As infecções causadas por Campylobacter spp. são relatadas como causa freqüente de gastrenterites de origem alimentar em vários países do mundo. As espécies bacterianas termofílicas pertencentes ao gênero Campylobacter, principalmente Campylobacter jejuni e Campylobacter coli, têm sido isoladas de fezes de animais e estão associadas à contaminação da carne durante o processo de abate. Estas duas espécies são as mais freqüentemente envolvidas nos casos de campilobacteriose humana veiculada por alimentos. O presente estudo pretendeu avaliar a presença e a população de Campylobacter spp. no abate de bovinos e cortes refrigerados de aves e bovinos comercializados na cidade de São Paulo/SP. Um total de 198 animais foi amostrado no couro logo após a sangria, na carcaça imediatamente após a esfola e após a evisceração. As amostras foram obtidas através da técnica de swab na região do peito abrangendo uma área de 400 cm2. Foram analisados também 120 cortes refrigerados de frango e 100 cortes de carne bovina, assim distribuídos: 40 amostras de asa, 20 de coxa com sobrecoxa, 20 de coxa, 20 de coxinha da asa, 20 amostras de peito; 20 de patinho bovino (M. biceps femoris), 20 de contrafilé (M. longissimus dorsi), 20 de coxão mole (M. semi membranosus), 20 de lagarto (M. semitendinosus) e 20 de alcatra (M. glutaes medius). As amostras foram analisadas segundo os métodos ISO 10272-1 e 2 e os isolados obtidos foram confirmados como Campylobacter pela técnica de PCR. Campylobacter foi isolado em 22,7% (45/198) das amostras de couro bovino, ou seja, apenas no ponto antes da esfola, e C. jejuni foi a única espécie encontrada. Nas amostras de cortes de frango Campylobacter foi isolado em 14,2% (17/120) das amostras. A espécie prevalente em frangos foi C. coli (88%), seguido de C. jejuni (12%). Campylobacter spp. não foi isolado dos cortes bovinos. A população de Campylobacter spp. foi < 13 UFC/cm2 em carcaças bovinas, < 2 UFC/g em amostras de frango e < 10 UFC/cm2 em cortes bovinos. A susceptibilidade de 120 isolados de frango e couro bovino foi determinada frente a 8 agentes antimicrobianos usando o método de disco-difusão. A resistência às quinolonas (ác. nalidíxico e ciprofloxacina) foi frequentemente observada nas cepas de C. jejuni (72,2%) e C. coli (50,8%) isoladas dos frangos. Entre os isolados de C. jejuni obtidos do couro bovino maior taxa de resistência foi observada para estreptomicina (32%), seguida da eritromicina (16%) e do ácido nalidíxico (14%). / Campylobacter spp. infections are reported as a frequent cause of foodborne gastroenteritis in many countries. The thermophilic bacterial species belonging to the genus Campylobacter, particularly Campylobacter jejuni and Campylobacter coli have been isolated from feces of animals and are associated with the contamination of meat during the slaughtering process. These two species are the most frequently involved in cases of human campylobacteriosis conveyed by food. The aim of the present study was to evaluate the presence and population of Campylobacter spp. during cattle slaughter and in refrigerated chicken and beef cuts commercialized in the city of Sao Paulo/SP. A total of 198 animals were sampled in the hide after bleeding, the carcass immediately after skinning and after evisceration. Samples were obtained by swab technique in the chest area encompassing an area of 400 cm2. We also analyzed 120 refrigerated chicken cuts and 100 beef cuts. The samples were analyzed according to ISO 10272-1 and 2 methods and the isolates were confirmed as Campylobacter by PCR technique. Campylobacter was isolated only in the hide samples (45/198), and C. jejuni was the only species found. Campylobacter was isolated in 14.2% (17/120) of chicken samples. The most prevalent species in chickens was C. coli (88%), followed by C. jejuni (12%). Campylobacter spp. was not isolated from beef cuts. The counts of Campylobacter spp. was < 13 CFU/cm2 in bovine carcasses, < 2 CFU/g in chicken samples and < 10 CFU/cm2 in beef cuts. The susceptibility to 8 antimicrobial agents of 120 isolates of chicken and bovine hide was determined using the disk-diffusion method. The resistance to quinolones (ciprofloxacin and nalidixic acid) was frequently observed in strains of C. jejuni (72.2%) and C. coli (50.8%) isolated from chickens. Among strains of C. jejuni obtained from bovine hide highest resistance rate was observed to streptomycin (32%), followed by erythromycin (16%) and nalidixic acid (14%).

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-07-29T11:13:47Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PesquisaCampylobactersppGranjas.pdf: 649227 bytes, checksum: c39eeff01123d4d81e68a9da4c4b01ec (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-08-28T14:41:49Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PesquisaCampylobactersppGranjas.pdf: 649227 bytes, checksum: c39eeff01123d4d81e68a9da4c4b01ec (MD5) / Made available in DSpace on 2014-08-28T14:41:49Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PesquisaCampylobactersppGranjas.pdf: 649227 bytes, checksum: c39eeff01123d4d81e68a9da4c4b01ec (MD5) Previous issue date: 2007 / As infecções de origem alimentar no homem, causadas por Campylobacter spp., resultam em grandes perdas econômicas e estão relacionadas à produção e o abate de frangos, etapas importantes na disseminação dessas bactérias. Baseando-se na importância do Campylobacter spp. na saúde pública e tendo em vista os dados constantes na literatura de que as aves comercializadas estão constantemente contaminadas com esse agente, sentiu-se a necessidade de realizar um estudo envolvendo a criação e o abate de frangos na região amazônica para que medidas profiláticas e de controle possam ser adotadas. O trabalho teve como objetivo estudar a ocorrência de Campylobacter spp. em granjas e abatedouro avícolas na mesorregião metropolitana de Belém – PA; isolar e identificar as espécies de Campylobacter spp. e identificar as fontes de contaminação nas granjas e os pontos críticos no abate. Foi coletado um total de 120 amostras em três granjas avícolas: 30 amostras de “swab” cloacal, 30 amostras de cama de frango, 30 amostras de ração e 30 amostras de água dos bebedouros. No abatedouro, foram colhidas 126 amostras: 36 amostras de água em 12 pontos diferentes da linha de abate e mais 30 amostras de pele do conjunto peito/ pescoço, 30 amostras de fígado e 30 amostras de moela. As amostras foram colhidas entre os meses de janeiro e maio de 2007 para o isolamento e identificação das espécies de Campylobacter spp. As amostras foram processadas na Seção de Bacteriologia e Micologia do Instituto Evandro Chagas – IEC da Secretaria de Vigilância em Saúde (SVS), Ministério da Saúde, Ananindeua – PA. Campylobacter spp. foi isolado em 33,3% (40/120) das amostras das granjas. Não houve diferença significativa (p>0,05) entre os percentuais de isolamentos positivos entre as três granjas pesquisadas. Ao analisar as freqüências dos isolados de Campylobacter spp. para cada tipo de amostra das granjas, observou-se que 96,6% (29/30) das amostras de “swab” cloacal, 33,3% (10/30) das amostras de cama e 3,3% (1/30) das amostras de água foram positivas para Campylobacter spp. Não foi isolada a bactéria nas amostras de ração. Campylobacter jejuni foi identificado bioquimicamente em 82,5% (33/40) das cepas isoladas nas granjas. No abatedouro, todas as cepas isoladas foram identificadas como C. jejuni., sendo isolado a bactéria em 8,73% (11/126) das amostras provenientes da linha de abate. Ao analisar as freqüências dos isolados de C. jejuni para cada tipo de amostra do abatedouro, observou-se que 27,8% (10/36) das amostras de água foram positivas para C. jejuni, seguido pela moela com 3,3% (1/30) das amostras positivas. Não foi isolado Campylobacter spp. nas amostras de fígado e de pele do conjunto peito/ pescoço. Houve diferença significativa (p<0,0001) entre os isolamentos positivos, negativos e os tipos de amostras processadas nas granjas e no abatedouro. As principais fontes de contaminação nas granjas foram o “swab” cloacal, a cama e, em menor escala, a água. Os principais pontos críticos observados no abatedouro foram a água, seguido pela moela. C. jejuni foi identificado em elevado percentual entre as cepas isoladas nas granjas e em todas as cepas do abatedouro. / The human infections of food origin caused by Campylobacter spp. result in high economic losses and these infections are correlated to poultry flock and slaughter, important steps in Campylobacter spp. dissemination. Based on the importance of this microorganism in public health and in the literature data that show the high poultry contamination by Campylobacter spp., we realize a study involving the flock and the poultry slaughter in Amazon region for adoption of prophylactic and control measures. The objective of this work was to investigate the occurrence of Campylobacter spp. in poultry flock and slaughterhouse in Amazon region and to isolate and to identify the Campylobacter species and to identify the sources of infection in the flock and the critical points in the slaughterhouse. We collected 120 samples in three flocks: 30 cloacal swab samples, 30 poultry litter samples, 30 feed samples and 30 water samples. In the slaughterhouse, 126 samples were collected: 36 water samples from 12 different points in the abattoir, 30 neck/ chest skin samples, 30 liver samples and 30 gizzard samples. The samples were collected from January to May 2007. The samples were processed in the Bacteriology and Micology Laboratory in Evandro Chagas Institute – Health Surveilance Office – Brazil’s Health Ministery. Campylobacter spp. was isolated in 33,3% (40/120) flock samples. There was no significant difference (p>0,05) between positive isolates in the three flocks. Campylobacter spp. was isolated in 96,6% (29/30) cloacal swab samples, 33,33% (10/30) poultry litter samples and 3,3% (1/30) water samples. There were no positive samples in the feed. Campylobacter jejuni was identified by biochemistry reactions in 82,5% (33/40) of isolates from flocks. In the slaughterhouse, all isolates were identified as C. jejuni. This microorganism was isolated in 8,73% (11/126) of slaughterhouse samples. C. jejuni was isolated in 27,8% (10/36) water samples and in 3,3% (1/30) gizzard samples. Campylobacter spp. was not isolated in liver samples neather in neck/ chest skin samples. There was significant difference (p<0,0001) between positive, negative isolates and among all kinds of samples collected in the flocks and at the slaughterhouse. The infections sources identified in the flocks were the cloacal swab, the poultry litter and the water. In the slaughterhouse, the critical points identified were the water and the gizzard. C. jejuni was identified in high levels in the flocks and in all isolates from the slaughterhouse.

Today, thermophilic Campylobacter spp. (besides Salmonella) represent one of the most common sources of human bacterial gastrointestinal infection. The main source of human C. jejuni infections is the consumption of insufficient heated chicken meat. Quantitative data on the occurrence of thermophilic Campylobacter spp. on poultry carcasses and poultry meat are needed to perform quantitative risk assessments and to verify the effect of different intervention strategies. The aims of the investigations presented here were (i) to generate accurate qualitative and quantitative data on the occurrence of Campylobacter spp. on within the primary production stage in at the abattoir and food, (ii) to study the behaviour of nine genotypically different C. jejuni strains in chicken meat juice supplemented with different concentrations of sodium chloride, curing salt and sodium nitrite, (iii) to detect the Campylobacter genotype distribution in poultry flocks by applying AFLP analysis and to describe a potential carry-over of Campylobacter strains among sequential and adjacent poultry flocks. For the above mentioned purposes, a number of samples (171 neck skin samples, 1047 samples of different turkey meat products and 112 turkey minced meat samples from an abattoir) were investigated in accordance with themethod of ISO / TS 10272-2: 2006 and ISO10272-1: 2006 to the quantitative and qualitative presence of Campylobacter spp.. The Campylobacter-strains were inoculated in chicken juice at initial concentrations of 102 and 104 CFU/ml and incubated for 24h at 42°C. Furthermore, 18 flocks of four poultry species were monitored to investigate the distribution and spread of Campylobacter genotypes between sequential and adjacent flocks. Caecal and liver samples were obtained at frequent intervals from birds of all flocks and these samples were examined for Campylobacter. The amplified fragment length polymorphism (AFLP) analysis was performed to genotype Campylobacter isolates. The prevalence of Campylobacter on neck skin was 83.0 % and the mean number was 2.00 log10 cfu/g. For turkey meat samples with skin (wings and thighs) the detected prevalence was 68.2 % and mean number 1.73 log10 cfu/g, respectively. Turkey meat samples without skin (breast filet) showed a prevalence of 79.0 % and a mean number of 1.58 log10 cfu/g. No Campylobacters were detectable in the turkey minced meat samples. Large variations between the detectable numbers of Campylobacter spp. were observed (maximum number up to 3.98 log10 cfu/g for turkey meat with skin) and confirm the importance of an early detection (before or during slaughter and processing) of these heavily contaminated slaughter lots. Whereas the strains multiplied in the media supplemented without additional of NaCl or with 1% NaCl, the bacterial population was significantly reduced when 2% NaCl was added. Growth did not occur and the cell number gradually decreased in chicken meat juice containing 3% NaCl. Significant differences in the survival potential among the different strains were only visible in the extreme condition of 3% NaCl supplementation. There was no different behaviour of the strains under the influence of NaCl compared with the behaviour in meat juice containing curing salt. The addition of sodium nitrite did not alter the survival. Of the 1643 caecal and liver samples investigated, 452 (27.5%) caecal samples and 11 (0.7%) liver samples contained Campylobacter. Of the caecal isolates 76.3% were identified as C. jejuni and 23.7% were identified as C. coli. Poultry flocks were largely colonized by more than one AFLP type and an intense exchange of Campylobacter genotypes between different poultry flocks occurred. The results show clearly that poultry and poultry meat are regarded as one of the main sources of thermophilic Campylobacter spp. infection in humans in the food chain. This is evident not only from the high rate of occurrence of these pathogens, but also from the often-high quantitative exposure samples. The risk of a foodborne infection is also enhanced by the comparatively very low minimum infectious dose for humans. At present, a complete elimination of thermophilic Campylobacter spp. from the food chain appears practically unreachable. This difficulty is reduced by the results of genetic strain diversity, because they suggest the existence of a variety of input sources. / hermophile Campylobacter (C.) spp. stellen heute neben den Salmonellen eine der häufigsten Ursachen für bakteriell bedingte Magen-Darm-Erkrankungen beim Menschen dar. Unzureichend erhitztes Geflügelfleisch und Geflügelfleischprodukte stellen eine der Hauptinfektionsquellen für humane C. jejuni-Infektionen dar. Quantitative Daten über die Belastung von Geflügelkarkassen und Geflügelfleisch mit thermophilen Campylobacter spp. werden benötigt, um einerseits quantitative Risikobewertungen durchführen zu können, andererseits aber auch den Erfolg verschiedener Interventionsmaßnahmen messen zu können. Zur Minderung der Zahl alimentär bedingter humaner Campylobacter-Infektionen spielt neben der Senkung der Campylobacter-Belastung von Nutztieren und der Vermeidung von Kreuzkontaminationen auch die Reduktion des Vorkommens des Erregers in der Lebensmittelkette durch technologische Prozesse eine große Rolle. Campylobacter spp. sind während der Be- und Verarbeitung von Lebensmitteln verschiedensten Stressoren ausgesetzt. Ziele der hier vorgestellten Untersuchungen waren, (i) Exakte qualitative und quantitative Daten zum Vorkommen von Campylobacter spp. in der Primärproduktion, auf dem Schlachthof und in Lebensmitteln zu ermitteln. (ii) Die Tenazität ausgewählter, genetisch unterschiedlicher C. jejuni-Stämme gegenüber verschiedenen Natriumchlorid-, Pökelsalz- und Natriumnitrit-Konzentrationen im Geflügelfleischsaftmodell zu untersuchen. (iii) Die Verwandtschaftsgrade der isolierten Stämme mit Hilfe einer molekularbiologischen Fingerprintingmethode (AFLP-Typisierung) darzustellen sowie das Vorkommen von thermophilen Campylobacter in verschiedenen Geflügelarten in einem Betrieb zu ermitteln. Dazu wurden 171 Halshautproben, 783 Proben verschiedener Putenfleischerzeugnisse und 233 Putenhackfleischproben aus einem Schlacht- und Zerlegebetrieb in Anlehnung an die Methode ISO/TS 10272-2: 2006 und ISO10272-1:2006 auf das quantitative und qualitative Vorkommen von Campylobacter spp. untersucht. Der Keimzahlverlauf wurde in experimentell kontaminiertem Geflügelfleischsaft (Zusatz von C. jejuni: 102 und 104 KbE/ml) über einen Zeitraum von 24 h (Bebrütungstemperatur 37°C) untersucht. 19 verschiedene Wirtschaftsgeflügel-Herden wurden untersucht, um die Verteilung und Ausbreitung von Campylobater-Genotypen zwischen sequentiellen und angrenzenden Herden festzustellen. Blinddarm- und Leber-Proben wurden in kurzen Abständen von Vögeln aller Herden gewonnen und untersucht . Für die Genotypisierung von Campylobacter spp. wurde die AFLP-Methode eingesetzt. Im Ergebnis wurden auf 83,0 % der Halshautproben Campylobacter spp. nachgewiesen, wobei der Mittelwert der quantitativen Belastung von Putenhalshautproben bei 2,00 log10 KbE/g lag. Putenfleischproben mit Haut waren zu 54,8 % Campylobacter positiv. Hier betrug die quantitative Belastung 1,79 log10 KbE/g. Bei Putenfleisch ohne Haut lagen die Nachweisraten bei 52,2,% bzw. 2,03 log10 KbE/g. In keiner der Putenhackfleischproben war Campylobacter nachweisbar. Große Schwankungen in der quantitativen Belastung (Maximalwerte bis 4,0 log10 KbE/g bei Putenfleisch mit Haut) bestätigen die Notwendigkeit, vor allem die stark belasteten Schlachtpartien schon vor bzw. während der Schlachtung und Verarbeitung identifizieren zu können. In Geflügelfleischsaft ohne bzw. mit Zusatz von 1% NaCl konnten sich alle Stämme vermehren, während das Wachstum bei 2% NaCl-Zusatz gehemmt wurde. Darüber hinaus konnte bei höherer NaCl-Konzentration (3%) eine Reduktion der Keimzahl nach 6 h Bebrütung bzw. ein Absterben von C.jejuni nach 24 h festgestellt werden. Dabei zeigten die Stämme im Geflügelfleischsaft mit 3% NaCl-Zusatz signifikante Unterschiede in ihrer Absterberate. Es konnte gezeigt werden, dass unterschiedlich ausgeprägte Salztoleranzen innerhalb der untersuchten Stämme mit unterschiedlichem Genotyp existieren, diese jedoch nur unter Extremsituationen signifikant ausgeprägt waren. Durch die Zugabe von praxisüblichen Pökelsalzkonzentrationen anstelle von Kochsalz und von Natriumnitrit konnte das Verhalten von C. jejuni in keinem Versuchsansatz beeinflusst werden. 452 Caecalproben (27,5%) und 11 Leberproben (0,7%) von insgesamt 1643 Caecal- und Leberproben wurden positiv auf Campylobacter spp. getestet. Von den 1643 aus dem Caecum stammenden getesteten Isolaten wurden 76,3% der Isolate als C. jejuni und (23,7%) der Isolate als C. coli identifiziert. Die AFLP- Analyse zeigte einen signifikanten Unterschied in der Diversität der AFLP-Typen aus den individuellen Herden und Proben aus unterschiedlichen Herden. Dies deutet auf eine große Zahl von Infektionsquellen hin. Die Ergebnisse belegen insgesamt sehr deutlich, dass Geflügel- Geflügelfleisch eine bedeutsame Quelle des Eintrags von Campylobacter-Keimen in die Nahrungskette ist. Das geht nicht nur aus der hohen Rate des Vorkommens dieser Erreger hervor, sondern auch aus der oftmals hohen quantitativen Belastung der Proben. Das Risiko einer Lebensmittelinfektion wird zudem durch die vergleichsweise sehr geringe minimale Infektionsdosis für den Menschen erhöht. Eine vollständige Elimination von thermophilen Campylobacter spp. aus der Lebensmittelkette erscheint derzeit praktisch nicht erreichbar. Diese Schwierigkeit wird durch die Ergebnisse zur genetischen Stammdiversität untersetzt, denn sie legen die Existenz einer Vielzahl unterschiedlicher Eintragsquellen nahe.

Une exposition aux viandes comporte un risque pour la santé, et les maladies transmises par ces viandes causent un fardeau important mondialement. En Afrique centrale, le gibier est une viande communément consommée en zone urbaine. L’absence d’information sur le niveau de consommation de gibier, ainsi que sur sa contamination, limite l’évaluation des risques sanitaires associés au gibier. Une étude transversale a visé la description du niveau de consommation des viandes parmi 205 ménages de Port-Gentil (Gabon), ainsi que certains déterminants de la consommation de ces viandes. Une seconde étude transversale a quantifié la contamination musculaire de gibier vendu à Port-Gentil par Salmonella, Campylobacter et Shigella. Sur une base de trois jours, 86% des ménages ont consommé de la volaille, 84% du poisson, 44% du bœuf, 25% du porc et 24% du gibier. La consommation de gibier fut plus fréquente le dimanche et parmi les ménages à revenu élevé. Le gibier fut principalement acquis en carcasse entière sans conservation particulière, mais toujours consommé bouilli. Des trois bactéries ciblées, seule Salmonella a été isolée parmi un de 128 échantillons de gibier. Ces études fournissent des informations utiles pour mieux comprendre les facteurs de risque pour la santé associés à la consommation de viandes au Gabon. Des études sur la contamination des viandes, notamment celles des carcasses de gibier, seront nécessaires pour mieux apprécier les risques spécifiques à chaque différente bactérie pathogène. / Meat poses some risks to human health and meat-borne diseases constitute a high burden worldwide. In central Africa, bushmeat is commonly consumed in the urban setting. A lack of information on bushmeat consumption and contamination limits the evaluation of risks to human health linked to bushmeat. A cross-sectional survey was conducted among 205 households of Port-Gentil (Gabon) to quantify relative consumption levels of different meat types and to explore certain determinants of meat consumption. A separate cross-sectional study aimed to determine the prevalence of Campylobacter, Salmonella and Shigella within bushmeat sold in markets of Port-Gentil. Based on a three-day recall period, 86% of household consumed poultry compared to 84% for fish, 44% for beef, 25% for pork and 24% for bushmeat. Bushmeat consumption was more important on Sundays and within high monthly income households. Most bushmeat was acquired as whole carcasses without formal meat conservation methods, but all bushmeat was boiled prior to consumption. One Salmonella was detected among one of 128 bushmeat samples, whereas no Campylobacter or Shigella were detected. This study provides useful information to help better understand risk factors associated with the consumption of bushmeat in Gabon. Further studies on bacterial contamination of meat, including bushmeat carcasses, are required to better understand potential health risks specific to different bacterial pathogens.

Campylobacter jejuni is the commonest cause of bacterial foodborne gastroenteritis in industrialised countries. Despite its significance, it remains unclear how C. jejuni is disseminated in the environment, whether particular strains are more pathogenic than others, and by what routes this bacterium is transmitted to humans. One major factor hampering this knowledge is the lack of a standardised method for fingerprinting C. jejuni. Therefore, the overall aim of this project was to develop systematic and novel genotyping methods for C. jejuni. Chapter Three describes the use of single nucleotide polymorphisms (SNPs) derived from the multilocus sequence typing (MLST) database of C. jejuni and the closely related Campylobacter coli for genotyping these pathogens. The MLST database contains DNA sequence data for over 4000 strains, making it the largest comparative database available for these organisms. Using the in-house software package "Minimum SNPs", seven SNPs were identified from the C. jejuni/C. coli MLST database that gave a Simpson's Index of Diversity (D), or resolving power, of 0.98. An allele-specific real-time PCR method was developed and tested on 154 Australian C. jejuni and C. coli isolates. The major advantage of the seven SNPs over MLST is that they are cheaper, faster and simpler to interrogate than the sequence-based MLST method. When the SNP profiles were combined with sequencing of the rapidly evolving flaA short variable region (flaA SVR) locus, the genotype distributions were comparable to those obtained by MLST-flaA SVR. Recent technological advances have facilitated the characterisation of entire bacterial genomes using comparative genome hybridisation (CGH) microarrays. Chapter Four of this thesis explores the large volume of CGH data generated for C. jejuni and eight binary genes (genes present in some strains but absent in others) were identified that provided complete discrimination of 20 epidemiologically unrelated strains of C. jejuni. Real-time PCR assays were developed for the eight binary genes and tested on the Australian isolates. The results from this study showed that the SNP-binary assay provided a sufficient replacement for the more laborious MLST-flaA SVR sequencing method. The clustered regularly interspaced short palindromic repeat (CRISPR) region is comprised of tandem repeats, with one half of the repeat region highly conserved and the other half highly diverse in sequence. Recent advances in real-time PCR enabled the interrogation of these repeat regions in C. jejuni using high-resolution melt differentiation of PCR products. It was found that the CRISPR loci discriminated epidemiologically distinct isolates that were indistinguishable by the other typing methods (Chapter Five). Importantly, the combinatorial SNP-binary-CRISPR assay provided resolution comparable to the current 'gold standard' genotyping methodology, pulsed-field gel electrophoresis. Chapter Six describes a novel third module of "Minimum SNPs", 'Not-N', to identify genetic targets diagnostic for strain populations of interest from the remaining population. The applicability of Not-N was tested using bacterial and viral sequence databases. Due to the weakly clonal population structure of C. jejuni and C. coli, Not-N was inefficient at identifying small numbers of SNPs for the major MLST clonal complexes. In contrast, Not-N completely discriminated the 13 major subtypes of hepatitis C virus using 15 SNPs, and identified binary gene targets superior to those previously found for phylogenetic clades of C. jejuni, Yersinia enterocolitica and Clostridium difficile, demonstrating the utility of this additional module of "Minimum SNPs". Taken together, the presented work demonstrates the potentially far-reaching applications of novel and systematic genotyping assays to characterise bacterial pathogens with high accuracy and discriminatory power. This project has exploited known genetic diversity of C. jejuni to develop highly targeted assays that are akin to the resolution of the current 'gold standard' typing methods. By targeting differentially evolving genetic markers, an epidemiologically relevant, high-resolution fingerprint of the isolate in question can be determined at a fraction of the time, effort and cost of current genotyping procedures. The outcomes from this study will pave the way for improved diagnostics for many clinically significant pathogens as the concept of hierarchal combinatorial genotyping gains momentum amongst infectious disease specialists and public health-related agencies.

Made available in DSpace on 2011-05-04T12:36:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 / A campilobacteriose, apesar de pouco estudada, pode representar um problema de Saúde Pública em nosso País, tendo em vista as precárias condições sanitárias de boa parte da população. Entretanto, sua aquisição, manutenção e transmissão no Brasil só poderão ser estimadas e valorizadas a partir de investigações criteriosas, incluindo não só as pesquisas zoonóticas em humanos e animais, mas também relacionadas ao ambiente. Durante o período de agosto de 2007 a dezembro de 2008 investigou-se, a presença de Campylobacter spp. como potencial indicador microbiológico de contaminação em amostras de água do baixo curso do rio São João RJ. O sítio de estudo, localizado na Região dos Lagos, serve como modelo de regiões que apresentam importantes atividades turísticas e econômicas, possuindo grande afluxo populacional, que promove desequilíbrios e impactos ao ecossistema. O trabalho incluiu também a pesquisa deste microrganismo na água de abastecimento da população local, poços, e fezes de voluntários, tendo sido isolados 28 representantes deste gênero. No processo de isolamento e identificação realizou-se a metodologia clássica fenotipica seguida da análise genotípica onde foram caracterizadas as seguintes espécies: C. jejuni (50 por cento), C. coli (21,43 por cento) e Campylobacter sp. (28,57 por cento). O alto índice de isolamento deCampylobacter no rio São João, (em todos os pontos de coleta) indica acontaminação por essas bactérias e sua possível manutenção natural no ambiente. Todavia sua total ausência na água distribuída pela rede pública sugere efetividade no processo de tratamento, já que a água é retirada da lagoa de Juturnaíba, um dos pontos que apresentou maior percentual de positividade para este gênero dentro do nosso estudo. O estabelecimento no futuro de um manejo mais adequado para esse manancial representará a melhoria da qualidade de vida da população do entorno, diretamente exposta, proporcionando também a redução dos problemas de saúde pública decorrentes do uso de água contaminada. / The campylobacteriosis, although little studied, may represent a public health problem in our country, given the precarious health conditions of much of the population. However, its acquisition, maintenance and transmission in Brazil can only be estimated and valued from research on, including not only the research zoonoses in humans and animals, but also related to the environment. During the period August 2007 to December 2008 it was investigated, the presence of Campylobacter spp. as a potential indicator of microbiolocal contamination in water samples in the lower course of the São João river - RJ. The site of study at Lake´s Region, serves as a model for regions that have major economic and tourist activities, with major population influx, which promotes imbalances and impacts on ecosystems. The work also included a search of this microorganism in the water supply of the local population, wells, and feces of volunteers, 28 isolates were representatives of this genus. In the process of isolation and identification was held on classic phenotypic methods followed by genetic analysis which were characterized the species: C. jejuni (50%), C. coli (21.43%) and Campylobacter sp. (28.57%). The high rate of isolation of Campylobacter in the river São João, (in all the collection points) indicates that contamination by these bacteria and their possible retention in the natural environment. However its total absence in the water distributed by public, suggests efficacy in the treatment process, due to the fact that water is removed from the lagoon of Juturnaíba, a point which had a higher percentage of positivity for this genus within our study. The future establishment of a more appropriate management for this source will improve the quality of life of the surrounding area, directly exposed, providing also the reduction of public health problems arising from the use of contaminated water.

Campylobacter spp., principalmente as espécies C. coli e C. jejuni, são a causa mais comum de doença bacteriana veiculada por alimentos na Europa, Estados Unidos e alguns outros locais do mundo. No Brasil, há uma escassez de estudos de C. coli, o que dificulta avaliar a dimensão do envolvimento dessa bactéria como causadora de doença nos seres humanos e em animais, bem como, determinar o impacto de sua presença em alimentos e no meio ambiente. O objetivo desse trabalho foi caracterizar molecularmente linhagens de C. coli isoladas de origens diversas no Brasil pela pesquisa da presença de genes relacionados à virulência por PCR, perfil de sensibilidade a antimicrobianos e pela análise da similaridade genotípica por métodos de tipagem molecular. Adicionalmente, o Índice de Discriminação (D) de tais metodologias foi verificado. Foram estudadas 63 linhagens de C. coli, isoladas de humanos (12), animais (21), alimentos (10) e ambiente (20), entre os anos de 1995 e 2011, nos Estados do Rio de Janeiro, São Paulo e Minas Gerais. Todas as linhagens apresentaram os genes flaA, cadF e sodB. O gene cdtB foi detectado em 20 (31,7%) linhagens, o gene flhA foi detectado em 11 (17,5%) linhagens, o gene dnaJ foi encontrado em 10 (15,9%) linhagens, o gene pldA foi detectado em sete (11,1%) linhagens, o gene iamA foi detectado em três (4,8%) linhagens, os genes cdtC e docA foram encontrados em duas (3,2%) linhagens, os genes cdtA e crsA foram encontrados em uma (1,6%) linhagem e os genes ciaB, wlaN, virB11 e racR não foram detectados. Dentre as 63 linhagens estudadas, 42 foram susceptíveis a todos os antimicrobianos testados. Das 21 linhagens resistentes, 10 (15,9%) foram resistentes a tetraciclina e doxaciclina, seis (9,5%) foram resistentes a ciprofloxacina e uma (1,6 %) foi resistente a eritromicina. Somente quatro (6,3%) linhagens foram resistentes a pelo menos duas diferentes classes de antibióticos testados simultaneamente. O dendrograma de similaridade genética de Pulsed field gel electrophoresis (PFGE) agrupou as 63 linhagens estudadas em dois grupos principais denominados PFGE-A e PFGE-B com similaridade genômica de 44,9% entre eles. Entretanto, algumas linhagens isoladas de humanos, animais, ambiente e alimentos apresentaram uma alta similaridade genotípica acima de 80% entre elas e, foram agrupadas em sete subgrupos denominados PFGE-A1 a PFGE-A7. O dendrograma de similaridade genômica das sequências da SVR do gene flaA agrupou as linhagens ii estudadas em dois grupos principais designados SVR-A e SVR-B, com similaridade acima de 83,1 % entre eles. Ademais, o depósito das sequências da SVR do gene flaA no banco de dados online demonstrou que os alelos 30 e o 1647 foram os mais frequentemente encontrados e permitiu a comparação das linhagens estudadas com os alelos descritos no banco de dados. Sete alelos, dentre os 22 encontrados, não haviam sido previamente descritos. A análise do locus CRISPR por HRMA dividiu as linhagens de C. coli em quatro diferentes perfis de melting. O Multilocus sequence typing (MLST) foi utilizado para tipar 20 linhagens de C. coli e foram obtidos 18 STs diferentes dos quais apenas dois já haviam sido previamente descritos. O D das metodologias de PFGE, sequenciamento da SVR do gene flaA, análise do locus CRISPR por HRMA e MLST foi de 0,986, 0,916, 0,550 e 0,989, respectivamente. Pode- se concluir que o potencial patogênico das linhagens de C. coli não foi evidenciado o que pode estar relacionado ao fato da maioria dos estudos envolvendo patogênese terem sido realizados para a espécie C. jejuni. Algumas linhagens apresentaram-se resistentes aos antimicrobianos testados, o que é preocupante uma vez que tais linhagens podem disseminar genes de resistência a outras isoladas de diversas fontes. Os resultados gerados pelos métodos de tipagem molecular por PFGE e sequenciamento da pequena região variável (SVR) do gene flaA demonstraram uma alta similaridade genotípica entre algumas linhagens de C. coli, sugerindo que uma possível contaminação tenha ocorrido entre linhagens isoladas de fontes clínicas e não clínicas ao longo de 16 anos no Brasil. Ademais, a análise dos alelos da SVR do gene flaA nos permitiu concluir que os alelos prevalentes nas linhagens estudadas diferem daqueles encontrados nos países Europeus. Os dados obtidos por MLST sugerem que as linhagens estudadas possuem uma grande diversidade genética entre si e em comparação com as linhagens isoladas em diferentes locais do mundo. Finalmente, as técnicas de MLST e PFGE foram as mais eficientes e adequadas na genotipagem das linhagens de C. coli estudadas. / Campylobacter spp., mainly the C. coli and C. jejuni species, are the most common cause of bacterial disease conveyed by food in Europe, United States, and other places worldwide. In Brazil, there is a paucity of studies on C. coli, which makes it difficult to evaluate the involvement of this bacterium as a cause of diseases in humans and animals, as well as to determine the impact of its presence in food and the environment. The aim of this study was to molecularly characterize C. coli strains isolated from diverse origins in Brazil by searching for the presence of virulence-related genes by PCR, antimicrobial sensitivity profile, and analysis of the genotypic similarity by molecular typing methods. Addicionaly, the Discriminatory Index (D) of those methodologies was acessed. Sixty-three C. coli strains isolated from humans (12), animals (21), food (10), and the environment (20) between 1995 and 2011, in the States of Rio de Janeiro, São Paulo, and Minas Gerais were studied. All strains presented the flaA, cadF and sodB genes. The cdtB gene was detected in 20 (31.7%) strains; the flhA gene was detected in 11 (17.5%) strains; the dnaJ gene was detected in 10 (15.9%) strains; the pldA gene was detected in 7 (11.1%) strains ; the iamA gene was detected in three (4.8%) strains; the cdtC and docA genes were found in two (3.2%) strains; the cdtA and crsA were found in one (1.6%) strain and the ciaB, wlaN, virB11 and racR genes were not detected. Among the 63 strains studied, 42 were susceptible to all antimicrobials tested. Of the 21 resistant strains, 10 (15.9%) were resistante to tetracycline and doxaciclyne, six (9.5%) showed resistance to ciprofloxacin, and one (1.6%) was resistant to erythromycin. Only four (6.3%) strains were simultaneously resistant to at least two different classes of the antibiotics tested. The dendrogram of genetic similarity of Pulsed field gel electrophoresis (PFGE) grouped the 63 strains studied into two groups namely PFGE-A and PFGE-B with a genomic similarity of 44.9% among them. However, some strains isolated from humans, animals, the environment and food presented a high genotypic similarity above 80% and were subdivided into seven groups designated as PFGE-A1 to PFGE-A7. The dendrogram of genetic similarity of the SRV-flaA gene sequences grouped the strains studied into two groups namely SVR-A and SVR-B, with similarity above 83.1% among them. Besides, the deposit of the SVR sequences of the flaA gene in the online database showed that the alleles 30 and 1647 were the iv most frequently found and allowed the comparison between the strains studied with the alleles described in the database. Seven alleles, among the 22 found have never been described before. The CRISPR locus analysis divided the C. coli strains into four different melting profiles. The Multilocus sequence typing (MLST) was used to type 20 C. coli strains and revealed 18 different STs among which just two had been previously described. The D of PFGE, SVR- flaA sequence, HRMA of CRISPR locus analysis and MLST was 0.986, 0.916, 0.550 and 0.989, respectively. In conclusion, the pathogenic potential of the C. coli strains was not highlighted, which could be related to the fact that the majority of the pathogenicity studies were performed with C. jejuni species. Some strains showed resistance to the antibiotics tested what is a concern once those strains may spread the resistance genes to other strains isolated from different sources. The results obtained by PFGE and SVR-flaA sequence showed a high genomic similarity among some C. coli strains which may suggest that a possible contamination may have occurred among clinical and non-clinical sources during 16 years in Brazil. Furthermore, the analysis of SVR- flaA alleles allowed the conclusion that the prevalent alleles in the strains studied were different from those found in European countries. The data obtained by MLST suggests that the strains studied had a high genomic diversity among them and in comparison with strains isolated from different places worldwide. Finally, the MLST and PFGE technicques were the most efficient and adequate in genotyping the C. coli strains studied.

As infecções causadas por Campylobacter spp. são relatadas como causa freqüente de gastrenterites de origem alimentar em vários países do mundo. As espécies bacterianas termofílicas pertencentes ao gênero Campylobacter, principalmente Campylobacter jejuni e Campylobacter coli, têm sido isoladas de fezes de animais e estão associadas à contaminação da carne durante o processo de abate. Estas duas espécies são as mais freqüentemente envolvidas nos casos de campilobacteriose humana veiculada por alimentos. O presente estudo pretendeu avaliar a presença e a população de Campylobacter spp. no abate de bovinos e cortes refrigerados de aves e bovinos comercializados na cidade de São Paulo/SP. Um total de 198 animais foi amostrado no couro logo após a sangria, na carcaça imediatamente após a esfola e após a evisceração. As amostras foram obtidas através da técnica de swab na região do peito abrangendo uma área de 400 cm2. Foram analisados também 120 cortes refrigerados de frango e 100 cortes de carne bovina, assim distribuídos: 40 amostras de asa, 20 de coxa com sobrecoxa, 20 de coxa, 20 de coxinha da asa, 20 amostras de peito; 20 de patinho bovino (M. biceps femoris), 20 de contrafilé (M. longissimus dorsi), 20 de coxão mole (M. semi membranosus), 20 de lagarto (M. semitendinosus) e 20 de alcatra (M. glutaes medius). As amostras foram analisadas segundo os métodos ISO 10272-1 e 2 e os isolados obtidos foram confirmados como Campylobacter pela técnica de PCR. Campylobacter foi isolado em 22,7% (45/198) das amostras de couro bovino, ou seja, apenas no ponto antes da esfola, e C. jejuni foi a única espécie encontrada. Nas amostras de cortes de frango Campylobacter foi isolado em 14,2% (17/120) das amostras. A espécie prevalente em frangos foi C. coli (88%), seguido de C. jejuni (12%). Campylobacter spp. não foi isolado dos cortes bovinos. A população de Campylobacter spp. foi < 13 UFC/cm2 em carcaças bovinas, < 2 UFC/g em amostras de frango e < 10 UFC/cm2 em cortes bovinos. A susceptibilidade de 120 isolados de frango e couro bovino foi determinada frente a 8 agentes antimicrobianos usando o método de disco-difusão. A resistência às quinolonas (ác. nalidíxico e ciprofloxacina) foi frequentemente observada nas cepas de C. jejuni (72,2%) e C. coli (50,8%) isoladas dos frangos. Entre os isolados de C. jejuni obtidos do couro bovino maior taxa de resistência foi observada para estreptomicina (32%), seguida da eritromicina (16%) e do ácido nalidíxico (14%). / Campylobacter spp. infections are reported as a frequent cause of foodborne gastroenteritis in many countries. The thermophilic bacterial species belonging to the genus Campylobacter, particularly Campylobacter jejuni and Campylobacter coli have been isolated from feces of animals and are associated with the contamination of meat during the slaughtering process. These two species are the most frequently involved in cases of human campylobacteriosis conveyed by food. The aim of the present study was to evaluate the presence and population of Campylobacter spp. during cattle slaughter and in refrigerated chicken and beef cuts commercialized in the city of Sao Paulo/SP. A total of 198 animals were sampled in the hide after bleeding, the carcass immediately after skinning and after evisceration. Samples were obtained by swab technique in the chest area encompassing an area of 400 cm2. We also analyzed 120 refrigerated chicken cuts and 100 beef cuts. The samples were analyzed according to ISO 10272-1 and 2 methods and the isolates were confirmed as Campylobacter by PCR technique. Campylobacter was isolated only in the hide samples (45/198), and C. jejuni was the only species found. Campylobacter was isolated in 14.2% (17/120) of chicken samples. The most prevalent species in chickens was C. coli (88%), followed by C. jejuni (12%). Campylobacter spp. was not isolated from beef cuts. The counts of Campylobacter spp. was < 13 CFU/cm2 in bovine carcasses, < 2 CFU/g in chicken samples and < 10 CFU/cm2 in beef cuts. The susceptibility to 8 antimicrobial agents of 120 isolates of chicken and bovine hide was determined using the disk-diffusion method. The resistance to quinolones (ciprofloxacin and nalidixic acid) was frequently observed in strains of C. jejuni (72.2%) and C. coli (50.8%) isolated from chickens. Among strains of C. jejuni obtained from bovine hide highest resistance rate was observed to streptomycin (32%), followed by erythromycin (16%) and nalidixic acid (14%).

Today, thermophilic Campylobacter spp. (besides Salmonella) represent one of the most common sources of human bacterial gastrointestinal infection. The main source of human C. jejuni infections is the consumption of insufficient heated chicken meat. Quantitative data on the occurrence of thermophilic Campylobacter spp. on poultry carcasses and poultry meat are needed to perform quantitative risk assessments and to verify the effect of different intervention strategies. The aims of the investigations presented here were (i) to generate accurate qualitative and quantitative data on the occurrence of Campylobacter spp. on within the primary production stage in at the abattoir and food, (ii) to study the behaviour of nine genotypically different C. jejuni strains in chicken meat juice supplemented with different concentrations of sodium chloride, curing salt and sodium nitrite, (iii) to detect the Campylobacter genotype distribution in poultry flocks by applying AFLP analysis and to describe a potential carry-over of Campylobacter strains among sequential and adjacent poultry flocks. For the above mentioned purposes, a number of samples (171 neck skin samples, 1047 samples of different turkey meat products and 112 turkey minced meat samples from an abattoir) were investigated in accordance with themethod of ISO / TS 10272-2: 2006 and ISO10272-1: 2006 to the quantitative and qualitative presence of Campylobacter spp.. The Campylobacter-strains were inoculated in chicken juice at initial concentrations of 102 and 104 CFU/ml and incubated for 24h at 42°C. Furthermore, 18 flocks of four poultry species were monitored to investigate the distribution and spread of Campylobacter genotypes between sequential and adjacent flocks. Caecal and liver samples were obtained at frequent intervals from birds of all flocks and these samples were examined for Campylobacter. The amplified fragment length polymorphism (AFLP) analysis was performed to genotype Campylobacter isolates. The prevalence of Campylobacter on neck skin was 83.0 % and the mean number was 2.00 log10 cfu/g. For turkey meat samples with skin (wings and thighs) the detected prevalence was 68.2 % and mean number 1.73 log10 cfu/g, respectively. Turkey meat samples without skin (breast filet) showed a prevalence of 79.0 % and a mean number of 1.58 log10 cfu/g. No Campylobacters were detectable in the turkey minced meat samples. Large variations between the detectable numbers of Campylobacter spp. were observed (maximum number up to 3.98 log10 cfu/g for turkey meat with skin) and confirm the importance of an early detection (before or during slaughter and processing) of these heavily contaminated slaughter lots. Whereas the strains multiplied in the media supplemented without additional of NaCl or with 1% NaCl, the bacterial population was significantly reduced when 2% NaCl was added. Growth did not occur and the cell number gradually decreased in chicken meat juice containing 3% NaCl. Significant differences in the survival potential among the different strains were only visible in the extreme condition of 3% NaCl supplementation. There was no different behaviour of the strains under the influence of NaCl compared with the behaviour in meat juice containing curing salt. The addition of sodium nitrite did not alter the survival. Of the 1643 caecal and liver samples investigated, 452 (27.5%) caecal samples and 11 (0.7%) liver samples contained Campylobacter. Of the caecal isolates 76.3% were identified as C. jejuni and 23.7% were identified as C. coli. Poultry flocks were largely colonized by more than one AFLP type and an intense exchange of Campylobacter genotypes between different poultry flocks occurred. The results show clearly that poultry and poultry meat are regarded as one of the main sources of thermophilic Campylobacter spp. infection in humans in the food chain. This is evident not only from the high rate of occurrence of these pathogens, but also from the often-high quantitative exposure samples. The risk of a foodborne infection is also enhanced by the comparatively very low minimum infectious dose for humans. At present, a complete elimination of thermophilic Campylobacter spp. from the food chain appears practically unreachable. This difficulty is reduced by the results of genetic strain diversity, because they suggest the existence of a variety of input sources. / hermophile Campylobacter (C.) spp. stellen heute neben den Salmonellen eine der häufigsten Ursachen für bakteriell bedingte Magen-Darm-Erkrankungen beim Menschen dar. Unzureichend erhitztes Geflügelfleisch und Geflügelfleischprodukte stellen eine der Hauptinfektionsquellen für humane C. jejuni-Infektionen dar. Quantitative Daten über die Belastung von Geflügelkarkassen und Geflügelfleisch mit thermophilen Campylobacter spp. werden benötigt, um einerseits quantitative Risikobewertungen durchführen zu können, andererseits aber auch den Erfolg verschiedener Interventionsmaßnahmen messen zu können. Zur Minderung der Zahl alimentär bedingter humaner Campylobacter-Infektionen spielt neben der Senkung der Campylobacter-Belastung von Nutztieren und der Vermeidung von Kreuzkontaminationen auch die Reduktion des Vorkommens des Erregers in der Lebensmittelkette durch technologische Prozesse eine große Rolle. Campylobacter spp. sind während der Be- und Verarbeitung von Lebensmitteln verschiedensten Stressoren ausgesetzt. Ziele der hier vorgestellten Untersuchungen waren, (i) Exakte qualitative und quantitative Daten zum Vorkommen von Campylobacter spp. in der Primärproduktion, auf dem Schlachthof und in Lebensmitteln zu ermitteln. (ii) Die Tenazität ausgewählter, genetisch unterschiedlicher C. jejuni-Stämme gegenüber verschiedenen Natriumchlorid-, Pökelsalz- und Natriumnitrit-Konzentrationen im Geflügelfleischsaftmodell zu untersuchen. (iii) Die Verwandtschaftsgrade der isolierten Stämme mit Hilfe einer molekularbiologischen Fingerprintingmethode (AFLP-Typisierung) darzustellen sowie das Vorkommen von thermophilen Campylobacter in verschiedenen Geflügelarten in einem Betrieb zu ermitteln. Dazu wurden 171 Halshautproben, 783 Proben verschiedener Putenfleischerzeugnisse und 233 Putenhackfleischproben aus einem Schlacht- und Zerlegebetrieb in Anlehnung an die Methode ISO/TS 10272-2: 2006 und ISO10272-1:2006 auf das quantitative und qualitative Vorkommen von Campylobacter spp. untersucht. Der Keimzahlverlauf wurde in experimentell kontaminiertem Geflügelfleischsaft (Zusatz von C. jejuni: 102 und 104 KbE/ml) über einen Zeitraum von 24 h (Bebrütungstemperatur 37°C) untersucht. 19 verschiedene Wirtschaftsgeflügel-Herden wurden untersucht, um die Verteilung und Ausbreitung von Campylobater-Genotypen zwischen sequentiellen und angrenzenden Herden festzustellen. Blinddarm- und Leber-Proben wurden in kurzen Abständen von Vögeln aller Herden gewonnen und untersucht . Für die Genotypisierung von Campylobacter spp. wurde die AFLP-Methode eingesetzt. Im Ergebnis wurden auf 83,0 % der Halshautproben Campylobacter spp. nachgewiesen, wobei der Mittelwert der quantitativen Belastung von Putenhalshautproben bei 2,00 log10 KbE/g lag. Putenfleischproben mit Haut waren zu 54,8 % Campylobacter positiv. Hier betrug die quantitative Belastung 1,79 log10 KbE/g. Bei Putenfleisch ohne Haut lagen die Nachweisraten bei 52,2,% bzw. 2,03 log10 KbE/g. In keiner der Putenhackfleischproben war Campylobacter nachweisbar. Große Schwankungen in der quantitativen Belastung (Maximalwerte bis 4,0 log10 KbE/g bei Putenfleisch mit Haut) bestätigen die Notwendigkeit, vor allem die stark belasteten Schlachtpartien schon vor bzw. während der Schlachtung und Verarbeitung identifizieren zu können. In Geflügelfleischsaft ohne bzw. mit Zusatz von 1% NaCl konnten sich alle Stämme vermehren, während das Wachstum bei 2% NaCl-Zusatz gehemmt wurde. Darüber hinaus konnte bei höherer NaCl-Konzentration (3%) eine Reduktion der Keimzahl nach 6 h Bebrütung bzw. ein Absterben von C.jejuni nach 24 h festgestellt werden. Dabei zeigten die Stämme im Geflügelfleischsaft mit 3% NaCl-Zusatz signifikante Unterschiede in ihrer Absterberate. Es konnte gezeigt werden, dass unterschiedlich ausgeprägte Salztoleranzen innerhalb der untersuchten Stämme mit unterschiedlichem Genotyp existieren, diese jedoch nur unter Extremsituationen signifikant ausgeprägt waren. Durch die Zugabe von praxisüblichen Pökelsalzkonzentrationen anstelle von Kochsalz und von Natriumnitrit konnte das Verhalten von C. jejuni in keinem Versuchsansatz beeinflusst werden. 452 Caecalproben (27,5%) und 11 Leberproben (0,7%) von insgesamt 1643 Caecal- und Leberproben wurden positiv auf Campylobacter spp. getestet. Von den 1643 aus dem Caecum stammenden getesteten Isolaten wurden 76,3% der Isolate als C. jejuni und (23,7%) der Isolate als C. coli identifiziert. Die AFLP- Analyse zeigte einen signifikanten Unterschied in der Diversität der AFLP-Typen aus den individuellen Herden und Proben aus unterschiedlichen Herden. Dies deutet auf eine große Zahl von Infektionsquellen hin. Die Ergebnisse belegen insgesamt sehr deutlich, dass Geflügel- Geflügelfleisch eine bedeutsame Quelle des Eintrags von Campylobacter-Keimen in die Nahrungskette ist. Das geht nicht nur aus der hohen Rate des Vorkommens dieser Erreger hervor, sondern auch aus der oftmals hohen quantitativen Belastung der Proben. Das Risiko einer Lebensmittelinfektion wird zudem durch die vergleichsweise sehr geringe minimale Infektionsdosis für den Menschen erhöht. Eine vollständige Elimination von thermophilen Campylobacter spp. aus der Lebensmittelkette erscheint derzeit praktisch nicht erreichbar. Diese Schwierigkeit wird durch die Ergebnisse zur genetischen Stammdiversität untersetzt, denn sie legen die Existenz einer Vielzahl unterschiedlicher Eintragsquellen nahe.

D. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Salmonella enterica serovar Typhimurium and Campylobacter jejuni are the leading causes of Salmonellosis and Campylobacteriosis that is characterised by gastroenteritis. These waterborne diseases can be easily prevented by home water treatment methods such as solar disinfection (SODIS). The SODIS process involves placing microbiologically unsafe water in clear plastic or glass bottles and exposing them to direct sunlight for approximately six to eight hours. SODIS kills microbes through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating. The result is microbiologically safe water. Continuous drinking of SODIS treated water may confer some immunological effects on the consumer. These immunological effects have not been thoroughly explored. Therefore, the objectives of this study were to firstly, characterise the effects of solar irradiation on the viability of S. Typhimurium and C. jejuni; secondly, to determine the cytotoxicity and modulation of cell death of solar irradiated S. Typhimurium and C. jejuni on macrophages. Thirdly, to analyse the chemokine and cytokine profiles of macrophages infected with solar irradiated S. Typhimurium and C. jejuni. Lastly, to analyse the host-cell interactions of macrophages infected with solar-irradiated and non-solar irradiated S. Typhimurium and C. jejuni using a proteomic approach. In all the experiments, S. Typhimurium and C. jejuni were (i) heat/chemically treated, (ii) solar and non-solar irradiated for 4 and 8 hours. A murine macrophage cell line RAW264.7 was co-cultured with the differentially treated bacteria species for 3 and 24 hours. Appropriate controls were included. The impact of solar irradiated S. Typhimurium and C. jejuni on intracellular growth, proliferation, cytotoxicity, and apoptosis on macrophages was assessed. Intracellular growth of the both bacterial species was assessed with the gentamicin protection assay, and cytotoxicity was determined by Lactate Dehydrogenase Assay (LDH). The macrophages treated with solar irradiated S. Typhimurium and C. jejuni showed no intracellular growth after 48 hours post-infection. However, the non-irradiated S. Typhimurium survived within the macrophages and were highly toxic to the macrophages (average cytotoxicity of 91%±32). The non-solar irradiated C. jejuni were metabolically active but non-culturable, whereas the solar-irradiated C. jejuni was metabolically inactive. Thus, solar irradiated C. jejuni showed a lower percentage cytotoxicity (2.57% ± 0.32%) in comparison to non-solar irradiated C. jejuni at 24 hours post-infection (p.i.) (30.28% ± 0.05%). Flow cytometric analysis showed that the non-irradiated S. Typhimurium brought about a statistically significant increase in the percentage of necrotic cells (48% ± 2.99%), whereas bacteria irradiated for 8 hours produced a lower percentage of necrotic cells (25% ± 5.87%). The heat/chemical attenuated samples had the lowest percentage of necrotic cells (21.15% ± 5.36%) at 24 h p.i. Macrophages treated with solar irradiated and non-solar irradiated C. jejuni did not induce necrosis, but apoptotic cell death. At 24 h p.i., the highest proportion of apoptotic cell death was observed in macrophages treated with non-solar irradiated C. jejuni whereas the solar irradiated C. jejuni showed a lower percentage of apoptotic cell death. Therefore, there is great possibility that S. Typhimurium and C. jejuni could become avirulent after SODIS treatment and this could prevent gastroenteritis in consumers of SODIS-treated water. The activation of macrophages infected with solar irradiated S. Typhimurium and C. jejuni was also assessed in this study. The production of nitric oxide (NO) was determined using the Greiss Reagent Assay, whereas the production of chemokines, cytokines, and growth stimulating factors by the RAW264.7 cells in vitro was measured using the Luminex 200. The results showed that both solar and non-solar irradiated S. Typhimurium inhibited the production of nitric oxide in the RAW264.7 cells. The heat/chemically attenuated S. Typhimurium induced a significant increase (p<0.0.5) in the production of NO2− in the macrophages when compared to the unstimulated RAW264.7. The chemokine and cytokine levels produced by the macrophages were similar in the solar inactivated S. Typhimurium and the live untreated S. Typhimurium. However, macrophages treated with heat/chemically attenuated S. Typhimurium showed an anti-inflammatory response by inhibiting the production of pro-inflammatory cytokines such as IL-1, IL-1, IL-2, IL-6, and IL-17 in macrophages. The macrophages treated with solar and non-solar irradiated C. jejuni possibly produced an anti-inflammatory effect since the amount of pro-inflammatory cytokines in the samples was significantly reduced during the late infection period (24 h p.i.). This study also analysed the proteomic profiles of macrophages treated with LPS, non-solar irradiated, solar irradiated, heat/ chemical inactivated S. Typhimurium, and C. jejuni. This was carried out using SWATH-mass spectrophotometry-based proteomics. Proteins were extracted from infected macrophages after 24 hours p.i. HILIC-based sample clean-up and digestion, DDA LCMS-MS (spectral library), SWATH LCMS-MS, and data processing were carried out. A total of 15,077 peptides matching to 2,778 proteins were identified at 1% FDR with numerous differentially expressed proteins (DEPs) detected in macrophages treated with lipopolysaccharide (LPS), non-solar irradiated C. jejuni (NS), heat-attenuated C. jejuni (HA) and 4h-solar irradiated (SI4) and 8h-solar irradiated (SI8) C. jejuni, respectively. Pathway analysis revealed that most of the upregulated proteins in macrophages treated with solar irradiated C. jejuni were involved in oxidation-reduction processes, endoplasmic reticulum stress, transport, antigen processing and presentation of exogenous peptide antigens via MHC class I (TAP-dependant) and ATP-biosynthetic processes. The KEGG-pathways also revealed the roles of some upregulated proteins in lysosomal and phagosome pathways. In conclusion, our results revealed that there is coordinated up-regulation of MHC-I processing pathways occurred at 24 h p.i. It is likely that proteins from solar irradiated C. jejuni may undergo proteasomal degradation, and the peptides are transported to the endoplasmic reticulum (ER) and loaded onto MHC-I molecules. Peptide loading results in class I complexes consolidation and transit to the cell surface where antigens can be presented to circulating CD8 + T cells. Additionally, solar irradiated C. jejuni also undergoes degradation in the phagosome. The phagosome has the potential to create antigens that can be expressed on the cell surface of macrophages to stimulate different lymphocytes and induce appropriate immune responses, thus, connecting the innate to adaptive immunity, and this could also have health benefits via the consumption of SODIS treated water. However, proteomic analysis of S. Typhimurium showed no significant differentially expressed proteins in macrophages treated with LPS, non-solar irradiated, and solar irradiated S. Typhimurium. This may be due to an overestimation of the extracted protein. However, DEPs in macrophages treated with heat-attenuated S. Typhimurium showed that macrophages may have adapted an anti-inflammatory M2 phenotype because the IFN-γ signalling pathway was downregulated. This may have contributed to non-expression of the chemokine IFN-γ in RAW264.7 cells. Moreover, proteins such as Hmox1 and Sqstm1 were upregulated, and this is also characteristic of M2 macrophages. This study provided new insights on the effect of solar irradiated Salmonella Typhimurium and Campylobacter jejuni on the proliferation and activation of macrophages in vitro.