Incidence of porcine circovirus type 2 and porcine parvoviruses in swine herds of some communities in Eastern Cape, South Africa
- Authors: Afolabi, Kayode Olayinka
- Date: 2018
- Subjects: Swine -- Diseases Swine -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/9691 , vital:34820
- Description: Porcine circovirus type 2 (PCV2) is one of the swine pathogens of global economic importance. Since its first detection in early 1990s as the main etiologic agent of porcine multisystemic wasting syndrome (PMWS) and many other porcine circovirus-associated diseases (PCVAD), the virus has been extensively studied and has been found to be present in virtually all the pig producing countries of the world. As a viral pathogen that brings about clinical diseases aided by co-infecting pathogens, the involvement of many other viral agents including porcine parvoviruses (PPVs) have caught the attention of stakeholders worldwide. However, no surveillance study of the viral pathogens has been carried out in South Africa as there are little or no information on their prevalence in the swine herds of the country. This present study therefore aimed at detection and molecular characterization of PCV2 and PPVs in swine herds of some selected communities in Eastern Cape Province, South Africa. A total of 375 field samples were collected from seven commercial and communal farms from three District Municipalities of Eastern Cape, South Africa between 2015 and 2016. Structured questionnaires were also administered to each farm at the time of sample collection to obtain some important information relating to health status and farm management practices in the sampled farms. With the aid of conventional PCR method, 339 samples were initially screened for the presence of PCV2; positive amplicons were sequenced and obtained partial genomes of the virus were preliminarily analyzed. In order to obtain the complete genomes of the virus, four overlapping primer pairs were used to amplify the full-genome of PCV2 from the initial positive samples; amplified genomes were sequenced using the Sanger methods, sequenced PCV2 genomes were assembled and characterized. Furthermore, the prevalences of some designated PPVs in the sampled farms were obtained using 110 samples randomly selected from the previously archived samples and screened with 6 different primer pairs specific for the detection of 7 PPVs. All the amplified parvoviruses’ genomes were sequenced; their sequenced partial genomes were subsequently base-culled and analysed. The data obtained revealed that 54/339 (15.93 percent) samples from the swine herds were positive for PCV2; whereas the degree of occurrence of the viral pathogen as observed at farm level ranges from approximately 5.6 to 60 percent. The majority 15/17 (88 percent) of the analyzed partial sequences were found clustering with other PCV2b strains in the phylogenetic analysis. More interestingly, two other sequences obtained were also found clustering within PCV2d genotype in the initial screening and analysis. Furthermore, 15 complete PCV2 genomes were successfully amplified, sequenced and assembled. NJ and ML phylogenetic analysis of the complete ORF2 gene and full genomes unanimously showed 11 of the assembled genomes belonging to genotype PCV2b. Another 3 of the characterized sequences formed clade with other reference mutant PCV2b and PCV2b subtype 1C (PCV2d) strains from different parts of the world. The last sequence however, clustered with other reference strains belonging to PCV2 intermediate clade 2 (PCV2-IM2) recently identified in a global PCV2 strains phylogenetic analysis. Other genetic analyses including multiple sequence alignment and p-distance analysis also confirmed the outcomes of the phylogenetic analyses of the complete capsid gene and fullgenomes of the virus. On the other hand, the findings of the molecular profiling for PPVs showed that all the screened parvoviruses were present in the study area, having prevalence of 29.1 percent (PPV1), 21.8 percent (PPV2), 5.5 percent (PPV3), 43.6 percent (PPV4), 21.8 percent (PBo-likeV) and 44.6 percent for PBoV1 and PBoV2. Double infection of the screened PPVs was observed to be very rampant among the pigs as high as 20/110 (18.2 percent) for PPV2/PPV4 and PPV4/PBoV; followed by 19/110 (17.3 percent) of the samples for PPV1/PPV4 and PPV1/PBoV. Three of the viruses were found simultaneously in 19 of the screened samples representing 17.3 percent, whereas 8 (7.3 percent) samples were positive for four of the viruses. Phylogenetic analyses of PPV1, PPV2 and PBoVs 1 and 2 were conducted with two major clades homologous for each of them. This is the first report of PCV2 in swine herds of the Province and the first detection of PCV2b, PCV2d and PCV2-IM2 strains in South African swine herds. It follows the first reported case of PCV2a in an outbreak of porcine multisystemic wasting syndrome (PMWS) in Gauteng Province, South Africa over two decades ago. Also, this is the first major epidemiologic study on PPVs in the country following the initial case study of 1975. These findings confirmed the presence of the allimportant viral pathogens among pigs and also give preliminary insights into the possibility of co-infections of the pathogens in the studied area. This could however result in a serious large scale outbreak of devastating disease(s) associated with the viral pathogens, thereby ultimately resulting in huge economic losses if no appropriate measures are taken to effectively curb their spread across the country.
- Full Text:
- Date Issued: 2018
- Authors: Afolabi, Kayode Olayinka
- Date: 2018
- Subjects: Swine -- Diseases Swine -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/9691 , vital:34820
- Description: Porcine circovirus type 2 (PCV2) is one of the swine pathogens of global economic importance. Since its first detection in early 1990s as the main etiologic agent of porcine multisystemic wasting syndrome (PMWS) and many other porcine circovirus-associated diseases (PCVAD), the virus has been extensively studied and has been found to be present in virtually all the pig producing countries of the world. As a viral pathogen that brings about clinical diseases aided by co-infecting pathogens, the involvement of many other viral agents including porcine parvoviruses (PPVs) have caught the attention of stakeholders worldwide. However, no surveillance study of the viral pathogens has been carried out in South Africa as there are little or no information on their prevalence in the swine herds of the country. This present study therefore aimed at detection and molecular characterization of PCV2 and PPVs in swine herds of some selected communities in Eastern Cape Province, South Africa. A total of 375 field samples were collected from seven commercial and communal farms from three District Municipalities of Eastern Cape, South Africa between 2015 and 2016. Structured questionnaires were also administered to each farm at the time of sample collection to obtain some important information relating to health status and farm management practices in the sampled farms. With the aid of conventional PCR method, 339 samples were initially screened for the presence of PCV2; positive amplicons were sequenced and obtained partial genomes of the virus were preliminarily analyzed. In order to obtain the complete genomes of the virus, four overlapping primer pairs were used to amplify the full-genome of PCV2 from the initial positive samples; amplified genomes were sequenced using the Sanger methods, sequenced PCV2 genomes were assembled and characterized. Furthermore, the prevalences of some designated PPVs in the sampled farms were obtained using 110 samples randomly selected from the previously archived samples and screened with 6 different primer pairs specific for the detection of 7 PPVs. All the amplified parvoviruses’ genomes were sequenced; their sequenced partial genomes were subsequently base-culled and analysed. The data obtained revealed that 54/339 (15.93 percent) samples from the swine herds were positive for PCV2; whereas the degree of occurrence of the viral pathogen as observed at farm level ranges from approximately 5.6 to 60 percent. The majority 15/17 (88 percent) of the analyzed partial sequences were found clustering with other PCV2b strains in the phylogenetic analysis. More interestingly, two other sequences obtained were also found clustering within PCV2d genotype in the initial screening and analysis. Furthermore, 15 complete PCV2 genomes were successfully amplified, sequenced and assembled. NJ and ML phylogenetic analysis of the complete ORF2 gene and full genomes unanimously showed 11 of the assembled genomes belonging to genotype PCV2b. Another 3 of the characterized sequences formed clade with other reference mutant PCV2b and PCV2b subtype 1C (PCV2d) strains from different parts of the world. The last sequence however, clustered with other reference strains belonging to PCV2 intermediate clade 2 (PCV2-IM2) recently identified in a global PCV2 strains phylogenetic analysis. Other genetic analyses including multiple sequence alignment and p-distance analysis also confirmed the outcomes of the phylogenetic analyses of the complete capsid gene and fullgenomes of the virus. On the other hand, the findings of the molecular profiling for PPVs showed that all the screened parvoviruses were present in the study area, having prevalence of 29.1 percent (PPV1), 21.8 percent (PPV2), 5.5 percent (PPV3), 43.6 percent (PPV4), 21.8 percent (PBo-likeV) and 44.6 percent for PBoV1 and PBoV2. Double infection of the screened PPVs was observed to be very rampant among the pigs as high as 20/110 (18.2 percent) for PPV2/PPV4 and PPV4/PBoV; followed by 19/110 (17.3 percent) of the samples for PPV1/PPV4 and PPV1/PBoV. Three of the viruses were found simultaneously in 19 of the screened samples representing 17.3 percent, whereas 8 (7.3 percent) samples were positive for four of the viruses. Phylogenetic analyses of PPV1, PPV2 and PBoVs 1 and 2 were conducted with two major clades homologous for each of them. This is the first report of PCV2 in swine herds of the Province and the first detection of PCV2b, PCV2d and PCV2-IM2 strains in South African swine herds. It follows the first reported case of PCV2a in an outbreak of porcine multisystemic wasting syndrome (PMWS) in Gauteng Province, South Africa over two decades ago. Also, this is the first major epidemiologic study on PPVs in the country following the initial case study of 1975. These findings confirmed the presence of the allimportant viral pathogens among pigs and also give preliminary insights into the possibility of co-infections of the pathogens in the studied area. This could however result in a serious large scale outbreak of devastating disease(s) associated with the viral pathogens, thereby ultimately resulting in huge economic losses if no appropriate measures are taken to effectively curb their spread across the country.
- Full Text:
- Date Issued: 2018
Laccase production by selected bacteria species isolated from some aquatic and terrestrial milieu of the Eastern Cape : applications in wastewater treatment
- Authors: Unuofin, John Onolame
- Date: 2018
- Subjects: Laccase Sewage -- Purification -- Biological treatment Water -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/9626 , vital:34810
- Description: Aromatic pollutants are a diverse group of chemicals which are continuously produced from industrialization, urbanization and sophistication in technological advancement. Pristine water source polluted by these chemicls makes the water unsafe for human consumption, and as well disrupts the trophic structure of the aquatic milieu. Physico-chemical treatment techniques employed so far have been accompanied by major drawbacks which have overriden the relative successes recorded, hence, greener, simpler and more efficient methods of pollutant transformation is imperative. The prospect of enzymatic treatment of pollutants has gradually been receiving growing attention in contemporary times due to the their environmental friendliness and production economic feasiblity. Laccase, a multicopper oxidase has heightened its appeal towards environmental and biotechnological applications due to its broad substrate specificity and its requirement of atmospheric molecular oxygen as a cosubstrate and the discharge of water as the byproduct. Hence, this present study was designed to evaluate the biotechnological potentials of laccases produced by some bacteria species from some aquatic biomes of the Eastern Cape Province, South Africa. The laccase-producing bacteria were isolated from selected environmental samples by selective enrichment using selective aromatic compounds as sole carbon source and subsequently, laccase-screening phenolic substrates. The laccase-producing bacteria were identified by molecular techniques as proteobacteria belonging to the following genera: Achromobacter, Bordetella, Citrobacter, Pseudomonas and Stenotrophomonas. Optimisation of laccase production in a submerged fermentation was by traditional and statistical methods, where four isolates (Hb9c; Achromobacter xylosoxidans HWN16, Hb16c; Bordetella bronchisepta HSO16, Berl11b2; Stenotrophomonas maltophilia BIJ16, Ie1c; Citrobacter freundii LLJ16) were evaluated for the fermentative production of laccase from lignocellulosic agroindustrial residues. Predictions from statistical optimisation showed that weakly acidic conditions (pH 5) and low agitation speed (100 rpm) were required for maximum laccase production from mandarin peelings (0.5 g/200 mL) and NaNO3 (0.25 g/200 mL) in Hb9c, maize stover (0.50 g/200 mL) and NaNO3 (0.050 g/200mL) in Berl11b2 while a lower agitation speed (50 rpm) was required for maximum laccase output from 2.0 g/200 mL maize stover and 0.050 g/200 mL KNO3 in Ie1c. However, 2.50 g/200 mL wheat bran, 0.050 g/200 mL yeast extract and 50 rpm agitiation under acidic conditions (pH3) yielded maximum laccase titres in Hb16c. Further characterisation of Hb9c and Ie1c laccase secretions portrayed their polyextremotolerant capacities. They were active at a broad range of tempertaure (0-90 degreesC); with optima at 70°C (Hb9c) and 60°C (Ie1c), pH (3-11); with optima at pH 6 (Ie1c) and pH 8 (Hb9c), respectively, and were equally thermo- and pH-stable. Their activities were either improved or left unabated by high concentrations of cations, detergents, and chloride. In addition, catalytic activities of Hb9c and Ie1c laccase secretions increased when they were preincubated with 2 – 20 percent of fluoride, a potent inhibitor. Consequently, a molecular perspective depicted the isolates to have multiple homologous laccase encoding genes. The enzymes were successfully immobilised on solid supports comprising gelatin and Na-alginate with a recovery of cca. 85 percent residual activity after 8 cycles of oprertional stability experiments. The immobilised laccases were remarkable in the decolourisation of synthetic dyes, albeit, free forms also elicited satisfactory performances. Ultimately, the application of free laccases in denim bleaching, individually or with a blend of a mediator, ABTS, showed that denim colours could be bleached without the need for chemical bleaching agents. The results obtained suggest the bacteria laccases produced from lignocellulosic wastes may serve as potent degraders of phenolic pollutants in water and, may also contribute to the bioeconomy and promote greener techniques for industrial applications.
- Full Text:
- Date Issued: 2018
- Authors: Unuofin, John Onolame
- Date: 2018
- Subjects: Laccase Sewage -- Purification -- Biological treatment Water -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/9626 , vital:34810
- Description: Aromatic pollutants are a diverse group of chemicals which are continuously produced from industrialization, urbanization and sophistication in technological advancement. Pristine water source polluted by these chemicls makes the water unsafe for human consumption, and as well disrupts the trophic structure of the aquatic milieu. Physico-chemical treatment techniques employed so far have been accompanied by major drawbacks which have overriden the relative successes recorded, hence, greener, simpler and more efficient methods of pollutant transformation is imperative. The prospect of enzymatic treatment of pollutants has gradually been receiving growing attention in contemporary times due to the their environmental friendliness and production economic feasiblity. Laccase, a multicopper oxidase has heightened its appeal towards environmental and biotechnological applications due to its broad substrate specificity and its requirement of atmospheric molecular oxygen as a cosubstrate and the discharge of water as the byproduct. Hence, this present study was designed to evaluate the biotechnological potentials of laccases produced by some bacteria species from some aquatic biomes of the Eastern Cape Province, South Africa. The laccase-producing bacteria were isolated from selected environmental samples by selective enrichment using selective aromatic compounds as sole carbon source and subsequently, laccase-screening phenolic substrates. The laccase-producing bacteria were identified by molecular techniques as proteobacteria belonging to the following genera: Achromobacter, Bordetella, Citrobacter, Pseudomonas and Stenotrophomonas. Optimisation of laccase production in a submerged fermentation was by traditional and statistical methods, where four isolates (Hb9c; Achromobacter xylosoxidans HWN16, Hb16c; Bordetella bronchisepta HSO16, Berl11b2; Stenotrophomonas maltophilia BIJ16, Ie1c; Citrobacter freundii LLJ16) were evaluated for the fermentative production of laccase from lignocellulosic agroindustrial residues. Predictions from statistical optimisation showed that weakly acidic conditions (pH 5) and low agitation speed (100 rpm) were required for maximum laccase production from mandarin peelings (0.5 g/200 mL) and NaNO3 (0.25 g/200 mL) in Hb9c, maize stover (0.50 g/200 mL) and NaNO3 (0.050 g/200mL) in Berl11b2 while a lower agitation speed (50 rpm) was required for maximum laccase output from 2.0 g/200 mL maize stover and 0.050 g/200 mL KNO3 in Ie1c. However, 2.50 g/200 mL wheat bran, 0.050 g/200 mL yeast extract and 50 rpm agitiation under acidic conditions (pH3) yielded maximum laccase titres in Hb16c. Further characterisation of Hb9c and Ie1c laccase secretions portrayed their polyextremotolerant capacities. They were active at a broad range of tempertaure (0-90 degreesC); with optima at 70°C (Hb9c) and 60°C (Ie1c), pH (3-11); with optima at pH 6 (Ie1c) and pH 8 (Hb9c), respectively, and were equally thermo- and pH-stable. Their activities were either improved or left unabated by high concentrations of cations, detergents, and chloride. In addition, catalytic activities of Hb9c and Ie1c laccase secretions increased when they were preincubated with 2 – 20 percent of fluoride, a potent inhibitor. Consequently, a molecular perspective depicted the isolates to have multiple homologous laccase encoding genes. The enzymes were successfully immobilised on solid supports comprising gelatin and Na-alginate with a recovery of cca. 85 percent residual activity after 8 cycles of oprertional stability experiments. The immobilised laccases were remarkable in the decolourisation of synthetic dyes, albeit, free forms also elicited satisfactory performances. Ultimately, the application of free laccases in denim bleaching, individually or with a blend of a mediator, ABTS, showed that denim colours could be bleached without the need for chemical bleaching agents. The results obtained suggest the bacteria laccases produced from lignocellulosic wastes may serve as potent degraders of phenolic pollutants in water and, may also contribute to the bioeconomy and promote greener techniques for industrial applications.
- Full Text:
- Date Issued: 2018
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