Masters Degrees (Microbial, Biochemical and Food Biotechnology)
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Browsing Masters Degrees (Microbial, Biochemical and Food Biotechnology) by Author "Bragg, R. R."
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Item Open Access Antibody fragments as a possible therapeutic treatment for infectious bronchitis in poultry(University of the Free State, 2018-09) Coetzee, Janetta Magrieta; Bragg, R. R.; Boucher, C. E.; Van der Westhuizen, W. A.Infectious bronchitis virus (IBV), a coronavirus, is the etiological agent for infectious bronchitis (IB), an acute respiratory disease of poultry. Infectious bronchitis is a notifiable disease, and taking in consideration that poultry is the second most consumed meat within South Africa, it highlights the importance of monitoring IBV outbreaks. Recombinant single chain variable fragments (scFv) have been used in therapeutic treatments for various human and veterinarian viruses, including other coronaviruses, such as SARS-CoV and MERS-CoV. The study aims to select scFv against the IBV antigen, with the use of phage display technology and commercial ELISA plates coated with the most prevalent IBV strains namely, H120 and M41. It has been proven that the S1 protein induces the binding of neutralising antibodies which provides protection against lethal CoV infections, thus, indicating a possible application for a therapeutic treatment. In this study, phage clones were selected from a human domain (dAb) library (Source BioScience, Australia). Panning was repeated three times using commercial ELISA plates coated with M41 and H120 IBV strains. Positive monoclonal phage clones were retrieved from the polyclonal mix by a sandwich ELISA and sequenced. The selected scFvs were expressed by Isopropyl β-D-1 thiogalactopyranoside induction and purified by immunoprecipitation with the Pierce Anti-c-Myc Agarose kit (Thermo Scientific, USA). After purification, binding ability of the scFvs were determined by means of a direct competitive ELISA. The neutralising ability of the scFvs was then determined by a virus neutralisation assay in ovo with the Avipro IBV H120 strain (Lohmann Animal Health Gmbh, Germany). This was performed in 9-day old SPF eggs over a time-period of six days. One set of eggs were injected with a dilution range of the IBV H120 strain and another set by a mixture of 2 μg/ml scFv with the IBV H120 strain. The end-point titres were determined and compared by the Spearman-Karber method (Spearman, 1908). Statistical analysis was performed using the student t-test with a p-value of 0.05. Round 1 of panning resulted in a total of 1.2 x 106 phages/ml and after round 3 a total of 3.0 x 1010 phages/ml were obtained. A total of 96 phage clones were manually selected from which only 12.5% showed a positive result during the sandwich ELISA. The 12 positive clones were sequenced and analysed based on nucleotide and amino acid composition. A total of five scFvs contained a complete variable heavy (VH) chain sequence, two of which was identical. This resulted in four unique and complete scFv sequences. These sequences showed a high variance in the nucleotide composition through- out the sequence. However, variation in the amino acid composition was only observed in the third complementary determining region. The scFvs were expressed and resulted in concentrations ranging from 204.38 μg/ml to 265.07 μg/ml. Detection of IBV antigen binding ability of the purified scFvs was conducted by a direct competitive ELISA. However, no statistical difference in absorbance values were observed, indicating insufficient binding of the scFvs. The in ovo virus neutralisation assay resulted in a one log reduction of the end-point titres. Statistical analysis proved one of the reductions to be statistically significant with a p- value less than 0.05, resulting in a partial neutralisation effect from the scFv. In conclusion, the selection process showed a progressive enrichment of antigen specific clones from the dAb library. The scFvs were successfully expressed, purified and characterised in terms of binding ability.Item Open Access Characterization of the putative haemagglutinin in haemophilus paragallinarum(University of the Free State, 2001-12) Barnard, Tobias George; Van Heerden, E.; Albertyn, J.; Bragg, R. R.Haemophilus paragallinarum , the causative agent of infectious coryza (IC), an acute respiratory disease in chickens and fowl, was first isolated in 1931 by De Blieck (1932). The first serious, documented outbreak in South Africa occurred in 1968 (Buys, 1982) on a multi-age layer-farm, soon the bacterium spread to most large production sites and established itself as the most common bacterial infection in layers (Bragg, 1995). The disease has a low mortality rate but leads to a drop in egg production of up to 40 % in layer hens and increased culling in broilers and thus poses significant financial liability to chicken farmers (Arzay, 1987; Bragg, 1995). One of the reasons for the success of survival for this bacterium is that after recovering from infection, birds become carriers of the bacterium, therefore aiding the spread of H. paragallinarum (De Blieck, 1948). Secondly, the bacterial strain belongs to one of nine serovars, which makes combating the spread of the disease through inactivated vaccination ineffective especially due to low cross protection among these serovars. (Rimler et al., 1977; Kume et al., 1980a). Various potential factors have been identified as potential virulence factors, e.g. the haemagglutinin protein. This protein plays a crucial role in adherence of the bacteria to the host's cells and is considered a possible virulence factor (Sawata et al., 1982; Yamaguchi et al., 1989). Sawata and co-workers (1982) reported at least three different haemagglutinins from H. paragallinarum strain 221 with one, HA-L, being serovar specific with the other common types shared by the different serovars in one serogroup.It would therefore be important to understand the working and interaction of the various virulence factors of H. paragallinarum, especially the haemagglutinins, in order to combat this bacterium.Item Open Access Charactt[sic]erization of a plasmid conferring NAD independence in Haemophilus paragallinarum(University of the Free State, 2003-05) Van Zyl, Anna Elizabeth; Albertyn, J.; Bragg, R. R.; Van Heerden, E.English: Members of the family Pasteurellaceae are classified in part by whether or not they require NAD+ supplement for growth on laboratory media. It is known that this phenotype is determined by a plasmid whose presence allows NAD+-independent growth of Haemophilus paragallinarum. In this study, this 6-kb plasmid, which was previously shown to be responsible for NAD+ independent growth of H. paragallinarum on defined media, was isolated. Isolated plasmid DNA was shredded by sonification and subcloned into vector PGEM-T easy. The recombinant plasmid was transformed into E.coli the transformants isolated were sequenced. Sequence analysis revealed one open reading frame of 1119bp that is predicted to encode a protein with a molecular mass of 43kD. Compared with the sequence databases, this protein was found to have significant sequence homology to Quinolinate phosphoribosyltransferase of Bacillus anthracis this enzyme is responsible for the production of nicotinic acid mononucleotide (NAMN) from nicotinate and quinolinate. A 3284bp nucleotide fragment of the plasmid revealed four additional open reading frames. Proteins encoded on this fragment of the plasmid all have significant homology to proteins from H. influenzae of which all have functions related to production and immunity of the bacteriocin haemocim. This bacteriocin produced by most type b-encapsulated strains of H. influenzae, is toxic to virtually all non-type b strains of H. influenzae idependent of encapsulation. This bacteriocin is thought to inhibit DNA synthesis, of susceptible strains. Purification of this bacteriocin and testing its toxicity to other pathogens as a possible antimicrobial drug might form the bases of a future study. Previous work has indicated that plasmid bearing strains of H. paragallinarum are less virulent, thus creating the possibility that more virulent wild type strains can be transformed and used as live vaccines. The influence of transformation with this plasmid on other members of the family Pasteurellaceae and the possibility of creating live vaccines should be further investigated Since species of the genus Haemophilus cannot easily be transformed with plasmid, this naturally occurring plasmid could be modified to create a vector, which has specific application in the transformation of Haemophilus species.Item Open Access Cloning and characterization of the capsule transport gene region from Haemophilus paragallinarum(University of the Free State, 2001-11) De Smidt, Olga; Albertyn, J.; Bragg, R. R.; Van Heerden, E.English: Haemophilus paragallinarum causes an acute respiratory disease of chickens known as infectious coryza (IC), a disease first recognized as a distinct entity in the late 1920's. Since the disease proved to be infectious and primarily affected nasal passages, the name "infectious coryza" was adopted (Blackall, 1989). Infectious coryza may occur in both growing chickens and layers. The major economic effect of the disease is an increased culling rate in meat chickens and a reduction in egg production (10-40%) in laying and breeding hens. The disease is limited primarily to chickens and has no public health significance (Yamamoto, 1991). The most common clinical signs are a nasal discharge, conjunctivitis, and swelling of the sinuses and face. Various sulfonamides and antibiotics are useful in alleviating the severity and course of infectious coryza; however, none of the therapeutic agents has been found to be bactericidal. Relapse often occurs after treatment is discontinued, and the carrier state is not eliminated (Yamamoto, 1991). All the commercially available bacterins against IC, consist of inactivated broth cultures of a combination of two or three different serotypes. Although vaccines against IC have been used in South Afr ica since 1975, it became apparent in the 1980s that the vaccines were becoming less effective in controlling the disease (Bragg et al., 1996). This could have been due to the emergence of a previously unknown serovar, or even serogroup and the possibility of changes in the population dynamics. Vaccine efficiency is therefore a problem and an alternative to available vaccines is needed. Capsules have long been associated with virulence properties of bacteria. The role that the capsule play in the virulence of bacterial species related to H. paragallinarum has been investigated by several workers (Kroll et al., 1988; Inzana et al., 1993; Boyce and Adler, 2000). Mutation, deletion or allelic exchange of gene/s involved in the transport of capsule polysaccharides in related species like Haemophilus influenza, Actinobacillus pleuropneumoniae and Pasteurella multocida, resulted in organisms with reduced virulence. The noncapsulated mutants of Actinobacillus pleuropneumoniae reported by Inzana et al. (1993) showed extreme stability and induceda protective immune response without any symptoms of disease. This not only proves the capsule�s involvement in virulence of bacteria but also offers the opportunity to investigate the possibility of producing live vaccines. The aim of this study was an attempt to understand the genetic organization of the capsular genes of H. paragallinarum in comparison to related HAP organisms and the possibility of producing a mutant lacking the capsule. The goals were: 1. Isolation and cloning of the capsule transport gene locus. 2. Sequencing and characterization of the locus 3. Transplacement of a gene/s to produce a noncapsulated mutant of H. paragallinarum.Item Open Access Cloning of the XynA gene from Thermomyces lanuginosus and expression in Saccharomyces cerevisiae(University of the Free State, 2001-11) Nel, Sanet; Albertyn, J.; Van Heerden, E.; Bragg, R. R.English: The xylanase from Thermomyces lanuginosus (XynA) was cloned into two shuttle vectors, pRS416 (single copy vector) and pRS426 (multi-copy vector) adjacent to a PDC1 promoter (designated pRS416:XynA and pRS426:XynA). An expression cassette for this xylanase was constructed by cloning of the XynA gene into a modified a-agglutinin (Aga1) gene from Saccharomyces cerevisiae. This modification entailed the deletion of the binding domain coding region of the Aga1, and the cloning of the XynA gene into this deleted binding domain region, which is flanked by a stalk-like protein coding region. This fusion protein was cloned into two shuttle vectors (pRS416 and pRS426), flanking the PDC1 promoter (designated pRS416:Aga1::XynA and pRS426:Aga1::XynA). The aim of the cassette was to immobilize the expressed enzyme on the cell surface of the yeast cell with the expression of the xylanase on the stalk of the Aga1, however, extracellular secretion of the enzyme was obtained upon expression. Enzyme assays performed on pRS416:XynA and pRS426:XynA yielded very low activity [0.1505 U/ml (2.5088 nKat/ml) and 0.0909 U/ml (1.5153 nKat/ml) respectively], whereas pRS416:Aga1::XynA and pRS426:Aga1::XynA yielded activities of 1.7035 U/ml (28.3973 nKat/ml) and 1.7319 U/ml (28.8707 nKat/ml) respectively. The partial characterization of this extracellular secreted recombinant xylanase (pRS416:Aga1::XynA and pRS426:Aga1::XynA) yielded an optimum temperature of 70 °C and an optimum pH of 6.0-7.0. Thermal stability for the recombinant xylanase was determined for temperatures 50 °C, 60 °C and 70 °C, and the activation energy for pRS416:Aga1::XynA and pRS426:Aga1::XynA were calculated as 34.86 kJ/mol and 53.59 kJ/mol respectively.Item Open Access The development of a molecular serotyping system and an investigation into the presence of prophages in Avibacterium paragallinarum serogroups(University of the Free State, 2014-01) Coetsee, Elke; Bragg, R. R.; Boucher, C. E.Avibacterium paragallinarum is an avian pathogen that causes the upper respiratory disease Infectious coryza (IC) in chickens. This disease has the ability to cause vast economic losses due to a decrease in egg production. To date the factors contributing to pathogenicity, immunogenicity and serotyping are still not clearly understood. Vaccine failures are a major problem that occurs due to no or poor cross-protection occurring especially between the C-serovars of A. paragallinarum. This problem will be overcome by having a more accurate serotyping technique available for the diagnosis of IC. Therefore one of the aims of this study was the development of a molecular serotyping technique, where a serotyping PCR was developed which distinguished between the Modesto (C-2) and SA-3 (C-3) A. paragallinarum isolates which is the major cause of IC in South Africa. Reported in a recently published article was the presence of a HP2-like and Mu-like phage within the genome of the Modesto (C-2) strain of A. paragallinarum. Therefore another major question addressed during this study was whether there are prophage genes present in all of the reference isolates as well as in field isolates of A. paragallinarum and what the effect of these phages might be on the virulence and pathogenicity of these isolates. Phage genes that are important during lysogeny were selected and screened for by means of PCR. From the results it was able to determine that some of these genes are present in some of these isolates but no discernible patterns were detected in terms of the effect on pathogenicity. Therefore future studies will be conducted to mainly focus on the effect these phages might have on the virulence and pathogenicity as well as whether these phages are responsible for the occurrence of different A. paragallinarum serovars.Item Open Access An epidemiological survey of Newcastle disease virus in South Africa(University of the Free State, 2001-05) Mashope, Barbara Keitumetse; Albertyn, J.; Bragg, R. R.; Van Heerden, E.English:The primary objective in this study was the investigation of the molecular epidemiology of NDV strains isolated during epizooties in South Africa in 1998. Isolates purported to be NDV were collected from the Onderstepoort Veterinary Institute, the University of Pretoria, and EarlyBird Farms, Standerton. Four of the twenty-two isolates collected were identified as NDV, and successfully grouped into genotype VIIb, previously described by Herezeg et al., (1999). The isolates were propagated in 9-10 day-old embryonated SPF eggs. Embryo mortality was observed, allantoic fluid harvested, and genomic RNA extracted using TRIZOL ® reagent, containing guanidine thiocyanate. An RT-PCR based detection method was used to screen the isolates received (Ballagi- Pordány et al., 1996). The optimised method entailed initial reverse transcription in a reaction mixture containing IIlM random hexamers p(dN)6, 200 U Moloney Murine Leukemia (M-ML V) RT and 25 U RNasin. Aliquots of 5 ul, of the cDNA mix were used as template in subsequent PCR amplification reactions. A 1349bp region of the fusion protein gene was amplified using primers ONDVlaa and ONDV 4aa. Fusion protein amplicons were obtained from field isolates, M89/98 (Ost Pool I), M89/98 (Av Pool 2), M57/98, and M308/98 obtained from OP. Restriction enzyme profiles of the fusion protein amplicons using restriction enzymes HinfI, BstO I, and Rsa I displayed fragment patterns that allowed their grouping into genotype VIIb, described by Herezeg et al., (1999). Non-group specific bands observed upon digestion of the amplicon of strain M308/98 with Hinf I released fragments not consistent with those observed in genotype VIIb isolates. These fragments suggest the presence of mutations in this area of the genome. Amplification of a region comprising 88% of the matrix protein gene was facilitated using primers Ml and M2. These amplicons were subjected to RE analysis using restriction enzymes Mbt) I and Hinf I. Analysis of the restriction profiles produced revealed that these four strains were not re-isolated forms of the commonly used live vaccine LaSota strain. A region of the fusion protein gene encoding the fusion protein cleavage activation site was amplified by means of a nested peR, and sequenced. Initial peR amplified a region spanning from the M gene nt 778 to F gene nt 545 using primers Kl and K2. These amplicons were purified and used as template in a nested peR using primers MV1 (M gene nt 1163) and B2 (F gene nt 470). Sequence analysis revealed that the amino acid sequence at the fusion protein cleavage site of strains M89/98 (Ost Pool I), M57/98,and M308/98 was RRQKR -l-F, indicative of velogenie strains. Strain M89/98 (Av Pool 2) displayed a sequence at the fusion protein cleavage site that is characteristic of lentogenie strains GRQGR-l-L. This finding suggests that genotype VIIb isolates are heterogeneous, composed of strains of varying pathogenicity . Phylogenetic analysis based on a 378nt long region of the nested peR amplicon allowed the grouping of all these isolates into genotype VIIb. A 1.5-1% divergence was observed between group VIIb isolates collected in 1993, and 1995, and those used in this study, collected in 1998. This genetic distance is consistent with a 0.5-1% divergence in strains per year. The remaining field strains not detected by RT-peR, but that displayed embryo mortality indicative of pathogenic agents were subjected to HAlHI tests. Strains M193/98 (Ost Pool 6), M183/98 (Ost Liv B), and M193/98 (Ost Pool 5) agglutinated chicken erythrocytes. Haemagglutination was not inhibited by anti-NDV serum. These results led to the suspicion of the presence of an unidentified pathogen, most likely avian influenza, as all three samples were collected from ostrich hosts, from which this virus has previously been isolated in South Africa.Item Open Access Establishment of serological and molecular techiques to investigate diversity of psittacine beak and feather disease virus in different psittacine birds in South Africa(University of the Free State, 2004) Kondiah, Kulsum; Bragg, R. R.; Albertyn, J.Psittacine beak and feather disease (PBFD) is a readily recognizable disease of wild and captive psittacines in Australia but it is also a problem worldwide wherever captive species are bred. The disease caused by beak and feather disease virus (BFDV) is characterized by the progressive development of feather dystrophy and loss. Although the occurrence of PBFD in South Africa has been reported only recently, it is already rampant and threatens the extinction of the endangered Cape parrot and black-cheeked lovebird. Currently no vaccine for PBFD is commercially available but the loss of approximately 10-20% of breeding stocks of psittacines annually in South Africa alone is enough to realise the importance of one. Genetic and antigenic differences in BFDV are significant aspects for production of a vaccine but the lack of a culture system for BFDV has limited studies into its genetics, antigenicity and pathogenicity. The objective of the study thus became to establish techniques that could be used to investigate genetic and antigenic differences that may be present in BFDV in South African psittacines. Molecular investigations involved the testing dried blood samples for BFDV nucleic acid using polymerase chain reaction (PCR). A region within the Rep gene was amplified and digested with HaeIII to yield restriction length fragment polymorphisms (RFLPs). Six RFLPs were identified, cloned, sequenced (UFS 1- 6) and phylogenetically analysed. BFDV was purified from body organs of PBFD- affected birds by cesium chloride density gradient centrifugation and fractions tested by PCR and haemagglutination (HA) assays. BFDV-specific antibodies were raised in two rabbits by inoculation with purified BFDV in Freund’s incomplete adjuvant and tested by haemagglutination inhibition (HI) assays and an enzyme-linked immunosorbent assay (ELISA). HA and HI assays were attempted using erythrocytes from African grey parrots and Brown-headed parrots. HI assays were also used to test parrot sera for the presence of antibodies to BFDV. UFS 1-6 were closely related to known BFDV isolates and UFS 1 may represent a unique genotype in South Africa as it was separated from its closely related isolates by a 90% bootstrap value. UFS 3, 4 and 5 isolated from budgerigars belong to the budgerigar lineage as they clustered with isolate BG3-NZ. Together with three previously identified genotypes, UFS 1 indicates the introduction of BFDV into southern Africa on four separate occasions. Purification of BFDV from organs was successful but yielded low titres possibly because low quantities of virus were present in the organs or because little virus was circulating in the bird upon its death. PCR and HA assays confirmed the presence of BFDV in fractions; the two results correlated well. HA and HI assays were successfully established using erythrocytes from African grey parrots and Brown-headed parrots. Antibodies to BFDV were successfully detected in sera of three parrots by the HI assay. However, the assay could not detect non-psittacine raised antibodies (rabbit-raised) due to non-specific reactions. BFDV-specific antibodies were successfully raised in rabbits and were verified by the use of an ELISA. The high level of genetic diversity observed in the study compels further investigation into the genetics of BFDV as such levels of diversity may become a limiting factor in the applicability of PCR as a diagnostic test. The entire diversity of BFDV has not been studied and future work may lead to the identification of more BFDV strains, an important factor in vaccine development. The rabbit- raised antibodies together with the HA and HI assays and ELISA can be used to study the antigenic differences that may be present in known BFDV isolates that may also lead to the identification of more strains of the virus.Item Open Access Evaluation of the optical density reading for the determination of shelf life of whole chicken carcasses by the enumeration of the microbial contamination levels(University of the Free State, 2003-11) Banda, Thabo; Bragg, R. R.; Viljoen, B. C.English: Increased media and public interest over meat contamination have highlighted the need for continual improvements in this regard in the poultry industry. In spite of all the effort and money placed into research on microbial contamination of poultry carcasses, it is still not possible to produce carcasses, which are free of spoilage and pathogenic microorganisms. Carcasses are mostly contaminated during processing, contributing to the presence of high number of mesophilic microorganisms. The spoilage microorganisms cause consumers to reject the product due to appearance, off odour or undesirable flavour whereas the pathogenic microorganisms may lead to health hazards. The Standard plate counts methods have been the method of choice for the enumeration of contaminating microbial populations in the poultry industry. However, these methods are laborious and time consuming. In this study, optical density (OD) reading as a microbial enumeration method on poultry carcasses was established and evaluated for its application as a rapid, alternative method. In chapter 2, the dominant microorganisms were isolated and identified from eviscerated poultry carcasses. The carcasses were sampled by whole carcass rinse method and bacteria were isolated on Plate Count Agar at 27oC for 48 h. A total of five different species were identified, based on their prevalence in the sample and were identified by conventional methods. The identification of the isolates was further confirmed by API techniques. The dominant species were Escherichia coli, Shewanella putrefaciens, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus. Mean counts (log10 cfu/g) of these organisms were 3.08, 1.48, 0.85, 1.74, and 1.21 respectively. In chapter 3, OD readings as an enumeration method was established. This required the establishment of a number of parameters, which include: 1) Determining the wavelength to be used throughout this study. This achieved by scanning the medium through the spectra of 200-700 nm and a wavelength of 420 nm was selected as the optimal wavelength. 2) Determination of incubation time, which was achieved by the construction of separate growth curves for each isolates and determine the time when all isolates were in the exponential phase of growth. A time of 4 h was selected. 3) Evaluation of the repeatability of the OD readings of each isolate. Both pure culture and mixed culture were evaluated. The coefficients of variation for repeatability evaluations had coefficients of variation less than 15% for pure culture. Similar coefficients of variation values was obtained with mixed cultures and most values were even below 10%. 4) The correlation of OD reading evaluated after 4 h incubation at 37oC to standard plate counts (incubated in PCA at 37oC for 24 h). The scatter plots graph obtained in this study had had a positive strong correlation, which was above 0.9 for all isolates, except for Serratia marcescens, which had the correlation coefficient of 0.886. The high repeatability and correlation showed high potential of the OD in enumeration, hence the further objective of the involved evaluating the method on chicken carcasses. In chapter 4, carcasses were sterilized, followed by artificially inoculation with known microorganisms at different load. Sterilization was evaluated by examining different concentrations of Virukill solution at different concentration. Virukill is a non-toxic, highly effective disinfectant. High concentration (1, 2 & 3 v/v%) of this disinfectant was effective in eradicating all microbial population under the applied conditions. It was established that at these high levels, substantial residual effects were found which prevented the re-inoculation of chicken carcasses. With lower concentrations, it was established that a 0.3 v/v% of Virukill eradicated all microorganisms and did not have residual effect, thus allowing re-inoculation of the carcasses with known concentrations of bacteria. A correlation coefficient between standard plate counts and the OD reading method of 0.903 and 0.968 were found for mixed cultures determined by whole carcass rinse and neck skin sampling methods, respectively. A. hydrophila, had correlation coefficient of 0.849 and 0.985 for whole carcass rinse and neck skin respectively. Similarly Serratia marcescens had 0.993 and 0.940 for whole rinse and neck skin respectively. The results found in this study clearly show that bacterial enumeration through the use of OD readings is capable of reducing time and labour required to obtain the initial microbial load after processing of the carcasses. The OD method evaluated on artificially contaminated carcasses is promising. The method shows great potential for enumeration of bacteria during routine evaluation at the poultry processing plant.Item Open Access The identification of unknown poultry viruses through established methods(University of the Free State, 2009-03-31) Lee, Ji-Yun; Bragg, R. R.English: The poultry industry suffers severely every year due to bacterial and viral infections. Hence, great effort has been incorporated into isolating and identifying the different microorganisms that afflict poultry with their disease. The efficiency of the vaccines provided against such infections is only as good as the research of the responsible bacterium or virus. There are cases, however, when “unknown” viruses create havoc within industry. This is mainly due to the a) virulent nature of the virus, b) frequency of infection and c) lack of knowledge about the virus. The unknown virus may be a truly novel virus or an existing virus that has mutated whilst replicating. It is thus important to refine the procedure of isolation and identification of the poultry viruses in order for outbreaks of an unknown virus to be dealt with quickly and efficiently. In this study, the isolation and passaging of two unknown virus samples as Newcastle disease virus (NDV) but failed to react with ND-specific PCR primers and did not haemagglutinate red blood cells were investigated These samples were two of a group of viruses submitted as NDV to the Veterinary Biotechnology Laboratory of the University of the Free State by the Onderstepoort Veterinary Institute. The aim of this study was to investigate into the identity of the two unknown virus samples, D1446/95 and 834/05, using various methods of identification. Procedures performed on this virus include:(presumed to be Newcastle propagation in embryonated SPF eggs and primary chicken embryo fibroblast (CEF) cell cultures; Disease Virus), purification of virus samples for ultrastructure identification (ultrastulu studies as by transmission electron microscopy (TEM) and negative staining;) serology work as per haemagglutination and mean death time (MDT) studies as well as restriction transcriptase polymerase chain reaction (RT-PCR) work. The virus samples were cultivated in embryonated SPF eggs via the allantoic cavity route. The embryo morality was observed and recorded, and the allantoic fluid containing the virus was successfully harvested. CEF primary cell cultures that were prepared in-house were inoculated with the individual samples separately. The virus samples showed cytopathic effects (CPE) after inoculation and incubation of the cell cultures. These CPE indicated that the virus samples infected the CEF monolayer and could be grown on CEF cell cultures as well. However, the cultivation of the virus samples in SPF eggs was preferred due to the difficulty that the primary cell cultures presented in a laboratory that is not directed at cell culture production and use. Through haemagglutination (HA) tests the virus samples were found to be unable to haemagglutinate chicken red blood cells (RBCs). It is speculated through this result that the virus samples are not classical NDV. However, it is entirely possible that the viruses agglutinate the RBCs of other mammalian or avian species. It is possible that these viruses may be evolved from classical NDV into, for example, Goose paramyxovirus The ultrastructure of the virus sample D1446/95 as observed through transmission electron microscopy, found an enveloped virus that measures about 100 nm to 150 nm in diameter. Although the ultrastructure of the virus does not conclusively identify the virus, it helps in preliminarily grouping it to other known viruses with similar ultrastructure and eliminating it from groups of viruses that have obvious differences in ultrastructure. Investigation into the virulence of the virus samples using the mean death time (MDT) indicated that the virus sample D1446/95 was moderately virulent with an MDT of 71,75 hours and the sample 834/05 was highly virulent with an MDT of 60 hours. As NDV ranges from being highly virulent (velogenic strains) to having low virulent (lentogenic strains) the MDT results were compared to the standard of the MDT of NDV. The standard of MDT for NDV states that velogenic strains take less than 60 hours to kill; mesogenic strains kill between 60 to 90 hours and lentogenic strains take longer than 90 hours to kill the SPF embryos. After the extraction of RNA from the harvested virus sample using TRIzol reagent, the viral RNA of D1446/95 and 834/05 were amplified using RT-PCR, primer sets designed based on Goose paramyxovirus ZJ1 and the Access RT-PCR System (Promega). The RT-PCR products were run and observed on 1% gels using gel electrophoresis. The RT-PCR products of the two virus samples resulted in multiple bands for the two virus samples that indicated that they are probably not classical NDV, but may possibly be NDV of other species perhaps even of goose NDV. However, this is still to be investigated further. Thus, through this study it was found that the unidentified virus samples D1446/95 and 834/05 are most likely not classical NDV, but rather could be a variation of classical NDV that may be branching off into paramyxovirus of other poultry or mammalian species as was found in the study of Goose paramyxovirus.Item Open Access Improving the current diagnostic strategy for beak and feather disease virus in parrots(University of the Free State, 2014) Munsamy, Yuri; Bragg, R. R.; Boucher, C. E.English: Beak and feather disease (BFD), caused by Beak and feather disease virus (BFDV) is a dermatological condition afflicting parrot species. It is becoming increasingly difficult to ignore not only the significant negative economic impact that the virus has on the parrot breeding industry but also the detrimental effect it has on the survival of the endemic Cape parrot (Poicephalus robustus). The virus, a member of the Circoviridae, is known to possess a non-enveloped, circular, single-stranded DNA genome. Two major open reading frames (ORFs) encode the replication associated protein (Rep) and the coat protein (CP). The study was set out to evaluate and improve the current diagnostic strategy for BFDV, with both molecular and serological techniques. The following objectives were attempted: 1. To evaluate polymerase chain reaction (PCR) and quantitative real-time polymerase chain reaction (qPCR) as diagnostic tools for BFDV. Detection of BFDV with conventional PCR is not always sensitive, especially in birds without clinical symptoms. Furthermore, genetic variance was suggested to have a detrimental effect on primer hybridisation. A real-time assay was designed to address these problems. It amplified a 115 bp fragment of ORF V1 and was able to quantify viral load. 2. To recombinantly express BFDV coat protein. A sustainable source of the main immunogen, coat protein, was needed for use in serological test development. Bacterial expression of BFDV CP was unsuccessful; however, BFDV CP from an alternative expression study was used as a serological diagnostic antigen. 3. To develop serological diagnostic tests for BFDV. A novel slide agglutination test was developed and will serve as an initial screening tool in serological diagnosis. Steps were made in the development of a competitive Enzyme Linked Immunosorbent Assay (ELISA) for a quantitative indication of immune response to BFDV. A significant proportion of asymptomatic BFDV infections exist. Using a combination approach of both molecular and serological tests increases the capacity to detect infections or exposure to virus. New techniques described should be used in conjunction with existing tests and should not completely replace conventional techniques for diagnosis of BFDV infection or detection of exposure to the virus.Item Open Access An investigation into the efflux mechanism against quaternary ammonium compounds as a contribution of bacterial resistance(University of the Free State, 2014-01) Coetzee, Marisa; Bragg, R. R.; Jansen, A. C.; Boucher, C. E.Bacterial infections, a major problem in the poultry industry, are controlled through the use of antibiotics. Due to the increase in antibiotic resistance and the restrictions placed on the use of antibiotics in animals, the poultry industry is slowly heading for a post-antibiotic era. The use of disinfectants, like quaternary ammonium compounds (QACs), could possibly be the last resort in the fight against bacterial infections. Resistance against QACs has been observed but needs to be investigated in order to prevent similar resistance problems. The overall aim of this study was to understand bacterial resistance to QACs, using the following objectives: To examine the presence of qac resistance genes in field isolates and determining if the number of genes present confer higher resistance. To study the expression of one of these qac resistance genes in the presence of increasing QAC concentration. To study the efflux system, to determine the uptake and efflux of disinfectant, and to determine if the bacteria causes structural changes of the disinfectant. Bacterial resistance is conferred through acquisition of resistance genes; therefore qac resistance genes were studied to understand resistance to QACs. Screening of field isolates using conventional PCR for qac resistance genes (smr, qacJ, qacH and qacG) showed that one strain could contain more than one resistance gene. This could not be correlated with the minimum inhibitory concentration (MIC) of the three QACs tested and possession of more genes did not necessarily make the strain more resistant. Staphylococcus aureus strains known to contain at least one of the resistance genes were also screened for these qac genes. Conventional PCR showed that all the genes could only be detected in the strain when it was exposed to QAC. Conversely, real-time PCR showed that the genes could be detected even in the absence of QAC, and was detected in susceptible strains as well. Therefore containing the genes does not necessarily confer resistance. Relative quantitative real-time PCR was used to determine the expression of the qacJ gene in the S. aureus strains VB3_qacJ and ATCC 25923. It was hypothesised that the expression of the qacJ gene would increase with increasing didecyldimethylammonium chloride (DDAC) concentration. However, in the VB3_qacJ strain there was no significant difference in expression when induced with different DDAC concentrations. Expression of known qac resistance genes needs to be investigated simultaneously to determine whether a relationship exists between the qac genes conferring resistance. The mechanism of resistance is mainly efflux of the disinfectants; thereby reducing the disinfectant concentration inside the cell. The efflux mechanism of S. aureus was also studied using liquid chromatography-mass spectrometry (LC-MS). The hypothesis was that the bacteria are able to alter the structure of QAC before extruding it from the cell. Samples were prepared for LC-MS using solid phase extraction (SPE). The SPE protocol was optimised for extraction DDAC from growth media. It was shown that the DDAC concentration in the growth media decreased after 18 hrs of growth. It was not determined if structural changes of DDAC occurred. Resistance to disinfectants is much more complex than originally thought, and therefore resistance is still not fully understood. Further research is required to understand the full mechanism of resistance against QACs, so that similar problems associated with antibiotic resistance can be prevented.Item Open Access The isolation and characterisation of a Psittacine Adenovirus from infected parrots in South Africa(University of the Free State, 2007-11) Mfenyana, Nandipha; Bragg, R. R.English: Incidences of an Adenoviral infection have been recently reported in South African psittacine birds. These birds have been diagnosed by histopathology post-mortem. This Psittacine Adenovirus (PsAdV) has been reported as the second most deadly disease-causing virus in psittacine species with Psittacine Beak and Feather Disease Virus (PBFD) being the first. High mortalities with no symptoms have been observed amongst the birds. There is limited information on the distribution and antigenic characterisation of PsAdV in South Africa. This prompted the investigation into the isolation and characterisation of this PsAdV by virtue of molecular and conventional techniques. Two birds suspected of being exposed to PsAdV were donated to the laboratory. Histopathological tests were already performed on these birds by a consulting veterinarian who found evidence supporting the Adenoviral infection. The livers of the parrots received were homogenised and DNA was then extracted from the suspension. These birds were from the same parrot breeding farm in the Free State region and their deaths occurred around September a year apart, inferring a seasonal occurrence of the infected birds. A polymerase chain reaction (PCR) was established as a rapid test to confirm the Adenoviral infection amongst the birds received. A primer pair amplifying the hexon gene loop1 variable region (L1) located in two conserved pedestral regions was modified using an existing primer pair as this gene codes for the hexon protein where the group, subgroup and type specific antigenic determinants are located. PCR resulted in the expected amplicon size of ~587bp for all the samples tested with the use of a Fowl Adenovirus (FAdV) received from the Faculty of Veterinary Science, Onderstepoort (Pretoria) as a positive control. The positive DNA products were then subjected to restriction fragment length polymorphism (RFLP) in order to investigate differences in the restriction profiles. The suspected PsAdV samples provided a similar profile compared to the different one elicited by the fowl sample where only two bands were similar to the other profile with two extra unique bands observed. These samples were then ligated into the pGem-Teasy vector and the positive clones were selected and sent to Inqaba Biotech for sequencing. The results where blasted on the NCBI GenBank Database and a high degree of similarity was found with the PsAdV sequences already on the database. Both the nucleotide and amino acid sequence alignments performed using DNAssist v2.2 showed a high homology with all the sequences. There were some differences observed and with the protein alignment the different amino acids observed were of the same group of amino acids. This suggested that the sequences were of the same genogroup. A neighbour-joining phylogenetic tree was constructed and this showed 4 major clusters where one of them represented the PsAdV sequences. The PsAdV sequences were separated from the other clusters by a 100% bootstrap value. The two minor branches within this cluster separating the UFS sequences from the reference PsAdV sequences suggested that the UFS samples could be different isolates. Attempts to cultivate the virus in primary chicken embryonated liver cells and SPF embryonated eggs were successful and a cytopathic effect (CPE) typical of Adenoviruses was seen in the cells. With the SPF eggs definite differences were observed between the infected embryos and the negative control embryos. These were also typical of Adenoviruses, particularly of group I Aviadenoviruses (AAVs). We propose that further studies be concentrated on the development of antibodies against this PsAdV in order to establish serological techniques so to be able to differentiate between different serogroups and isolates. We also propose the development of a vaccine against this psittacine Adenovirus so to put biosecurity measures in place.Item Open Access Isolation and characterisation of bacteriophages and their potential use for the control of bacterial infectons in poultry(University of the Free State, 2009-05-29) Kakoma, Kasweka; Bragg, R. R.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.Item Open Access Molecular characterization of Mycoplasma gallisepticum strains from South African poultry(University of the Free State, 2007-11) Mathengtheng, Lehlohonolo; Bragg, R. R.English: Mycoplasma gallisepticum is the most pathogenic avian Mycoplasma species and leads to great economic losses worldwide (Kleven, 2003). Prevention and control of this organism has been achieved by vaccination and antibiotic administration. Ferraz and Danelli (2003) reported on the most widely used live vaccines which are MG-F, Ts-11 and 6/85. The MG-F strain is currently not registered for use in South Africa. M. imitans is the most closely related organism to M. gallisepticum and was tentatively identified as M. gallisepticum until Bradbury and co-workers (1993) defined it as a different species. However, this species is still misidentified as M. gallisepticum due to serological cross-reactivity and M. gallisepticum specific primers also detecting M. imitans (Markham et al., 1999). While M. gallisepticum has been characterized in some countries, very little information is documented on the South African isolates. It therefore became the aim of this study to investigate the presence and diversity of this organism in Southern Africa, investigate the presence of M. imitans as well as to establish the relatedness of the Southern African strains to those isolated outside Africa. Samples were collected from serologically positive birds from different farms in South Africa and Zimbabwe. Two PCR (Polymerase Chain Reaction) assays were optimized, one to detect M. gallisepticum of a specific gene Mgc2, the other to detect both M. gallisepticum and M. imitans. A total of five samples were detected with the Mgc2-PCR, while almost all samples could be detected with the 16S rRNA gene-PCR. This is a possible indication of a different M. gallisepticum isolate that can be detected on the 16S rRNA gene level but not at the Mgc2 level, or the isolate could indeed be M. imitans. In a study by Woese and co-workers (1985), the Mgc2 gene was implicated as one of the rapidly mutating genes due to adaptation, a possible reason for the non- detection of Mgc2 but the detection of the 16S rRNA gene. Of the sequenced 16S rRNA gene-PCR amplicons, M. gallisepticum and M. imitans had the highest homology. However, there are only two complete sequences of the 16S rRNA genes that belong to two M. gallisepticum strains deposited in Genbank, thus limiting the information on the isolates detected. Restriction Fragment Length Polymorphisms (RFLPs) were performed and well optimized. Ts-11 gave the expected profile while tested samples could not be placed in any of the reference groups. It was observed, however, that the RFLP profile of one of the amplicons was similar to the 6/85 vaccine strain and subsequent results correlated with this finding. Two of the amplicons could be sequenced and further analyzed. A phylogenetic tree showed one of the amplicons clustering away from the other Mycoplasma species though its sequence was found to be that of M. gallisepticum or M. imitans. In another tree, one of the amplicons showed more homology to the pathogenic strains while the other showed more homology to the vaccine strain 6/85. The DGGE analyses showed that the amplicons consist of a mixed template which was the reason why some samples could not be properly sequenced. This might be an indication of mixed infection within the flocks. However, it was expected of the 16S rRNA to give these results. Furthermore, the DGGE results indicated that the vaccine strain Ts-11 is used to vaccinate some of the flocks, while other flocks are infected with wild-type Mycoplasma. The results also suggest the possibility of the presence of two copies of the amplified region of the 16S rRNA gene in this vaccine strain. The study has confirmed the presence of M. gallisepticum and the possible presence of M. imitans. Different yet closely related Mycoplasma isolates are also present in South Africa. These could represent novel strains or species and warrant further investigation.Item Open Access A molecular study of Mycoplasma gallisepticum field isolates from poultry in southern Africa(University of the Free State, 2012-07) Moretti, Serena Angela; Bragg, R. R.; Boucher, C. E.English: Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in both chickens and turkeys. Little research has been done to characterize the MG field strains present in southern Africa. Field evidence however, suggested breaks in the vaccinations against MG, despite the proper cold chain and correct administration. Molecular methods were used to screen various poultry farms in South Africa and Zimbabwe for MG. Isolates were further characterized by gene-targeted sequencing (GTS). Portions of the cytadhesin pvpA, gapA, mgc2 genes and the uncharacterized surface lipoprotein gene designated MGA_0319 were sequenced and analysed. Three MG strains were identified in this study: a Ts-11-like strain; a strain showing closest percentage identity to the atypical MG RV-2 strain found in Israel, however with the lack of a large deletion within the pvpA region; and lastly an isolate from Zimbabwe, likely to be a novel MG strain. The latter contained a unique, large, in-frame nucleotide insertion in the mgc2 gene. Antigenically-significant variation (determined in silico) within the membrane surface proteins of this isolate was found compared to the MG Rlow strain used as an inactive vaccine on the poultry farm. It is thus postulated that by MG altering its antigenic profile, it allows effective avoidance of immune recognition and antibodies produced as a result of vaccination with inactivated and possibly live MG vaccines. Further research is however needed to substantiate this claim.Item Open Access The potential of neokestose as a prebiotic for broiler chickens(University of the Free State, 2008) Van der Westhuizen, René Johan; Kilian, S. G.; Bragg, R. R.English: The inulin neoseries, trisaccharide, neokestose was produced by the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma Y4-3) during growth on sucrose. To produce neokestose, whole cells harvested from the late exponential growth phase were incubated for 36 to 40 h at 25 oC in 0.2 M citratephosphate buffer (pH 7) containing 220 g.l-1 sucrose. Neokestose made up about 50 % of this mixture, which was purified equally well by both a carbon:celite chromatography as well as a batch filtration process, when eluting with similar amounts of water followed by a 50 % ethanol elution step. A final product was combined from various purification runs which consisted of 82.6 % neokestose, 8.7 % sucrose, 7.6 % GF3, 1.2 % glucose and 0.1 % fructose. Lactobacillus and Bifidobacterium genera are considered part of the beneficial group in in the intestine of animal and man. Bifidobacterium levels were higher than Lactobacillus levels in the caeca of New Hampshire layers, whereas in this study only Lactobacillus species were found in broilers. The reason for the absence of the Bifidobacterium species in the caecum of broilers was not determined. The prebiotic effect was evaluated on 5 week old broiler caecal material in vitro over 24 hours based on the viable levels of the total anaerobic bacteria, Lactobacillus and coliforms. The prebiotic effect was also evaluated on viable levels of added Salmonella Typhi, Escherichia coli and Campylobacter jejuni. Volatile fatty acids and pH were measured. The effect of neokestose on these groups was compared to that of inulin, a known prebiotic, and glucose. The total anaerobe and Lactobacillus levels increased over 24 hours for neokestose, inulin and glucose. Although there was no significant difference between the treatments higher levels were found for neokestose and glucose than for inulin. A decrease in the viable levels of E. coli, S. Typhi and C. jejuni were seen over 24 hours. The production of acetic acid, butyric acid and propionic acid was not significantly different for the treatments and the control. The pH decrease over 24 hours for the treatments was significantly different from the control, which indicated that lactate (not measured) production was probably higher in the neokestose, inulin and glucose treatments. In vivo tests are, however, required to fully evaluate the prebiotic and “bifidogenic” effect of neokestose for broilers.