Staphylococcus aureus adlb gene is associated with high prevalence of intramammary infection in dairy herds of northern Italy: A cross-sectional study.

Staphylococcus aureus is a major mastitis pathogen in dairy cattle worldwide, responsible for substantial economic losses. Environmental factors, milking routine, and good maintenance of milking equipment have been described as important factors to prevent intramammary infections (IMI). Staphylococcus aureus IMI can be widespread within the farm or the infection can be limited to few animals. Several studies have reported that Staph. aureus genotypes differ in their ability to spread within a herd. In particular, Staph. aureus belonging to ribosomal spacer PCR genotype B (GTB)/clonal complex 8 (CC8) is associated with high within-herd prevalence of IMI, whereas other genotypes are generally associated with individual cow disease. The adlb gene seems to be strictly related to Staph. aureus GTB/CC8, and is a potential marker of contagiousness. We investigated Staph. aureus IMI prevalence in 60 herds in northern Italy. In the same farms, we assessed specific indicators linked to milking management (e.g., teat condition score and udder hygiene score) and additional milking risk factors for IMI spread. Ribosomal spacer-PCR and adlb-targeted PCR were performed on 262 Staph. aureus isolates, of which 77 underwent multilocus sequence typing. In most of the herds (90%), a predominant genotype was identified, especially Staph. aureus CC8 (30%). In 19 of 60 herds, the predominant circulating Staph. aureus was adlb-positive and the observed IMI prevalence was relevant. Moreover, the adlb gene was detected only in genotypes of CC8 and CC97. Statistical analysis showed a strong association between the prevalence of Staph. aureus IMI, the specific CCs, and carriage of adlb, with the predominant circulating CC and presence of the gene alone explaining the total variation. Interestingly, the difference in the odds ratio obtained in the models for CC8 and CC97 suggests that it is carriage of the adlb gene, rather than the circulation of these CCs per se, that leads to higher within-herd prevalence of Staph. aureus. In addition, the model showed that environmental and milking management factors had no or minimal effect on Staph. aureus IMI prevalence. In conclusion, the circulation of adlb-positive Staph. aureus strains within a herd has a strong effect on the prevalence of IMI. Thus, adlb can be proposed as a genetic marker of contagiousness for Staph. aureus IMI in cattle. However, further analyses using whole-genome sequencing are required to understand the role of genes other than adlb that may be involved in the mechanisms of contagiousness of Staph. aureus strains associated with high prevalence of IMI.

New ternary powder metallurgy Ti alloys via eutectoid and isomorphous beta stabilisers additions.

A group of new ternary Ti alloys bearing eutectoid and isomorphous beta stabilising elements was created to be manufactured through the conventional powder metallurgy route. The effect of the simultaneous addition of the same amount of Mn and Nb on the manufacturability, properties, and hardening behaviour was investigated. The ternary alloys are composed of the α-Ti and ß-Ti phases and have a lamellar microstructure resulting from the slow cooling upon sintering. However, the size of the equiaxed α grains and of the α + ß lamellae is monotonically reduced, especially the interlamellar spacing, as the amount of alloying elements increases. Due to their physical properties, Mn enhances and Nb hinders densification during sintering resulting in a decreasing trend of the relative density with the alloying elements content. Consequently, the resistance to plastic deformation increases (UTS, 514-726 MPa), the ductility decreases (elongation, 13.2-2.6%), and the fracture mode changes from intergranular to transgranular. The new ternary alloys share the same hardening mechanism, but the amount of deformation after necking is, generally, higher for lower amounts of Mn and Nb.

Fabrication and characterisation of low-cost powder metallurgy Ti-xCu-2.5Al alloys produced for biomedical applications.

Ti and Ti-based materials are of growing interest as biocompatible structural materials in a wide range of biomedical applications. Traditionally, one of the main factors hindering the wider use of this class of materials has been the relatively high manufacturing cost. Today, Ti-6Al-4V remains the most widely used material for dental and orthopaedic implants. However, the presence of cytotoxic vanadium in its composition casts doubt on the safety of using this alloy as biomedical material. This study aims to study the microstructural features and mechanical properties of ternary alloys Ti-xCu-2.5Al (where x = 0.5-5 wt%Cu) obtained by powder metallurgy (PM) methods. The attractiveness of this group of materials lies in its economy due to the significantly lower cost of Cu compared to vanadium and the intrinsic advantages of PM. The obtained samples demonstrated increasing tensile strength and Vickers hardness values with increasing Cu content, from 640 MPa to 195 HV to 800 MPa and 250HV, respectively. At the same time, an inverse relationship was observed for the elongation. A higher content of ß-stabiliser is accompanied by the formation of a more significant number of spherically shaped pores and a refined lamellar structure which are responsible for the changes in mechanical properties.

Transmission patterns of a Mycobacterium avium subsp. paratuberculosis clone within a single heard investigated by Whole Genome Sequencing.

Mycobacterium avium subsp. paratuberculosis (MAP) is characterized by a low genomic rate of mutation. Current subtyping tools, such as Mini-Micro-satellite analyses, do to have not sufficient discriminatory power to disclose MAP's evolution on small spatial and temporal scales. The aim of the study was to investigate the population structure of MAP inside a single dairy herd using whole genome sequencing (WGS) approaches. For this purpose, the genomes of 43 field isolates, recovered from the faeces of 36 cows of the same dairy herd from 2012 to 2016, were sequenced by WGS. The isolates' genomes showed a low number (43) of polymorphic sites (SNPs), confirming the clonal origin of the herd infection. However, despite the limited genomic diversity found in WGS, the phylogenetic analysis was discriminatory enough to detect the presence of different genomic clades and sub-clades inside the herd population. In addition, the phylodynamic reconstruction showed the existence of an ancestor clade from which the other clades and sub-clades originated. Moreover, by reconstructing the putative within-herd transmission networks using WGS data, we demonstrated that: (i) in a herd where MAP is endemic, multiple isolates recovered from a single animal and differing from each other by few (three/four) SNPs can originate from different transmission or passive shedding events and not from intra-host evolution; and (ii) variability of minisatellites coupled with a few microsatellites does not represent reliable tracers of within-herd infection chains. Our findings show that WGS, coupled with relevant epidemiological information, represents a valuable tool to work out fine epidemiological and micro-evolutionary relationships such as those at herd-level scale.

Ternary Ti alloys functionalised with antibacterial activity.

Prosthesis bacterial infection occurring during surgery is a rising health issue. Pathogenic bacterial infection causes inflammation, interferes with the healing process, inhibits osteogenesis and, eventually, leads to implant failure. These issues can be tackled either by applying coatings or developing multifunctional (i.e. structural and antibacterial) materials. In this work, ß eutectoid bearing functionalised Ti alloys were designed and manufactured via the cost-effective press and sinter powder metallurgy route. The systematic analysis of the ternary Ti-xCu-yMn alloys shows that the mechanical properties proportionally increase with the amount of alloying elements added. All the ternary Ti-xCu-yMn alloys have strong antibacterial activity against Escherichia coli with respect to the negative control (i.e. pure Ti). Our study demonstrates that ternary Ti-xCu-yMn alloys are promising candidates for structural prostheses functionalised with antibacterial capability.

Behaviour of novel low-cost blended elemental Ti-5Fe-xAl alloys fabricated via powder metallurgy.

Ti alloys, generally made via wrought metallurgy, are commonly used as biomedical materials. The manufacturing of such alloys via powder metallurgy offers the possibility to reduce the cost as well as to develop innovative compositions not otherwise achievable. The aim of this study is to understand the effect that the progressive addition of Al has on the physical and mechanical behaviour of the low-cost powder metallurgy Ti-5Fe alloy for structural biomedical implants. Specifically, Ti-5Fe-xAl (x = 1-6 w.%) alloys were developed combining blending elemental and cold pressing plus vacuum sintering to further limit the manufacturing costs as Al is lighter and cheaper than Ti. This investigation demonstrates that the amount of Al added significantly changes the thermodynamics of the sintering process and induces microstructural modifications such as grain refinement. These effects jointly with the Al solid solution strengthening leads to progressively stronger and harder (but less ductile) α+ß Ti alloys characterised by the typical α+ß lamellar microstructure with mechanical behaviour suitable for a variety of structural biomedical implants.

Development of Cu-bearing powder metallurgy Ti alloys for biomedical applications.

Cu-bearing Ti alloys could be used as structural biomedical materials where the releasing of Cu ions is beneficial to lower infection incidences associated with surgical implants. The manufacturing of these alloys via powder metallurgy techniques can lower the production costs. In this study three ternary Cu-bearing Ti-xAl-yCu alloys were produced using conventional powder metallurgy. The mechanical properties increase with the amount of alloying elements. Samples of each composition were also forged to clarify the effect of subjecting them to hot deformation. Forging the samples improved the strength of the alloys due to the reduction of porosity and the refinement of the microstructural features. It is found that Ti-2Al-1Cu is the most ductile, Ti-6Al-4Cu is the strongest and Ti-10Al-5Cu has a purely elastic behaviour. Some of these powder metallurgy Ti-xAl-yCu alloys have better overall mechanical behaviour than their cast counterparts and therefore are valuable alternative to produce medical and dental implants with improved properties and reduced cost.

Low-cost powder metallurgy Ti-Cu alloys as a potential antibacterial material.

Ti and Ti alloys are extensively used in biomedical applications due to their excellent biocompatibility and mechanical properties but their high-cost of production is still a limiting factor. It has been reported that the addition of Cu to Ti enables the creation of Ti alloys exhibiting antibacterial properties. Therefore, in this study Ti-Cu alloys (Cu = 0.5, 2.5 and 5 in wt.%) with potential antibacterial activity were fabricated by powder metallurgy (i.e. cold press and vacuum sintering) to reduce the production costs. As many biomaterials are employed as structural components, the Ti-Cu alloys were also subjected to ß forging in order to improve their mechanical properties. It is found that the studied Ti-Cu alloys have superior mechanical properties to other commonly used Ti-based materials and are, thus, potential candidate for biomedical applications. Moreover, among the tested materials, the ß forged Ti-5Cu alloys has tensile strength of 904 MPa, elongation of 6.7%, and Vickers hardness of 302 HV, which are comparable to those of the Ti-6Al-4V, and comprises the Ti2Cu phase (confirmed by the XRD) as microstructural feature, which is fundamental to guarantee antibacterial properties.

Mechanical properties and microstructure of Ti-Mn alloys produced via powder metallurgy for biomedical applications.

Titanium and especially its alloys are highly employed materials in biomedical applications because of their balanced mechanical properties and biocompatibility. Ti-Mn alloys (1, 5, and 10 wt%. Mn) were produced by powder metallurgy as a potential alternative material for biomedical applications. Two sets of samples were produced, one set as-sintered and the other was beta (ß) forged. For the as-sintered samples with a content of up to 10 wt% Mn, the tensile strength ranged from 606 to 1070 MPa. On the other hand, for the ß forged alloys the tensile strength ranged from 728 to 1224 MPa and the maximum value was for Ti-5Mn. Forged Ti-5Mn exhibits a good balance of mechanical properties such as ultimate tensile strength (1224 MPa), elongation (4.6%) and Vickers hardness (415 HV). The purely elastic properties of the Ti-10Mn alloy is attributed to the effects of the omega (ω) phase, the formation of which is due to the high amount of beta stabiliser added to Ti.

Assessment of Salmonella survival in dry-cured Italian salami.

The inactivation of Salmonella during curing of Italian traditional pork salami was investigated. A total of 150 batches of ground raw meat (GRM) used for salami manufacturing by four producers were tested for Salmonella by real-time PCR followed by ISO 6579 cultural confirmation and MPN enumeration. Salami produced with Salmonella positive GRMs were re-tested at the end of their curing period. Aw, pH and NaCl content were also measured. Detection of Salmonella was performed testing both 25 and 50g of the samples. By Real-Time PCR 37% of the GRMs resulted positive, but cultural detection of Salmonella was obtained in 14% of the samples only. Salmonella enumeration ranged from 31 MPN/g to <1.3 MPN/g. The difference between testing 50g and 25g of the samples was statistically significant (p value≤0.01). In particular, ISO-50g detected Salmonella in 100% of all positive samples, vs. 62% of ISO-25g. Salami made of the contaminated GRMs were 29% Salmonella-positive, as most batches of salami produced with Salmonella-positive GRMs resulted negative after regular curing (20-48days). Overall, 13% of salami produced with Salmonella-contaminated GRMs were positive. They belonged to six batches, which turned out negative after prolonged curing ranging between 49 and 86days. Salmonella enumeration in salami ranged from 8.7 MPN/g to <1.3 MPN/g. Unlike GRMs, no significant difference was observed between the ISO-50g and the ISO-25g in detecting Salmonella in cured salami (p value: >0.05). The most common Salmonella serovars in GRMs were Derby (52%), Typhimurium monophasic variant 4, (Barbuti et al., 1993), 12:i:- (19%) and Stanley (10%). Salmonella Derby (56%), London, Branderup, Panama (13%, respectively) and Goldcoast (6%) were most frequent in cured salami. The study showed negative correlation between real-time CT values and cultural confirmation of Salmonella, as well as the importance of sample size for Salmonella detection. Among considered factors with possible effect on the occurrence of Salmonella in salami, statistical analysis revealed a role for aw in salami and for Salmonella load in GRMs, while pH and NaCl content did not significantly affect the probability of finding Salmonella in dry-cured salami in the context of this study. In particular the lower aw values due to longer curing were associated with lower Salmonella presence in traditional dry-cured salami.

Evaluation of the mechanical properties of powder metallurgy Ti-6Al-7Nb alloy.

Titanium and its alloys are common biomedical materials owing to their combination of mechanical properties, corrosion resistance and biocompatibility. Powder metallurgy (PM) techniques can be used to fabricate biomaterials with tailored properties because changing the processing parameters, such as the sintering temperature, products with different level of porosity and mechanical performances can be obtained. This study addresses the production of the biomedical Ti-6Al-7Nb alloy by means of the master alloy addition variant of the PM blending elemental approach. The sintering parameters investigated guarantee that the complete diffusion of the alloying elements and the homogenization of the microstructure is achieved. The sintering of the Ti-6Al-7Nb alloy induces a total shrinkage between 7.4% and 10.7% and the level of porosity decreases from 6.2% to 4.7% with the increment of the sintering temperature. Vickers hardness (280-300 HV30) and tensile properties (different combination of strength and elongation around 900MPa and 3%) are achieved.

The role of heterogeneity on the invasion probability of mosquito-borne diseases in multi-host models.

Heterogeneity in transmission and stochastic events can play a significant role in shaping the epidemic dynamics of vector-borne infections, especially in the initial phase of an outbreak. In this work, by using multi-type branching process methodologies, we assess how heterogeneities in transmission among a large number of host groups can affect the invasion probabilities of a mosquito-borne disease. We show with both analytical and numerical methods that heterogeneities in transmission can shape the invasion probabilities differently from how they affect the basic reproduction number (R0). In particular, we find that, while R0 always increases with the heterogeneity, the invasion probability after the introduction of infected hosts can decrease with the increase of transmission heterogeneity, even approaching zero when the number of host groups is very large. In addition, we show that the invasion probability via infected vectors is always larger than via infected hosts when heterogeneous transmission is sufficiently high. Our findings suggest that, for multi-species infections (e.g. West Nile fever and Rift Valley fever) or for single-species infections with patchy host distribution, the introduction of primary infected vectors may represent a higher risk for major outbreaks occurrence than introductions of infected hosts.

Feasibility study of the production of biomedical Ti-6Al-4V alloy by powder metallurgy.

Titanium and its alloys are characterized by an exceptional combination of properties like high strength, good corrosion resistance and biocompatibility which makes them suitable materials for biomedical prosthesis and devices. The wrought Ti-6Al-4V alloy is generally favored in comparison to other metallic biomaterials due to its relatively low elastic modulus and it has been long used to obtain products for biomedical applications. In this work an alternative route to fabricate biomedical implants made out of the Ti-6Al-4V alloy is investigated. Specifically, the feasibility of the conventional powder metallurgy route of cold uniaxial pressing and sintering is addressed by considering two types of powders (i.e. blended elemental and prealloyed). The characterization of physical properties, chemical analysis, mechanical behavior and microstructural analysis is carried out in-depth and the properties are correlated among them. On the base of the results found, the produced alloys are promising materials for biomedical applications as well as cheaper surgical devices and tools.

Mechanical behaviour of pressed and sintered CP Ti and Ti-6Al-7Nb alloy obtained from master alloy addition powder.

The Ti-6Al-7Nb alloy was obtained using the blending elemental approach with a master alloy and elemental titanium powders. Both the elemental titanium and the Ti-6Al-7Nb powders were characterised using X-ray diffraction, differential thermal analysis and dilatometry. The powders were processed using the conventional powder metallurgy route that includes uniaxial pressing and sintering. The trend of the relative density with the sintering temperature and the microstructural evolution of the materials sintered at different temperatures were analysed using scanning electron microscopy and X-ray diffraction. A minimum sintering temperature of 1200°C has to be used to ensure the homogenisation of the alloying elements and to obtain a pore structure composed of spherical pores. The sintered samples achieve relative density values that are typical for powder metallurgy titanium and no intermetallic phases were detected. Mechanical properties comparable to those specified for wrought Ti-6Al-7Nb medical devices are normally obtained. Therefore, the produced materials are promising candidates for load bearing applications as implant materials.

Mechanical behaviour of pressed and sintered titanium alloys obtained from master alloy addition powders.

The fabrication of the workhorse Ti-6Al-4V alloy and of the Ti-3Al-2.5V alloy was studied considering the master alloy addition variant of the blending elemental approach conventionally used for titanium powder metallurgy. The powders were characterised by means thermal analysis and X-ray diffraction and shaped by means of uniaxial pressing. The microstructural evolution with the sintering temperature (900-1400 °C) was evaluated by SEM and EDS was used to study the composition. XRD patterns as well as the density by Archimedes method were also obtained. The results indicate that master alloy addition is a suitable way to fabricate well developed titanium alloy but also to produce alloy with the desired composition, not available commercially. Density of 4.3 g/cm³ can be obtained where a temperature higher than 1200 °C is needed for the complete diffusion of the alloying elements. Flexural properties comparable to those specified for wrought Ti-6Al-4V medical devices are, generally, obtained.

Mechanical behaviour of pressed and sintered titanium alloys obtained from prealloyed and blended elemental powders.

The applicability of irregular prealloyed Ti-6Al-4V powder for the fabrication of titanium products by pressing and sintering and its employment as a master alloy to obtain the Ti-3Al-2.5V alloy was studied. To this end, the starting powders were characterised by dilatometry, differential thermal analysis and XRD. Green samples were obtained by cold uniaxial pressing, and the evolution of the microstructure over the sintering temperature range 900-1400°C was studied. The variation of the final density and mechanical properties with the sintering temperature was considered. Based on the study carried out, it can be stated that more reliable powders are needed to open the titanium market to new applications. A relative density of 95% and diverse microstructural features and mechanical properties equivalent to those of biomedical devices can be obtained by the pressing and sintering route.

Mechanical properties and microstructural evolution of vacuum hot-pressed titanium and Ti-6Al-7Nb alloy.

Hot-pressing is a powder metallurgy process where loose powder is loaded into a mould, usually of graphite, and sintered by the simultaneous application of high temperature and pressure. In this study elemental titanium and Ti-6Al-7Nb alloy powders are hot-pressed under different conditions in order to study the influence of the processing parameters on the microstructure and mechanical properties. The samples are characterised in terms of relative density, microstructure, XRD, percentage of interstitials, three-point bending test and hardness. Relative densities as high as 99% are obtained, the oxygen and carbon content remains almost constant but nitrogen percentage increases. This is due to the interaction with the BN coated mould and leads to the formation of a reacted layer in the surface, composed by different titanium compounds, which greatly affect the mechanical properties. Nevertheless, the removal of this reacted layer leads to an important improvement of the ductility, especially for elemental titanium.

Effects of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. I: empirical assessment.

Tick borne encephalitis (TBE) is endemic to eastern and central Europe with broad temporal and spatial variation in infection risk. Although many studies have focused on understanding the environmental and socio-economic factors affecting exposure of humans to TBE, comparatively little research has been devoted to assessing the underlying ecological mechanisms of TBE occurrence in enzootic cycles, and therefore TBE hazard. The aim of this study was to evaluate the effect of the main ungulate tick hosts on the pattern of tick infestation in rodents and TBE occurrence in rodents and questing adult ticks. In this empirical study, we considered three areas where endemic human TBE occurs and three control sites having no reported human TBE cases. In these six sites located in Italy and Slovakia, we assessed deer density using the pellet group count-plot sampling technique, collected questing ticks, live-trapped rodents (primarily Apodemus flavicollis and Myodes glareolus) and counted ticks feeding on rodents. Both rodents and questing ticks were screened for TBE infection. TBE infection in ticks and rodents was positively associated with the number of co-feeding ticks on rodents and negatively correlated with deer density. We hypothesise that the negative relationship between deer density and TBE occurrence on a local scale (defined by the minimum overlapping area of host species) could be attributed to deer (incompetent hosts) diverting questing ticks from rodents (competent hosts), know as the 'dilution effect hypothesis'. We observed that, after an initial increase, the number of ticks feeding on rodents reached a peak for an intermediate value of estimated deer density and then decreased. Therefore, while at a regional scale, tick host availability has already been shown to be directly correlated with TBE distribution, our results suggest that the interactions between deer, rodents and ticks are much more complex on a local scale, supporting the possibility of a dilution effect for TBE.

Effect of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. II: population and infection models.

Tick-borne encephalitis is an emerging vector-borne zoonotic disease reported in several European and Asiatic countries with complex transmission routes that involve various vertebrate host species other than a tick vector. Understanding and quantifying the contribution of the different hosts involved in the TBE virus cycle is crucial in estimating the threshold conditions for virus emergence and spread. Some hosts, such as rodents, act both as feeding hosts for ticks and reservoirs of the infection. Other species, such as deer, provide important sources of blood for feeding ticks but they do not support TBE virus transmission, acting instead as dead-end (i.e., incompetent) hosts. Here, we introduce an eco-epidemiological model to explore the dynamics of tick populations and TBE virus infection in relation to the density of two key hosts. In particular, our aim is to validate and interpret in a robust theoretical framework the empirical findings regarding the effect of deer density on tick infestation on rodents and thus TBE virus occurrence from selected European foci. Model results show hump-shaped relationships between deer density and both feeding ticks on rodents and the basic reproduction number for TBE virus. This suggests that deer may act as tick amplifiers, but may also divert tick bites from competent hosts, thus diluting pathogen transmission. However, our model shows that the mechanism responsible for the dilution effect is more complex than the simple reduction of tick burden on competent hosts. Indeed, while the number of feeding ticks on rodents may increase with deer density, the proportion of blood meals on competent compared with incompetent hosts may decrease, triggering a decline in infection. As a consequence, using simply the number of ticks per rodent as a predictor of TBE transmission potential could be misleading if competent hosts share habitats with incompetent hosts.

A new procalcitonin optical immunosensor for POCT applications.

A new immunosensor for the determination of procalcitonin was developed. A sandwich assay format was implemented on a polymethylmetacrylate optical biochip, opportunely shaped in order to obtain several flow channels and potentially suitable for point of care testing applications. The sandwich format makes use of two new rat monoclonal antibodies. The capture antibody was covalently immobilised on the surface of the plastic chip, and the detection antibody was labelled with DY647 dye. Different combinations of capture and detection antibodies were investigated, and particular attention was devoted in order to avoid the non-specific adsorption. A limit of detection of 0.088 mg L(-1) was achieved within the working range of 0.28-50 mg L(-1) in buffer samples. The assay was also implemented in human serum, and 0.2 and 0.7-25 mg L(-1) were the attained limit of detection and working range, respectively.