Ocorrência de Listeria monocytogenes em frangos alternativos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção de adenovírus humanos em amostras de água superficial e esgoto não tratado oriundas de diversos ecossistemas aquáticos da cidade de Belém-PA

Os vírus entéricos são importantes agentes de doenças de veiculação hídrica. Entre esses, os adenovirus humanos (HAdV) assumiram importância por serem um dos principais causadores de gastrenterite em crianças menores de cinco anos e pela sua maior resistência a fatores físicos e químicos em detrimento a outros vírus no ambiente. Várias pesquisas têm demonstrado ausência de relação entre a presença de bactérias indicadoras e vírus. Diante disso, diversos autores têm sugerido a inclusão desses agentes como potenciais indicadores de contaminação viral e fecal da água. O objetivo desse trabalho foi detectar a presença de HAdV em amostras de água e esgoto não tratado oriundas de diversos ecossistemas aquáticos da cidade de Belém-PA. Foram selecionados seis pontos de amostragem, dentre eles um esgoto não tratado: Esgoto do UNA e cinco coleções hídricas: Porto do Açaí, Ver-o-Peso, Igarapé Tucunduba, Lago Bolonha e Lago Água Preta. Foi feita uma coleta mensal de dois litros de água em cada ponto durante 24 meses consecutivos, de nov/2008 a out/2010, totalizando 144 amostras. Foi utilizada água destilada autoclavada para controle negativo de cada ponto em todos os testes utilizados. As amostras foram concentradas pelo método de adsorção-eluição e posteriormente centrifugadas para a obtenção de dois mL. O DNA foi extraído pelo kit comercial Qiagen. Para a detecção molecular foram empregadas a Reação em Cadeia Mediada pela Polimerase (PCR convencional) e a PCR em tempo real, sendo utilizados iniciadores e sondas específicos que amplificam um gene do hexon de 301 e 96 pb, respectivamente. Visando-se melhorar o produto amplificado para sequenciamento genômico, algumas amostras positivas pela PCR convencional foram submetidas à Nested-PCR com a utilização de mais um par de iniciadores que amplificam uma região interna de 171 pb. Amostras de água e esgoto foram sequenciadas, analisadas e comparadas a outras obtidas no GeneBank. Os HAdV foram detectados em 59% (85/144) das amostras de água superficial e esgoto não tratado, sendo que a positividade obtida pela PCR convencional foi de 22,9% (33/144) e pela PCR em tempo real de 58,7% (84/143). A primeira detectou o agente apenas nas amostras do igarapé Tucunduba (62,5%) e do esgoto do UNA (75%) e a segunda em amostras provenientes dos seis pontos de coleta (com uma variação de 25 a 100%). O agente foi detectado em todos os 24 meses do estudo, estando presentes em pelo menos dois pontos, mensalmente. A PCR em tempo real se mostrou mais sensível nesse estudo, tendo encontrado o agente em 36,4% (52/143) das amostras não detectadas pela PCR convencional. Das oito amostras genotipadas todas pertencem à espécie F, sendo quatro referentes ao sorotipo 40 e quatro ao 41. Nossos resultados confirmam a alta circulação desse patógeno nas águas superficiais e esgoto da cidade, sugerindo a inclusão dos HAdV como bons indicadores de contaminação viral e fecal da água. A pesquisa desses vírus em ambientes aquáticos é pioneira em Belém e tais resultados são de relevante importância para as políticas de saúde pública e ambiental, servindo como base para estudos complementares nessa área.

Segurança, imunogenicidade e eficácia protetora de duas doses da vacina RIX4414 contendo rotavírus atenuado de origem humana

OBJETIVO: Determinar a segurança, imunogenicidade e eficácia de duas doses da vacina contra o rotavírus em lactentes brasileiros saudáveis. MÉTODOS: Foi realizado um estudo randomizado, multicêntrico, duplo-cego e controlado por placebo no Brasil, México e Venezuela. Os lactentes receberam duas doses orais de vacina ou placebo aos 2 e 4 meses de idade, juntamente com as imunizações de rotina, exceto a vacina oral contra poliomielite (VOP). O presente estudo relata apenas os resultados obtidos em Belém, Brasil, onde o número de indivíduos por grupo e os títulos da vacina viral foram os seguintes: 194 (10^4,7 unidades formadoras de focos - UFF), 196 (10^5,2 UFF), 194 (10^5,8UFF) e 194 (placebo). A resposta de anticorpos anti-rotavírus (anti-RV) foi avaliada em 307 indivíduos. A gravidade clínica dos episódios de gastroenterite (GE) foi determinada através de um escore com 20 pontos, onde um valor ≥ 11 foi considerado como GE grave. RESULTADOS: As taxas de sintomas gerais solicitados foram semelhantes tanto nos indivíduos que receberam a vacina como naqueles a quem se administrou placebo. Aos 2 meses após a segunda dose, ocorreu resposta em termos de IgA sérica para RV em 54,7 a 74,4% dos vacinados. Não houve interferência na imunogenicidade das vacinas de rotina. A eficácia da vacina contra qualquer gastroenterite por rotavírus (GERV) foi de 63,5% (IC95% 20,8-84,4) para a maior concentração (10^5,8 UFF). A eficácia foi de 81,5% (IC95% 44,5-95,4) contra GERV grave. Em sua maior concentração (10^5,8 UFF), a RIX4414 conferiu uma proteção de 79,8% (IC95% 26,4-96,3) contra GERV grave causada pela amostra G9. CONCLUSÕES: A RIX4414 foi altamente imunogênica com baixa reatogenicidade, e não interferiu na resposta sérica à difteria, tétano, coqueluche, hepatite B e antígenos Hib. Duas doses da RIX4414 conferiram proteção significativa contra a GE grave causada pelo RV.

Eficácia dos fungos nematófagos Duddingtonia flagrans e Arthrootrys robusta na profilaxa das infecções naturais por nematódeos gastrointestinais em ovinos

Pós-graduação em Medicina Veterinária - FMVZ

Variação da ecogenicidade difusa em parênquima hepático homogêneo relacionada aos achados bioquímicos e hematológicos em cães

The present study evaluated the liver with homogeneous parenchyma in dogs, with diffuse reduced echogenicity (G1), increased echogenicity (G2) and normal echogenicity (G3) by ultrasound examination associating the findings on animal profile, liver size and hematology and biochemistry results. Blood samples obtained by cephalic or jugular venipunture were submitted for hemogram and ALT, ALP, total proteins, albumin, globulin, urea and creatinine analysis. Among the 30 dogs in G1, 30 (100%) presented portal venous and gall bladder wall clarity, 23 (76.67%) presented normal liver size and edges, 15 (50%) were from 1 to 6 years old and 8 (26.67%) belonged to the lhasa apso breed. No predispositions were found according to gender, as well as no significant alterations in biochemical and hematological exams were observed in G1. The most prevalent disease found in this group was gastroenteritis (43.33%). Among the 30 dogs in G2, 27 (90%) presented hepatomegaly and rounded hepatic edges, 18 (60%) were over 9 years old, 16 (53.33%) were female and 9 (30%) belonged to the poodle breed. The laboratorial findings related to this group were increased ALT and ALP serum activity, decreased levels of total protein, albumin, globulin, erythrocytes and hematocrit, as well as leukocytosis with neutrophilia and a left shift, eosinopenia, lymphopenia and monocytosis. The most prevalent diseases found in this group were metabolic disorders (54%), such as diabetes mellitus and hyperadrenocorticism, and chronic hepatopathies (17%) due to prolonged and continuous use of corticoid and anticonvulsive drugs. Among the 30 dogs in the control group (G3), normal liver size and edges were presented in 22 (73.33%). In this group, no alterations were seen in laboratorial exams.

Parâmetros clínicos e laboratoriais relacionados ao prognóstico em cães com gastroenterite hospitalizados

Pós-graduação em Cirurgia Veterinária - FCAV

Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Distribution of non-LEE-encoded type 3 secretion system dependent effectors in enteropathogenic Escherichia coli

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vaccination of sheep and cattle against haemonchosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Absentismo laboral por patología odontológica y médica en una industria de gran porte del sector mecánico y mueblero

Introduction: The current Brazilian legislation states that health services must be performed by the Specialized Service of Safety Engineering and Labor Medicine (SESMT). Objective: To analyze the dental and medical reasons for absenteeism, checking the interference of factors such as age, gender and position of the worker, and most of the reasons that led the employee to miss work. Material and methods: The research appears as a descriptive study with a quantitative approach. The dental and medical certificates and statements of attendance were analyzed, duly approved, lodged in a acrylic industry in the city of Araçatuba-SP, from January to July 2011. Results: Of the total number of certificates (n = 1841), only 103 (5.6%) were for dental reasons. Predominance of the age group of 20-29 years, males and with non-administrative function. The reasons most dental and medical certificates stated that led the workers to miss working days were "inclusive and impacted teeth" and "diarrhea and gastroenteritis of presumed infectious origin", respectively. As to factors related to absenteeism, there was a severity index of 2.83, 1.33 frequency and average duration of absences of 2.12. Conclusions: It is concluded that dental reasons had little weight on total absences due to illness that caused the temporary leave of the worker for a shorter period. The age and function variables influence the occurrence of work absenteeism.

Surtos de gastroenterite relacionados ao consumo de laticínios no estado de São Paulo no período de 2000 a 2010

Milk is considered a nutritionally complete food but it is also an excellent substrate for microbial growth. Therefore, it may pose a risk to consumers’ health. The aim of this study was to collect data on the occurrence of gastroenteritis outbreaks caused by the intake of milk and dairy products in the state of São Paulo, from 2000 to 2010. The data were collected from the records on foodborne illness outbreaks kept by the Division of Waterborne and Foodborne Diseases of the Center for Epidemiological Surveillance of the Department of Health of the State of São Paulo. A total of 239 outbreaks were notifi ed. They involved 2.418 cases related to the consumption of dairy products during the study period. The etiological agents were identifi ed in 79 outbreaks (33.0%). They were mostly bacteria (84.8%), especially Staphylococcus aureus. Most outbreaks occurred at home (59.4%) and during the months of January (14.2%) and July (12.5%). Most (24.1%) of the affected individuals aged 20 to 49 years. The results shows the need of better manufacturing and handling practices, as well as increasing the awareness of the population and relevant agencies about the importance of notifying cases and implementing preventive measures to ensure food safety.

Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Indagine sulla presenza di Helicobacter pullorum in allevamenti avicoli italiani

From September 2005 to December 2006, in order to define the prevalence of Helicobacter pullorum in broiler chickens, laying hens and turkey, a total of 365 caecum contents of animals reared in 76 different farms were collected at the slaughterhouse. A caecum content of a ostrich was also sampled. In addition, with the aim of investigating the occurrence of H. pullorum in humans, 151 faeces were collected at the Sant’Orsola-Malpighi University Hospital of Bologna from patients suffering of gastroenteritis. A modified Steele–McDermott membrane filter method was used. Gram-negative curved rod bacteria were preliminary identified as H. pullorum by a PCR assay based on 16S rRNA, then subjected to a RFLP-PCR assay to distinguish between H. pullorum and H. canadensis. One isolate from each farm was randomly selected for phenotypic characterization by biochemical methods and 1D SDSPAGE analysis of whole cell proteins profiles. Minimum Inhibitory Concentration (MIC) for seven different antibiotics were also determined by agar dilution method. Moreover, to examine the intraspecific genomic variability, two strains isolated from 17 different farms were submitted to genotyping by Pulse-Field Gel Electrophoresis (PFGE). In order to assess the molecular basis of fluorquinolone resistance in H. pullorum, gyrA of H. pullorum CIP 104787T was sequenced and nucleotide sequences of the Quinolone Resistance Determining Region (QRDR) of a total of 18 poultry isolates, with different MIC values for ciprofloxacin and nalidixic acid, were compared. According to the PCR and PCR-RFLP results, 306 out of 366 animals examined were positive for H. pullorum (83,6%) and 96,1% of farms resulted infected. All positive samples showed a high number of colonies (>50) phenotipically consistent with H. pullorum on the first isolation media, which suggests that this microrganism, when present, colonizes the poultry caecum at an elevate load. No human sample resulted positive for H. pullorum. The 1D SDS-PAGE whole protein profile analysis showed high similarity among the 74 isolates tested and with the type strain H. pullorum CIP 104787T. Regarding the MIC values, a monomodal distribution was found for ampicillin, chloramphenicol, gentamicin and nalidixic acid, whereas a bimodal trend was noticed for erythromycin, ciprofloxacin and tetracycline (indicating an acquired resistance for these antibiotics). Applying the breakpoints indicated by the CSLI, we may assume that all the H. pullorum tested are sensitive only to gentamicin. The intraspecific genomic variability observed in this study confirm that this species don’t have a clonal population structure, as motioned by other autors. The 2490 bp gyrA gene of H. pullorum CIP104787T with an Open Reading Frame (ORF) encoding a polypeptide of 829 amino acids was for the first time sequenced and characterized. All ciprofloxacin resistant poultry isolates showed ACA®ATA (Thr®Ile) substitution at codon 84 of gyrA corresponding to codons of gyrA 86, 87 and 83 of the Campylobacter jejuni, H. pylori and Escherichia coli, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin resistant phenotype of poultry isolates. This is the first report of isolation of H. pullorum in turkey and in ostrich, indicating that poultry species are the reservoir of this potential zoonotic microorganisms. In order to understand the potential role as food-borne human pathogen of H. pullorum, further studies must be carried on.

Epidemiologia e caratterizzazione molecolare di virus emergenti responsabili di Zoonosi: Calcivirus e Virus dell'Epatite E

Il virus dell’Epatite E (HEV) e i calicivirus (norovirus e sapovirus) causano rispettivamente epatite acuta e gastroenterite. Questi virus sono considerati agenti eziologici emergenti rappresentando un problema di sanità pubblica e di sicurezza alimentare. Per HEV, è ormai confermata la trasmissione zoonotica, e il suino è considerato il principale serbatoio asintomatico. Norovirus e sapovirus infettano sia i bambini che gli adulti. Sebbene questi virus siano stati identificati anche negli animali, la possibile trasmissione zoonotica non è stata dimostrata in modo conclusivo. Il lavoro sperimentale condotto durante il Dottorato di Ricerca è stato focalizzato sullo studio degli aspetti biologici ed epidemiologici dell’infezioni causate da HEV e da calicivirus. Per la prima volta in Italia, i risultati ottenuti hanno dimostrato la presenza del virus HEV nei fegati di suini in fase di macellazione ed hanno confermato, attraverso la ricerca di anticorpi, un’elevata esposizione degli animali al virus. Inoltre, mediante la produzione di antigeni e reattivi immunologici, sono stati messi a punto test diagnostici per la ricerca di anticorpi contro HEV nel suino e nei cinghiali. Il lavoro svolto per la ricerca di calicivirus nel suino e nel bovino ha dimostrato la circolazione dei sapovirus in popolazioni di suini asintomatici e la presenza di norovirus nei vitelli affetti da diarrea acuta.Sono stati inoltre sviluppati reattivi immunologici, utilizzando proteine del capside di norovirus umano e bovino espresse con il sistema ricombinante baculovirus. Questi hanno permesso di evidenziare la presenza di anticorpi contro norovirus umano e bovino, in sieri di veterinari professionalmente esposti. Inoltre, sono stati utilizzati per sviluppare metodi per la concentrazione dei virus da matrici a bassa concentrazione.Infine, le VLP sono state utilizzate per valutare l’attivazione del sistema immunitario umano ex vivo. I risultati hanno dimostrato che le VLP di NoV stimolano il sistema immunitario attivando risposte di tipo Th1 e Th2 .

Food safety and zoonotic enteric pathogens: sources, risk factors and transmission routes of human salmonellosis and campylobacteriosis

Salmonella and Campylobacter are common causes of human gastroenteritis. Their epidemiology is complex and a multi-tiered approach to control is needed, taking into account the different reservoirs, pathways and risk factors. In this thesis, trends in human gastroenteritis and food-borne outbreak notifications in Italy were explored. Moreover, the improved sensitivity of two recently-implemented regional surveillance systems in Lombardy and Piedmont was evidenced, providing a basis for improving notification at the national level. Trends in human Salmonella serovars were explored: serovars Enteritidis and Infantis decreased, Typhimurium remained stable and 4,[5],12:i:-, Derby and Napoli increased, suggesting that sources of infection have changed over time. Attribution analysis identified pigs as the main source of human salmonellosis in Italy, accounting for 43–60% of infections, followed by Gallus gallus (18–34%). Attributions to pigs and Gallus gallus showed increasing and decreasing trends, respectively. Potential bias and sampling issues related to the use of non-local/non-recent multilocus sequence typing (MLST) data in Campylobacter jejuni/coli source attribution using the Asymmetric Island (AI) model were investigated. As MLST data become increasingly dissimilar with increasing geographical/temporal distance, attributions to sources not sampled close to human cases can be underestimated. A combined case-control and source attribution analysis was developed to investigate risk factors for human Campylobacter jejuni/coli infection of chicken, ruminant, environmental, pet and exotic origin in The Netherlands. Most infections (~87%) were attributed to chicken and cattle. Individuals infected from different reservoirs had different associated risk factors: chicken consumption increased the risk for chicken-attributed infections; animal contact, barbecuing, tripe consumption, and never/seldom chicken consumption increased that for ruminant-attributed infections; game consumption and attending swimming pools increased that for environment-attributed infections; and dog ownership increased that for environment- and pet-attributed infections. Person-to-person contacts around holiday periods were risk factors for infections with exotic strains, putatively introduced by returning travellers.