Campylobacter is the commonest cause of human acute bacterial-enteritis in the developed world (ACMSF, 1993). Over the last ten years Great Britain has experienced an increase in the number of reported cases of campylobacter associated illness over the last ten years. There are numerous under reporting issues associated with campylobacter-related illness, and as such the actual number of cases that occur each year is unknown and the magnitude of the public health risk posed by this organism can only be hypothesised. Infection with campylobacter has been linked in epidemiological studies with the consumption of poultry, in particular chicken meat. A quantitative risk assessment (QRA) model has been produced to investigate this issue. Through the use of appropriate modelling techniques and collected data the QRA model assesses the risk of human infection with campylobacter consequent upon the consumption of a chicken meal. The model describes each of the stages of the chicken supply chain and the mechanisms by which the chicken/chicken product becomes contaminated was investigated thus allowing the identification of mitigation strategies, which can reduce such contamination. Model results estimate that the risk of infection with campylobacter associated with the consumption of a single serving of chicken has a mean value ranging from 0.040 to 0.070 with a 95th percentile ranging from 0.098 to 0.160. These results have been used as a benchmark to which the impact of mitigation strategies are compared. Results clearly show that a reduction in the national flock prevalence, combined with a reduction in the within flock prevalence of positive flocks can have a dramatic impact upon the risk of infection. Further, freezing of chicken meat prior to consumption also considerably reduces the estimates risk.

Orientador: Tereza Cristina Cardoso da Silva / Banca: Vera Cláudia Lorenzetti Magalhães Curci / Banca: Maria Cecília Rui Luvizotto / Resumo: O presente estudo descreve o desenvolvimento e a aplicação da reação de transcrição reversa in situ em cadeia da polimerase (RT-PCR in situ), para detectar a co-infecção experimental de perus de 1-dia de idade com Coronavirus (TCoV) e Astrovirus (TAstV-2) isolados de casos clínicos no Brasil. A primeira etapa da reação consistiu na preparação específica de sondas de DNA biotiniladas homólogas ao gene da polimerase viral do TAstV-2 e da região 3'UTR do TCoV. Foram utilizados cortes histológicos de intestino correspondendo ás regiões do íleo, junção íleo-ceco e ceco para avaliar a reação de RT-PCR in situ. Para permeabilização tecidual uma digestão foi aplicada com 10 μg/μl proteinase K por 30 min. Na etapa de hibridização, as sondas de DNA homólogo às regiões genômicas virais ligadas à biotina foram diluídas na concentração de 2μg/μl na solução de hibridização e incubadas overnight à 42ºC. Em seguida, uma diluição ótima do anticorpo monoclonal anti-biotina acoplado a fosfatase alcalina e a peroxidase foram aplicados, para TAstV-2 e TCoV, respectivamente. O substratos diaminobenzidina 3,3 (DAB) e FastRed Ò foram utilizados para identificar a hibridização das regiões homólogas correspondentes ao TCoV e TAstV-2, respectivamente. A reação positiva foi visualizada por deposição de pigmentos vermelhos (TAstV-2) e marrom acastanhado (TCoV). Em relação à localização dos genes virais amplificados, foram confirmados nas células da base (células caliciformes) e ao longo das vilosidades intestinais principalmente no citoplasma dos enterócitos de forma difusa para ambos os vírus. Marcações positivas também foram evidenciadas na submucosa próximas às regiões com intensa congestão vascular... (Resumo completo, clicar acesso eletrôncio abaixo) / Abstract: This study describes the development and application of the reaction in situ reverse transcription polymerase chain reaction (RT-PCR in situ) to detect co-infection of turkeys to experimental 1-day-old with Coronavirus (TCoV) and Astrovirus ( TAstV-2) isolated from clinical cases in Brazil. The first step of the reaction has been to prepare specific biotinylated DNA probes homologous to the viral polymerase gene of TAstV-2 and the 3'UTR region of TCoV. We used histological sections of intestine corresponding to the regions of the ileum, ileum-cecum junction and cecum to evaluate the reaction of RT-PCR in situ. For permeabilization tissue digestion was applied with 10 g / uL proteinase K for 30 min. In step hybridization, DNA probes homologous to the viral genomic regions linked to biotin were diluted to the concentration of 2μg/μl in hybridization solution and incubated overnight to 42 ° C. Then, an optimal dilution of monoclonal anti-biotin coupled to alkaline phosphatase and peroxidase were applied to TAstV-2 and TCoV, respectively. 3.3 The substrate were used to identify the hybridization ofÒdiaminobenzidine (DAB) and FastRed homologous regions corresponding to TCoV and TAstV-2, respectively. The positive reaction was visualized by deposition of red pigment (TAstV-2) and brown brown (TCoV). Concerning the location of the amplified viral genes was confirmed by the base cells (goblet cells) and along the intestinal villi in the cytoplasm of enterocytes diffusely to both viruses. Tags positive were also demonstrated in the submucosa close to the areas with intense vascular congestion. In conclusion, the RT-PCR in situ standard in this study showed good ability to detect viral RNA promoting a desirable... (Complete abstract click electronic access below) / Mestre

La enterotoxetemia, enfermedad nfecciosa más importante en crías de alpaca, es causante de altas tasas de mortalidad y se caracteriza por ocasionar brotes con muerte súbita sin permitir la aplicación de tratamientos convencionales. El control está supeditado a la aplicación de medidas preventivas de manejo y a la aplicación de inmunoprofilaxis. En e presente trabajo se ensayaron tres programas de vacunación anticlostridial durante las tres campañas de parición ( 2001, 2002 y 2003) en una Unidad de Producción de una Empresa de Propiedad Social del departamento de Puno. Se empleó un anacultivo a base de cepas del Clostridium perfringes predominantemente de origen ovino ( cepas A,B;C y D) más una cepa tipo A aislada de alpaca. El efecto de la vacuna se realizó comparando tasas de mortalidad total y asociada a enterotoxemia ocurrida antes de la introducción de la vacuna(año 2000) con los 3 años con programa de vacunación (años 2001, 2002 y 2003). El programa # 1 (año 2000) con los 3 años con programa de vacunación (años 2001, 2002, 2003). El programa #1 (año 2001) administró la vacuna al 100% de las madres gestantes (dos dosis).y al 100% de las crías (una dosis). El programa #2 (año 2002) vacun´solamente al 100% de las crías (dos dosis) y en el programa #3(año 2003) se procedió a vacunar solamente al 100% de las madres gestantes (una dosis). El efecto de la vacuna fue evaluado comparando los índices de mortalidad neonatal total y asociadas a enterotoxemia, básicamente relizando diagnóstico de campo. La aplicación de la vacuna redujo lamortalidad neonatal total progresivamente, desde 33.41% (año 2000), hasta alcanzar el 25.17% (año 2001), 23.72% (año 2002) y el 9.38 % (año 2003). La 19.45% (año 2000) hasta 7.18%, 9.1% y 0.98% para los años vacunados (2001, 2002 y 2003). Comparando los diferentes programas de vacunación, el análisis de diferencia estadística de proporciones determinó que el anacultivo (vacuna) fue de progrma aplicado. Sin embargo, la reducción fue más palpable en aquellos programas que involucraron vacunar madres gestantes. / Enterotoxemia, the most mportant infectious disease in alpacas juvenile, is the cause og high mortaly and to present outbreaks with suden death that does not allow the application of conventional treatments. Cntrol depends on preventive handlng measures and the application of inmunoprofilaxis. The present work applied three programs of anticlostridial vaccnation during three campaigns of birth (2001, 2002 and 2003) in a Social propriety Enterprise´s Unite ofProduction in Puno. We employed an anacultivo with strains of Clostridium perfringens predominantly of ovine origin and a strain type A isolated enterotoxemia Mortality that occurred before the introduction of the vaccine (year 2000) with the three years with program of vaccination ( years 2001, 2002 AND 2003). In program #1 (year 2001) the vaccine was administered to 100# of pregnant mothers( two juveniles were vaccinated (100%) (two doss) and n program #3 (year 2003) only pregnant mothers were vaccinated (100%) ( a dose vaccinated). The application of the vaccine equally reduced the tital neonatal mortality associated with enterotoxemia from 19,45% (year 2000) to 25.17 % (year 2001), 23.72% (year 2002) and 9.38% (year 2003). The vaccine equally reduced the mortality associated wirh enterotoxemia from 19.45%(year 2000) to 7.18% ,9,1% and 0,98% in the years in wich vaccination was given (2001, 2002and 2003). Comparng the different programs of vaccination, the anlysis of statistical difference of proportions detrmined that the anaculativo (vaccine) was wffective for the control of enterotoxemia in alpacas independently of the type of application program. Nevertheless, the reduction in mortality was greater in the programs that involved vaccinating pregnant mothers.

The entezopathogenicity and antigens of Campylobacter-like organisms and Campylobacter app associated with the proliferative enteropathies were investigated. Two gnotobiotic pigs exposed orally to a filtered suspension of intestinal mucosa designated 284/86 from a naturally infected pig subsequently developed lesions of proliferative enteritis. Culture of the successful mucosal inoculum only revealed a moderate number of C. coli, however an apparently greater number of Campylobacter-like organisms was evident in smears of this inoculum. The pathogenesis of porcine proliferative enteritis was clearer from the results of this study. Ten days after infection, curved bacilli had colonised the ileal and large intestinal crypts. Attachment and entry of Campylobacter-like organisms into crypt enterocytes was also evident, with some proliferation of both bacteria within cells and of the enterocytes themselves. Twenty days after infection there was similar intracellular colonisation of bacteria and proliferative activity, although no luminal bacteria were evident. A moderate sub-acute inflammatory reaction was evident throughout. Conventional hamsters dosed with C. jejuni developed varying degrees of localised acute intestinal inflammation. Hamsters dosed with C. hyointestinalis or C. cola did not develop any lesions. Lesions of proliferative enteritis were detected in hamsters dosed with porcine tissue 284/86. Numerous intra-cytoplasmic Campylobacter-like organisms were detected within enterocytes in affected portions of intestine. Weanling hamsters this proved to be susceptible to the agent of porcine proliferative enteritis by cross-species transmission. Whole cell antigen preparations were made of various Camoylobacter sp. -Indirect immunofluorescence assays incorporating rabbit antisera to each Campylobacter sp gave specific endpoints for each antiserum of 1: 160 to 1: 320. Rabbit antisera prepared to Campylobacter-like organisms partly purified from proliferative enteritis mucosa, by a homogenisation and filtration technique, also gave specific reactions in this assay, up to 1: 610. Intracellular Campylobacter-like organisms were also compared in gel electrophoresis protein profiles and immunoblotting reactions to Campylobacter spp. The intracellular organisms tested had a distinctive protein profile dissimilar to the profiles of the known Campylobacter spp. In immunoblotting reactions, each of the Camoylobacter sp antisera reacted strongly with homologous antigens, but none reacted with Camcylobacter-like organisms prepared from lesions, except for a minor reaction seen with one serum. Similarly antisera to Campylobacter-like organisms showed a strong reaction to 25K to 27K components of homologous antigens, with only minor reactions to various other components of the cultivated Campylobacter app. Therefore it is likely that the intracellular Campylobacter-like organisms have a distinctive antigenic profile and that the 25 and 27K components are major antigenic components. Mouse monoclonal antibodies were produced that were apparently specific to the intracellular Camoylobacter-like organisms. Immunoblotting results showed that these antibodies only bound to a 251 to 27K outer membrane component present in the intracellular organisms. Reactions with this component could not be detected in assays with normal pig intestine, or Camoylobacter sp antigen. Restriction endonuclease digestion of Camoylobacter sp with Bgl II gave suitably resolved DNA fragments of between 2kb and 25kb. Patterns obtained with Bgl II digestion of Camoylobacter sp were dissimilar to those of Camoylobacter-like organisms, and each Camoylobacter sp had a characteristic distinct pattern. Digestion of DNA from porcine tissue samples with Bgl II produced a diffuse smear of fragmented DNA bands between 0.5 and19kb, with no recognizable "ladder" effect. The genome of the Carylobacter-like organisms within enterocytes in proliferative enteritis therefore is different to that of known Camoylobacter spp associated with the disease. This suggests that the differences in antigenic structures between these bacteria axe due to genetic differences. Only a limited number of strains were examined. Looking at the evidence provided by this study, the overall tenor of the results suggests that the intracellular organisms could be a separate, new species of Camp lobacter. If indeed the intracellular organisms are a single, new Cammylobacter PGS/ABST ecies, then a new name may be proposed, such as "C. intracellulare". Verifica nthis side Oni of the validity of such a proposal would require further DNA studies.

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Thesis (M.S.)--Auburn University, 2008. / Abstract. Includes bibliographical references (p. 33-36).

Thesis (doctoral)--Groningen, 1982.

Inaug.-diss.-Bern. / "Literaturverzeichnis": p. 22.

Proefschrift Rijksuniversiteit Groningen. / Met lit.opg. - Met samenvatting in het Nederlands.

Proefschrift Rijksuniversiteit Groningen. / Met lit.opg.