EMERGING PATHOGENS IN VERTEBRATES: BIOLOGY, GENOMICS AND INFECTIVITY OF BACTERIA ASCRIBED TO THE MIDICHLORIACEAE FAMILY

This PhD thesis is the outcome of a wider project focused on studying the biology, genomics and infectivity of bacteria ascribed to the Midichloriaceae family. Midichloriaceae are a group of bacteria extremely diversified in terms of the ecological and taxonomic diversity of their hosts. Members of the family Midichloriaceae can be considered as a wide bacterial group, still to be investigated under several aspects. In particular, the association of members of the family Midichloriaceae with parasites (such as ticks, fleas and parasitic amoebae) suggests that these bacteria could be potentially infectious to vertebrates (including humans) and even responsible for medical and veterinarian diseases. The researchers conducted in this PhD project have been focused on three main topics: i) possible involvement of a midichloriacea in a disease (red mark syndrome; RMS) affecting rainbow trout; ii) in-depth analysis of different aspects concerning the hard tick Ixodes ricinus and its intramitochondrial symbiont Midichloria mitochondrii (i.e. antibiotic treatment of the arthropod, salivary glands vs. ovaric tissue comparative proteomics, transmission of the symbiont to the vertebrate host); iii) presence of M. mitochondrii-related bacteria in different tick species. A brief description of each topic of the work done is here reported: i) RMS is a chronic skin disease of unknown etiology affecting farmed rainbow trout Oncorhynchus mykiss in Europe. It consists of single or multiple skin lesions usually localized on the trunk of fish approaching market size. Many authors suppose that a bacterial infection could be the cause of the disease. Some studies have reported a possible correlation between the presence of a Rickettsia-like organism (subsequently ascribed to the family Midichloriaceae) and RMS skin lesions. Our work concerning the possible involvement of a midichloriacea in RMS in rainbow trout resulted in the detection of bacteria of this family, besides in skin lesions, in various internal organs and in the bloodstream. No bacteria were found in unaffected fish and in healthy skin samples from affected individuals. Bacteria were detected also in fish ponds water were RMS-affected fish are farmed. These results give support to the hypothesis of the involvement of these bacteria in developing RMS. It is possible that some free-living eukaryotes could host or be the vector of this midichloriacea. ii) M. mitochondrii is the most prevalent symbiont of the hard tick I. ricinus, present in 100% of eggs and adult females of wild I. ricinus populations. This bacterium is intracellular and is the only known symbiont able to invade the mitochondria of the host cells. However, the role that M. mitochondrii plays in the host metabolism has yet to be elucidated. In addition, multiple lines of evidence indicate the possibility of the transmission of this bacterium to the vertebrate host during the tick blood meal. In order to investigate the role of M. mitochondrii in the biology of the tick host, we performed an antibiotic treatment on I. ricinus individuals, with the aim of reducing/eliminating the symbiont, and to potentially observe the dynamic of bacterial infection in the tick host. We microinjected engorged adult females of I. ricinus with tetracycline, and we allowed the resulting larvae to feed on gerbils treated with the same antibiotic. The amount of M. mitochondrii was evaluated at different stages of the experiment using molecular techniques. In addition we evaluated the presence/absence of the symbiont DNA in the blood of gerbils used for the larval feeding. The performed treatments did not allow to eliminate the symbiont population from the host tick, however it allowed to reduce the multiplication that occurs after the larval blood meal. These results open the way for future experiments, using different antibiotic molecules, different administration methods and antibiotic administration on subsequent tick stages, to fulfill the goal of eliminating M. mitochondrii from the host I. ricinus, a major step in our understanding of the impact of this bacterium on ticks. In order to investigate, from a proteomic point of view, the tick I. ricinus and its symbiont, the protein profile of the ovary tissue (OT) and of salivary glands (SG) of adult females of this tick species were generated. To compare the OT and SG profiles, 2-DE profiling followed by LC-MS/MS protein identification were performed. We detected 21 spots showing significant differences in the relative abundance between the OT and SG, ten of which showed 4- to 18-fold increase/decrease in density. This work allowed to establish a method to characterize the proteome of I. ricinus, and to detect multiple proteins that exhibit a differential expression profile in OT and SG. Additionally, it was possible to use an immunoproteomic approach to detect a protein from the symbiont. Finally, the method developed will pave the way for future studies on the proteomics of I. ricinus, with the goals of better understanding the biology of this vector and of its symbiont M. mitochondrii. The transmission of M. mitochondrii after I. ricinus bite has been evidenced through direct and indirect evidences in human and other vertebrates by different authors. However, the time of seroconversion against the antigens of this symbiont is still unknown, as well as the kinetic of the antibody response against M. mitochondrii. In this work we performed an experimental infestation of rabbits with wild I. ricinus ticks (harboring M. mitochondrii) and I. ricinus from a lab colony (free of the symbiont). We were able to show the first seroconversion of a vertebrate model against M. mitochondrii. The seroconversion occurs approximately around the first and the second week after tick infestation, with duration of at least one month after infestation. It has been observed that M. mitochondrii represent an actual pack of antigens that can persist in the vertebrate host for a prolonged time. M. mitochondrii DNA was also observed circulating in blood of infested rabbits. The flagellar protein FliD of M. mitochondrii can be considered an interesting marker for I. ricinus bite, at least concerning adult females bite. However, the antibody response against this protein suggests that a possible screening for this marker should be considered within a limited time after tick infestation. Additional markers both from M. mitochondrii and I. ricinus should be considered in view of developing a possible marker for I. ricinus bite detection. iii) The multiple screening of 17 tick species allowed the detection and quantification of bacteria of the family Midichloriaceae in seven of them, including the first report of a representative of this family in a soft tick species (Argasidae), Ornithodoros maritimus. Based on sequence identity and phylogenetic analysis we propose that all these bacterial symbionts of ticks could be members of the genus Midichloria. The performed screening highlights different prevalence levels and variable bacterial loads in different tick species including one, Ixodes aulacodi, where the bacterium is present in all examined individuals, like in I. ricinus. This result prompts us to hypothesize different roles of Midichloria bacteria in different tick species

Comparison of Italian and Hungarian Black Spot Ranking

AbstractBlack spot ranking is an important tool for finding the sites with potential safety improvement on the road network. The EU Directive on Road Infrastructure Safety Management also demands the ranking of high accident concentration sites. This paper gives an introduction to localizing high accident concentration sites and the indicators used by Italy and Hungary. Accident and traffic volume data are gathered for motorway sections from both countries. Safety ranking is made using two conventional indicators, absolute number of accidents and accident rate. A more sophisticated ranking using the Empirical Bayes method is applied. Expected average crash frequency with Empirical Bayes adjustment is calculated. Based on the estimation of the crash frequency, the Critical Crash Rate (CCR) was added to identify and rank black spots. This additional performance measure is able to take into account traffic volume as required by the EU Directive. Results of the Empirical Bayes method are compared with the conventional procedures. It is concluded that the results are not comparable; inasmuch as there are modifications in the order of black spots. Based on the comparison of results recommendations are given to change the practice in both countries

Sensor-based pavement diagnostic using acoustic signature for moduli estimation

AbstractThe diffusion of smart infrastructures for smart cities provides new opportunities for the improvement of both road infrastructure monitoring and maintenance management.Often pavement management is based on the periodic assessment of the elastic modulus of the bound layers (i.e., asphalt concrete layers) by means of traditional systems, such as Ground Penetrating Radar (GPR) and Falling Weight Deflectometer (FWD). Even if these methods are reliable, well-known, and widespread, they are quite complex, expensive, and are not able to provide updated information about the evolving structural health condition of the road pavement. Hence, more advanced, effective, and economical monitoring systems can be used to solve the problems mentioned above.Consequently, the main objective of the study presented in this paper is to present and apply an innovative solution that can be used to make smarter the road pavement monitoring. In more detail, an innovative Non-Destructive Test (NDT)-based sensing unit was used to gather the vibro-acoustic signatures of road pavements with different deterioration levels (e.g. with and without fatigue cracks) of an urban road. Meaningful features were extracted from the aforementioned acoustic signature and the correlation with the elastic modulus defined using GPR and FWD data was investigated.Results show that some of the features have a good correlation with the elastic moduli of the road section under investigation. Consequently, the innovative solution could be used to evaluate the variability of elastic modulus of the asphalt concrete layers, and to monitor with continuity the deterioration of road pavements under the traffic loads

How Different Stocking Densities Affect Growth and Stress Status of Acipenser baerii Early Stage Larvae

In the present study, a multidisciplinary approach was used in order to evaluate growth, muscle development, and stress status in Siberian sturgeon Acipenser baerii larvae at schooling (T1) and complete yolk sac absorption (T2), reared at three stocking densities (low, medium, and high). Larvae growth, morphological muscle development, and whole-body cortisol levels were assessed. The expression of genes involved in the growth process (igf1 and igf2), in the myogenesis (myog), and in the regulation of cellular stress (glut1, glut2, glut4, and hsp70) was analyzed using a quantitative PCR. Larvae reared at lower densities showed a higher Specific Growth Rate and showed a physiological muscle development. Cortisol levels were low and did not differ significantly, both in different time sampling and across densities, suggesting that either the considered densities are not stressors in this species in the early stages of development or the hypothalamus-pituitary-adrenal (HPA) axis is not yet fully mature. Gene expression of glut1, igf1, and igf2 showed an up-regulation in both developmental stages at all the rearing densities considered, while myog significantly up-regulated at T1 at the highest density. Considering all of the results, it would seem that lower densities should be used in these stages of development, as these showed a higher growth rate, even if it is not economically feasible in commercial hatcheries. Therefore, choosing an intermediate stocking density could be a good compromise between larval performance and economical feasibility

Red mark syndrome: Is the aquaculture water microbiome a keystone for understanding the disease aetiology?

Aquaculture significantly contributes to the growing demand for food worldwide. However, diseases associated with intensive aquaculture conditions, especially the skin related syndromes, may have significant implications on fish health and industry. In farmed rainbow trout, red mark syndrome (RMS), which consists of multiple skin lesions, currently lacks recognized aetiological agents, and increased efforts are needed to elucidate the onset of these conditions. Most of the past studies were focused on analyzing skin lesions, but no study focused on water, a medium constantly interacting with fish. Indeed, water tanks are environmental niches colonized by microbial communities, which may be implicated in the onset of the disease. Here, we present the results of water and sediment microbiome analyses performed in an RMS-affected aquaculture facility, bringing new knowledge about the environmental microbiomes harbored under these conditions. On the whole, no significant differences in the bacterial community structure were reported in RMS-affected tanks compared to the RMS-free ones. However, we highlighted significant differences in microbiome composition when analyzing different samples source (i.e., water and sediments). Looking at the finer scale, we measured significant changes in the relative abundances of specific taxa in RMS-affected tanks, especially when analyzing water samples. Our results provide worthwhile insight into a mostly uncharacterized ecological scenario, aiding future studies on the aquaculture built environment for disease prevention and monitoring

Alcohol imagery on popularly viewed television in the UK

Background: Exposure to alcohol consumption and product imagery in films is associated with increased alcohol consumption among young people, but the extent to which exposure also occurs through television is not clear. We have measured the occurrence of alcohol imagery in prime-time broadcasting on UK free-to-air television channels. Methods: Occurrence of alcohol imagery (actual use, implied use, brand appearances or other reference to alcohol) was measured in all broadcasting on the five most popular UK television stations between 6 and 10 p.m. during 3 weeks in 2010, by 1-min interval coding. Results: Alcohol imagery occurred in over 40% of broadcasts, most commonly soap operas, feature films, sport and comedies, and was equally frequent before and after the 9 p.m. watershed. Brand appearances occurred in 21% of programmes, and over half of all sports programmes, a third of soap operas and comedies and a fifth of advertising/trailers. Three brands, Heineken, Budweiser and Carlsberg together accounted for ∼40% of all brand depictions. Conclusions: Young people are exposed to frequent alcohol imagery, including branding, in UK prime-time television. It is likely that this exposure has an important effect on alcohol consumption in young people

Exploration of the TRIM fold of MuRF1 using EPR reveals a canonical antiparallel structure and extended COS-box

MuRF1 (TRIM63) is a RING-type E3 ubiquitin ligase with a predicted tripartite TRIM fold. TRIM proteins rely upon the correct placement of an N-terminal RING domain, with respect to C-terminal, specific substrate-binding domains. The TRIM domain organization is orchestrated by a central helical domain that forms an antiparallel coiled-coil motif and mediates the dimerization of the fold. MuRF1 has a reduced TRIM composition characterized by a lack of specific substrate binding domains, but contains in its helical domain a conserved sequence motif termed COS-box that has been speculated to fold independently into an α-hairpin. These characteristics had led to question whether MuRF1 adopts a canonical TRIM fold. Using a combination of electron paramagnetic resonance, on spin-labeled protein, and disulfide crosslinking, we show that TRIM63 follows the structural conservation of the TRIM dimerization domain, observed in other proteins. We also show that the COS-box motif folds back onto the dimerization coiled-coil motif, predictably forming a four-helical bundle at the center of the protein and emulating the architecture of canonical TRIMs.publishe

Detection and genetic characterization of domestic cat hepadnavirus in cats with cavitary effusions

: After the identification of the novel domestic cat hepadnavirus (DCH) in 2018, its potential pathogenetic role in feline hepatic diseases has been suggested. Following the detection of DCH in a cat's serum and peritoneal effusion, the aim of this study was to retrospectively investigate the presence of DCH in cats with and without cavitary effusions along with DCH presence in effusions. Stored serum and effusion samples from cats with and without effusions admitted to the Veterinary Teaching Hospital of Lodi (Italy) in 2020-2022 were included based on results of hematobiochemical parameters. Effusions were classified based on cytological and physicochemical findings. The likelihood of liver damage was estimated based on clinical and laboratory findings. Samples were tested for DCH presence by quantitative PCR (qPCR). Positive samples were subjected to whole genome sequencing and phylogenetic analysis. DCH was detected in both serum and peritoneal effusion samples of 2/72 (2.8%) enrolled cats, included in the group with effusions (2/33; 6.1%), with one cat showing inflammatory and the other non-inflammatory effusion. Both DCH-positive cats belonged to the group with a likelihood of liver damage (2/22, 9.1%). Phylogeny showed that the DCH sequences from this study clustered with the prototypic Australian strain but were not included in the clade with other Italian DCH sequences. Results suggest the circulation of different DCH variants in Italy and show the presence of DCH in effusion samples from DCH-positive cats, mirroring the presence of HBV in body fluids from HBV-infected humans. Further studies are still recommended to define the pathogenic role of DCH in cats

Demodicosis in a captive African straw-coloured fruit bat (Eidolon helvum).

Demodicosis is most frequently observed in the domestic dog (Canis familiaris), but it has rarely been reported in bats (Chiroptera). The overpopulation of Demodex spp. that causes dermatological changes is generally associated with a compromised immune system. We describe the gross and histological features of generalized demodicosis in an adult female African straw-coloured fruit bat (Eidolon helvum) drawn from a captive research colony. The histology of the lesions revealed comedones and follicular infundubular cysts harbouring numerous Demodex spp. mites, eliciting a minimal inflammatory response in the adjacent dermis. The histological examination of a full set of tissues did not reveal clear evidence of immunosuppression, although a clinical history of recent abortion and possible stressors due to captivity could be considered risk factors for the demodicosis. Attempts to determine the Demodex species using PCR on DNA extracted from the formalin fixed paraffin embedded tissue failed. This is the first clinical and histological description of demodicosis in Eidolon helvum