCC for its higher specificity, but careful cost/benefit evaluations will have to be made.

Evaluation of a bovine cathelicidin ELISA for detecting mastitis in the dairy buffalo: Comparison with milk somatic cell count and bacteriological culture.

A recently developed bovine cathelicidin (CATH) ELISA was evaluated in the dairy buffalo (Bubalus bubalis) by testing 618 quarter milk samples from a herd with subclinical mastitis cases. Somatic cell count (SCC) and bacteriological culture (BC) were carried out on the same samples for comparison. Out of 618 quarters, 258 (41.75%) were positive to CATH, 289 (46.76%) had SCC > 200,000 cells/mL, and 457 (73.95%) were positive to BC. The most prevalent microorganism was Staphylococcus aureus (SAU, 35.76% of all quarters), followed by non-aureus staphylococci (NAS, 22.17% of all quarters). Clinical mastitis quarters were only 7 (1.13%). CATH levels were significantly higher in clinical quarters and in high SCC, BC-positive quarters than in healthy, low SCC, BC-negative quarters. The highest median values were observed for SAU and the lowest for NAS. Differences among microorganism classes were generally more significant for SCC than for CATH. Test characteristics of the CATH ELISA, evaluated by considering as true positives all BC-positive quarters with SCC > 200,000 cells/mL (N = 242), and as true negatives all sterile quarters with SCC < 200,000 cells/mL (N = 44), were as follows: sensitivity 57.85%, specificity 84.09%, positive predictive value 95.24%, negative predictive value 26.62%, accuracy 61.89%. Therefore, the bovine CATH ELISA showed a fair sensitivity and a good specificity in detecting water buffalo mastitis.

Milk cathelicidin and somatic cell counts in dairy goats along the course of lactation.

This research communication reports the evaluation of cathelicidin in dairy goat milk for its relationship with the somatic cell count (SCC) and microbial culture results. Considering the limited performances of SCC for mastitis monitoring in goats, there is interest in evaluating alternative diagnostic tools. Cathelicidin is an antimicrobial protein involved in innate immunity of the mammary gland. In this work, half-udder milk was sampled bimonthly from a herd of 37 Alpine goats along an entire lactation and tested with the cathelicidin ELISA together with SCC and bacterial culture. Cathelicidin and SCC showed a strong correlation (r = 0.72; n = 360 milk samples). This was highest in mid-lactation (r = 0.83) and lowest in late lactation (r = 0.61), and was higher in primiparous (0.80, n = 130) than in multiparous goats (0.71, n = 230). Both markers increased with stage of lactation, but cathelicidin increased significantly less than SCC. In addition, peak level in late lactation was lower for cathelicidin (5.05-fold increase) than for SCC (7.64-fold increase). Twenty-one (5.8%) samples were positive to bacteriological culture, 20 for coagulase-negative staphylococci and one for Streptococcus spp.; 18 of them were positive to the cathelicidin ELISA (85.71% sensitivity). Sensitivity of SCC >500 000 and of SCC >1 000 000 cells/ml was lower (71.43 and 23.81%, respectively). Therefore, the high correlation of cathelicidin with SCC during the entire lactation, along with its lower increase in late lactation and good sensitivity in detecting intramammary infection (IMI), indicate a potential for monitoring subclinical mastitis in dairy goats. However, based on this preliminary assessment, specificity should be improved (40.41% for cathelicidin vs. 54.57 and 67.85% for SCC >500 000 and >1 000 000 cells/ml, respectively). Therefore, the application of cathelicidin for detecting goat IMI will require further investigation and optimization, especially concerning the definition of diagnostic thresholds.

Structural and Immunodiagnostic Characterization of Synthetic Antigen B Subunits From Echinococcus granulosus and Their Evaluation as Target Antigens for Cyst Viability Assessment.

Background: Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Methods: Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. Conclusions: The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.

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 "local breeds" 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, 1day after calving, 7-10days after calving and 30days 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-1ß, TNF-α, 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-1ß, TNF-α, 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.

Cathelicidin production and release by mammary epithelial cells during infectious mastitis.

Cathelicidins are well-characterized antimicrobial peptides (AMPs) that are present in significant amounts in mastitic milk. Neutrophils are believed to be the main producers of these AMPs, while the role of mammary epithelial cells (MECs) in their production and release is still unclear. In this work, cathelicidin production patterns were investigated in mammary tissues of ewes infected by Staphylococcus aureus, Streptococcus uberis, or Mycoplasma agalactiae, with a combined approach including immunohistochemistry, immune-colocalization, and fluorescent in situ hybridization. Our results confirm that MECs produce and release cathelicidins in response to different mastitis pathogens. As opposed to neutrophils, however, MECs do not seem to store the preformed protein precursor in their cytoplasm, but appear to synthesize and release it only upon exposure to the microorganisms. Cathelicidin production by MECs appears to occur before leukocyte influx in the milk, suggesting a role for these cells in the initial response of the mammary epithelium to microbial infection. Once in the milk, infiltrating neutrophils release massive amounts of cathelicidin by degranulation and production of neutrophil extracellular traps, acting as the main contributor for cathelicidin abundance in mastitic milk. Taken together, our results support the active contribution of MECs to cathelicidin production and release, and reinforce the value of cathelicidins as sensitive and pathogen-independent mastitis markers.

Mycoplasma lipoproteins are major determinants of neutrophil extracellular trap formation.

Neutrophil granulocytes are paramount to innate responses as major effectors of acute inflammation. Among the various strategies enacted by neutrophils to eliminate microbes NETosis is a novel distinct antimicrobial activity in which an interlacement of chromatin fibres rich in granule-derived antimicrobial peptides and enzymes is extruded (NETs, neutrophils extracellular traps ). NETs contribute to the pathogenesis of acute and chronic inflammatory disorders. The interactions of mycoplasmas and innate immune cells, in particular neutrophil granulocytes, are poorly defined. Here, we describe NET formation in vivo in the mammary gland and milk of sheep naturally infected by Mycoplasma agalactiae. Also, we assess the contribution of liposoluble proteins, the most abundant component of the Mycoplasma membrane, in inducing NETosis. We demonstrate that Mycoplasma liposoluble proteins induce NET release at levels comparable to what observed with other stimuli, such as lipopolysaccharides and phorbol 12-myristate 13-acetate. Stimulation of neutrophils with synthetic diacylated lipopeptides based on the M. agalactiae P48, P80, and MAG_1000 proteins, combined in a mix or used individually, suggests that NETosis might not be dependent on a specific lipopeptide sequence. Also, NETosis is partially abolished when TLR2 is blocked with specific antibodies. The results presented in this work provide evidences for the mechanisms underlying NET activation in mycoplasma infections, and on their contribution to pathogenesis of mycoplasmosis.

Zn2+ Uptake in Streptococcus pyogenes: Characterization of adcA and lmb Null Mutants.

An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized null deletion mutants of the adcA and lmb genes of Streptococcus pyogenes strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in Streptococcus pneumoniae. Null adcA and lmb mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn2+ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to lmb, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and null mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the null adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

Diagnostic Accuracy of Antigen 5-Based ELISAs for Human Cystic Echinococcosis.

BACKGROUND: Clinical diagnosis and follow up of cystic echinococcosis (CE) are based on imaging complemented by serology. Several immunodiagnostic tests are commercially available, but the development of new tools is still needed to overcome the lack of standardization of the target antigen, generally consisting of a crude extract of Echinococcus granulosus hydatid cyst fluid. In a previous work, we described a chromatographic method for the preparation of a highly enriched Antigen 5 fraction from hydatid cyst fluid. The high reactivity of patient sera against this preparation prompted us to evaluate further this antigen for the serodiagnosis of CE on a larger cohort of samples. METHODOLOGY/PRINCIPAL FINDINGS: A total of 327 sera from CE patients with heterogeneous conditions for cyst stage, cyst number, organ localization, drug therapy, and surgical intervention, together with 253 sera from healthy controls, were first analyzed by an ELISA based on the Ag5 preparation in two different experimental setups and, in parallel, by a commercial ELISA routinely used in clinical laboratories for CE serodiagnosis. The Ag5 ELISAs revealed different sensitivity (88.3% vs 95.3%) without significant differences in specificity (94.1% vs 92.5%), for the two setups, respectively. Moreover, possible relationships between the Ag5 ELISA absorbance results and clinical variables were investigated. Chi squared test, bivariate logistic regression and multiple regression analyses highlighted differences in the serology reactivity according to pharmacological treatment, cyst activity, and cyst number. CONCLUSIONS/SIGNIFICANCE: The two Ag5 ELISAs revealed different performances depending on the setup. The good diagnostic sensitivity and the high reliability of the Ag5 preparation method make this antigen a promising candidate for the serodiagnosis of CE. Further studies will be needed to evaluate the ability of our test to provide useful information on specific CE clinical traits.

Diversity of cwp loci in clinical isolates of Clostridium difficile.

An increased incidence of Clostridium difficile infection (CDI) is associated with the emergence of epidemic strains characterized by high genetic diversity. Among the factors that may have a role in CDI is a family of 29 paralogues, the cell-wall proteins (CWPs), which compose the outer layer of the bacterial cell and are likely to be involved in colonization. Previous studies have shown that 12 of the 29 cwp genes are clustered in the same region, named after slpA (cwp1), the slpA locus, whereas the remaining 17 paralogues are distributed throughout the genome. The variability of 14 of these 17 cwp paralogues was determined in 40 C. difficile clinical isolates belonging to six of the currently prevailing PCR ribotypes. Based on sequence conservation, these cwp genes were divided into two groups, one comprising nine cwp loci having highly conserved sequences in all isolates, and the other five loci showing low genetic conservation among isolates of the same PCR ribotype, as well as between different PCR ribotypes. Three conserved CWPs, Cwp16, Cwp18 and Cwp25, and two variable ones, Cwp26 and Cwp27, were characterized further by Western blot analysis of total cell extracts or surface-layer preparations of the C. difficile clinical isolates. Expression of genetically invariable CWPs was well conserved in all isolates, whilst genetically variable CWPs were not always expressed at comparable levels, even in strains containing identical sequences but belonging to different PCR ribotypes. This is the first report on the distribution and variability of a number of genes encoding CWPs in C. difficile.

Surface interactome in Streptococcus pyogenes.

Very few studies have so far been dedicated to the systematic analysis of protein interactions occurring between surface and/or secreted proteins in bacteria. Such interactions are expected to play pivotal biological roles that deserve investigation. Taking advantage of the availability of a detailed map of surface and secreted proteins in Streptococcus pyogenes (group A Streptococcus (GAS)), we used protein array technology to define the "surface interactome" in this important human pathogen. Eighty-three proteins were spotted on glass slides in high density format, and each of the spotted proteins was probed for its capacity to interact with any of the immobilized proteins. A total of 146 interactions were identified, 25 of which classified as "reciprocal," namely, interactions that occur irrespective of which of the two partners was immobilized on the chip or in solution. Several of these interactions were validated by surface plasmon resonance and supported by confocal microscopy analysis of whole bacterial cells. By this approach, a number of interesting interactions have been discovered, including those occurring between OppA, DppA, PrsA, and TlpA, proteins known to be involved in protein folding and transport. These proteins, all localizing at the septum, might be part, together with HtrA, of the recently described ExPortal complex of GAS. Furthermore, SpeI was found to strongly interact with the metal transporters AdcA and Lmb. Because SpeI strictly requires zinc to exert its function, this finding provides evidence on how this superantigen, a major player in GAS pathogenesis, can acquire the metal in the host environment, where it is largely sequestered by carrier proteins. We believe that the approach proposed herein can lead to a deeper knowledge of the mechanisms underlying bacterial invasion, colonization, and pathogenesis.

Influence of Moraxella sp. colonization on the kidney proteome of farmed gilthead sea breams (Sparus aurata, L.).

BACKGROUND: Currently, presence of Moraxella sp. in internal organs of fish is not considered detrimental for fish farming. However, bacterial colonization of internal organs can affect fish wellness and decrease growth rate, stress resistance, and immune response. Recently, there have been reports by farmers concerning slow growth, poor feed conversion, and low average weight increase of fish farmed in offshore floating sea cages, often associated with internal organ colonization by Moraxella sp. Therefore, presence of these opportunistic bacteria deserves further investigations for elucidating incidence and impact on fish metabolism. RESULTS: A total of 960 gilthead sea breams (Sparus aurata, L.), collected along 17 months from four offshore sea cage plants and two natural lagoons in Sardinia, were subjected to routine microbiological examination of internal organs throughout the production cycle. Thirteen subjects (1.35%) were found positive for Moraxella sp. in the kidney (7), brain (3), eye (1), spleen (1), and perivisceral fat (1). In order to investigate the influence of Moraxella sp. colonization, positive and negative kidney samples were subjected to a differential proteomics study by means of 2-D PAGE and mass spectrometry. Interestingly, Moraxella sp. infected kidneys displayed a concerted upregulation of several mitochondrial enzymes compared to negative tissues, reinforcing previous observations following lipopolysaccharide (LPS) challenge in fish. CONCLUSIONS: Presence of Moraxella sp. in farmed sea bream kidney is able to induce proteome alterations similar to those described following LPS challenge in other fish species. This study revealed that Moraxella sp. might be causing metabolic alterations in fish, and provided indications on proteins that could be investigated as markers of infection by Gram-negative bacteria within farming plants.