Survey on the spirilar flora of Lagomorphs

Members of the genera Campylobacter and Helicobacter have been in the spotlight in recent decades because of their status as animals and/or humans pathogens, both confirmed and emerging, and because of their association with food-borne and zoonotic diseases. First observations of spiral shaped bacteria or Campylobacter-like organisms (CLO) date back to the end of the 19th century, however the lack of adequate isolation methods hampered further research. With the introduction of methods such as selective media and a filtration procedure during the 1970s led to a renewed interest in Campylobacter, especially as this enabled elucidation of their role in human hosts. On the other hand the classification and identification of these bacteria was troublesome, mainly because of the biochemical inertness and fastidious growth requirements. In 1991, the taxonomy of Campylobacter and related organisms was thoroughly revised, since this revision several new Campylobacter and Helicobacter species have been described. Moreover, thanks to the introduction of a polyphasic taxonomic practice, the classification of these novel species is well-founded. Indeed, a polyphasic approach was here followed for characterizing eight isolates obtained from rabbits epidemiologically not correlated and as a result a new Campylobacter species was proposed: Campylobacter cuniculorum (Chapter 1). Furthermore, there is a paucity of data regarding the occurrence of spiral shaped enteric flora in leporids. In order to define the prevalence both of this new species and other CLO in leporids (chapter 2), a total of 85 whole intestinal tracts of rabbits reared in 32 farms and 29 capture hares, epidemiologically not correlated, were collected just after evisceration at the slaughterhouse or during necroscopy. Examination and isolation methods were varied in order to increase the sensibility level of detection, and 100% of rabbit farms resulted positive for C. cuniculorum in high concentrations. Moreover, in 3.53% of the total rabbits examined, a Helicobacter species was detected. Nevertheless, all hares resulted negative both for Campylobacter or Helicobacter species. High prevalence of C. cuniculorum were found in rabbits, and in order to understand if this new species could play a pathological role, a study on some virulence determinants of C. cuniculorum was conducted (Chapter 3). Although this new species were able to adhere and invade, exert cytolethal distending toxin-like effects although at a low titre, a cdtB was not detected. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly levels of particular virulence-associated factors although, cell adhesion and invasion occurred. Furthermore, antibiotic susceptibility was studied (chapter 4) in Campylobacter and in Escherichia coli strains, isolated from rabbits. It was possible to find acquired resistance of C. cuniculorum to enrofloxacin, ciprofloxacin and erytromycin. C. coli isolate was susceptible to all antimicrobial tested and moreover it is considered as a wild-type strain. Moreover, E. coli was found at low caecal concentration in rabbits and 30 phenotypes of antibiotic resistance were founded as well as the high rate of resistances to at least one antibiotic (98.1%). The majority of resistances were found from strains belonging to intensive farming system. In conclusion, in the course of the present study a new species isolated from rabbits was described, C. cuniculorum, and its high prevalence was established. Nevertheless, in hare samples no Campylobacter and Helicobacter species were detected. Some virulence determinants were further analyzed, however further studied are needed to understand the potential pathogenicity of this new species. On the other hand, antimicrobial susceptibility was monitored both in C. cuniculorum and indicator bacteria and acquired resistance was observed towards some antibiotics, indicating a possible role of rabbitries in the diffusion of antibiotic resistance. Further studies are necessary to describe and evaluate the eventual zoonotic role of Campylobacter cuniculorum.

Patologie da Porcine Circovirus tipo 2 (PCV2) nel suino

The main work involved the PMWS (Post-weaning multisystemic Wasting Syndrome), caused by PCV-2 (Porcine Circovirus type 2) that involved post-weaned pigs. Merial Italy has funded a study activity in which groups of 3-5 animals were sampled for lungs, tracheo-bronchial and superficial inguinal lymph nodes, ileum and tonsils. The protocol applied can be identified as a more diagnostic potential on the individual than on the group. PNP. Another investigation has been conducted to study proliferative and necrotizing pneumonia (PNP), a form of interstitial pneumonia in weaning and post-weaning pigs characterized by hypertrophy and hyperplasia of type II pneumocytes, coagulative necrosis and granular debris within alveolar spaces. Many studies suggest porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) as the main causes of the disease, but Aujeszky disease virus (ADV) and swine influenza virus (SIV) are also considered. An immunohistochemical study was carried out to evaluate the role of these viruses in PNP lesions in Italy. PNP results primarily associated with PRRSV, even if co-infection is characterized by more severe histological features. Reproductive pathology. A major risk factor for PCV2 infection is a viraemic episode taking place in pregnant sows with low antibody titer which is transmitted by specific PCV2 products of conception. PCV2 can infect the fetus even by vehicles through infected semen or ova, or as a result of infection of the genital tract. An investigation was carried out to identify the presence and localization of PCV2 in the genital tracts of sows experimentally infected with PCV2 and in their fetuses. The results obtained suggest that: conventional sows can be infected by intrauterine exposition; low antibody titres increase the probability of infection; PCV2 infection close to insemination time reduces the pregnancy rate; placental lesions may represent an additional cause of fetal suffering.

Rilievi anatomo-patologici e batteriologici condotti sull’apparato gastroenterico di una metapopolazione di cinghiali (Sus scrofa)

Nell’ambito della patologia gastroenterica del suino sono comprese alcune malattie sostenute da batteri spirillari gram negativi, di cui sono disponibili numerose trattazioni riguardanti, soprattutto, l'aspetto epidemiologico e patogenetico. Per alcuni di questi agenti microbici, e per le relative manifestazioni patologiche, poco si conosce nel cinghiale selvatico, animale correlato filogeneticamente al suino domestico, ma compreso in un’ecologia completamente differente. Da queste premesse è nato un approccio di ricerca e studio del comportamento di questi microrganismi in una metapopolazione di cinghiali, abbattuti durante il piano di controllo della popolazione densità-dipendente nel Parco dei Gessi e Calanchi dell’Abbadessa (BO), cercando di rapportare le conoscenze riportate in letteratura sul suino domestico con quanto è scaturito dalle indagini condotte sul cinghiale selvatico. In particolare è stata indagata con metodica immunoistochimica la presenza di Lawsonia intracellularis, patogeno del suino responsabile di Enterite Proliferativa (EP), in secondo luogo sono state condotte indagini batteriologiche e istologiche da stomaco e intestino, finalizzate all’isolamento di microrganismi spirillari dei generi Campylobacter e Helicobacter, da correlare all’eventuale presenza di lesioni infiammatorie e ulcerative gastriche o enteriche valutate secondo sistemi a punteggio ottenuti dalla bibliografia o realizzati in base alla tipologia di infiltrato cellulare e alla sua localizzazione. In ultimo, a fini comparativi con uno studio condotto nel 2002-2004 nello steso Parco Regionale, sono stati monitorati i livelli di antibioticoresistenza di indicatori fecali usando metodiche internazionali standardizzate (Escherichia coli e Enterococcus faecium.) nonché su un numero significativo di isolati di Campylobacter lanienae, per ottenere indicazioni preliminari sull’andamento nei 10 anni trascorsi dello stato di inquinamento da farmaco del Parco stesso. I risultati ottenuti permettono di ampliare le conoscenze sulla flora enterica del cinghiale selvatico e pongono questioni di sicurezza pubblica sulla gestione dei mammiferi selvatici.

Metagenomic trajectory of gut microbiome in the human lifespan

Co-evolving with the human host, gut microbiota establishes configurations, which vary under the pressure of inflammation, disease, ageing, diet and lifestyle. In order to describe the multi-stability of the microbiome-host relationship, we studied specific tracts of the bacterial trajectory during the human lifespan and we characterized peculiar deviations from the hypothetical development, caused by disease, using molecular techniques, such as phylogenetic microarray and next-generation sequencing. Firstly, we characterized the enterocyte-associated microbiota in breast-fed infants and adults, describing remarkable differences between the two groups of subjects. Successively, we investigated the impact of atopy on the development of the microbiome in Italian childrens, highlithing conspicuous deviations from the child-type microbiota of the Italian controls. To explore variation in the gut microbiota depending on geographical origins, which reflect different lifestyles, we compared the phylogenetic diversity of the intestinal microbiota of the Hadza hunter-gatherers of Tanzania and Italian adults. Additionally, we characterized the aged-type microbiome, describing the changes occurred in the metabolic potential of the gut microbiota of centenarians with respect to younger individuals, as a part of the pathophysiolology of the ageing process. Finally, we evaluated the impact of a probiotics intervention on the intestinal microbiota of elderly people, showing the repair of some age-related dysbioses. These studies contribute to elucidate several aspects of the intestinal microbiome development during the human lifespan, depicting the microbiota as an extremely plastic entity, capable of being reconfigured in response to different environmental factors and/or stressors of endogenous origin.

Relevance of the pig stomach for the detection of dietary factors and gut maturation and control

In this thesis two approaches were applied to achieve a double general objective. The first chapter was dedicated to the study of the distribution of the expression of genes of several bitter and fat receptor in several gastrointestinal tracts. A set of 7 genes for bitter taste and for 3 genes for fat taste was amplified with real-time PCR from mRNA extracted from 5 gastrointestinal segments of weaned pigs. The presence of gene expression for several chemosensing receptors for bitter and fat taste in different compartments of the stomach confirms that this organ should be considered a player for the early detection of bolus composition. In the second chapter we investigated in young pigs the distribution of butyrate-sensing olfactory receptor (OR51E1) receptor along the GIT, its relation with some endocrine markers, its variation with age, and after interventions affecting the gut environment and intestinal microbiota in piglets and in different tissues. Our results indicate that OR51E1 is strictly related to the normal GIT enteroendocrine activity. In the third chapter we investigated the differential gene expression between oxyntic and pyloric mucosa in seven starter pigs. The obtained data indicate that there is significant differential gene exression between oxintic of the young pig and pyloric mucosa and further functional studies are needed to confirm their physiological importance. In the last chapter, thymol, that has been proposed as an oral alternative to antibiotics in the feed of pigs and broilers, was introduced directly into the stomach of 8 weaned pigs and sampled for gastric oxyntic and pyloric mucosa. The analysis of the whole transcript expression shoes that the stimulation of gastric proliferative activity and the control of digestive activity by thymol can influence positively gastric maturation and function in the weaned pigs.