Escherichia coli diarrhoea is the most important source of mortality in piglets. The most frequently isolated strain in enterotoxigenic E. coli diarrhoea is F4ab/ac. Recent studies in South Africa reported non-fimbrial strains such as PAA and EAST-1 to be prevalent. The objective of the study was to determine whether there are breed differences among pigs with respect to E. coli adhesion phenotypes and correlate them to polymorphisms at selected candidate genes in the South African population. A total of 225 pigs aged 3-12 weeks of the imported (Large White, Landrace and Duroc), local and crossbreds, were sampled from the Eastern Cape and Limpopo provinces of South Africa and genotyped for PCR-RFLP polymorphisms at four candidate genes associated with E. coli F4ab/ac resistance/susceptibility. These genes were Mucin 4 (MUC4), Mucin 13, (MUC13), Mucin 20 (MUC20) and Transferrin Receptor (TFRC). The TFRC and MUC13 genes were less polymorphic, the C allele was close to fixation and the homozygous CC genotype was the most frequent in all three pig populations. There was a significant difference (P <0.05) in allelic and genotypic distribution amongst breeds for the TFRC locus. The g.8227G>C polymorphism in MUC4 segregated in all three breeds and the marker was moderately polymorphic. There was a significant difference (P <0.05) in genotypic distribution amongst breeds for MUC4.The g.191C>T polymorphism in MUC20 segregated in the local and crossbred pigs and was close to fixation in the imported pigs. There was a significant difference (P <0.05) in allelic and genotypic distribution amongst breeds for MUC20, which was moderately polymorphic. There was a reduction in heterozygosity in both the TFRC and MUC13 loci, although MUC4 and MUC20 genes had higher heterozygosity levels. The MUC4 gene had a negative FIS value, indicating outbreeding at this locus. The MUC20, MUC13 and TFRC genes had a positive FIS value, indicating inbreeding at these loci. Overall, the studied population was outbred. Imported pigs in TFRC and MUC20 deviated from Hardy-Weinberg equilibrium (HWE). All breeds were in HWE at the MUC4 and MUC13 genes. There was no linkage disequilibrium observed amongst the analysed loci. iv A total of 109 piglets of three breeds (Large White, indigenous and crossbred) aged 3-5 weeks, were investigated for the susceptibility to E. coli F4, PAA strains and EAST-1 toxin. Adhesion tests were conducted on pig intestinal cells, which were viewed under a phase contrast microscope. Three phenotypes were identified as, adhesive, weakly adhesive and non-adhesive. There was a significant association (P <0.05) between breed and level of adherence of the F4 and PAA strains. Highest frequencies of adhesion phenotypes were observed in the indigenous pigs for both F4 and PAA E. coli strains. Large White pigs had the lowest frequency of non-adhesion in F4 and PAA E. coli strains. The F4 strain had a higher (P <0.05) level of adherence compared to PAA and EAST-1 in Large White pigs. Age of pigs had a significant effect on the level of E. coli adherence in indigenous and crossbred pigs (P <0.05). Adhesion of F4 and EAST-1 was higher in weaned indigenous and crossbred pigs, respectively, than in suckling piglets. There was no significant difference between F4 adhesion and the genotypes at all four candidate genes genotypes. The study showed that both imported and local pig populations carry receptors and are susceptible to F4, PAA and EAST-1 E. coli infections. Indigenous pigs were less susceptible than Large White to E. coli infection. Although polymorphic and segregating in the populations, the MUC4 g.8227G>C and MUC20 g.191C>T mutations were not associated with the adhesion phenotypes and cannot be used in the selection of susceptible animals. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.

Este trabalho realizou importantes avanços na elucidação do potencial das toxinas termo-lábeis do tipo II (LT-IIs) como adjuvantes por via intradérmica e transcutânea. Os dados gerados indicam que LT-IIb e LT-IIc nativas atuam como potentes adjuvantes vacinais por via intradérmica induzindo respostas imunológicas antígeno específicas, como medido pela produção de anticorpos sistêmicos (IgG) e ativação de linfócitos T CD8+ citotóxicos. Soma-se ao potencial adjuvante demonstrado para as LT-IIs por essa via a baixa reatogenicidade das moléculas, com menores níveis de edema e reduzida migração leucocitária para o sítio de inoculação em comparação a LT-I. Os resultados indicam também que o efeito adjuvante das LTs aplicadas por via transcutânea pode estar relacionado à capacidade de ligação ao gangliosídeo GM1 já que a toxina LT-IIb, incapaz de interagir com este receptor, não apresenta efeito adjuvante por essa via. O conjunto de dados apresentados abre perspectivas para o emprego das LT-IIs nativas como adjuvantes parenterais em vacinas para animais e humanos. / This work has made significant advances in the understanding of the potential of type II heat-labile toxins (LT-IIs) as vaccine adjuvants by intradermal and transcutaneous route. The generated data indicate that native forms of LT-IIb and LT-IIc act as potent vaccine adjuvants when intradermally injected inducing antigen-specific immune responses, as measured by the generation of systemic serum antibody (IgG) and activation of cytotoxic CD8+ T lymphocytes. Besides the adjuvant effects, LT-IIs show reduced side effects, measured by the lesser edema formation and reduced leukocytes migration to the site of injection, in comparison to LT-I. The results also indicate that the adjuvant activity of LTs applied transcutaneously may be related to the the ganglioside GM1 binding property since the LT-IIb toxin, unable to interact with this receptor, has no adjuvant effect by this route. The presented data set opens prospects for the employment of native LT-IIs as parenteral adjuvants in vaccines for animals and humans.

Les diarrhées post-sevrages causées par des infections à Escherichia coli entérotoxinogène positif pour le fimbriae F4 (ETEC F4), entraînent des pertes économiques importantes chez les producteurs de porc. Depuis quelques années, l’utilisation de probiotiques, comme additif alimentaire pour prévenir ce type d’infection entérique et réduire les traitements aux antimicrobiens, suscite un intérêt grandissant en production porcine. Le but du présent travail est de déterminer l’influence de l’administration des probiotiques Pediococcus acidilactici (PA) et Saccharomyces cerevisiae boulardii (SCB) sur la colonisation et l’attachement des ETEC F4, l’accumulation de fluide intestinal et l’expression de cytokines dans l’iléon de porcelets sevrés. Dès la naissance, différentes portées de porcelets ont été affectées aux traitements suivants : PA, SCB, PA + SCB, témoin et témoin avec antibiotiques (ATB). Une dose quotidienne de probiotiques (1 × 109 UFC) a été administrée aux porcelets des groupes probiotiques durant la lactation et après le sevrage. Sept jours après le sevrage, à 28 jours d’âge, des porcelets positifs pour le récepteur intestinal spécifique pour F4 ont été infectés oralement avec une souche ETEC F4. Les porcelets ont été euthanasiés 24 heures après l’infection (jour 29) et différents échantillons intestinaux ont été prélevés. Chez les porcelets recevant des probiotiques, l’attachement des ETEC F4 à la muqueuse iléale était significativement diminué chez les groupes PA ou SCB en comparaison avec le groupe ATB. Finalement, l’expression de cytokines intestinales était plus élevée chez les porcs du groupe PA + SCB en comparaison avec les porcelets témoins. En conclusion, les résultats de cette étude suggèrent que l’administration de probiotiques pourrait être une alternative pour limiter les infections à ETEC F4 chez le porc. / Postweaning diarrhea (PWD) associated with F4-positive enterotoxigenic Escherichia coli (ETEC F4) causes important economic losses in swine production. Since a couple of years, the use of probiotics as feed additives to prevent such enteric infections and reduce the use of antimicrobial treatments, has gained in interest. The aim of the present study is to evaluate the effects of Pediococcus acidilactici (PA) and Saccharomyces cerevisiae boulardii (SCB), on ETEC F4 colonization and attachment, accumulation of intestinal fluid and cytokines expression in weaned pig’s ileum. At birth, different litters of pigs were allocated to the following treatments: PA, SCB, PA + SCB, control (CTRL) and control with antibiotics (ATB). Probiotics (1 × 109 CFU) were administered daily to probiotics group during the lactation period and after weaning. One week after weaning, at 28 days of age, all F4-receptor-positive pigs were orally challenged with an ETEC F4 strain. Pigs were slaughter 24 hours later (day 29) and different intestinal samples were collected. In pigs treated with PA or SCB, the attachment of ETEC F4 to the ileal mucosa was significantly reduced in comparison with the ATB group. Finally, intestinal cytokines were upregulated in PA + SCB group in comparison with the CTRL group. In conclusion, these results suggest that administration of probiotics could be an alternative to attenuate ETEC F4 infection in pigs.

Les diarrhées post-sevrages causées par des infections à Escherichia coli entérotoxinogène positif pour le fimbriae F4 (ETEC F4), entraînent des pertes économiques importantes chez les producteurs de porc. Depuis quelques années, l’utilisation de probiotiques, comme additif alimentaire pour prévenir ce type d’infection entérique et réduire les traitements aux antimicrobiens, suscite un intérêt grandissant en production porcine. Le but du présent travail est de déterminer l’influence de l’administration des probiotiques Pediococcus acidilactici (PA) et Saccharomyces cerevisiae boulardii (SCB) sur la colonisation et l’attachement des ETEC F4, l’accumulation de fluide intestinal et l’expression de cytokines dans l’iléon de porcelets sevrés. Dès la naissance, différentes portées de porcelets ont été affectées aux traitements suivants : PA, SCB, PA + SCB, témoin et témoin avec antibiotiques (ATB). Une dose quotidienne de probiotiques (1 × 109 UFC) a été administrée aux porcelets des groupes probiotiques durant la lactation et après le sevrage. Sept jours après le sevrage, à 28 jours d’âge, des porcelets positifs pour le récepteur intestinal spécifique pour F4 ont été infectés oralement avec une souche ETEC F4. Les porcelets ont été euthanasiés 24 heures après l’infection (jour 29) et différents échantillons intestinaux ont été prélevés. Chez les porcelets recevant des probiotiques, l’attachement des ETEC F4 à la muqueuse iléale était significativement diminué chez les groupes PA ou SCB en comparaison avec le groupe ATB. Finalement, l’expression de cytokines intestinales était plus élevée chez les porcs du groupe PA + SCB en comparaison avec les porcelets témoins. En conclusion, les résultats de cette étude suggèrent que l’administration de probiotiques pourrait être une alternative pour limiter les infections à ETEC F4 chez le porc. / Postweaning diarrhea (PWD) associated with F4-positive enterotoxigenic Escherichia coli (ETEC F4) causes important economic losses in swine production. Since a couple of years, the use of probiotics as feed additives to prevent such enteric infections and reduce the use of antimicrobial treatments, has gained in interest. The aim of the present study is to evaluate the effects of Pediococcus acidilactici (PA) and Saccharomyces cerevisiae boulardii (SCB), on ETEC F4 colonization and attachment, accumulation of intestinal fluid and cytokines expression in weaned pig’s ileum. At birth, different litters of pigs were allocated to the following treatments: PA, SCB, PA + SCB, control (CTRL) and control with antibiotics (ATB). Probiotics (1 × 109 CFU) were administered daily to probiotics group during the lactation period and after weaning. One week after weaning, at 28 days of age, all F4-receptor-positive pigs were orally challenged with an ETEC F4 strain. Pigs were slaughter 24 hours later (day 29) and different intestinal samples were collected. In pigs treated with PA or SCB, the attachment of ETEC F4 to the ileal mucosa was significantly reduced in comparison with the ATB group. Finally, intestinal cytokines were upregulated in PA + SCB group in comparison with the CTRL group. In conclusion, these results suggest that administration of probiotics could be an alternative to attenuate ETEC F4 infection in pigs.

PhD (Microbiology) / Department of Microbiology / Diarrhoea is still a major problem worldwide. Enteric pathogens such as Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC) and Enterotoxigenic E. coli (ETEC) have been reported to cause diarrhoea in children under the age of 5 years. The incidences of these pathogens are due to factors such as poor water quality, sanitation and hygiene practices. Infections with these pathogens result in diarrhoea and have been reported to result in severe disease outcomes more especially in children under 2 years of age. EPEC infections have been well studied using in vitro analyses, with studies highlighting the adherence traits, proteins and virulence genes involved in pathogenesis and inflammatory responses. EPEC is characterized by localized adherence with microcolony formation at the site of infection. In vivo studies have reported on human EPEC infection. However, the current animal models have not been able to replicate clinical outcomes (such as diarrhoea and weigh loss) of EPEC infection similar to humans. Therefore, there is still a need for a suitable small animal model that mimic clinical outcomes of human EPEC infections in vivo. Children living in poor environmental conditions are more susceptible to diarrhoeal pathogens. Furthermore, the incidences of children being exposed to co-infections (more than one pathogen at the same time) is relatively high. The EAEC/EPEC (A/P) and EPEC/ETEC (P/T) co-infections have been increasingly detected in children with and without diarrhoea. It has been suggested that patients infected with these co-infections might result in severe disease outcome than those infected with single pathogens. Pathogens are constantly evolving and the microbe-microbe interaction in the host can result in these pathogens competing for the same niche and thus result in increased virulence. Interaction of co-infections can lead to increased inflammatory responses thus affecting the infected host. The first objective of this study was to develop an EPEC murine model using weaned C57BL/6 mice that have been pretreated with antibiotic cocktail. Mice were orally infected with wild-type (WT) typical EPEC, bfp- and escN mutant strains. The WT had transient weight loss and wet stools with mucous; and the bfp- infected mice also had transient weight loss and bloody stool appearance. Increase in inflammatory biomarkers MPO, LCN-2, CRP, IL-6 and SAA were observed in the WT and bfp- infected mice. The mice infected with escN mutant did not exhibit any weight changes and the stools were similar to the uninfected mice. Furthermore, no inflammatory biomarkers were observed in mice infected with the escN mutant. Metabolic perturbations were observed in WT EPEC infected mice at day 3 post infection with the TCA cycle metabolites (reduced succinate, citrate, fumarate, cis-aconitate) being excreted at lower quantities indicating that the energy production in the infected mice was greatly affected. The second objective of this study was to determine the interaction between the P/T coinfections using in vitro and in vivo analyses. In vitro, human colorectal tumour 8 (HCT-8) cells were infected with single strains of ETEC, EPEC and both the pathogens and incubated for 3 hours. After infection the cells were analysed for bacterial adherence using real-time PCR. The single strains adhered at the same rate similar to the P/T coinfected cells. IL-8, as a marker of inflammatory response, was measured using ELISA. The results indicated that the P/T co-infected cells had a significant increase in IL-8 response higher than the single infections. The P/T co-infections were further analysed in vivo using the EPEC murine model developed in this study. Interestingly, mice infected with P/T co-infections developed severe diarrhoea accompanied with significant increased weight loss and some mice died during the 3-day infection period. The inflammatory responses MPO, LCN-2 and SAA were higher in the co-infected mice indicating a synergistic effect. The bfp and eltA virulence genes were significantly increased in the P/T co-infections. The third objective of this study was to determine the interaction between A/P coinfections using in vitro and in vivo analyses. HeLa cells and HCT-8 cells were infected with EAEC, EPEC and both the pathogens at the same time in order to determine adherence and inflammatory responses. EAEC adherence was higher than EPEC and A/P co-infections adherence. A/P co-infections did not have increased IL-8 response in HCT-8 cells when compared to EAEC alone. The virulence genes involved in EPEC adherence and Type 3 Secretion System (bfp, eae, tir, ler, per, espB and espA) were significantly reduced in A/P co-infected cells. An interesting adherence trait was observed between the A/P co-infections in HeLA cells, EAEC was found to adhere around EPEC altering the localized adherence pattern. The A/P co-infections were further analysed using the EPEC murine model developed in this study. The A/P infected mice had diminished weight changes and EAEC shedding was enhanced when EPEC was present. Faecal inflammatory biomarkers MPO and LCN-2 in A/P infected mice did not have any additive effect. The findings of this study contributed significantly to the knowledge of human EPEC infection in weaned C57BL/6 mice, highlighting clinical outcomes, inflammatory responses and metabolic perturbations. Furthermore, this study also highlighted the interaction of P/T and A/P co-infections using in vitro and in vivo analyses in order to determine the disease severity and outcomes. It was observed in this study that coinfections can result in either synergistic or antagonistic effects. Further studies are therefore, required in order to understand the underlying mechanisms that are involved during co-infections and this can further assist in the development of therapeutic interventions. / NRF

Clostridium perfringens entérotoxinogène figure parmi les principales causes de toxi-infection alimentaire dans le monde. L’utilisation d’une approche par culture bactérienne classique pour isoler C. perfringens entérotoxinogène dans la viande de volailles est courante. Cependant, cette méthode d’isolement de ce microorganisme s’appuie sur le phénotype d’une double hémolyse attribuable, alors que peu des souches entérotoxinogènes de C. perfringens n’en produisent. Cet aspect complique ainsi l’étude des réservoirs de cet important pathogène. Les objectifs de cette étude étaient de valider la capacité d’une sonde marquée à la digoxigénine à détecter le gène cpe codant pour l’entérotoxine de C. perfringens et de valider l’utilisation d'une approche d'isolement combinée à l'hybridation sur colonie pour détecter C. perfringens entérotoxinogène. Des échantillons de liquides de rinçage de carcasses de poulets de chair ont été utilisés pour la réalisation de la présente étude. L’ADN et colonies lysées de souches entérotoxingènes de C. perfringens, et des échantillons de liquides de rinçage de carcasses de poulets de chair ont été soumis à la méthode d'hybridation sur colonie afin d'identifier la présence du gène cpe de C. perfringens. Les résultats de cette étude ont montré que la sonde d’ADN synthétisée est spécifique et sensible, permettant ainsi la détection du gène cpe de C. perfringens à partir de séquences d’ADN et de colonies lysées d’une souche contrôle de C. perfringens positifs au gène cpe, mais aussi à partir de colonies isolées des échantillons de liquides de rinçage de carcasses de poulets de chair contaminés artificiellement par C. perfringens entérotoxinogène. Notre étude suggère que cette méthode d’isolement combinée à l’hybridation sur colonie permet de récupérer C. perfringens entérotoxinogène à partir d’échantillons de liquides de rinçage de carcasses de poulets de chair. / Enterotoxigenic Clostridium perfringens is one of the main causes of foodborne illness worldwide. The use of a conventional bacterial culture approach to isolate enterotoxigenic C. perfringens from poultry meat is common. However, this method relies on the phenotype of attributable double hemolysis, whereas few enterotoxigenic strains of C. perfringens produce it. This aspect complicates the study of the reservoirs of this important pathogen. The objectives of this study were to validate the ability of a digoxigenin-labeled probe to detect the cpe gene encoding C. perfringens enterotoxin and to validate the use of an isolation approach combined with hybridization on colony to detect enterotoxigenic C. perfringens. DNA and pure colonies of enterotoxigenic strains of C. perfringens, and samples of broiler carcass rinses were subjected to the colony hybridization method to identify the presence of the cpe gene encoding the C. perfringens enterotoxin. The results of the present study revealed that the synthesized DNA probe is sensitive, thus allowing the detection of the cpe gene of C. perfringens from DNA sequences and from lysed colonies of a control strain of C. perfringens positive for the cpe gene. The probe also hybridized to the DNA of lyzed colonies isolated from carcass rinsates artificially contaminated with cpe-positive C. perfringens. Our study suggests that this isolation method combined with colony hybridization allows the recovery of enterotoxigenic C. perfringens from broiler carcass rinse fluid samples.