Heterologous expression and characterisation of a keratinase produced by Chryseobacterium carnipullorum.

Chryseobacterium carnipullorum 9_R23581T, isolated from raw chicken meat, was evaluated for its potential to degrade keratin found in feathers. The focus of this study was to heterologously express and characterise a keratinolytic enzyme produced by C. carnipullorum. Chryseobacterium carnipullorum secretes proteolytic enzymes that have feather degrading capabilities during its exponential growth phase. This study concluded that the most likely main component of the keratinolytic enzymes of C. carnipullorum was peptidase M64, a serine-endopeptidase with a molecular weight in crude form of 49.46 kDa. Primers were designed on the selected gene of interest, which was amplified from the genome of C. carnipullorum (accession number NZ-FRCD01000002.1). The gene coding for peptidase M64 was further cloned, propagated and expressed in E. coli BL21 [DE3] cells. Purification was by Immobilised Metal Affinity Chromatography (IMAC). The molecular weight of the keratinase was about 50 kDa after purification while its optimum temperature and pH were 50 °C and 8.5, respectively. The activity of this keratinase was inhibited by phenylmethylsulfonyl fluoride (PMSF) and it was enhanced by the presence of divalent metal ions such as Mg2+ and Ca2+. Enzyme activity was further assayed by application to chicken feathers and observed degradation was an indication of keratinolytic potential.

Bacterial resistance to Quaternary Ammonium Compounds (QAC) disinfectants.

Control of bacterial diseases has, for many years, been dependent on the use of antibiotics. Due to the high levels of efficacy of antibiotics in the past other disease control options have, to a large extent, been neglected. Mankind is now facing an increasing problem with antibiotic resistance. In an effort to retain some antibiotics for human use, there are moves afoot to limit or even ban the use of antibiotics in animal production. The use of antibiotics as growth promoters have been banned in the European Union and the USA. The potential ban on the use of antibiotics to treat diseases in production animals creates a dilemma for man-suffer significant problem with bacterial infection or suffer from a severe shortage of food! There are other options for the control of bacterial diseases. These include vaccine development, bacteriophage therapy, and improved biosecurity. Vaccine development against bacterial pathogens, particularly opportunistic pathogens, is often very challenging, as in many cases the molecular basis of the virulence is not always clearly understood. This is particularly true for Escherichia coli. Biosecurity (disinfection) has been a highly neglected area in disease control. With the ever-increasing problems with antibiotic resistance-the focus should return to improvements in biosecurity. As with antibiotics, bacteria also have mechanisms for resistance to disinfectants. To ensure that we do not replace one set of problems (increasing antibiotic resistance) with another (increasing resistance to disinfectants) we need to fully understand the modes of action of disinfectants and how the bacteria develop resistance to these disinfectants. Molecular studies have been undertaken to relate the presence of QAC resistance genes in bacteria to their levels of sensitivity to different generations of QAC-based products. The mode of action of QAC on bacteria has been studied using NanoSAM technology, where it was revealed that the QAC causes disruption of the bacterial cell wall and leaking of the cytoplasm out of the cells. Our main focus is on the control of bacterial and viral diseases in the poultry industry in a post-antibiotic era, but the principles remain similar for disease control in any veterinary field as well as in human medicine.

Bacteriophages as potential treatment option for antibiotic resistant bacteria.

The world is facing an ever-increasing problem with antibiotic resistant bacteria and we are rapidly heading for a post-antibiotic era. There is an urgent need to investigate alterative treatment options while there are still a few antibiotics left. Bacteriophages are viruses that specifically target bacteria. Before the development of antibiotics, some efforts were made to use bacteriophages as a treatment option, but most of this research stopped soon after the discovery of antibiotics. There are two different replication options which bacteriophages employ. These are the lytic and lysogenic life cycles. Both these life cycles have potential as treatment options. There are various advantages and disadvantages to the use of bacteriophages as treatment options. The main advantage is the specificity of bacteriophages and treatments can be designed to specifically target pathogenic bacteria while not negatively affecting the normal microbiota. There are various advantages to this. However, the high level of specificity also creates potential problems, the main being the requirement of highly specific diagnostic procedures. Another potential problem with phage therapy includes the development of immunity and limitations with the registration of phage therapy options. The latter is driving research toward the expression of phage genes which break the bacterial cell wall, which could then be used as a treatment option. Various aspects of phage therapy have been investigated in studies undertaken by our research group. We have investigated specificity of phages to various avian pathogenic E. coli isolates. Furthermore, the exciting NanoSAM technology has been employed to investigate bacteriophage replication and aspects of this will be discussed.

A community approach for pathogens and their arthropod vectors (ticks and fleas) in cats of sub-Saharan Africa.

BACKGROUND: Arthropod-borne pathogens and their vectors are present throughout Africa. They have been well studied in livestock of sub-Saharan Africa, but poorly studied in companion animals. Given their socioeconomic importance, the African Small Companion Animal Network (AFSCAN), as part of the WSAVA Foundation, initiated a standardized multi-country surveillance study. METHODS: In six countries (Ghana, Kenya, Nigeria, Tanzania, Uganda, and Namibia) in both rural and urban settings, 160 infested cats were sampled to assess their ectoparasite community (ticks and fleas), as well as the micro-parasite prevalence within those ectoparasites (60 and 118 pools of ticks and fleas, respectively) and blood (276 cats, including 116 non-infested). RESULTS: Almost two thirds of all infested cats originated from Tanzania and Kenya. Despite the large macro-geographical variation, no consistent difference was found in ectoparasite diversity and numbers between East and West Africa. Far more flea-infested than tick-infested cats were found. The most dominant ectoparasite was Ctenocephalides felis. Among the ticks, the exophilic Haemaphysalis spp. were the commonest, including species that are not typically linked with companion animals (Haemaphysalis spinulosa and Haemaphysalis elliptica). The most prevalent pathogens found in the blood and fleas were Bartonella henselae and Mycoplasma haemofelis. In the ticks, the dog-associated Hepatozoon canis was most commonly found. A high degree of co-parasitism was found in all countries and habitats. CONCLUSIONS: Our continent-wide standardized field study highlights the cat's potential to serve as a reservoir of pathogens that can be transmitted to humans or livestock, especially when cats are expected to become more commonly kept in African villages and towns.

Regulation of outer-membrane proteins (OMPs) A and F, during hlyF-induced outer-membrane vesicle (OMV) biosynthesis.

BACKGROUND: Gram-negative bacteria actively secrete outer membrane vesicles into the surrounding environment and these vesicles have been shown to play various physiological and protective roles such as carrying antibiotic-degrading enzymes and acting as decoys against host defences, therefore promoting the pathogenesis of the bacterium. It has been shown that avian pathogenic Escherichia coli species can increase vesicle biosynthesis through the acquisition of the hlyF gene but the effect this has on the cell by scavenging outer-membrane associated proteins (OmpA, OmpF) into the vesicles during vesicle release have not yet been investigated. RESULTS: Relative quantitative real-time PCR data obtained from hlyF expressing and non-expressing cells showed that during hlyF induction, ompF showed a nearly 2-fold down regulation relative to the non-expressing cells during the entire 24 hours, while ompA was expressed at the same level as the non-expressing cells during the first 8 hours of expression. At 24 hours post-hlyF expression, ompA was up-regulated 4-fold. CONCLUSIONS: The regulatory effects of the newly described outer-membrane vesicle biosynthesis-related gene, hlyF, on E. coli has not previously been investigated. As hlyF-induced vesicles contain OmpA and OmpF scavenged from the bacterial outer-membrane, potential regulatory effects on the host was investigated. An increase in ompA expression and an insignificant decrease in ompF expression was observed during hlyF induction demonstrating that hlyF-related biosynthesis is not related to decreased ompA expression, which is one of the potential mechanisms discussed in literature for biosynthesis. Outer-membrane vesicle biosynthesis during hlyF over-expression could potentially be accomplished through a different mechanism(s).