Proteomic and Molecular Analysis of Methicillin Resistance and Selected Toxigenic genes in Coagulase-negative Staphylococcus spp From Food and Animal Sources.

While most coagulase-negative Staphylococcus (CNS) are apathogenic, recent evidence suggests some food and animal derived CNS isolates may carry and express virulence factors including classical enterotoxins, toxic shock syndrome, and methicillin resistance genes. The present study was designed to assess the potential role of CNS in the epidemiology of foodborne illnesses and to determine the likelihood of food and domestic animals as transmission vehicles of methicillin resistance. Of the animal-derived food samples tested, 27.3% (39/143) were Staphylococcus-positive compared to only 9.5% (23/242) of the plant foods. A total of 92 Staphylococcus spp cultured from 385 food (62/92) and 30 diagnostic animal (30/92) samples were tested by the polymerase chain reaction (PCR) for classical enterotoxin (sea, seb, sec, sec/, and see), toxic shock syndrome toxin-1 (tsst-1), and mecA genes. All PCR-positive isolates were further tested by immunoblotting for production of the corresponding toxin. Susceptibility patterns of both CNS and coagulase-positive Staphylococcus (CPS) were assessed for ~- lactam antimicrobial agents and the isolates analyzed for presence of the mecA gene. Of all study isolates, 20/92 (21.7%) were CPS and 72/92 (78.3%) CNS. Of the 20 CPS isolates, 15.4% (2/13) 5. aureus cultured from steak were sec positive while only 1/7 (14.3%) CPS (5. aureus) from a diagnostic feline sample was positive for both sec and tsst-1 genes. Both toxigenic 5. aureus isolates from steak and a diagnostic feline sample also expressed detectable amounts of SEC and TSST-1 toxins. On the other hand, 1.4% (1/49) of the CNS (5. lugdunensis) from strawberries was positive for the sec gene but negative for SEC toxin and 2/49 (2.8%) of the CNS (5. hominis) gave unexpected base pair PCR products with see primers. All CNS isolates were generally susceptible to test ~-lactam antimicrobial agents; 80% of the CPS isolates were resistant to penicillin and amoxicillin of which 1/20 (5%) were positive for the mecA gene. Based on this data, 3/20 (15%) of CPS and 3/72 (4.2%) of CNS isolates were positive for toxigenic genes thus underscoring the potential role food-derived CNS isolates may have in the epidemiology of foodborne illnesses. Although only 5% of the CPS and none of the study CNS isolates expressed mecA gene, 80% of the CPS were resistant to penicillin, suggesting other mechanisms of drug resistance. The presence of mecApositive CPS and toxin producing Staphylococcus isolates underscore the public health significance of organisms from this genus

Experimental evaluation of inactivated and live attenuated vaccines against Mycoplasma mycoides subsp. mycoides

AbstractThe current control method for contagious bovine pleuropneumonia (CBPP) in Africa is vaccination with a live, attenuated strain of Mycoplasma mycoides subsp. mycoides (Mmm). However, this method is not very efficient and often causes serious adverse reactions. Several studies have attempted to induce protection using inactivated mycoplasma, but with widely contradictory results. Therefore, we compared the protective capacity of the live T1/44 vaccine with two inactivated preparations of Mmm strain Afadé, inoculated with an adjuvant. Protection was measured after a challenge with Afadé. The protection levels were 31%, 80.8% and 74.1% for the formalin-inactivated, heat-inactivated and live attenuated preparations, respectively. These findings indicate that low doses of heat-inactivated Mmm can offer protection to a level similar to the current live attenuated (T1/44) vaccine formulation

Contagious bovine pleuropneumonia: identification of candidate vaccine molecules through inhibition of pathogen-host cell interaction with monoclonal antibodies

Contagious bovine Pleuropneumonia (CBPP) is a severe respiratory disease of cattle that causes economic loses of up to 40 million Euros annually in sub-Saharan Africa. The preferred method of control is vaccination however the currently available vaccine confers insufficient and short term immunity hence there is need to develop a better vaccine. The disease is caused by Mycoplasma mycoides subsp. mycoides (Mmm) a pathogen that belongs to a group of closely related pathogens, the M. mycoides cluster. Mmm only causes pneumonia in cattle and related species but not in sheep or goats, suggesting that there must be a specific interaction between bovine lung cells and Mmm. Calves younger than six months on the other hand do not develop pneumonia but rather arthritis and associated lameness. The aim of thesis is to assess the adherence of Mmm to host cells and to further identifiy Mmm specific monoclonal antibodies that block this binding. This is the first study to describe the adhesion process for Mmm. Our hypothesis is that prevention of binding between Mmm and bovine lung epithelial cells in vivo will prevent severe disease. The identification of the Mycoplasma adhesin(s) that mediate this interaction could be candidate vaccine targets. The specific objectives of this study are to assess the manifestation of CBPP in 7 month old calves and to compare in- contact and intubation methods of infection, establish an indirect flow cytometry assay to measure Mycoplasma adherence to different host cells and produce a panel of Mmm- specific monoclonal antibodies, and select for mAbs that inhibit Mmm adherence to bovine lung epithelial cells; select carbohydrate substances with different functional groups and test their ability to inhibit Mmm cyto-adherence. The antigens to antibodies that inhibit adherence could be potential vaccine candidatesAcknowledgements........................................ i List of abbreviations................................... ii Summary................................................. iv Samenvatting............................................ vi Chapter 1: Literature review on Mycoplasma mycoides subsp. mycoides................................................ 1 Aim and objectives of the thesis........................ 17 Chapter 2: Manifestation of contagious bovine pleuropneumonia in 7- month-old calves infected via bronchoscope............................................ 19 Chapter 3: Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells................. 29 Chapter 4: Characterization of Mmm cyto-adherence through inhibition by specific antibodies and carbohydrate substances.............................................. 45 Chapter 5:Conclusions and perspectives.................. 63 References.............................................. 68 Racheal Aye CV.......................................... 84nrpages: 83status: publishe

Manifestation of contagious bovine pleuropneumonia in seven month-old calves infected via bronchoscope and in contact transmission

Mycoplasma mycoides subsp. mycoides (Mmm) causes contagious  bovine pleuropneumonia (CBPP) in adult cattle however calves younger than 6 months develop mostly arthritis and associated lameness, but very few lesions in the lungs, suggesting that Mycoplasma does not easily colonize lungs of young calves. The aim of this study was to assess effects of CBPP in 7-month-old calves following infection by in-contact transmission; and manifestation in adult cattle and calves infected via intubation method. Both adult and seven month old calves developed respiratory indications of CBPP. Although none of the adult cattle developed arthritis, 7/10 and all 5 calves infected by intubation and in-contact transmission methods respectively developed arthritis. The results show that 7-month-old calves, whether infected by intubation or in-contact method develop respiratory signs similar to adult cattle as well as arthritis as seen in calves younger than six months. This suggests that at seven months, calves have developed susceptibility to lung lesions but still have a propensity for damage in the joints like very young calves.Key words: Contagious bovine pleuropneumonia, Mycoplasma mycoides subsp. mycoides, Calves, intubation, in-contact transmission Manifestation de la pleuropneumonie contagieuse bovine chez des veaux de sept mois infectes avec un bronchoscope et par contactMycoplasma mycoides sous-espèce mycoides (Mmm) est responsable de la pleuropneumonie contagieuse bovine (PPCB) chez les bovins adultes, mais les veaux de moins de 6 mois développent essentiellement de l’arthrite et de la boiterie associée, mais très peu de lésions pulmonaires, ce qui porte à croire que Mycoplasma ne colonise pas facilement les poumons des jeunes veaux. Le but de cette étude était d’évaluer les effets de la PPCB chez les veaux de 7 mois infectés par contact, et les manifestations chez les bovins adultes et les veaux infectés par intubation. Les veaux adultes et ceux de sept mois ont développé des signes respiratoires de PPCB. Bien qu’aucun des bovins adultes n’ait développé de l’arthrite, 7/10 et tous les 5 veaux infectés par intubation et transmission par contact ont respectivement développé de l’arthrite. Les résultats montrent que les veaux de 7 mois, qu’ils soient infectés par intubation ou par contact, développent des signes respiratoires similaires à ceux des bovins adultes, tels que l’arthrite chez les veaux de moins de six mois. Ceci donne à penser qu’à sept mois les veaux ont développé une susceptibilité aux lésions pulmonaires, mais ont toujours une propension d’endommagement des  articulations comme les très jeunes veaux.Mots-clés : pleuropneumonie contagieuse bovine, Mycoplasma mycoides sous-espèce mycoides, veaux, intubation, transmission par contact

Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells.

BACKGROUND Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains. RESULTS There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor. CONCLUSIONS Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP

Malaria exposure drives both cognate and bystander human B cells to adopt an atypical phenotype

Atypical memory B cells (aMBCs) are found in elevated numbers in individuals exposed to malaria. A key question is whether malaria induces aMBCs as a result of exposure to Ag, or non-Ag-specific mechanisms. We identified Plasmodium and bystander tetanus toxoid (TT) specific B cells in individuals from areas of previous and persistent exposure to malaria using tetramers. Malaria-specific B cells were more likely to be aMBCs than TT-specific B cells. However, TT-specific B cells from individuals with continuous exposure to malaria were more likely to be aMBCs than TT-specific B cells in individuals from areas where transmission has ceased. Finally, sequences of BCRs specific for a blood stage malaria-Ag were more highly mutated than sequences from TT-specific BCRs and under strong negative selection, indicative of ongoing antigenic pressure. Our data suggest both persistent Ag exposure and the inflammatory environment shape the B-cell response to malaria and bystander Ags