Isolation and characterisation of bacteriophages and their potential use for the control of bacterial infectons in poultry
| dc.contributor.advisor | Bragg, R. R. | |
| dc.contributor.author | Kakoma, Kasweka | |
| dc.date.accessioned | 2015-08-20T12:41:52Z | |
| dc.date.available | 2015-08-20T12:41:52Z | |
| dc.date.issued | 2009-05-29 | |
| dc.description.abstract | English: Avian pathogenic Escherichia coli (APEC), the causative agent of colibacillosis belong to the family enterobacteriaceae. The disease is manifested as localised or systemic infections which include peritonitis, airsacculitis, omphalitis, swollen head syndrome and colisepticaemia in poultry. Colibacillosis results in increased mortality, condemnations of carcasses at slaughter, reduced feed conversion and production due to morbidity and increased cost of treatment. The standard method for the control of colibacillosis has been the use of antibiotics either as a treatment or as prophylaxis through the addition of antibiotics into the animal feed at sub-minimal levels. The efficacy of antibiotics has drastically reduced over the years due to the emergence and increased prevalence of antibiotic resistance. The use of some antibiotics and antibiotic growth promoters such as fluoroquinolones has been banned and further bans are still impending. Therefore, colibacillosis can potentially cripple the poultry industry nationally and worldwide, if traditional treatment options are ineffective. There is a great demand for potential alternatives to the use of antibiotics and lytic bacteriophages offer attractive advantages over antibiotics in this regard. This study focused on the isolation and characterisation of lytic bacteriophages with the aim of determining their potential to control E. coli infections, in poultry. Nine bacteriophages were successfully isolated from sewage samples and five from chicken faecal matter. The lytic patterns of the phages against E. coli K12 and E. coli strains isolated from diseased poultry (presumed to be pathogenic) were determined. Among the phages isolated from sewage samples, 31-5, E13/2, E13/5 and BS4 lysed four or more of the E. coli strains. The widest host strain range was exhibited by E13/2 which infected 8 of the 11 strains. Thus, this isolate may hold the greatest potential for therapy. In contrast, isolates from chicken faecal matter had a very narrow host strain range, lysing a maximum of two E. coli strains including the original host. An interesting observation was made in that the laboratory strain; E. coli K12 was resistant to lysis by four out of the five isolates (from chicken faecal matter). This is an important factor to consider in any potential form of treatment where only the pathogenic strains are targeted while the normal microbiota should remain unaffected. The varied lytic patterns observed for most of the isolates further demonstrate the importance of employing phage cocktails rather than single preparations to treat or prevent colibacillosis. Additionally, some of the isolates may prove useful in phage typing due to their high discriminatory nature in lysis. Although much has been reported on APEC-specific coliphages isolated from sewage, this is one of a few studies where they have been successfully isolated from chicken faecal matter. Examination of morphological characteristics by transmission electron microscopy revealed the presence of a majority of tailed phages which are members of the Myoviridae and Siphoviridae families. For some viruses definite conclusions could not be made concerning their identity and they were therefore only tentatively placed into certain families. Molecular analyses included polymerase chain reaction (PCR) using primers specific for the g23 sequence of T4-type phages and the DNA polymerase of T7-like podophages. Although the identity of every phage isolate could not be inferred, the relatedness between isolates SK4 and SK5 was established using the PCR technique of random amplified polymorphic DNA (RAPD). Phylogenetic investigations revealed that 31-5 and SK5 were closely related followed by E13/5 and SK4. In conclusion, this study demonstrated that some of the isolated phages have the potential to prevent, eliminate or reduce APEC in poultry. Further research is however, necessary to fully characterise the phage isolates and perform efficacy tests in vivo. | en_ZA |
| dc.description.abstract | Afrikaans: Voël potogeniese Escherichia coli (VPEC), wat die siekte colibacillosis veroorsaak, behoort in die familie Entrobacteriaceae. Die siekte, wat in pluimvee voorkom, manifesteer as ʼn lokale of sistematiese infeksie wat peritonitis, airsacculitis, omphilais, geswelde kop sindroom en colisepicaemia insluit. Colibacillosis ly tot verhoogde mortaliteit, afkeuring van karkasse tydens slagting, verlaagde voer omskakeling en produksie as gevolg van morbiditeit en die verhoogde koste van behandeling. Die standaard metode vir die beheer van colibacillosis is die gebruik van antibiotika beide as behandeling of as profilakse deur die toevoeging van subminimal vlakke van antibiotika in die voer. Die effektiwiteit van antibiotika het in die afgelope jare drasties afgeneem as gevolg van teenwoordigheid van antibiotika weerstandige bakterieë. Die gebruik van sommige antibiotika en antibiotiese groei stimuleerders soos fluoroquinolones is al verban en nog verdere verbanning van ander antibiotika word verwag. Dus kan colibacillosis die pluimvee industrie kniehalter op buite nasionale as internasionale vlak as die tradisionele behandeling onefektief is. Daar is ʼn groot aanvraag na alternatiewe behandelings in die plek van antibiotika en litiese bakteriofae bied ʼn baie aantreklike voordeel bo antibiotika. Hierdie studie fokus op die isolasie en karakterisering van litiese bakteriofae met die potensiaal om E. coli infeksies in pluimvee te beheer. Nege bakterifage was suksesvol geïsoleer uit riool en vyf vanaf hoender fekale materiaal. Die litiese patrone van die fage teenoor E. coli K12 en E. coli geïsoleer vanuit siek pluimvee (aangeneem as patogeniese E. coli) was bepaal. Die fage wat uit riool geïsoleer was, 31-5, E12/2, E12/5 en BS4 het vier en meer van die E. coli stamme liseer. Die E12/2 het die grootste gasheer reeks gehad wat 8 van die 11 stamme infekteer het. Hierdie isolaat toon die grootste potensiaal vir terapeutiese gebruik. In teenstelling hiermee het die isolate vanaf hoender fekale materiaal ‗n nouer gasheer reeks wat ‗n maksimum van twee E. coli stamme, insluitend die oorspronklike gasheer, liseer. ‗n Baie interessante observasie was geneem met die laboratorium stam, E. coli K12, wat bestand was teen lise van vier van die vyf isolate (hoender fekale materiaal). Dit is ‗n belangrike faktor wat in ag geneem moet word wanneer gekyk word na behandeling van slegs die patogeniese stamme waar die normale mikro-organisme moet ongeaffekteer bly. Die verskeidenheid litiese patrone gesien met die meeste van die isolate demonstreer verder die noodsaaklikheid van die gebruik van verskillende fage as behandeling in stede van ‗n enkele faag om colibacillosis te voorkom. Sommige van die fage kan ook gebruik word tydens faag tipering as gevolg van hul wye verskeidenheid in litiese patrone. Alhoewel daar al baie inligting geraporteer was op VPEC-spesifieke colifage geïsoleer uit riool, is hierdie die eerste studie waar fage suksesvol geïsoleer was uit hoender fekale materiaal. Tydens morfologiese karakterisering met behulp van transmissie elektron mikroskopie, is die teenwoordigheid van fage met sterte meestal gevind wat deel van die Myoviridae en die Siphoviridae families uitmaak. Vir sommige van die virusse kon daar nie ʼn definitiewe gevolgtrek geneem word inverband met hul identiteit en was gevolglik net tentatief geplaas in sekere families. Die molekulêre analise sluit in die polimirase ketting reaksie (PKR) waar primers spesifiek vir die g23 volgorde van die T4-tipe fage en die DNS polimerase van T7- tipe podofage gebruik was. Alhoewel die identiteit van al die fage wat geïsoleer was nie toegeken kon word nie, kon die verwantskap tussen die isolate SK4 en SK5 wel deur die gebruik van PKR tegniek, lukraak vermeerdering polimorfiese DNS (RAPD), bepaal word. Filogenetiese ondersoek het aangedui dat 31-5 en SK5 naby verwant is, gevolg deur E13/5 en SK4. In opsomming, die studie demonstreer dat sommige van die geïsoleerde fage het die potensiaal om VPEC in pluimvee te voorkom, elimineer en te verminder. Verdere ondersoek is egter nodig om die fage verder te karakteriseer en om effektiwiteit toetse in vivo te doen. | |
| dc.identifier.uri | http://hdl.handle.net/11660/992 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Bacteriophages -- Therapeutic use | en_ZA |
| dc.subject | Poultry -- Diseases -- Treatment | en_ZA |
| dc.subject | Bacterial diseases | en_ZA |
| dc.subject | Dissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2009 | en_ZA |
| dc.title | Isolation and characterisation of bacteriophages and their potential use for the control of bacterial infectons in poultry | en_ZA |
| dc.type | Dissertation | en_ZA |